Research Report 2010 - MDC

Research Report 2010MAX DELBRÜCK CENTERFOR MOLECULAR MEDICINEBERLIN-BUCHMEMBER OF THEHELMHOLTZ ASSOCIATION

Research Report 2010

ContentInhaltContentInhalt..................................................................................................................................................................................................IIDirector's introduction by Walter RosenthalBuilding our future on existing strengths: Extending the path from basic scienceto clinical medicineEinleitung Walter RosenthalDen Weg von der Grundlagenforschung in die klinische Medizin ausbauen:Entwicklung und Stärkung des vorhandenen Potentials.............................................................................................................................................................................................................. VIIICardiovascular and Metabolic Disease ResearchHerz-Kreislauf- und Stoffwechselerkrankungen ....................................... 1Coordinator: Thomas E. WillnowBasic Cardiovascular FunctionMolecular Cardiovascular ResearchThomas E. Willnow ...................................................................................................................................................................... 6Molecular Mechanisms in Embryonic Forebrain DevelopmentAnnette Hammes (Delbrück Fellow).................................................................................................................................... 10Molecular Muscle PhysiologyIngo L. Morano............................................................................................................................................................................... 12Neuromuscular and Cardiovascular Cell BiologyMichael Gotthardt ....................................................................................................................................................................... 16Epithelial Morphogenesis and Zebrafish GeneticsSalim Seyfried ................................................................................................................................................................................ 18Angiogenesis and Cardiovascular PathologyFerdinand le Noble....................................................................................................................................................................... 20Molecular and Cellular Basis of Embryonic DevelopmentFrancesca M. Spagnoli................................................................................................................................................................ 22Developmental Biology and Pathophysiology of the KidneyKai M. Schmidt-Ott ...................................................................................................................................................................... 24Genetics and Pathophysiology of Cardiovascular DiseasesSystems Biology of Gene Regulatory ElementsNikolaus Rajewsky........................................................................................................................................................................ 26Medical Genomics and Genetics of Complex Cardiovascular DiseasesNorbert Hübner ............................................................................................................................................................................ 30Hypertension, Vascular Disease, Genetics, and NephrologyFriedrich C. Luft.............................................................................................................................................................................. 32II Content

Mechanisms of Hypertension-Induced Target Organ DamageDominik N. Müller (Delbrück Fellow)................................................................................................................................... 36Clinical CardiologyRainer Dietz..................................................................................................................................................................................... 38Cardiovascular Molecular GeneticsLudwig Thierfelder....................................................................................................................................................................... 42Magnetic ResonanceThoralf Niendorf ........................................................................................................................................................................... 46Cardiovascular HormonesMichael Bader ................................................................................................................................................................................ 48Mobile DNAZsuzsanna Izsvák .......................................................................................................................................................................... 52Genetics of Allergic DiseaseYoung-Ae Lee.................................................................................................................................................................................. 54Cell Signaling and Mass SpectrometryMatthias Selbach.......................................................................................................................................................................... 56microRNAs and Mechanisms of Metabolic DiseasesMatthew Poy .................................................................................................................................................................................. 58Mathematical Modelling of Cellular SystemsJana Wolf.......................................................................................................................................................................................... 60RNA Biology and Post-transcriptional regulationMarkus Landthaler....................................................................................................................................................................... 62Cancer ResearchKrebsforschungsprogramm ..................................................................................................................... 65Coordinator: Claus ScheidereitSignaling Pathways, Cell and Tumor BiologySignal Transduction in Tumor CellsClaus Scheidereit .......................................................................................................................................................................... 78Signals Provided by Wnt/β-catenin and Met/Gab1/Shp2 in Development and CancerWalter Birchmeier ........................................................................................................................................................................ 82Signaling Mechanisms in Embryonic Stem CellsDaniel Besser (Delbrück Fellow)............................................................................................................................................. 86Genetics of Tumor Progression and MetastasisUlrike Ziebold ................................................................................................................................................................................. 88Cell Differentiation and TumorigenesisAchim Leutz .................................................................................................................................................................................... 90Cancer, Stem Cells, and Transcription FactorsFrank Rosenbauer ........................................................................................................................................................................ 94Content III

Surgical OncologyPeter M. Schlag.............................................................................................................................................................................. 96Cancer Genetics and Cellular Stress Responses in Pathogenesis andTreatment of Lymphatic MalignanciesClemens A. Schmitt ..................................................................................................................................................................... 100Mechanisms of Protein Quality ControlThomas Sommer .......................................................................................................................................................................... 102Nuclear Signaling and Chromosomal DomainsHarald Saumweber...................................................................................................................................................................... 106Structural and Functional GenomicsMacromolecular Structure and InteractionUdo Heinemann ........................................................................................................................................................................... 108Structure and Mechanism of Membrane-remodeling G-proteinsOliver Daumke ............................................................................................................................................................................... 112Bioethics and Science CommunicationChristof Tannert ............................................................................................................................................................................ 114Tumor ImmunologyDifferentiation and Growth Control in Lymphocyte Development and ImmunopathogenesisMartin Lipp...................................................................................................................................................................................... 116Regulatory Mechanisms of Lymphocyte Trafficking in Homeostasis and ImmunopathogenesisUta E. Höpken (Delbrück Fellow) ........................................................................................................................................... 120Molecular Immunology and Gene TherapyThomas Blankenstein ................................................................................................................................................................. 122Molecular Cell Biology and Gene TherapyWolfgang Uckert........................................................................................................................................................................... 124Biology and Targeted Therapy of LymphomaBernd Dörken ................................................................................................................................................................................. 126Molecular ImmunotherapyAntonio Pezzutto .......................................................................................................................................................................... 130Clinical and Molecular OncologyPeter Daniel..................................................................................................................................................................................... 132Cellular Immunology of Autoimmune Reactions – Controlling the Balancebetween Effector and Suppressor T CellsKirsten FalkOlaf Rötzschke ............................................................................................................................................................................... 134Experimental PharmacologyIduna Fichtner................................................................................................................................................................................ 136IV Content

Function and Dysfunction of the Nervous SystemFunktion und Dysfunktion des Nervensystems .............................................. 141Coordinator: Carmen BirchmeierSignaling Pathways and Mechanisms in the Nervous SystemDevelopmental Biology/Signal TransductionCarmen Birchmeier...................................................................................................................................................................... 150Physiology and Pathology of Ion TransportThomas Jentsch ............................................................................................................................................................................ 154Neuronal ConnectivityFritz G. Rathjen .............................................................................................................................................................................. 158Molecular Physiology of Somatic SensationGary R. Lewin .................................................................................................................................................................................. 162Molecular Neurobiology of Cell-surface Channels and ReceptorsInes Ibañez-Tallon......................................................................................................................................................................... 166RNA Editing and Hyperexcitability DisordersJochen C. Meier.............................................................................................................................................................................. 168Signaling and Transport ProcessesBjörn Christian Schroeder ........................................................................................................................................................ 170Sensory NeurobiologyJan Siemens .................................................................................................................................................................................... 172Neural Circuits and BehaviorJames Poulet ................................................................................................................................................................................. 174Imaging of the Living BrainMolecular NeurologyFrauke Zipp ........................................................................................................................................................................................ 176Pathophysiological Mechanisms of Neurological and Psychiatric DisordersCellular NeurosciencesHelmut Kettenmann................................................................................................................................................................... 178Proteomics and Molecular Mechanisms of Neurodegenerative DisordersErich Wanker................................................................................................................................................................................... 182Aging-related Protein Misfolding and Detoxification MechanismsJan Bieschke (Delbrück Fellow)............................................................................................................................................... 186Content V

at the MDC. The role of the institute is to provide aframework in which researchers can carry out theirwork at the highest possible level and, ideally, totranslate their findings into new forms of prevention,diagnoses and therapies of diseases. To accomplishthese ambitious goals we must respond to changesand anticipate new opportunities – which, in the currentlandscape of research, means reworking some ofour internal infrastructures, as well as those that connectus to our partners in Berlin, Germany, and theinternational scientific community. In this brief introductionI would like to give readers a glimpse of thelogic behind our current activities and future plans.Several of the major, ongoing projects were conceivedand began maturing during the administration of mypredecessor, Walter Birchmeier, who served as theScientific Director of the MDC until the end of 2008and continues to head a research group within theinstitute. When I had the privilege of being named hissuccessor at the beginning of 2009, I stepped into aninstitute that is thriving and producing a steady outputof excellent scientific work. That state of affairsshould be considered part of Walter Birchmeier’slegacy. All of us who have worked with him know ofthe high priority he has placed on the quality of theMDC’s research and the continual need to recruit talentedyoung scientists from around the world. Thesuccess of that strategy is documented within thesepages.Research reports provide an institute-centricoverview of the state of the art in the main areas ofour research: cardiovascular and metabolic disease,cancer, and function and diseases of the nervous system.This work is going on at a time when the life sciencesare becoming very integrative. Over the lastdecade, advanced new methods have producedamazing amounts of data which cover aspects of lifethat have never been directly observable and arefinally allowing us to link the structures and functionsof molecules to the behavior of cells, embryonicdevelopment, and the processes that underlie lifethreateningdiseases. For example, until very recently,it was virtually impossible to capture quantitativedata about the proteins active in different types ofcells – a huge bottleneck in molecular biology.des MDC als Netzwerk und Infrastruktur, das die Interaktionder Wissenschaftler aus den unterschiedlichenDisziplinen untereinander fördert und den Wissenschaftlernmodernste Technologien und Infrastrukturenfür die Entwicklung neuer Methoden nicht nur inder Grundlagenforschung, sondern auch in der Prävention,Diagnostik und Therapie von Krankheiten zur Verfügungstellt.Um dieses anspruchsvolle Ziel zu erreichen, müssen wirauch unsere wissenschaftliche und organisatorischeInfrastruktur nachjustieren. Ebenso müssen wir unsereVerflechtung mit den wissenschaftlichen Partnern speziellin Berlin, in ganz Deutschland und in der internationalenwissenschaftlichen Gemeinschaft weiter entwickeln.Mit dieser kurzen Einführung möchte ich kurz das Konzeptunserer gegenwärtigen Arbeit und der zukünftigenEntwicklung des MDC skizzieren. Es wurde unterder Leitung meines Vorgängers, Walter Birchmeier, entworfen.Anfang 2009 hatte ich die Ehre, seine Nachfolgeals Direktor des MDC anzutreten, während WalterBirchmeier sich wieder ganz der wissenschaftlichenArbeit als Leiter seiner Forschungsgruppe widmete. Ichübernahm von ihm die Leitung eines Institutes, dessenMitarbeiter kontinuierlich wichtige wissenschaftlicheErkenntnisse gewinnen und in den besten Zeitschriftenveröffentlichen. Diese Leistung kann man als VermächtnisBirchmeiers an die nachfolgende jüngereGeneration ansehen: Wir alle haben in der Zusammenarbeitmit ihm erlebt, mit welcher Beharrlichkeit er diefür das MDC wichtigsten Ziele verfolgte: die Erhöhungder wissenschaftlichen Qualität und die Rekrutierungbegabter junger Forscher aus der ganzen Welt. DieserBericht dokumentiert auch den Erfolg seiner Arbeit alsDirektor des MDC.In den nachfolgenden Forschungsberichten werden diegewonnenen wissenschaftlichen Erkenntnisse in unserenHauptarbeitsgebieten kardiovaskuläre und metabolischeErkrankungen, Krebs sowie Erkrankungen desNervensystems dargestellt. Wie Sie in den einzelnenBerichten feststellen können, gibt es am MDC aber viele„Grenzgänger“; denn molekulare Fragestellungen lassensich häufig nicht auf einzelne Organe oder Krankheitenbeschränken.Darüber hinaus erleben wir gegenwärtig eine rasanteEntwicklung bei den verfügbaren Technologien undDirector's introduction IX

That barrier is now falling thanks to methods such asSILAC, a specialty of the laboratory of new group leaderMatthias Selbach. His lab is intensively collaboratingwith several others, including that of NikolausRajewsky, who has modified the method to investigatethe role of various types of small, noncoding RNAs in arange of developmental and cellular processes.Another example of transcending barriers is thesearch for multiple genes that contribute to complexhealth problems such as cardiovascular disease,Alzheimer’s, and many types of cancer. For many yearsit has been clear that some forms of these diseaseshave a genetic component, but the patterns by whichthey are inherited are obscure. Identifying the genesthat are responsible will likely require a huge effortinvolving many laboratories and clinics, very largegroups of patients and their families, new animalmodels, and sophisticated computer algorithms. TheMDC will play an active role in the establishment of ahuge national cohort organized by the HelmholtzAssociation in Germany, with the participation ofhundreds of thousands of randomly selected subjectsfrom all over the country, in a major project involvingHelmholtz partners such as the DKFZ, the HelmholtzCenter for Health and the Environment (HMGU) inMünich, the Helmholtz Center for Infectious Diseases(HZI) in Braunschweig, the German Center forNeurodegenerative Diseases (DZNE) in Bonn, andother national partner institutes. Regionally, the MDCwill cooperate with the Charité-Universitätsmedizinin Berlin, the German Institute for NutritionalResearch (DIfE) in Potstdam and the Robert KochInstitute (RKI) in the center of Berlin to recruitpatients from three regions within Berlin-Brandenburg: on the Charité Benjamin Franklin campusin the southwest, on the Charité Campus in thecity center, and on the Berlin-Buch campus in thenortheast. Locally, a total of 40,000 patients will berecruited. These people and the rest of the cohort willbe systematically studied and the results will providea basis for epidemiological research into the causes ofdiabetes, cardiovascular diseases, neurodegenerativediseases and cancer.Yet even prior to the establishment of this importantresource, our groups have made significant progressVerfahren, welche die Lebenswissenschaften insgesamtnachhaltig beeinflussen. Die Hochdurchsatzverfahren,die in kurzer Zeit eine riesige Menge an Daten liefern,die ein Jahrzehnt zuvor nicht vorstellbar waren, eröffnennicht nur ungeahnte Möglichkeiten, sondern stellendie Wissenschaftler auch vor neue Herausforderungen.Sie erlauben die komplette Bestandsaufnahmeeiner Zelle, Organ, Organismus zu einem bestimmtenZeitpunkt auf Genom-, Transkriptom-, Proteom- undMetabolomebene. Mit Hilfe eines umfangreichen ausgeklügeltenDatenmanagements und -analyse und derEntwicklung mathematischer Modelle können wirzukünftig eine Gesamtschau komplexer Prozesse erhalten,wie Embryonalentwicklung oder die Entstehungkomplexer Krankheiten. Ein wichtiger Baustein istdabei weiterhin die Aufklärung der Funktion einzelnerProteine in verschiedenen Zelltypen. Hochdurchsatzverfahrenerlauben auch hier jetzt eine Übersicht der Auswirkungenauf das Gesamtsystem – eine Möglichkeit,die der Molekularbiologie bisher weitgehend verschlossenwar. Ein wichtiger Beitrag ist die Weiterentwicklungder sogenannten SILAC-Methode durch unseren in2007 hinzu gekommenen NachwuchsgruppenleiterMatthias Selbach. Seine Gruppe kooperiert z.B. intensivmit Nikolaus Rajewsky. Die Anwendnung des weiterentwickeltenVerfahrens im Rahmen eines gemeinsamenProjektes hat entscheidend zum Verständnis derFunktion verschiedener Typen kleiner nicht kodierenderRNS-Moleküle beigetragen.Seit vielen Jahren ist es klar, dass die meisten Krankheitenkomplexe genetische und umweltbedingte Ursachenhaben. Daher wird die Helmholtz-Gemeinschaftgemeinsam mit Universitäten und außeruniversitärenForschungseinrichtungen in den nächsten Jahren einegroße nationale Kohorte aufbauen. Nach einer dreijährigenPlanungs- und Pilotphase sollen 200.000 Studienteilnehmeraus verschiedenen Regionen Deutschlandsrekrutiert, medizinisch untersucht und systematischweiter beobachtet werden. Die gewonnenenDaten dienen als Basis für zukünftige epidemiologischeUntersuchungen zu den Ursachen von Diabetes, Herz-Kreislauf-Erkrankungen, neurodegenerativen Erkrankungenund Krebs. Das MDC wird an der Rekrutierungder Großkohorte von 200.000 Probanden aktiv teilnehmen.Dies ist ein wichtiges Kooperationsprojekt mit denanderen Gesundheitszentren innerhalb der Helmholtz-Gemeinschaft, dem Deutschen Krebsforschungszen-X Director's introduction

in discovering genes that contribute to these diseasetypes. The groups of Norbert Hübner and FriedrichLuft, in a collaboration with Jan Monti of the Heliosclinic, discovered that a molecule called Ephx2 plays arole in the development of arrhythmic beating of theheart and to sudden death through heart failure inhypertensive rats. The groups also provide evidencethat Ephx2 plays a similar role in patients. This workbenefited enormously from Hübner’s studies of therat, one of the most important model organisms inmedical research, but which has lagged behindbecause of a lack of tools for genetic manipulationwhich are so highly developed in the mouse andother laboratory animals. The completion of the ratgenome in 2004 has given a major boost to laboratorieswhich combine computational and “wet lab”methods in the search for genes related to disease.New tools for the genetic manipulation of rats are theaim of the international, EU-funded consortiumEURATools, which is coordinated by Norbert Hübner.This type of work demonstrates a general principlethat has been successful at the MDC: combiningstudies of fundamental mechanisms and modelorganisms with work on patients. Thomas Willnow’sstudies of the family of low density lipoprotein receptor-relatedproteins (LRP) led to the discovery of amechanism involving sorLA, a receptor expressed inneurons, and the accumulation of amyloid plaques –proteins which aggregate between nerve cells in thedevelopment of Alzheimer’s disease. Systematic workon ion channels and transporters from the lab ofThomas Jentsch, who holds a joint appointment fromthe MDC and FMP, has revealed several mutationswhich cause changes in the structure of the inner earand lead to congenital deafness.The interface between clinical groups and basicresearch laboratories steadily produces findings thatare having a direct impact on the diagnosis and treatmentof disease. Over the past two years Peter Schlag,who has a joint appointment at the Charité and MDC,has established a Charité Comprehensive CancerCenter in downtown Berlin to ensure a coordinatedapproach to diagnoses, therapies, and the medical,psychological, and social care of patients. At the sametime, Schlag’s group at the MDC has identified thetrum (DKFZ) in Heidelberg, dem Helmholtz ZentrumMünchen für Gesundheit und Umwelt (HMGU), demHelmholtz Zentrum für Infektionsforschung (HZI) inBraunschweig und dem Deutschen Zentrum für NeurodegenerativeErkrankungen (DZNE) in Bonn sowieanderen nationalen Partnerinstituten. Regional wirddas MDC in Zusammenarbeit mit der Charité – UniversitätsmedizinBerlin, dem Deutschen Institut fürErnährungsforschung (DIfE) in Potsdam und demRobert Koch-Institut (RKI) in Berlin-Mitte an drei Ortenim Großraum Berlin-Brandenburg Probanden rekrutieren:im Südwesten am Charité-Campus BenjaminFranklin, im Stadtzentrum am Campus Charité Mitteund im Nordosten am Campus Berlin-Buch. Insgesamtsollen 40.000 Probanden rekrutiert werden.Wie die nachfolgenden Beispiele zeigen, ist die amMDC betriebene molekularmedizinische Forschung dieunverzichtbare Basis und häufig der Ausgangspunktfür Patienten- und Probanden-orientierte Projekte. DieForschungsgruppen von Norbert Hübner und FriedrichLuft, in Zusammenarbeit mit Jan Monti, Helios-Klinikumin Berlin-Buch, konnten im Tiermodell der hypertensivenRatte mit dem sogenannten Ephx2-Molekülund in „knockout“-Mäusen einen Faktor identifizieren,der für die Entstehung einer Herzinsuffizienz verantwortlichist. Darüber hinaus konnten Hinweise dafürgefunden werden, dass Ephx2 eine ähnliche Rolle beiPatienten spielt. Dies ist ein gutes Beispiel für dieBedeutung von Krankheitsmodellen im Tier. Durch dieFertigstellung des Rattengenom-Projekts im Jahre2004, an dem Hübners Gruppe maßgeblich beteiligtwar, können genetische Veränderungen bei erkranktenTieren leichter identifiziert werden. Heute koordiniertHübner das von der EU geförderte Konsortium EURA-TOOLS, in dem wichtige neue Methoden der genetischenAnalyse der Ratte entwickelt werden.Erfolge dieser Art sind ein Ergebnis eines allgemeinenArbeitsprinzips des MDC: der Kombination von Grundlagenforschungin Zellen und an Modellorganismenmit der Analyse von klinischen Daten. So führten ThomasWillnows Untersuchungen der LRP-Rezeptorfamiliezur Entdeckung eines Mechanismus für die Akkumulationvon Amyloidplaques, wie sie in den Nervenzellenbei der Alzheimer-Krankheit eine Rolle spielen.An diesem Vorgang ist sorLA, ein Rezeptor dieser Familie,der in Neuronen exprimiert wird, ursächlich beteiligt.Director's introduction XI

protein MACC1 as a marker for metastatic cells in colorectalcancer (CRC). MACC1 is involved in regulatinginjury response and tissue growth in the HGF/METsignaling pathway, which has been intensively studiedin Walter Birchmeier’s lab. The finding shouldallow doctors to assess the risk that CRC in a patientwill develop into a dangerous metastatic form, whichhas important implications in designing an effective,individualized treatment for the disease. Additionally,it establishes a mechanistic link between tumors anda crucial pathway involved in human development,contributing to our understanding of “cancer stemcells.”Yet another example of this type of interface is thelong-term collaboration of the labs of ClausScheidereit (MDC) and Bernd Dörken (Charité/MDC),along with the group of Martin Lipp and many othercolleagues. They have discovered many links betweenthe behavior of the transcription factor NF-kB and thedevelopment of Hodgkin’s disease and other lymphomas.Here, too, the findings are exposing mechanismsthat should result in improved diagnosis andtreatment for cancer patients.Another important step in carrying out our mission isto ensure that discoveries are capitalized on; to developa small-molecule inhibitor into a drug, for example,requires years of work and significant investments,and often the “ball is fumbled” in the handofffrom basic science to industry. Even a very importantfinding may require many further steps of developmentbefore it is at a state that it has become a basisfor founding a new company or before it attracts theinterest of the pharmaceutical industry. To smooththe way, the BMBF has sponsored a competitive programcalled Go-Bio, which provides several years ofsupport to academic groups to bridge the gapbetween a basic science discovery and commercialization.Since some promising projects are not quiteat a state to apply, the MDC has developed an internalstimulus program called “pre-Go-Bio,” to whichgroups can apply for three-year funding to prepare foran application within the Go-Bio program or withinanother program with a similar intention. In 2008two groups were awarded pre-Go-Bio grants; twomore received funding under the program in 2009.Im Labor von Thomas Jentsch werden die Funktion unddie Auswirkungen einer Fehlfunktion von Ionenkanälenund -transportern erforscht. Dabei entdeckten die Wissenschaftlermehrere Mutationen in bestimmtenIonenkanälen, die für Veränderungen in der Strukturdes Innenohrs verantwortlich sind und zu angeborenerTaubheit führen. Thomas Jentsch gehört gleichzeitig zuunserem Nachbarinstitut, dem Leibniz-Institut fürMolekulare Pharmakologie (FMP). Die Forschungsgruppewird von beiden Instituten jeweils zur Hälfte finanziert.Eine Besonderheit, die die enge Zusammenarbeitzwischen FMP und MDC illustriert.Ein wichtiges Anliegen des MDC ist und bleibt die engeInteraktion mit klinischen Gruppen der Charité. Die beiuns auf dem Campus Berlin-Buch gemeinsam mit derCharité entwickelten Schnittstellen zwischen klinischenGruppen und experimentell orientierten Grundlagenwissenschaftlernermöglicht nachhaltige Auswirkungenauf die Diagnostik und Behandlung von Krankheiten.In den vergangenen zwei Jahren hat der Charité-Kliniker Peter Schlag, der auch Leiter einer Arbeitsgruppeam MDC ist, im Zentrum von Berlin das sog. CharitéComprehensive Cancer Center aufgebaut. Dieses Zentrumermöglicht ein koordiniertes Herangehen andiagnostische, therapeutische, psycho- und sozialmedizinischeProbleme der Patienten. Im Rahmen ihrerexperimentell-wissenschaftlichen Arbeit am MDC hatdie Gruppe von P. Schlag in Zusammenarbeit mit WalterBirchmeier das Protein MACC1 identifiziert, das alsdiagnostischer Marker für Dickdarm- und Rektumkrebsverwendet werden könnte. MACC1 spielt im HFG/MET-Signalweg, der von Walter Birchmeiers Gruppe intensivuntersucht wird, eine wichtige Rolle und reguliertdadurch Gewebswachstum MACC1 und kann in der Klinikbei der Abschätzung des Risikos einer metatstatischenAusbreitung des Krebses wertvolle Informationenliefern, die in Zukunft möglicherweise eine gezielte,individuell angepasste Therapie ermöglicht. MACC1spielt möglicherweise auch eine Rolle bei der Entstehungsogenannter Krebsstammzellen.Ein weiteres Ergebnis dieser Art von interdisziplinärerForschung liefert die seit langem bestehende Zusammenarbeitder Forschungsgruppen von Claus Scheidereit(MDC) und Bernd Dörken (Charité/MDC) sowieder Gruppe von Martin Lipp (MDC) und weiteren Kollegen.Sie haben zahlreiche Bindeglieder zwischen demXII Director's introduction

The ECRC offers research space and funding, on acompetitive basis, for research projects of the Charitéand the MDC aiming to develop new methods for diseaseprevention, diagnosis, and therapy. It providesaccess to out-patient clinics and an infrastructure forinvestigator-initiated studies (phase 1 and early phase2). For clinicians, the ECRC offers protected researchtime outside the constraints of clinical routines in thehospital, and close proximity to MDC research groups.Members of all the clinical departments of theCharité and MDC scientists working on relevant projectsare eligible to participate in ECRC activities, uponthe submission of a proposal and a positive evaluationthrough peer review. Ongoing projects cover allthe major research areas of the MDC (cancer, cardiovasculardisease, and diseases of the nervous system).The ECRC’s educational objectives address the factthat physicians and biologists are still trained in quitedifferent ways and new programs are necessary totrain the “physician-scientists” of the future.In brief, the key features of the ECRC are the following:· Clinicians or basic researchers can apply for independentproject groups to conduct a translationalproject within the ECRC. Funding is providedfor three years (extension possible). The fundingdescision is based on a competitive evaluationinvolving external experts.· Clinicians and researchers of the MDC can jointlysubmit proposals for collaborative projects whichcan be funded through the ECRC after a competitiveevaluation (KKP program). The projects areconducted at the ECRC and/or the MDC.· Within the ECRC, the MDC and Charité jointlysponsor a training program for clinicians (KAP) inbasic molecular biology, in which clinicians take abreak from their duties and join one of the MDC’slaboratories. This promotes long-term collaborationsbetween the home and host .· Clinicians direct out-patient clinics and carry outclinical studies within the ECRC;· Clinicians and scientists have access to clincicalresearch center (outpatients clinics, beds for clinständnisfür den gesunden und den kranken Organismusentwickeln muss, nicht so einfach zu bewerkstelligenist. Eine Lösungsmöglichkeit besteht in der Einrichtungvon Forschungsgruppen, in denen Grundlagenwissenschaftlerund Kliniker langfristig eng zusammenarbeiten.Gleichwohl bleibt ein „kultureller Graben“zwischen beiden Welten bestehen, der jedoch aufsystematische, pragmatische und effektive Weise überwundenwerden kann. Dies ist die Motivation für mehreregrößere langfristige Initiativen, die wir für dieZukunft des MDC für entscheidend halten. Ich werde siehier nur kurz skizzieren, ohne einer späteren genauerenBeschreibung im Bericht vorzugreifen.Das Experimentelle und Klinische Forschungs -zentrum (ECRC)Seit Gründung des MDC hat die Zusammenarbeit vonGrundlagenforschung und klinischer Medizin durcheine Serie von Maßnahmen intensive Förderung erfahren.Darauf baut das ECRC auf, bei dem es sich um einkooperatives Projekt mit der Charité-Universitätsmedizinhandelt, das 2007 auf dem Bucher Campus gegründetwurde, um die Translationsforschung zu unterstützen.Diese enge Partnerschaft mit der Charité ermöglichtes, dass zukünftige ECRC-Projekte Probanden undPatienten tendenziell aus allen Bereichen der Universitätsklinikenrekrutieren können. Die Kliniken behandelnetwa 120 000 Patienten stationär und betreuen ca. 600000 Personen ambulant. Dies ist ein bedeutendes Hintergrundpotentialfür ambitiöse Projekte.Es folgt eine kurze Beschreibung der laufenden Pro -jekte.Das ECRC bietet Forschungsinfrakstruktur und Finanzierungsmöglichkeiten,für die sich Forschungsprojekteder Charité und des MDC mit dem Ziel der Entwicklungneuer Methoden der Vorbeugung, Diagnostik und Therapievon wichtigen Krankheiten bewerben können.Hinzu kommt als Infrastruktur die Möglichkeit, Probandenohne Klinikaufnahme zu rekrutieren und Phase-1-und frühe-Phase-2-Studien zu betreiben. Für klinischeForschung im engeren Sinne bietet das ECRC die Möglichkeit,ärztlich kontrollierte Forschung jenseits derengen Rahmenbedingungen und Abläufe im Krankenhausund in enger räumlicher Nachbarschaft zu Forschergruppenauf dem MDC-Campus durchzuführen.XIV Director's introduction

cical trials) and technology platforms, (e.g. ultrahigh-fieldMR instruments, GMP facility, bio -banking).Currently, the ECRC consists of two buildings on theBerlin-Buch campus: the Research Building (“For -schungs haus”) of the Charité (former Robert-Rössle-Klinik) for clinical activities and a new building of theMDC for the ultrahigh-Field MR facility equipped with7 T and 3 T whole-body human MR scanners and a 9.4T animal MR scanner. Constructions for a third buildingfinanced by the MDC, comprising 2600 m 2 of laboratoryand office space, will begin in spring 2010,with a target completion date of 2012.A big step forward was the appointment of ThoralfNiendorf as group leader at the MDC, as director ofthe ultrahigh-Field MR facility and professor (W3,chair) for ultrahigh-Field MR at the Charité in August2009. A physicist by training, Thoralf Niendorf is aleading expert in MR imaging with a research experiencein academia and industry.The ECRC structure will be crucial in establishing andutilizing the Helmholtz Cohort, in which 200,000people will be recruited across Germany, including40,000 from the Berlin region.. We are currentlyrecruiting for a W3 professorship (chair) in epidemiology.Besides other research projects, the appointedscientist will administer the Berlin arm of the cohort.The Berlin Institute for Medical Systems Biology(BIMSB)The greatest challenge for today’s biomedical scienceis to take what we have learned about the discreteelements of biological systems – such as genes, RNAs,and proteins – and understand how they worktogether in extremely complex, dynamic networks toproduce higher levels of structure such as cells, tissues,and organisms. This integrative view is essentialto learning how biological processes change duringdisease. Obtaining it will require amassing and analyzinghuge amounts of data from high-throughputtechnology platforms, in vitro studies, work with cells,and genetic and developmental studies of completeorganisms with an aim to drawing parallels betweenother species and humans. The narrowing gapAn den Forschungsprojekten des ECRC können im Prinzipje nach Fragestellung alle Wissenschaftler des MDCund aller beteiligten Abteilungen der Charité teilnehmen.Die laufenden Projekte konzentrieren sich auf dieHauptrichtungen des MDC, nämlich Krebsforschung,Herz-Kreislauf-Forschung und Krankheiten des Nervensystems.Entsprechende Abteilungen der Charitésind beteiligt.Die Ausbildungspläne des ECRC konzentrieren sich aufdie Überbrückung des immer noch sehr verschiedenenAusbildungsganges von Ärzten und Biologen und zielenauf das Training eines klinisch-experimentell spezialisiertenNachwuchses für die Zukunft.Kurz zusammengefasst lässt sich das ECRC wie folgtcharakterisieren:· Kliniker oder Grundlagenforscher können sich zuunabhängigen Forschungsgruppen zusammenschließen,um ein Translationsprojekt durchzuführen.Finanzielle Förderung ist für drei Jahre vorgesehen,Verlängerung und Ausbau sind möglich.Der Zuschlag wird auf der Basis einer wettbewerblichenAusschreibung vergeben.· Kliniker oder Grundlagenforscher können kooperativeProjekte zur Förderung einreichen, die ebenfallsauf interner Ausschreibungsbasis vergebenwird (KKP-Programm). Je nach Sachlage werdensolche Projekte im ECRC oder in anderen Einheitendes MDC durchgeführt.· Das ECRC führt ein Ausbildungsprogramm (KAP)über molekulare Grundlagen der Biomedizindurch, das von MDC und Charité-Kliniken gemeinsamunterstützt wird. So können klinisch tätigeÄrzte für einige Zeit in MDC-Laboratorien arbeiten,wodurch zu langfristiger Zusammenarbeitvon gastgebender Abeilung und Gastwissenschaftlermotiviert wird.· Das ECRC führt klinische und tagesklinische Forschungsarbeitenmit Patienten unter Anleitungvon erfahrenen Klinikern durch.· Sowohl klinische als auch experimentelle Forscherhaben Zugriff zur klinischen Forschungskapazität(stationäre und tagesklinische Betreuung) und zurtechnischen Ausrüstung (z.B. ultrahochauflösendeMagnetresonanz, GMP-Infrastruktur, Biobankauswertung).Director's introduction XV

etween disciplines such as biology, chemistry,physics, mathematics, engineering and other fieldsare dissolving to make this one of the most excitingeras in science, and a time at which progress canoften only be made through a new type of interdisciplinarity.Our own history aptly reflects this: work carriedout 50 years ago by the physicist Max Delbrück,after whom the MDC is named, with the geneticistTimoffeéf Ressovsky played a central role in the establishmentof modern molecular biology. This trend isincreasing and we must ensure that young scientistsare given the tools and concepts to cope.Over the past two years our reflections about theseissues at the MDC have coalesced into a proposal forthe establishment of a new institute, the BerlinInstitute for Medical Systems Biology (BIMSB), as animportant extension of our activities in the field. Asolid scientific program has been established, and theproposed size and scientific program were very positivelyevaluated by an international panel of expertsfrom relevant fields in April 2009. More details on theresearch and ongoing projects of the BIMSB appearlater in the book. Here I will briefly report on the statusof the project as a whole.Support for the establishment of BIMSB comesthrough the BMBF Initiative “Innovation and leadingresearch in the new German states” as well as by theSenate of Berlin. The MDC provides administration,infrastructure, and staff in support of the projectsand new groups. Nikolaus Rajewsky played a centralrole in the development of the proposal and is thespeaker and scientific coordinator of the project. Aswell as recruiting new groups with a systems biologyorientation to the MDC, the project is implementingscientific collaborations with the Charité, theHumboldt University of Berlin (HU), the FreeUniversity of Berlin (FU), the Leibniz Institute forMolecular Pharmacology (FMP), the Max PlanckInstitute for Molecular Genetics, the MPI for InfectionBiology, and other research institutions. In agreementwith the partners of BIMSB, a site has been chosen inthe city center for the construction of a BIMSB building:on the north campus of the HU Berlin close to itsinstitute for theoretical biology, to Charité clinics, andthe “Bernstein Center for Computational Neuro -Gegenwärtig verfügt das ECRC auf dem Bucher Campusüber zwei Gebäude: das Forschungshaus der Charitéfür klinische Forschung (vormals Robert-Rössle-Klinik)und das neue Gebäude, das das ultra-hochauflösendeMR-Gerät mit 7 T und 3 T Ganzkörper MR Scannernund einem 9.4 T Scanner für experimentelle Tierversuchebeherbergt. Ein drittes, vom MDC finanziertesGebäude, das 2600 m 2 an Labor- und Büroflächeumfassen wird, wird im Frühjahr 2010 begonnen werden.Die Fertigstellung ist für 2012 geplant.Ein großer Schritt in Richtung auf dieses Ziel ist mit der2009 gelungenen Bestellung von Thoralf Niendorfgetan worden. Er ist MDC-Gruppenleiter für die hochauflösendeMR-Technik am ECRC und gleichzeitig W3-Professor an der Charité. Thoralf Niendorf ist ein führenderSpezialist für bildgebende MR-Technik und hatForschungserfahrung sowohl im industriellen als auchim akademischen Bereich.Die geplante ECRC-Struktur wird von hoher Bedeutungsein für den Aufbau und die Arbeit mit der Helmholtz-Kohorte, die 200 000 Probanden und Patienten ausDeutschland, einschließlich 40 000 Personen aus derBerliner Region.Das Berliner Institut für Medizinische System -biologie (BIMSB) des MDCVor der biomedizinischen Forschung steht gegenwärtigdie große Herausforderung, die Funktionsweise der einzelnenKomponenten biologischer Systeme (Gene, RNS,Proteine, usw.) in ihrem Zusammenwirken in komplexendynamischen Netzwerken höherer Struktureinheiten(Zellen, Gewebe, Organismen) des Lebens zu verstehen.Große Schritte im Erkenntnisgewinn werden oft durchein multidisziplinäres Herangehen erreicht. Dies hatschon unser Namenspatron, Max Delbrück, bewiesen,der als Physiker in Zusammenarbeit mit dem GenetikerTimoffeéf Ressovsky die Molekularbiologie vor nahezu50 Jahren begründet hat. Eine ganzheitliche Sicht istauch für das Verständnis der bei Krankheitsprozessenablaufenden Veränderungen essentiell. Mit den heutezur Verfügung stehenden neuen Technologien undHochdurchsatzverfahren sind wir in der Lage, sehrgroße Datenmengen zu erzeugen, die nur mittelsmathematischer Ansätze analysiert und verstandenXVI Director's introduction

science.” This location will permit stronger ties in theeducation of university and PhD students.Training the “systems biologists” of the future is achallenge that needs to be addressed in a wellthought-out way. Thus education is an importantcomponent of the BIMSB, and currently the mostimportant element is the joint PhD program establishedbetween the MDC and New York University.This offers an original model whereby two instituteson different continents can strengthen their ties andwork together in a practical way through sharing students,assigned to two labs, who split their timebetween them. Although the project formally startedonly this year, it has already produced a publication inNature Methods, and other papers are in the pipeline.The state of Berlin has declared its intention to providethe funds for the construction of the new building.In a new type of “integrative” lab, scientists usingexperimental approaches will team up with thoseusing theoretical approaches. In order to optimallyestablish the many themes, disciplines, and technologiesof this research area, 25 groups (including at leastfive W3 professorships) and five technology platformsare planned.The BIMSB will also provide a basis for the developmentof the German Center for CardiovascularResearch (Deutsches Zentrum für Herz-Kreislauf -forschung, DZHK; see below) through an emphasis oncardiovasular and metabolic diseases (particularly inthe area of metabolomics).German Center for Cardiovascular Research(Deutsches Zentrum für Herz-Kreislauf -forschung, DZHK ) and German Institute forCardiovascular Research (Deutsches Institut fürHerz-Kreislaufforschung, DIHK )Cardiovascular diseases are the most common causeof death worldwide. They arise from a complex mixtureof genetic and environmental factors – an enormouschallenge as researchers try to track them backto specific genes, mechanistic processes in cells andintricate body systems. Such multifactorial diseasespose special problems including finding patients witha particular profile, extrapolating from a very smallnumber of cases, and thoroughly integratingwerden können. Die immer stärkere Verschränkungverschiedener Wissenschaftsdisziplinen wie Biologie,Chemie, Physik, Mathematik, Ingenieurwissenschaftenund auch Sozialwissenschaften ist eine wichtige undspannende Entwicklung. Diese Entwicklung muss sichlangfristig auch in der Ausbildung junger Wissenschaftlerniederschlagen.In den vergangenen zwei Jahren hat das MDC ein Konzepterarbeitet, in dem diesen Entwicklungen Rechnunggetragen wird. Das MDC plant mit der Errichtungdes Berliner Institut für Medizinische Systembiologie(BIMSB) als Teil des MDC eine wichtige Ausweitungseiner Aktivitäten im Bereich Systembiologie. ImApril 2009 wurde das wissenschaftliche Konzepte voneinem internationalen Gremium weltweit anerkannterExperten aus den relevanten Disziplinen begutachtet.Details des Konzeptes und seiner gegenwärtigenVerwirklichung werden in diesem Bericht ebenfallsdargestellt. Hier beschränke ich mich auf einen kurzenBericht über den Entwicklungsstand dieses Institutes.Die Gründung des BIMSB wurde durch die Förderungdes BMBF über die Initiative „Innovation und Spitzenforschungin den Neuen Ländern“ und zusätzlicherMittel des Senats von Berlin ermöglicht. NikolausRajewsky hat diese Entwicklungen entscheidendvorangetrieben und übernahm die Funktion des Sprechersund Koordinators des Gesamtprojekts. Erste neueForschungsgruppen konnten bereits eingerichtet werden,weitere werden folgen. Das MDC wird seine engeZusammenarbeit mit den Berliner Universitäten undder Charité über das BIMSB vertiefen. Gemeinsam mitden Partnern wurde für das BIMSB ein Standort aufdem Nordcampus der Humboldt-Universität zu Berlinin der Nähe ihres Institutes für Theoretische Biologie,dem Campus Mitte der Charité und des Berliner „BernsteinZentrums für Computational Neuroscience”gefunden. Dieser Standort ermöglicht auch eine stärkereEinbindung in die Ausbildung von Studenten undDoktoranden.Die Ausbildung von “Systembiologen” ist ein wichtigesAnliegen. Ein besonderer Bestandteil des Ausbildungskonzeptesist ein gemeinsames Doktoranden-Programmvon BIMSB, MDC, und des Zentrums für FunktionelleGenomik, New York University. Dies ist ein neuartigesModell einer interkontinentalen Zusammenarbeitbei der Ausbildung von Doktoranden, die ihreDirector's introduction XVII

approaches from bioinformatics/systems biology,clinical, and experimental approaches. Germany has astrong reputation in cardiovascular research. Butthere remain clear deficits that need to be addressed,particularly as cardiovascular diseases have a lowerpriority in the national research agenda than otherhealth problems that pose a lesser burden on thehealth care system. Cardiovascular diseases must beapproached in a highly interdisciplinary way, througha meaningful blend of basic and clinical research, andour efforts can be improved through coordination,networking and the common use of infrastructure.Together with partners from medical faculties andthe German Society for Cardiology, the MDC is spearheadingan effort to substantially increase supportfor this research through the establishment of aGerman Center for Cardiovascular Research (Deut -sches Zentrum für Herz-Kreislaufforschung, DZHK). Itwill consist of a German Institute for CardiovascularResearch (Deutsches Institut für Herz-Kreis lauf -forschung, DIHK) as an extension of the MDC andintegrate excellent ongoing work across Germany byestablishing new interconnected partner institutes.They will be chosen and their budgets will be establishedthrough a transparent, competitive reviewprocess. The DIHK will be set up by the MDC in closecollaboration with the Charité. Besides its researchactivities, it will manage the administration of thepartner institutes and provide technology platformsas well as other infrastructures. The DIHK will be ableto draw on all the resources and infrastructure of theECRC and BIMSB.The overall mission of the DZHK can be summarizedas following:· To become the key national institution for basicand clinical cardiovascular research in Germany;· To establish and provide national technologicalplatforms for cardiovascular research;· To establish a nationwide training platform forgraduate and post-graduate;· To inform the public about scientific and medicalprogress in this area;· To intensify contacts with the pharmaceuticalindustry.Arbeiten zwischen jeweils zwei Forschungsgruppen anbeiden Standorten aufteilen. Obwohl das Projekt erst indiesem Jahr gestartet ist, konnten bereits eine Publikationin der Zeitschrift „Nature Methods“ und eine Reiheweiterer Manuskripte veröffentlicht werden.Das Land Berlin hat seine Absicht bekundet, die Mittelfür den Bau des neuen Gebäudes zur Verfügung zu stellen.Es wird sich durch neuartige „integrative Laboratorien“mit einer starken Verzahnung experimentellerund theoretischer Arbeitsplätze auszeichnen. DasGebäude wird Platz für bis zu 25 Forschergruppen(davon mindestens fünf W3-Professuren) und fünfTechnologieplattformen bieten.Das BIMSB wird darüber hinaus eine wichtige Unterstützungfür den Aufbau eines Deutschen Zentrums fürHerz-Kreislauf-Forschung (DZHK) bilden (siehe weiterunten), das sich auf die Erforschung kardiovaskulärerund metabolischer Erkrankungen konzentriert.Deutsches Zentrum für Herz-Kreislaufforschung(DZHK) und Deutsches Institut für Herz-Kreislaufforschung(DIHK)Erkrankungen des Herz-Kreislaufsystems sind die häufigsteTodesursache weltweit. Für die Forschung stellensie eine besondere Herausforderung dar, da zu ihneneine Vielzahl genetischer und umweltbedingter Faktorenbeitragen. Gerade in den letzten Jahren sind aberneue Ansätze (z.B. in der Systembiologie) entwickeltworden, die dem komplexen Charakter dieser ErkrankungenRechnung tragen.Auf dem Gebiet der kardiovaskulären Forschung leistendeutsche Arbeitsgruppen exzellente, international vielbeachteteBeiträge. Gleichwohl bleibt es eine wichtigeAufgabe, vorhandene Stärken besser zu nutzen undauszubauen. Inbesondere sind eine bessere Zusammenarbeitund Koordination bestehender Forschungsaktivitätenund der Aufbau neuer Forschungsgruppenund -infrastrukturen notwendig, um die Überführungvon Ergebnissen aus der Grundlagenforschung in dieKlinik zu fördern und die internationale Sichtbarkeitweiter zu steigern. Dabei müssen experimentelle undklinische Ansätze eng zusammengeführt werden.Darüber hinaus ist festzustellen, dass national derErforschung kardiovaskulärer Erkrankungen bislang einwesentlich geringerer Stellenwert zugemessen wurdeXVIII Director's introduction

A detailed proposal for the DZHK has been submittedto the Federal Science Ministry. A call for applicationsas partner institutes in the DZHK is envisaged for2010.The Development of an In Vivo PathophysiologyLaboratory for long-term studies at the MDCThe success of the MDC is largely based on the generationand study of animal models – including traditionalsystems such as the fly, mouse, zebrafish, and C.elegans, important biomedical organisms such as therat, and some more “exotic” animals including planariaand the naked mole rat (Heterocephalus glaber).Work on genetics and fundamental mechanismswithin these systems is proceeding at a rapid pace,but extrapolating this work to human medicinerequires extensive, creative, long-term phenotypingof animals. To study the impact of targeted changesin genes on the function of complex organ systemssuch as the cardiovascular or nervous systems, or theeffectiveness of new therapies in animal models,state-of-the-art methods must be used to investigateliving animals (in vivo technologies). This has twomain goals: to acquire new basic knowledge and todevelop and test new treatments.Since our facilities for this type of work are currentlyrather limited, we have developed a major proposalfor the construction of an In Vivo PathophysiologyLaboratory (IPL) which has received an enthusiasticreview and is considered essential by the ScientificAdvisory Board, external reviewers and within theHelmholtz Association.The IPL will accommodate research laboratories thatare implementing and further developing cuttingedgephenotyping tools (e.g., imaging and the in-vivorecording of cellular activity). Additionally, the facilitywill provide a large laboratory devoted to behavioraland physiological studies.The IPL will be an invaluable infrastructure that supportsall relevant MDC groups and projects such asthe DIHK. We foresee very fruitful collaborationsbetween the screening unit at the FMP and the imagingfacilities in continuing our studies of the effects ofprobes, inhibitors, and drug candidates on organismsals anderen Erkrankungen mit einer geringeren Belastungdes Gesundheitssystems.In Zusammenarbeit mit den medizinischen Fakultätenverschiedener Universitäten und der Deutschen Gesellschaftfür Kardiologie bemüht sich das MDC um einenAusbau und eine stärkere Vernetzung der Forschungdurch die Einrichtung eines Deutschen Zentrums fürHerz-Kreislaufforschung (DZHK). Dieses wird auseinem Deutschen Institut für Herz-Kreislaufforschung(DIHK) als Teil des MDC und mehreren Partnerinstitutionenin ganz Deutschland bestehen.Die Partnerinstitute werden in einem transparentenWettbewerb ausgewählt. Das MDC wird das DIHK inenger Abstimmung mit der Charité gründen. Nebenseinen Forschungsaufgaben wird es Plattformtechnologienund andere Infrastrukturen zur Verfügung stellen.Das DIHK wird ebenfalls die Ressourcen von ECRCund BIMSB nutzen können.Die Mission des DZHK kann wie folgt zusammengefasstwerden:· Es soll die nationale Leitinstitution für diekardiovaskuläre Grundlagen- und klinischeForschung in Deutschland werden.· Es soll national nutzbare technologischePlatt formen für die kardiovaskuläre Forschungbereit halten.· Es soll eine deutschlandweit nutzbare Plattformfür die Ausbildung von Graduierten und Postgraduiertenwerden.· Es soll die Öffentlichkeit über den wissenschaftlichenFortschritt und seine medizinischeAnwendung informieren.· Es soll auf seinem Wirkungsfeld den Kontakt zurpharmazeutischen Industrie intensivieren.Ein entsprechender detaillierter Vorschlag für dieErrichtung des DZHK wurde beim Bundesministeriumfür Forschung eingereicht. Die Einladung, Partnerinstitutim DZHK zu werden, wird an alle einschlägigenInstitutionen im Jahr 2010 ergehen.Errichtung eines In-vivo PathophysiologielaborsDer bisherige Erfolg des MDC beruht zum großen Teilauf die Erzeugung und Analyse von Modellorganis-Director's introduction XIX

von Humboldt Foundation (AvH). The award stipendhas enabled him to return to Germany and establisha research group at the MDC in May, 2009, after fouryears as postdoctoral fellow at the University ofCalifornia, San Francisco, USA.The MDC is financing two junior groups, those ofBjörn Christian Schroeder (previously of theUniversity of California, San Francisco) and JamesPoulet (École Polytechnique Fédérale de Lausanne,Switzer land), within the excellence cluster“NeuroCure: Towards a better outcome of neurologicaldisorders.” The groups started at the MDC in 2009.Schroeder is working on the Berlin-Buch campus andPoulet is at the Charité campus in Berlin-Mitte until anew building is constructed for NeuroCure in the citycenter.Markus Landthaler, of Rockefeller University in NewYork, is the first junior group leader to be recruitedwithin the framework of the BIMSB.The MDC continues to recruit scientists for ourongoing projects and new activities. Major ongoingrecruitments include filling professorships in theareas of Epidemiology, Metabolism, Cardiovascularresearch, and Systems Biology.The recruitment of excellent PhD students is also apriority for the institute and is based on internationalcalls and a rigorous selection procedure. The programsoffered to PhD students continue to expandand now include lectures, courses, seminars, researchvisits to other laboratories, etc. The MDC is now hometo several graduate schools that have been establishedthrough grants with the HelmholtzAssociation and have received a total of 7.2 Mio. ¤ fundingto run over the next six years. These include thegraduate school “Molecular Cell Biology,” which comprisesthe International PhD programme in MolecularCell Biology, the International Helmholtz ResearchSchools in Molecular Neurobiology (MolNeuro) andTranslational Cardiovascular and Metabolic Medicine(TransCard), and the MDC-NYU Exchange Program inSystems Biology. Each of these projects springs fromthe initiative of one or more of our research labs andaims to enhance our current offerings for PhD students.At the same time, we intend to better integratethese programs through a core PhD course held in thewinden, bevor das Vorhaben Realität werden kann. Wirhoffen sehr, dass es als prioritäre Maßnahme aufgenommenwird, die im Frühjahr 2010 vom Helmholtz-Senat festgelegt werden.Nach unserer Auffassung ist die Errichtung des IPLessentiell für die Zukunft des MDC. Eine eingehendephänotypische Charakterisierung existierender undneu zu entwickelnder Tiermodelle ist ein grundlegenderund unverzichtbarer Schritt für das bessere Verständnismenschlicher Krankheiten.Einwerbung von exzellenten jungen Wissen -schaftlern und die öffentliche Wahrnehmungdes MDC sind zwei Seiten einer MedailleUnter der Leitung von Walter Birchmeier hat das MDCneue hochtalentierte junge Wissenschaftler und exzellenteerfahrene Professoren gewinnen können. In denJahren 2008 und 2009 haben sieben neue Nachwuchsgruppenihre Arbeit am MDC aufgenommen.Jana Wolf, Humboldt-Universität zu Berlin, hat ihreForschungsgruppe im Rahmen der Helmholtz-Allianz„Systembiologie“ und durch zusätzliche Mittel überdas FORSYS-Programm des BMBF im Frühjahr 2008am MDC etabliert.Francesca M. Spagnoli, Rockefeller University, New Yorkund Matthew Poy, ETH-Zürich, haben ebenfalls in2008 ihre Arbeit am MDC aufgenommen. Im September2009 erhielt Francesca Spagnoli vom EuropäischenForschungsrat (engl. Abkürzung ERC) einen von 240vergebenen ERC starting grants von über 2500 Bewerbern.Jan Erik Siemens, University of California, San Francisco,der im Sommer 2008 den Sofia-Kovalevskaja-Preisder Alexander von Humboldt Stiftung erhielt, hat seineArbeit im Mai 2009 am MDC aufgenommen.Im Exzellenzcluster „NeuroCure: Towards a better outcomeof neurological disorders“ finanziert das MDCzwei Nachwuchsgruppen. Björn Christian Schroeder,University of California, San Francisco, und James Poulet,Ecole Polytechnique Fédérale de Lausanne, Schweiz,haben in 2009 ihre Arbeit am MDC gegonnen. BjörnSchroeder arbeitet am MDC in Berlin-Buch und JamesPoulet an der Charité, Campus Berlin-Mitte, bis dasneue NeuroCure Gebäude in Berlin-Mitte bezogenwerden kann.Director's introduction XXI

fall; the 2009 session lasted two weeks and comprisedtalks by the heads of core facilities and investigatorsfrom our research programs. Additionally,retreats from students of the MDC and FMP wereheld in 2008 and 2009, organized by the students andthe PhD programs.The MDC is mainly financed by public resources. It istherefore highly motivated to raise awareness of theinstitute among non-scientists, who will eventuallybe affected by our work and the applications it produces.In additions, these efforts are essential in givingyoung people a first-hand look at science and,hopefully, stimulating many of them to choose a scientificcareer. A major activity is the Gläsernes Labor,operated by the BBB Management GmbH on theBerlin-Buch campus with significant support fromthe MDC. It continues to welcome huge numbers ofvisitors, particularly school children, for hands-onexperiments in the lab and an encounter with modernbiology. In 2008 the teaching labs were fullybooked, receiving over 10,000 pupils and theirteachers.Our very active press office disseminates news aboutour findings to the media and coordinates our interactionswith the public in that regard. The publicationof Geneticists in Berlin-Buch, a collection of articlesabout three famous scientists associated with thecampus, explores our rich history. Finally, we arebringing out other unique publications to give theresearch community and the wider public insightsinto work and life on our campus. 2008 saw the publicationof the book Translations: From today’s scienceto tomorrow’s medicine in Berlin-Buch, a large, coffeetablebook with lavish photographs and a wide rangeof easy-to-understand stories from our current work.A last remarkTo conclude, I would like to emphasize that it hasbeen a pleasure – and a great challenge – to assumethe directorship of a thriving, growing. “Molecularmedicine” and “translational research” are buzzwordsthat can be heard across the world. As outlined above,at the MDC we are pursuing these themes in a verydynamic, flexible and concrete way. As we make thetransition from the current size and scope of ourMarkus Landthaler, Rockefeller University, New York, istder erste Nachwuchsgruppenleiter, der für das BIMSBgewonnen werden konnte.Die Auswahl exzellenter Doktorstudenten erfolgt amMDC über ein strenges Auswahlverfahren nach internationalerAusschreibung. Unser PhD-Programm bietetden Doktoranden mehrere Vorlesungsreihen, Kurseund Seminare, Kongressteilnahmen, Forschungsaufenthaltein anderen Laboren und vieles mehr. Ende 2009erfolgte bereits die achte Ausschreibung unseres PhD-Programms, das wir gemeinsam mit der Humboldt-Universität zu Berlin in 2001 gestartet haben. Das MDChat für alle Doktoranden, die ab Juli 2006 mit ihrerArbeit am MDC begonnen haben, ein einheitliches Curriculumetabliert. Mit zentralen Mitteln der Helmholtz-Gemeinschaft, die das MDC in den letzten zwei Jahrenfür drei Programme einwerben konnte, stehen nun insgesamt7,2 Mio. € über eine Laufzeit von insgesamt 6Jahren zusätzlich für eine strukturierte Ausbildung zurVerfügung. Zu diesen Programmen gehören die Helmholtz-Graduiertenschule„Molecular Cell Biology“, dasHelmholtz-Kolleg „Molecular Neurobiology“ und dasHelmholtz-Kolleg “Translational Cardiovascular andMetabolic Medicine – TransCard“. Im Gegensatz zu denKollegs, die thematisch fokussiert sind, ist die Graduiertenschuleals disziplinübergreifende Dachstrukturangelegt. Wie in den Jahren zuvor wurden auch in2008 und 2009 PhD-Retreats gemeinsam von Doktorandendes MDC und FMP organisiert.Das MDC wird vorwiegend mit öffentlichen Mittelnfinanziert. Wir sind deshalb sehr daran interessiert, derÖffentlichkeit die Ergebnisse unserer Arbeit näher zubringen. Wir hoffen auf diesem Weg, jungen Menscheneinen Einblick in die Wissenschaft aus erster Hand zubieten und sie zu einer wissenschaftlichen Laufbahn zuermutigen.Eine wichtige Aktivität ist hierbei die Unterstützungdes Gläsernen Labors, das auf dem Bucher Campus vonder BBB Management GmbH betrieben wird. Es wirdvor allem von Schulkindern besucht, die mit ersteneigenen molekularbiologischen Experimenten einenEinblick in die moderne Biologie erhalten. 2008 und2009 war das angebotene Programm durch ca. 10.000Schülerinnen und Schüler und ihren Lehrkräften vollständigausgebucht.XXII Director's introduction

organization to a larger, multi-faceted one, we aretaking care to tighten our interactions with our partners,on the regional, national and international levels,through activities such as the BIMSB and DIHK.These steps are necessary to achieve the primarygoals of our campus, to integrate new developments,and to preserve a multifaceted range of researchactivities (and here, we regard the establishment ofthe IPL as a necessity).These developments are only possible because wehave attained a critical mass that stimulates furthergrowth. And our efforts can only come to fruition in aculture which has a critical mass of superlative basicscience. By building on existing strengths, we aremoving in a clearly defined direction that promises tohave a major impact on the field of human health.Finally, excellent science requires excellent scientists.Thus increasing the attractiveness of the MDC to thebest scientists around the world is central to all themeasures we are planning.Unsere Stabstelle für Presse- und Öffentlichkeitsarbeitverfasst zahlreiche Berichte für die Medien und koordiniertunsere Interaktionen mit der Öffentlichkeit. Mitder Veröffentlichung des Buches „Genetiker in Berlin-Buch”, eine Sammlung von Artikeln zu drei berühmtenWissenschaftlern, die früher auf dem Campus gearbeitethaben, tragen wir unserer vielschichtigen VergangenheitRechnung. Für die Abbildung der Gegenwarthaben wir in 2008 das Buch: “Translations: Fromtoday’s science to tomorrow’s medicine in Berlin-Buch” veröffentlicht. Es ist eine umfangreiche, unterhaltendeGeschichten- und Anekdotensammlung mitallgemein verständlichen Darstellungen und eindrucksvollenFotos unserer Wissenschaftler und derenForschung.SchlusswortEs ist für mich eine große Freude und eine sehr an -spruchsvolle Aufgabe, die Leitung einer so erfolgreichenund dynamischen wissenschaftlichen Institution,wie sie das MDC darstellt, übernommen zu haben. DasMDC hat die Herausforderung, die mit den Schlagworten„Molekulare Medizin“ und „Translationsforschung“verbunden sind, seit seiner Gründung angenommenund mit großem Enthusiasmus verfolgt. Dabei hatsich eine dynamische Institutsstruktur herausgebildet,die – bei Beibehaltung des Grundkonzepts – neue Entwicklungenintegrieren kann. Dies ist eine wichtigeVoraussetzung für die aktuelle Herangehensweise:unter Beibehaltung einer facettenreichen Forschungseinheit(dazu ist der Neubau des IPL erforderlich!) dieregionale, nationale und internationale Verflechtung,u.a. durch den Aufbau von BIMSB und DIHK, voranzutreiben.Eine entscheidende Voraussetzung für die Verwirklichungder hier dargestellten Konzepte ist dadurchgegeben, dass das MDC eine kritische Masse an exzellenterForschung erreicht hat. Wir sind davon überzeugt,dass wir aufbauend auf unseren Stärken hervorragendeErgebnisse in der Gesundheitsforschung erreichenwerden. Vor allem aber muss es das Ziel des MDCbleiben, im weltweiten Wettbewerb die besten Wissenschaftlerfür sich zu gewinnen. Denn: Exzellente Forschunggedeiht dort, wo sich exzellente Forscher treffenund ihre Ideen gemeinsam umsetzen.Director's introduction XXIII

Cardiovascular andMetabolic Disease ResearchHerz-Kreislauf- und StoffwechselerkrankungenCoordinator: Thomas E. WillnowBasic Cardiovascular FunctionCoordinator: Thomas E. WillnowGenetics and Pathophysiology ofCardiovascular DiseasesCoordinator: Nikolaus Rajewsky

Cardiovascular and Metabolic Disease ResearchHerz-Kreislauf-Krankheiten und metabolisches Syndrom Thomas E. WillnowCardiovascular disease is the most commoncause of death worldwide. In industrializednations, this state of affairs has been the casesince early in the 20 th century. In the 21 st century, cardiovasculardisease has become global. For instance,almost half the disease burden in low-and-middleincomecountries of Europe and Central Asia nowcomes from cardiovascular diseases. In the UnitedStates, 60 million people have known cardiovasculardisease, 50 million have hypertension, 13 million havecoronary disease, and 5 million have had a stroke.These figures comprise 1 in 5 of the US population.Figures for the European Union are no different. Fivepercent of the US and EU population are known tohave type 2 diabetes mellitus. This “adult-onset” diabetesis now the most common cause of diabetes inchildren. Kidney disease leading to end-stage renalfailure is increasing exponentially and diabetes, coupledwith hypertension, is the most common cause.The number of people with a body mass index (BMI)>30 is approaching 30% of the population and halfthe population is overweight (BMI >25%). These figuresare alarming, since it could mean a reversal ofthe trend throughout the last century of an everincreasinglife expectancy in our societies.Learning to prevent and treat cardiovascular andmetabolic diseases will require understanding theunderlying genetic and pathophysiological mechanisms,and this approach offers a passport to in vivotranslational approaches. Thus research in this programaims at elucidating the genetic pathways whichregulate the normal function of the cardiovascularsystem and metabolism and whose defects causehuman diseases. Ultimately, the identification of suchdisease genes will lead to a better understanding ofpathological processes, to improved diagnoses, and tonew concepts in therapy.Toward these goals, we use functional genomicsapproaches to study disease processes in model systemsranging from the zebrafish, to the mouse andrat, and we compare our findings to studies conductedin humans (and vice versa). The studies are performedby group leaders at the MDC in close collaborationwith clinicians at the Charité, UniversityMedicine of Berlin.Weltweit sind Erkrankungen des Herz-Kreislauf-Systems inzwischen die häufigste Todesursache.Galt dies seit Beginn des 20. Jahrhundertszunächst nur für die Industrienationen, trifft dieserBefund im 21. Jahrhundert nunmehr global zu. So ist dieHälfte der Erkrankungen in den europäischen und zentralasiatischenGesellschaften mit geringem oder mittleremNationaleinkommen heute diesem Komplex von Krankheitenzuzurechnen.In den USA leiden 60 Millionen Menschen an Herz-Kreislauf-Krankheiten.50 Millionen haben Bluthochdruck, 13Millionen leiden an Herzkrankheiten, und 5 Millionen hatteneinen Schlaganfall. Damit ist also jeweils einer vonfünf Einwohnern der USA von einer Erkrankung des Herz-Kreislaufsystems betroffen. Hinzu kommt, dass 5 Prozentder US-Bürger an Diabetes vom Typ 2 leiden.Auch in der Europäischen Union verhält es sich nichtanders. Fünf Prozent der EU-Bürger leiden ebenfalls anDiabetes vom Typ 2. Diese typischerweise erst im Erwachsenenaltereinsetzende Form der Zuckerkrankheit istheute bereits die häufigste Form von Diabetes im Kindesalter.Als Folge nehmen Nierenerkrankungen bis hin zumNierenversagen exponentiell zu.Eine große Rolle spielt in diesem Zusammenhang auchdas Übergewicht. 30 Prozent der Bevölkerung erreichenden gefährlichen BMI-Index-Wert (body mass index) vonüber 30. Bereits 50 Prozent der Bevölkerung haben einenIndex von mehr als 25, was bereits Übergewicht anzeigt.Das sind alarmierende Befunde. Sie könnten den Trendständig steigender Lebenserwartung umkehren, der dasvergangene Jahrhundert gekennzeichnet hat.Um Erkrankungen des Herz-Kreislauf-Systems verhütenoder erfolgreich behandeln zu können, muss man die zuGrunde liegenden genetischen und pathophysiologischenMechanismen aufklären. Ziel unseres Forschungsprogrammsist es deshalb, genetische Netzwerke aufzuklären,welche die normale Funktion des kardiovaskulären Systemsregulieren und die, wenn sie defekt sind, zu Erkrankungenführen. Letztlich wird die Aufklärung solcherkrankheitsverursachenden Gene zu einem besseren Verständnispathologischer Prozesse, aber auch zu besserendiagnostischen Verfahren und schließlich zu neuen Therapiekonzeptenführen.Im Hinblick auf diese Zielsetzung betreiben wir funktionelleGenomforschung in Modellsystemen wie Zebrafisch,Maus oder Ratte. Wir vergleichen die gewonnenen Befundemit solchen, die am Menschen erhoben werden konn-2 Cardiovascular and Metabolic Disease Research

Our research activities are organized in two topics, (i)Basic Concepts of Cardiovascular Function and (ii) theGenetics and Pathophysiology of CardiovascularDisease. Both topics center around the comparativeanalysis of model systems and human diseases. Closeinteractions between scientists in these two topicspromote synergies between projects using forwardand reverse genetics. They stimulate exchangebetween projects in basic science and disease-orientedresearch and promote the transfer of new ideasand concepts from the “bench” to the “bedside”.Interestingly, many of the mechanisms that contributeto cardiovascular diseases also play a fundamentalrole in other body systems, such as the nervoussystem and the brain, so scientists from the programhave made important contributions to theMDC’s other main research topics.Topic 1: Basic concepts of CardiovascularFunctionThe aim is to characterize major regulatory pathwaysthat underlie normal cardiovascular function and toinvestigate these in cell culture and organ systems. Aswell as studying the adult organism, an emphasis isplaced on developmental pathways, as they representparadigms for differentiation processes in normaland diseased adult tissues. Major focuses of ourresearch activities in this topic are cardiovascular signaltransduction and cell biology as well as developmentand regeneration of the cardiovasculature.Some recent highlights from the program includeoutstanding work by some of our younger groupleaders. For example, in 2008 Michael Gotthardt’s labdiscovered that the Coxsackie-adenovirus receptor(CAR), primarily known for its role in allowing thevirus to infect cells, also plays a crucial role in heartdevelopment and function. CAR is a tight junctionprotein and ties cardiac cells together to help transmitelectrical signals between them. Defects in othercell-contact proteins have been linked to arrhythmia,but so far CAR had not been implicated. Using conditionalknockout mice, the lab discovered that a loss ofCAR disturbs the localization of ion channels calledconnexins, which apparently must be clustered in thecell membrane to coordinate the transmission often. Dieser wissenschaftliche Ansatz erfordert die engeZusammenarbeit grundlagen-orientierter Arbeitsgruppendes MDC mit klinischen Gruppen in der Charité –Universitätsmedizin Berlin.Unser Forschungsprogramm gliedert sich in zwei Teilgebiete:1) Grundlagen der Funktion des kardiovaskulärenSystems und 2) Genetik und Pathophysiologie kardiovaskulärerKrankheiten. Beide Teilgebiete konzentrieren sichauf die vergleichende Analyse von Modellsystemen undKrankheiten des Menschen. Die enge Zusammenarbeitentlang dieser Forschungsstrategien eröffnet Synergienzwischen solchen Projekten, die vom Gen auf das Merkmalzielen und denen, die vom (Krankheits-)Merkmalausgehend die genetischen Ursachen suchen. So ergibtsich ein logisches Ineinandergreifen von Grundlagenstudienund klinisch orientierter Forschung, was den Transfervon neuen Erkenntnissen vom „Labor“ zum „Krankenbett“in einer sinnvollen Weise befördert.Teilthema 1: Grundlagen der Funktion desHerz-KreislaufsystemsHier ist es das Ziel, die wichtigsten Regelkreise, die derFunktion des Herz-Kreislauf-System zugrunde liegen,aufzuklären. Sie sollen sowohl in der Zellkultur alsauch in intakten Organsystemen untersucht werden.Neben dem Studium des adulten Organismus werdenauch Regulationsnetzwerke der embryonalen Entwicklungbetrachtet, da sie modellhaft für Differenzierungsprozessein pathologisch veränderten Gewebendes Erwachsenen sind. Der Schwerpunkt der Untersuchungenauf diesem Gebiet liegt sowohl auf der Signaltransduktionin der Zelle als auch auf den Entwicklungs-und Regenerationsprozessen des kardiovaskulärenSystems.Herausragende Ergebnisse im Rahmen dieses Programmshaben insbesondere jüngere Forscher gruppenerbracht. Hier ist Michael Gotthards Gruppe zu nennen,die herausfand, dass der Rezeptor des Coxsackie-Adenovirus (CAR) ein wichtiger Faktor in der Entwicklungund Funktion des kardiovaskulären Systems ist.CAR ist ein „tight junction protein”, das Herzzellen miteinanderverbindet und diese zur Übertragung elektrischerSignale befähigt. Defekte in anderen Zellkontakt-Proteinen wurden bereits mit Herz-Arrhythmie in Verbindunggebracht; CAR gehörte jedoch bislang nichtzu diesen Kandidatenproteinen. Mit Hilfe des sogenannten„conditional knock-out” in der Maus hatte dieCardiovascular and Metabolic Disease Research 3

electrical signals. More recent work by the lab hasprovided insights into how infections by the coxsackievirus cause heart damage. The work emphasizesCAR’s significance as a potential target for therapies,while underlining the importance of understandingits functions in the healthy heart.Salim Seyfried’s group has been working on elucidatingthe mechanisms that control cardiac cell behaviorsduring early heart formation. Initially most cardiaccells are arranged within a flat sheet called theheart cone which undergoes a complex morphogenetictransformation to create the heart tube. Therole of cell migrations or epithelial bending in thisprocess has been unclear. Recent projects by the labhave shown how the differential expression of proteinsin the left and right sides of the heart field leadto asymmetries that are crucial in giving the organ itsproper shape. Time-lapse microscopy studies combinedwith the analysis of genetically modifiedzebrafish showed that cells migrate asymmetrically,which plays a central role in establishing differentdorsoventral regions of the heart tube. Another findingof the group was that cardiac cells initially acquiretheir shapes independently of the function of theearly heart. This finding revealed new connectionsbetween the way cardiac cells acquire shapes andcontribute to the hearts’ structure as a whole.Topic 2: Genetics and Pathophysiology ofCardiovascular DiseasesWithin this topic we aim to identify major genetic,genomic and pathophysiological mechanisms inpatients and in animal models of cardiovascular disease.Based on our findings, novel concepts forimproved diagnosis and therapeutic intervention areexplored.Over the past two years, several projects within theprogram have established new connections betweengenes and susceptibility to heart diseases and fundamentalmechanisms that play a role in their development.A collaboration between the groups of NorbertHübner, Friedrich Luft and clinician Jan Monti ofCharité/HELIOS has shown that the gene Ephx2 contributesto heart failure and the arrhythmic beatingthat signals sudden cardiac death. The researchersGruppe zuvor feststellen können, dass die Ausschaltungdes CAR-Gens die Lokalisierung bestimmter Proteine,den Connexinen, verändert. Connexine müssen offensichtlichals Cluster in der Zellmembran vorliegen, umdie Übertragung elektrischer Signale zu koordinieren.Neuere Ergebnisse lieferten Einsichten in die von Coxsackieverursachte Herzschädigung. Sie betonen dieBedeutung von CAR als möglichem Ansatzpunkt fürtherapeutische Eingriffe, aber auch für das Verständnisder Funktion des gesunden Herzens.Salim Seyfrieds Arbeitsgruppe untersuchte den Kontrollmechanismusfür das Verhalten von Herzzellenwährend der embryonalen Organbildung. Am Beginndes Prozesses sind die Herzzellen als flache Schichtangeordnet (heart cone), aus der durch komplexe morphologischeVeränderungen die Herzröhre entsteht.Wie dabei Wanderung und Richtungsänderung vonEpithelien zustande kommen, war bislang unklar. DieGruppe konnte zeigen, dass durch genau abgestimmteExpression von Proteinen auf der rechten und der linkenSeite des Herzfeldes eine Asymmetrie entsteht, dieentscheidend für die präzise Gestaltbildung des Organsist. Mikros kopische Zeitverlaufsstudien an genetischveränderten Zebrafischen zeigten den asymmetrischenWan derungsverlauf der Zellen, der eine zentrale Rolle inder Aus bildung der dorsoventralen Anordnung derHerz röhre spielt. Darüber hinaus fand die Arbeitsgruppeheraus, dass Herzzellen ihre ursprüngliche Gestaltunabhängig von der Funktion der ersten Herzanlageannehmen. Dieser Befund zeigt neue Wege für das Verständnisder Gestaltbildung der Herzzellen und ihresBeitrags zur Gestaltbildung des ganzen Organs auf.Teilthema 2: Genetik und Pathophysiologie vonHerz-Kreislauf-ErkrankungenZiel ist hier, die genetischen und pathophysiologischenMechanismen von kardiovaskulären Erkrankungen amMenschen und in Modellorganismen zu identifizieren.Unsere Befunde dienen der konzeptionellen Vorbe reitungneuer Diagnose- und Therapieverfahren.In den vergangenen zwei Jahren haben mehrere Forschungsprojektedes Programms neue Zusam menhängezwischen Genvarianten, die zu Herz erkrankungen disponieren,und grundlegenden Mecha nismen der embryonalenHerzentwicklung gefunden. Die Forschergruppen vonNorbert Hübner, Friedrich Luft und dem Kliniker Jan Monti(Charité/HELIOS) konnten in einer Gemeinschaftsarbeit4 Cardiovascular and Metabolic Disease Research

combined computation and laboratory experimentsin a study of a rat model for spontaneous hypertensiveheart failure (SHHF). By crossing SHHF rats withanother strain that is hypertensive but does notdevelop heart failure, they obtained a population ofrats with various degrees of heart failure while all animalshad high blood pressure. This allowed the scientiststo peel apart factors that contribute to highblood pressure and heart failure. Ephx2 modifies ahormone called EET in cardiac muscle, which plays arole in coordinating heart cell contraction, relaxesblood vessels and helps protect the organ after sometypes of heart attacks. Extending the work to humanpatients recovering from heart failure, they discoveredlow levels of Ephx2 in patient tissue. An improperlyfunctioning version of Ephx2 found in some peoplemay lead to more severe damage in the aftermathof heart failure.Another very active area within the program exploresfundamental mechanisms by which cells regulategene expression and the connections between thesesystems and heart functions and disease. One focusof the labs of Nikolaus Rajewsky and MatthiasSelbach is the way microRNAs regulate hundreds ofgenes. Combining a method called SILAC (whichlabels amino acids with a stable, non-radioactive isotope)with mass spectrometry, the researchersobtained the first quantitative measurements ofglobal changes in protein production in cells thatexpress particular microRNAs. Sometimes thesesmall molecules cause the destruction of messengerRNAs; in other cases they prevent the translation ofmRNAs into proteins. The scientists found that singlemicroRNAs use both methods as a sort of global “volumecontrol” for the output of hundreds of genes,allowing the cell to tune protein production up anddown. Prior to the development of this method, it wasdifficult to measure the amounts of molecules synthesizedby cells under various conditions. In manycases, diseases are thought to arise from a change inthe dosage of a particular protein found in certaintypes of cells, rather than its absolute presence orabsence. The work provides a new method to get ahandle on this issue.nachweisen, dass das Gen Ephx2 eine wichtige Rolle beider Entstehung der Herzinsuffizienz und der Arrhythmiespielt. Letztere ist ein Vorbote des plötzlichen Herztodes.Die Forscher kombinierten Computermodellstudien undExperimente an einem Rattenmodell für spontane hypertensiveHerzinsuffizienz (SHHF). Sie kreuzten SHHF-Rattenmit einem anderen Stamm, der zwar Bluthochdruck,aber keine Herzinsuffizienz entwickelt. Sie erhielten soeine Population von Ratten mit abgestuften Ausprägungenvon Herzinsuffizienz bei durchgehend bestehendemBluthochdruck. Die Analyse ergab damit verschiedeneUrsachen für Bluthochdruck und für Herzinsuffizienz. ImHerzmuskel wird die Wirkung eines Hormons mit derBezeichnung EET durch Ephx2 verändert. Dieses Hormonist wichtig für die Koordination von Herzmuskel -kontraktion und Blutgefäßerweiterung und bildet einenSchutzfaktor beim Herzinfarkt. Bei der Ausweitung dieserStudien auf Herzpatienten, die sich in der Erholungsphasenach einer akuten Herzinsuffizienz befanden, wurdenerniedrigte Ephx2-Werte im Pa tien ten gewebe beobachtet.Eine Fehlfunktion des Ephx2 könnte also bei manchenPatienten die Ursache für schwere Schädigungen in derErholungsphase nach einer Herzinsuffizienz sein.Ein weiteres sehr intensiv bearbeitetes Teilgebiet des Programmssind die fundamentalen Mechanismen derRegulation der Genexpression und ihrer Verbindung zuHerzfunktion und Herzversagen. Ein Schwerpunkt derStudien in den Laboratorien von Nikolaus Rajewsky undMatthias Selbach ist die Regulation der Expression vonHunderten von Genen durch microRNAs. Durch den koordiniertenEinsatz der SILAC-Methode (bei der gewisseAminosäuren durch stabile, nicht-radioaktive Isotopenmarkiert werden) und anschließender Massenspektrometriekonnten die Forscher erstmals quantitative Messungenvon Veränderungen der Gesamtproduktion von Proteinenin Zellen erhalten, die microRNS exprimieren. Ineinigen Fällen bewirken diese kleinen Moleküle die Zerstörungvon Boten-RNAs; in anderen Fällen hemmen siedie Translation der mRNA und damit die Entstehung derdurch sie kodierten Proteine. Es zeigte sich, dass manchemicroRNS beide Regulationsmechanismen nutzen, umeine Art „Gesamtkontrolle“ über die Ausbildung Hunderterverschiedener Proteine auszuüben. Solche wissenschaftlichenUntersuchungen waren vor der Entwicklungdes kombinierten SILAC-Verfahrens äußerst schwierig.Die neue Methode erlaubt nun einen Zugang zu diesenkomplizierten experimentellen Forschungsansätzen.Cardiovascular and Metabolic Disease Research 5

Basic Cardiovascular FunctionStructure of the GroupThomas E. WillnowGroup LeaderProf. Dr. Dr. Thomas E. WillnowScientistsDr. Tilman BreiderhoffDr. Anne-Sophie CarloDr. Juliane ReicheDr. Michael RoheDr. Vanessa SchmidtGraduate StudentsTilman Burgert*Safak Caglayan*Anna Christa*Molecular Cardiovascular ResearchVPS10P domain receptors such as SORLA and sortilin comprise a recently identified class ofintracellular sorting proteins that are predominantly expressed in neurons but also in nonneuronalcell types. VPS10P domain receptors were previously considered to be orphan receptorswith activities in neuronal protein trafficking that were poorly understood. However, newfindings revealed unexpected roles for these receptors as regulators of neuronal viability andfunction. Recent work from our laboratory has uncovered the molecular mechanisms of regulatedprotein transport and signaling through VPS10P domain receptors. Loss of this regulation maycontribute to devastating disorders of the nervous system, including Alzheimer disease, affectivedisorders, and cell death following spinal cord injury. We also obtained first encouraging dataconcerning possible roles for these receptors in regulation of the cardiovascular system.IntroductionThe VPS10P domain is a protein module that was firstrecognized in the vacuolar protein sorting 10 protein(VPS10P) in Saccharomyces cerevisiae. VPS10P is a sortingreceptor that directs the trafficking of lysosomalenzymes from the Golgi to the vacuole (the lysosome inYeast). Subsequently, this protein domain was found toconstitute the unifying structural feature of a newgroup of type 1-membrane receptors that are conservedthroughout evolution from baker’s yeast to man. Themembers of this gene family are now known as VPS10Pdomain receptors. Five receptors are found in vertebrates:sortilin, SORLA, SORCS1, SORCS2, and SORCS3(Fig. 1A).VSP10P domain receptors were initially considered arather peculiar group of sorting proteins with unknownfunction. However, the mammalian receptors of thegene family surfaced as potential disease genes in anumber of association studies in patients. These diseasesencompass Alzheimer’s disease (AD) and othertypes of age-related dementias (in which SORLA andSORCS1 have been implicated), bipolar disorders (inwhich SORCS2 has been implicated), as well as senescenceof the nervous system (in which sortilin has beenimplicated). In addition, several common cardiovascularand metabolic disorders involving VPS10P domainreceptors were identified including type 2 diabetes(which has been linked to SORCS1 and SORCS3) as well asdyslipidemia, and myocardial infarction (which havebeen linked to sortilin).Subcellular trafficking of VPS10P domainreceptorsThe trans-Golgi network (TGN) is an organelle that isimportant for the distribution of proteins between variouscellular compartments. It serves to direct newlysynthesized proteins into constitutive or regulatedsecretion. It sorts proteins into endosomal or lysosomalcompartments, and it participates in axonal transportand the action of signaling endosomes in neurons.Given its structural similarity to VPS10P, a sorting receptorin the TGN, a similar function for SORLA in Golgitrafficking had been anticipated. Our recent data on the6 Cardiovascular and Metabolic Disease Research

Helena Emich*Esther Kur*Daniel MilitzTechnical AssistantsChristine KruseKristin KampfMaria SchmeisserDonathe Vetter*Tatjana Pantzlaff*SecretariatVerona Kuhle*part of the period reportedFigure 1: Structural and cellular biology of VPS10P domain receptors. (A) Structural organization of VPS10P domain receptors from yeast (VPS10P)and humans (sortilin, SORLA, SORCS-1, -2, -3). (B) Cellular trafficking of SORLA. Newly synthesized receptors exit the trans-Golgi network (TGN) (1)and reach the cell surface via the constitutive secretory pathway (2). At the cell surface, they interact with the AP-2 complex to internalize viaclathrin-coated pits (3). Internalized receptors are returned to the TGN via retrograde sorting pathways, and continue subsequent shuttlingbetween endosomal and TGN compartments involving adaptor proteins GGA and PACS1 (4).cell biology of this receptor confirmed this notion (Fig.1B). SORLA is predominantly found intracellularly inendosomal vesicles and the TGN. In neurons, the receptoris concentrated in the cell body with no apparentpolarization. Receptor molecules at the plasma membraneexhibit rapid internalization that is mediated byan acidic cluster dileucine site in the cytoplasmic tail ofSORLA that interacts with the ubiquitous adaptor complexAP-2. From early endosomes, internalized receptorsare returned to the TGN (Fig. 1B). Using site-directedmutagenesis, we demonstrated that several cargoadaptors mediate the shuttling of SORLA between TGNand endosomes, including the clathrin adaptors GGA1, -2, -3 as well as PACS1 (Fig. 1B).SORLA, a key gene in Alzheimer’s diseaseSORLA is a 250-kDa protein widely expressed in neuronsof the cortex, hippocampus and cerebellum. Its involvementin Alzheimer’s disease (AD) was initially suggestedby the demonstration of low levels of Sorla geneexpression in patients suffering from the sporadic formof the disease. The association of inherited Sorla genevariants with occurrence of AD in several populationsfurther substantiated this notion. Now, our studieshave uncovered the molecular mechanism wherebySORLA controls the intracellular transport and processingof the amyloid precursor protein (APP) and contributesto AD progression.APP, a type-1 membrane protein is central to the pathologyof AD. APP is converted to a 40 to 42 amino acidCardiovascular and Metabolic Disease Research 7

Figure 2: SORLA and sortilin in neuronal protein transport and signaling. (A) Role of SORLA in trafficking and processing of APP. In neurons, newlysynthesized APP molecules traverse the trans-Golgi network (TGN) en route to the plasma membrane (1). The precursor proteins internalize fromthe cell surface (2) and traffic from early to late endosomes for processing into Aβ (3). SORLA acts as sorting receptor that traps APP in the TGN,reducing the number of precursor molecules that can reach the cell surface and enter endocytic processing pathways (4). In addition, APP moleculesthat reach the cell surface and enter early endosomes are shuttled by SORLA back to the TGN, further reducing the extent of Aβ production inlate endosomes (5). (B) Role of sortilin in (pro)neurotrophin action. Mature NGF binds to a heteromeric complex of TrkA and p75 NTR that stimulatessignaling pathways promoting cell survival. In contrast, proNGF engages a dual receptor complex consting of sortilin and p75 NTR that elicits neuronalcell death.amyloid β peptide (Aβ) through sequential cleavage byβ- and γ-secretases. In the brain, Aβ forms neurotoxicoligomers and amyloid plaques, the pathological hallmarksof AD. Using cell culture and transgenic mousemodels we elucidated the mechanisms of SORLA actionin neurodegenerative processes in molecular detail.According to our data, SORLA acts as intracellular sortingreceptor for APP that controls trafficking of the precursorprotein between TGN and early endosomes. Inparticular, SORLA blocks export of APP from the TGNinto post-Golgi compartments that harbor β-secretaseactivity and thus impairs formation of Aβ (Fig. 2A).Consequently, genetic overexpression of SORLA in culturedneurons results in reduced processing of APP intoAβ. In contrast, disruption of the receptor gene inmouse models of AD significantly increased Aβ productionand plaque formation, similar to the situation inpatients with sporadic AD.Sortilin controls neuronal cell deathAs part of a collaborative project with the laboratory ofAnders Nykjaer from Aarhus University, we uncoveredthe role of sortilin in the action of neurotrophins (NTs).NTs are growth factors that regulate neuronal survival,axon and dendrite specification, and target innervation.In mammals, the NT family includes nerve growth factor(NGF), brain-derived nerve growth factor (BDNF), aswell as NT-3 and -4/5. Their trophic action is mediatedby binding to receptor tyrosine kinases (called Trk receptors)and to the common p75 neurotrophin receptor(p75 NTR ). All NTs are synthesized as proneurotrophins(proNTs) that are subsequently processed to theirmature counterparts in the TGN. Paradoxically, whilemature neurotrophins promote neuronal survival bybinding to Trk receptors and p75 NTR , proneurotrophinsinduce apoptosis when released from cells.8 Cardiovascular and Metabolic Disease Research

Previously, we demonstrated that sortilin is an essentialcomponent for transmitting proNGF-induced deathsignals through p75 NTR (Fig. 2B). In this death receptorcomplex, sortilin specifically binds the pro-domain ofthe proneurotrophins with high affinity while p75 NTRsimultaneously engages the mature NGF domain. Theformation of this ternary receptor-ligand complex iscrucial for the pro-apoptotic function. In this model,sortilin acts as the crucial molecular switch thatenables neurons coexpressing p75 NTR and Trk receptorsto selectively respond to proNGF by apoptosis ratherthan survival (Fig. 2B).Now, we further substantiated the role of sortilin inproNT mediated cell death by generating a sortilin-deficientmouse model and by testing the contribution ofthe p75 NTR /sortilin receptor complex to neuronal viabilityin vivo. In the developing retina, sortilin -/- mice exhibitreduced neuronal apoptosis indistinguishable fromthat observed in p75 NTR-/- . Surprisingly, while sortilin deficiencydoes not impact on developmentally regulatedapoptosis of sympathetic neurons, it prevents their agedependentdegeneration. Furthermore, in an injury paradigmlesioned corticospinal neurons in sortilin -/- miceare protected from death. Thus, the sortilin pathwayplays distinct roles in proNT-induced apoptotic signalingin pathological conditions but also in specific stagesof neuronal development and ageing.Selected PublicationsNykjaer, A, Lee, R, Teng, TT, Nielsen, MS, Jansen, P, Madsen, P, Jacobsen, C,Kliemannel, M, Willnow, TE, Hempstead, B and Petersen, CM. (2004). Anovel neurotrophin receptor essential for proNGF induced neuronal celldeath. Nature 427, 843-848.Andersen, OM, Reiche, R, Schmidt, V, Gotthardt, M, Spoelgen, R, Behlke, J,von Arnim, CAF, Breiderhoff, T, Jansen, P, Wu, X, Bales, KR, Cappai, R,Masters, CL, Gliemann, J, Mufson, EJ, Hyman, BT, Paul, SM, Nykjær, N andWillnow, TE. (2005) SorLA/LR11, a neuronal sorting receptor that regulatesprocessing of the amyloid precursor protein. Proc. Natl. Acad. Sci. USA 102,13461-13466.Hammes, A, Andreassen, TK, Spoelgen, R, Raila, J, Huebner, N, Schulz, H,Metzger, J, Schweigert, FJ, Luppa, PB, Nykjaer*, A. and Willnow*, TE. (2005)Impaired development of the reproductive organs in mice lackingmegalin, an endocytic receptor for steroid hormones. Cell 122, 751-62.Jansen, P, Giehl, K, Nyengaard, JR, Teng, K, Lioubinski, O, Sjoegaard, SS,Breiderhoff, T, Gotthardt, M, Lin, F, Eilers, A, Petersen, CM, Lewin, GR,Hempstead, BL, Willnow, TE* and Nykjaer, A*. (2007) Roles for thepro-neurotrophin receptor sortilin in neuronal development, aging andbrain injury. Nature Neuroscience 10, 1449-57.Willnow*, TE, Petersen, CM and Nykjaer*, A. (2008). VPS10P domainreceptors – regulators of neuronal viability and function. Nature ReviewsNeuroscience 9, 899-909.* joint corresponding authorshipPerspectiveDysregulation of vesicular protein transport is emergingas a molecular mechanism of major importanceunderlying many disease processes. Obviously, intracellularsorting receptors of the VPS10P domain receptorgene family play key roles in these processes. Our futurework has yet to refine the molecular details how sortilinand SORLA affect neuronal viability and functionthrough their interactions with neurotrophin receptorsand APP. Furthermore, novel activities of the neuronalprotein transport machinery may be uncovered as welearn more about the orphan receptors SORCS1,SORCS2, and SORCS3. As well as in the nervous system,SORLA and sortilin are also distinctly expressed in nonneuronalcell types such as in kidney, liver and adiposetissue. Our preliminary studies have uncovered importantroles played by these receptors in renal ion transportand blood pressure regulation (SORLA) or in controlof systemic cholesterol homeostasis (sortilin). In thefuture, we expect to gain novel insights into proteinsorting pathways that may be central to the developmentof renal and metabolic disorders.Cardiovascular and Metabolic Disease Research 9

Annette Hammes(Delbrück Fellow)Structure of the GroupGroup LeaderAnnette HammesScientistOleg LioubinskiGraduate StudentsChandresh GajeraAnnabel ChristMolecular Mechanisms in EmbryonicForebrain DevelopmentHoloprosencephaly (HPE) is defined as a failure of the embryonic forebrain to separate intodiscrete hemispheres along the mid-sagittal axis. HPE is the most common forebrainanomaly in human embryos. Several pathways in neural tube patterning have been implicatedin the etiology of HPE, including megalin (LRP2), a low-density lipoprotein receptor-relatedprotein, expressed in the neuroepithelium during embryonic development. We have usedmegalin mutant mouse models to demonstrate that lack of megalin in the neuroepitheliumcauses HPE. Similarly, mutations in the human Megalin gene cause Donnai-Barrow syndrome inpatients, characterized by facial dysmorphology, ocular anomalies, and agenesis of the corpuscallosum – also features of holoprosencephaly. Our group is studying the mechanisms wherebymegalin regulates signaling pathways crucial for early forebrain development. Our aim is tounderstand the pathophysiological pathways underlying common forebrain midline defects inpatients.Identification of defects in the molecularpathways underlying holoprosencephaly(Annabel Christ, Oleg Lioubinski)Megalin -/- mice suffer from forebrain defects with conditionsassociated with holoprosencephaly (HPE). Onsetof the HPE phenotype becomes evident as early as midgestation.Megalin-deficient embryos at E 10.5 showsignificantly smaller telencephalic vesicles and animpaired subdivision of forebrain hemispheres.To reveal the underlying molecular defects of the HPEphenotype in megalin mutant mice we analyzed theexpression of marker genes involved in early forebraindevelopment and found major changes in the expressionand activity of key morphogens. We demonstratedthat bone morphogenetic protein 4 (BMP4) signaling inthe rostral and dorsal neuroepithelium is increased inmegalin -/- embryos as early as E 9.5. Sonic hedgehog(SHH) expression is lost in the ventral telencephalon atE 10.5, while fibroblast growth factor 8 (FGF8) is aberrantlyexpressed in the rostral midline of the forebrainat E 9.5. Other pathways, e.g. the WNT signaling, do notshow any obvious changes in megalin mutant mice.Recent unpublished findings from our lab now shedlight on the temporal onset of megalin function in forebrainpatterning.The mesodermal prechordal plate (PcP) anterior to thenotochord underlying the rostral diencephalic ventralmidline (RDVM) neuroepithelium in the forebrain is theessential organizing center for midline specification ofbrain, facial, and oral structures. SHH is secreted initiallyfrom the prechordal plate, subsequently the morphogenis found in the ventral midline of the forebrainneural plate where it induces its own expression andthat of other factors involved in early forebrain patterning,e.g. the transcription factor Six3.In our studies we detected SHH protein in the prechordalplate and the RDVM at E 8.25 (8 somites) in wildtype mice whereas in megalin mutant embryos SHHwas only found in the prechordal plate but not in theoverlying neuroepithelium (Figure 1). It is only at the 10somite stage (E 8.75) that the morphogen becomesdetectable in the RDVM neuroepithelium of megalin -/-mice. This intriguing observation suggests that megalindeficiency delays secretion and proper distribution of10 Cardiovascular and Metabolic Disease Research

SHH. In line with these findings expression of Six3, adownstream target of SHH, is significantly down-regulatedin the forebrain of megalin -/- embryos and Shhexpression fails to be established properly in the ventralmidline of the forebrain during later stages of development.It is likely that the delay and decrease in the signalingof SHH during the initial patterning of the ventralmedial forebrain neural plate has detrimental consequencesfor CNS development in megalin mutants.A role for megalin in adult neurogenesis(Chandresh Gajera)In the adult brain, megalin is expressed in ependymalcells, a specialized epithelial cell layer lining the brainventricles. We found the strongest expression of thereceptor in the lateral wall of the lateral ventricles comparedto other ventricular regions. The lateral cell layerplays a crucial role in controlling the generation ofadult neurons from neuronal precursor cells in the subventricularzone (SVZ) of the lateral ventricles. The SVZis one of two regions of adult neurogenesis in themammalian forebrain. Neuronal precursors generatedin this region migrate to the olfactory bulb where theydifferentiate into mature neurons. Interestingly, SHHstimulates adult neurogenesis in the SVZ, and this effectrequires repression of BMP activity via chordin and noggin,similar to the situation seen in the embryonic neuraltube. Ependymal cells secrete noggin and thereby diminishthe inhibitory effect of BMP4 on SVZ neurogenesis.Recently, we obtained exciting new results from theanalysis of adult megalin-deficient mice that providedirect evidence for a role of the receptor in adult neurogenesis.Megalin deficiency does not affect the cellulararchitecture of the SVZ in mice. Nevertheless, mutantanimals show a decrease in cell proliferation in the lateralwall of the lateral ventricles (based on BrdU incorporationexperiments) affecting the neuronal precursorpopulation in the SVZ. The most prominent differencebetween megalin-deficient and control mice is foundby staining for GFAP (glial fibrillary acidic protein), amarker for neuronal precursor cells. Megalin -/- mice showa weaker and altered staining pattern for GFAP in theSVZ. Furthermore, megalin mutant mice show a decreasein nestin, a marker of undifferentiated neuronal precursors,in Dlx2, a marker of rapidly dividing precursors, andin PSA-NCAM, a marker of migrating neuroblasts.To address the mechanism whereby megalin influencesthe proliferation of neuronal precursors in the SVZ wetested the hypothesis that megalin may regulate thelevels of neurogenesis by inhibiting BMP4 activity inthe SVZ. We tested the expression of BMP2/4 andAbnormal SHH protein expression in megalin mutant embryosImmunostainings for Sonic hedgehog (SHH, red color) demonstrateexpression of the morphogen on coronal sections of wild type miceat E 8.25 in the prechordal plate (PcP) and in the overlying rostraldiencephalic ventral midline (RDVM) neuroepithelium (arrow,boundary indicated by the dotted line). Megalin protein is localizedon the apical surface of the neuroepithelium (green color) in wildtype embryos. In somite matched megalin deficient embryos SHHprotein could be detected in the prechordal plate but SHH expressionfails to be established in the overlying neuroepithelium during thisstage of development. The inset shows a schematic illustration ofthe developing CNS at E 8.0 in a sagittal view with the mesodermderived prechordal plate (PcP, purple) and the more caudal notocord(No) underlying the neuroepithelium of the diencephalon (Di) andthe floorplate (FP), respectively. Te: telencephalon, End: endodermdownstream mediators of this pathway. In these experiments,we discovered that there are increased levels ofBMP2/4 protein specifically in SVZ in megalin mutantsas compared to controls. We also detected a robust upregulationof phospho-Smad 1/5/8, and ID3, downstreammediators and targets of BMP signaling, respectively.Taken together, these results provide conclusiveevidence that megalin is a major regulator of adultneurogenesis in the SVZ, acting by suppressing theBMP4 signaling pathway. Remarkably, a similar function(to suppress BMP4 to activate SHH-dependentneurogenesis) is also performed by the receptor in theneural tube, supporting the concept that adult neurogenesisin the SVZ recapitulates features seen duringformation of neurons in the embryonic brain.Selected PublicationsWillnow, TE, Hammes, A, Eaton, S. (2007) Lipoproteins and their receptorsin embryonic development – more than cholesterol clearance.Development, 134, 3239-3249Hammes, A, Andreassen, TK, Spoelgen, R, Raila, J, Hubner, N, Schulz, H,Metzger, J, Schweigert, FJ, Luppa, PB, Nykjaer, A, Willnow, TE. (2005) Role ofendocytosis in cellular uptake of sex steroids. Cell 122(5): 751-762.Spoelgen, R, Hammes, A, Anzenberger, U, Zechner, D, Andersen, OM,Jerchow, B, Willnow, TE. (2005) LRP2/megalin is required for patterning ofthe ventral telencephalon. Development 132(2): 405-414.Cardiovascular and Metabolic Disease Research 11

Ingo L. MoranoStructure of the GroupGroup LeaderProf. Dr. Ingo MoranoScientistsDr. Hannelore HaaseDr. Daria PetzholdDr. Andreas MargGraduate StudentsChristiane LookRomy SiegertJanine LossieInes PankonienLena MartinMaria BöhmerMolecular Muscle PhysiologyContraction of all muscle types is elicited by increasing myoplasmic Ca 2+ and interaction ofType II myosins with thin (actin) filaments. In striated muscle, Ca 2+ bind to troponin C,which turn the thin filament “on”, allowing myosin force-generating actin interactions. Insmooth muscle cells, Ca 2+ bind to calmodulin which then activate myosin light chain kinase.Phosphorylation of the 20kDa light chain of smooth muscle myosins then allows forcegeneratingactin interaction. We are studying the functional roles of subunits of key proteinsof Ca 2+ handling and force generation, i.e. the L-type Ca 2+ channel and type II myosins instriated and smooth muscle. Any change of these proteins by mutation, differential geneexpression, alternative splicing of the transcripts, or post-translational modification modulatestriated and smooth muscle function. Understanding muscle contraction regulation at themolecular and functional levels provide the opportunity to develop new therapeutic strategiesfor the treatment of cardiovascular and skeletal muscle dysfunction.Essential myosin light chain (ELC) functions in theheartDaria Petzhold, Janine Lossie, Maria Böhmer, BurcuSimsek; Ralf Meißner, Petra Sakel, Saskia ReichertIn the adult human heart two ELC isoforms areexpressed, namely an atrial-specific (MYL4, ALC-1, accessionNP_001002841) and a ventricular-specific (MYL3,VLC-1, accession NP_000249) isoform. ELCs bind withtheir N-terminus to actin and with their C-Terminus toIQ1 of the myosin lever arm (Figure 1). ALC-1 is preferentiallytargeted into sarcomeres of human and rodentcardiomyocytes. Most patients with hypertrophic cardiomyopathyand congenital heart diseases re-expresshALC-1 in their ventricles, partially replacing the VLC-1isoform. The VLC-1-to-ALC-1 isoform shift induced a pronouncedpositive inotropic effect.The molecular basis for the isoform-specific sarcomericsorting pattern and the molecular mechanisms of ALC-1 inotropy are not yet understood. In this project we testthe hypothesis that different binding affinities of the C-terminus of essential myosin light chain (ELC) isoformsto the IQ1 motif of the myosin lever arm provide amolecular basis for distinct sarcomeric sorting andinotropic activity.Hypertrophic Cardiomyopathy associates with fivemutations in the essential ventricular myosin lightchain gene (MYL3, AC_000135) (M149V, E143K, A57G,E56G, R154H). The pathomechanism of MYL3 mutations,however, is not yet understood. In this project, we willinvestigate the functional consequences of MYL3 mutations.We employ analytical ultracentrifugation, circulardichroism, and surface plasmon resonance spectroscopyto investigate structural properties, secondarystructures, and protein-protein interactions of recombinanthead-rod fragments of cardiac β-myosin heavychain and ELC isoforms. Cellular functions of ELC isoformswill be investigated by monitoring shorteningand intracellular free Ca 2+ (Fura-2) of adult rat cardiomyocytesinfected with adenoviral (Ad) vectors using ELCisoforms or β-galactosidase as expression cassettes. Wewill generate transgenic mouse lines overexpressingnormal or mutated ELC isoforms. In addition, we elaboratea structural model which explains the cis-inhibitoryaction of the IQ2 domain on myosin function.12 Cardiovascular and Metabolic Disease Research

Burcu SimsekRalf MeißnerTechnical AssistantsPetra SakelSteffen LutterKarin KarczewskiPetra DomaingSaskia ReichertSecretariatManuela KaadaFigure 1. 3D-model of the actomyosin complex. (A)Gauss–Connolly surfaces are used to visualize the molecularcomplex. Actin units are coloured orange, brown, and yellow.The myosin S1 head (green), the regulatory light chain (red), theshortened essential light chain (white), the 46 N-terminalresidues of A1 (blue), and clusters of acidic residues on actin(pink) are shown. (B) More detailed view on the potential interactionof N-terminal APKK of A1 with acidic residues on actin.Ionic interactions between lysine residues (K3 and K4) of APKKand acidic residues (E361 and E364) on actin were assumed.Figure 2. Proposed model for sympatheticcontrol of I CaL by ahnak1.Under basal conditions, I CaL carriedby the α1C-subunit is repressed bystrong ahnak1/β2-subunit binding(left panel). Upon sympathetic stimulation,PKA sites in ahnak1 and/or inβ2 become phosphorylated. Thisreleases the β2-subunit from ahnak1inhibition resulting in increased I CaL .The role of myomesin missense mutations on thegenesis of hypertrophic cardiomyopathyRomy Siegert (In collaboration with Cemil Öczelik,University Medicine Charité, Berlin and IrinaAgarkova, Universitäts-Spital, Zürich,)Hypertrophic cardiomyopathy (HCM) is a commoncause of sudden cardiac death in young people. Threemissense mutations in the myomesin gene haverecently been detected in patients with HCM. Thesemutations are located close to the myosin and titinbinding sites and the dimerization region. The aim ofthe project is to study the molecular pathomechanismscausing the development of HCM by myomesin mutations(e.g. V1490I). Therefore, functional properties ofmutated recombinant fusion proteins will analyzed, i.e.force measurements of poly-myomesin by atomic forcemicroscopy, structural properties by circular dichroismand melting curves, and cellular functions by transientexpression of normal and mutated myomesin domainsin cardiomyoblasts. Transgenetic rat lines which overexpressfunctionally relevant myomesin mutations willbe generated in order to investigate their potential tocause HCM.Cardiovascular and Metabolic Disease Research 13

Figure 3. Membrane resealing assay performed on wild-type mousesingle skeletal muscle fibres. Membrane damage (5x5µm) wasinduced with a two-photon confocal laser-scanning microscope (LSM510 META, Zeiss) coupled to a 488-nm Argon Ti:Sapphire Laser in thepresence of FM 1-43FX fluorescence dye. Top: Plot of fluorescenceintensity (n=10) against time in the presence (red line) and absence(black line) of Ca 2+ . Data are means +-SEM. Bottom: fluorescenceobtained in the presence (right) and absence (left) of Ca 2+ .Ahnak1 – a novel, prominent modulator of cardiacL-type Ca 2+ channelsHannelore Haase, Ines Pankonien, Karin Karczewski,Steffen Lutter (In collaboration with Nathan Dascal,University Tel Aviv, Israel)Ahnak1 is located at the sarcolemma and T-tubuli ofcardiomyocytes indirectly associated with the voltagedependentL-type Ca 2+ channel (L-VDCC) via its β2-subunit.The goal of this study will be understanding theinteractions of ahnak1 with L-VDCC, i.e. α1C and β2, andtheir modulation by β-adrenergic stimulation, mutations,and PKC activation (Figure 2). This will be achievedby heterologous expression of ahnak1 fragments inXenopus oocytes and HEK cells and the characterizationof their effects on cardiac and smooth muscle L-VDCC. Furthermore, reconstitution of β-adrenergicmodulation of L-VDCC in heterologous expression systems,and elucidation of the role of ahnak1, Cavβ andother signaling components in PKA and PKC modulationwill be studied.The functional role of the ahnak protein familyin adult skeletal muscle fibersAndreas Marg, Petra DomaingThe aim of the project is the elucidation of the role ofahnak protein family in skeletal muscle fibers. Thetransmembrane protein dysferlin seems to anchorahnak1 and ahnak2 to the sarcolemma, thus providing amembrane-stabilizing dysferlin-ahnak-actin complex.We investigate whether the ahnak protein family isimportant for membrane stability and Ca 2+ handling ofskeletal muscle fibers. A laser-assisted membraneresealing (Figure 3) and a Fura2-based fluorescenceassay of electrically stimulated enzymatically isolatedsingle skeletal muscle fibers from mouse Flexor digitorumbrevis will be applied.Identification of new adipocyte-derivedcardiodepressant factorsChristiane Look, Lena Martin (In collaboration withValeria Lamounier-Zepter, University Dresden)Recently, we showed that cultivated human adipocytesecrete factors which depress contraction of cardiomyocytes(Figure 4). The aim of this project is to identifyadipocyte-derived cardiodepressant factors. We willassess the role of different adipose tissue depots in producingthese cardiodepressant factors in our in vitroadipocyte/cardiomyocyte and adipocyte/isolated perfusedheart models. Finally, the adipocyte-derived cardiodepressantfactors will be analysed in vivo in microdialysissamples of subcutaneous adipose tissue ofobese patients as well as in serum of our obese patientcohort. The resulting data will allow the developmentof risk profiling and novel therapeutic strategies forheart dysfunction in obese patients.14 Cardiovascular and Metabolic Disease Research

Figure 4. Effect of adipocyte-conditionedmedium (AM) on shorteningamplitude (top trace) and Fura-2signal (bottom trace) of an adultrat cardiomyocyte. A, B)Representative chart recordings ofshortening amplitudes.Selected PublicationsLamounier-Zepter V, Look C, Alvarez J, Christ T, Ravens U, Schunck W-H,Ehrhart-Bornstein M, Bornstein SR, Morano I (2009) Adipocyte fatty acidbindingprotein suppresses cardiomyocyte contraction: a new linkbetween obesity and heart disease. Circ Res. 105:326-34.Tünnemann, G, Behlke, J, Karczewski, P, Haase, H. Cardoso, MCh, Morano, I.(2007). Modulation of muscle contraction by a cell permeable peptide. JMol Med.12:1405-12.Aydt EM, Wolff G, Morano I (2007) Molecular modelling of the myosin-S1(A1) isoform. J. Struct. Biol., 159:158-63.Haase H, Dobbernack G, Tünnemann G, Karczewski P, Cardoso C, PetzholdD, Schlegel WP, Lutter S, Pierschalek P, Behlke J, Morano I. (2006)Minigenes encoding N-terminal domains of human cardiac myosin lightchain-1 improve heart function of transgenic rats. FASEB J. 20:865-73.Haase H, Alvarez J, Petzhold D, Doller A, Behlke J, Erdmann J, Hetzer R,Regitz-Zagrosek V, Vassort G, Morano I. (2005) Ahnak is critical for cardiacCa(V)1.2 calcium channel function and its beta-adrenergic regulation.FASEB J. 19:1969-77.Patent applications„Behandlung der Dysfunktion der Herzkontraktilität oderHerzinsuffizienz, insbesondere bei übergewichtigen und adipösenPatienten mittels Hemmung von FABP4“. P1880EPCardiovascular and Metabolic Disease Research 15

Michael GotthardtStructure of the GroupGroup LeaderProf. Dr. med. Michael GotthardtScientistsMichael RadkeYu Shi*Padmanabhan Vakeel*Graduate StudentsChen ChenUta WrackmeyerUlrike LisewskiThirupugal GovindarajanKatharina Rost*Neuromuscular and CardiovascularCell BiologyOur long-term goal is to establish how mechanical input is translated into molecularsignals. We focus on titin, the largest protein in the human body and the multi -functional coxsackie-adenovirus receptor (CAR).To lay the groundwork for the in vivo analysis of titin’s multiple signaling, elastic, andadaptor domains, we have generated various titin deficient mice (knock-in and conditionalknockout animals) and established a tissue culture system to study titin’s muscle and nonmusclefunctions. We utilize a combination of cell-biological, biochemical, and genetic toolsto establish titin as a stretch sensor converting mechanical into biochemical signals.Using a comparable loss of function approach we have created a conditional knockout ofthe coxsackie-adenovirus receptor. With these mice, we have demonstrated that CAR iscrucial for embryonic development and determines the electrical properties of the heart.Titin based mechanotransductionMichael Radke, Thirupugal Govindarajan, Martin Liss,Padmanabhan VakeelTitin is a unique molecule that contains elastic springelements and a kinase domain, as well as multiplephosphorylation sites. Therefore, it has been frequentlyspeculated that titin and invertebrate giant titin-likemolecules could act as a stretch sensor in muscle. Morerecently, this concept has been supported by studies onhuman dilative cardiomyopathies which suggest animpaired interaction of titin with its regulatory ligandssuch as Tcap. So far it has remained unknown how thestretch signal is processed, i.e. how the mechanicalstimulus stretch is converted into a biochemical signal.To investigate the stretch signaling pathway, we applymechanical strain in vivo (plaster cast for skeletal muscle;aortic banding for the heart) and in tissue culture(cultivation of primary cells on elastic membranes). Theresulting changes in protein expression and localizationin our titin kinase and spring element deficient animalsare used to map the mechanotransduction pathway.Sarcomere assemblyNora Bergmann, Katharina Rost, ThirupugalGovindarajanOverlapping titin molecules form a continuous filamentalong the muscle fiber. Together with the multiplebinding sites for sarcomeric proteins, this makestitin a suitable blueprint for sarcomere assembly. Theuse of transgenic techniques does not only allow us toaddress the function of titin’s individual domains in sarcomereassembly, but also to follow sarcomere assemblyand disassembly using fluorescently tagged proteins.Understanding the structural and biomechanicalfunctions of titin will help elucidate the pathomechanismsof various cardiovascular diseases and ultimatelyaid the development of suitable therapeutic strategies.Smooth muscle and non-muscle titinsNora Bergmann, Katharina RostRecently titin has been proposed to perform non-musclefunctions following its localization to various cell16 Cardiovascular and Metabolic Disease Research

Martin Liss*Brian Anderson**Technical AssistantsBeate GoldbrichMandy Terne**Melanie Manzke**Mirko Wehle**SecretariatSylvia Olbrich*part of the period reported** guest, part of the period reportedSchematic diagram of the sarcomere.Titin forms a continuous filamentsystem along the muscle fiberoverlapping in the M-band (titin C-terminus) and in the Z-disc (N-terminus).The titin kinase is foundnear the edge of the M-band region,while the elastic PEVK resides in theI-band. Titin interacts with a plethoraof sarcomeric proteins, such as T-cap and C-protein.compartments such as the chromosomes of drosophilaneuroblasts and the brush border of intestinal epithelialcells. Titin has been implicated in cytokinesisthrough localization to cleavage furrows and in chromosomecondensation through localization to mitoticchromosomes. Drosophila melanogaster deficient in thetitin homologue D-titin show chromosome abnormalitiesand aneuploidity.Our preliminary data indicate that titin is present in virtuallyevery cell-type tested. Nevertheless, our knockoutof titin’s M-band exon 1 and 2 does not show an obviousnon-muscle phenotype, such as a defect in implantationor in cell-migration. Accordingly, we have extendedthe analysis of our titin knockout animals to actin-filamentdependent functions (assembly of the brush border)and generated additional titin deficient animals toestablish the role of titin in non-muscle cells.Functional analysis of the Coxsackie-AdenovirusReceptorYu Shi, Chen Chen, Uta Wrackmeyer, Ulrike LisewskiCAR was cloned as a receptor used by adeno- and coxsackievirusto enter cells but its physiological role hasremained obscure. Detailed information on the expressionpattern such as upregulation surrounding myocardialinfarction and a critical role in embryonic development(lethality in mid-gestation of the CAR knockout)are well established, but no information on its role inthe adult heart has been available.We have generated both tissue culture and animalmodels to study CAR’s function in cardiac remodeling,inflammatory cardiomyopathy, and basic cellularprocesses such as endocytosis and cell-cell contact formation.Our preliminary data suggest a critical role of CAR in theconduction of electrical signals from the atria to thecardiac ventricle. The inducible heart-specific knockoutof CAR has enabled us to completely block the entry ofcoxsackievirus into cardiomyocytes and prevent allsigns of inflammatory cardiomyopathy.Selected PublicationsShi, Y., Chen, C., Lisewski, U., Wrackmeyer, U., Radke, M., Westermann, D.,Sauter, M., Tschöpe, C., Poller, W., Klingel, K., Gotthardt, M. (2009) Cardiacdeletion of the Coxsackievirus-adenovirus-receptor abolishes CVB3infection and prevents myocarditis in vivo. JACC 7;53(14):1219-26Lisewski, U., Shi, Y., Wrackmeyer, U., Chen, C., Fischer, R., Schirdewan, A.,Juettner, R., Rathjen, F., Poller, W., Radke, M., Gotthardt, M. (2008) The tightjunction protein CAR regulates cardiac conduction and cell-cellcommunication. JExMed 205(10):2369-79Raddatz, K., Albrecht, D., Hochgraefe, F., Hecker, M., Gotthardt, M. (2008) Aproteome map of murine heart and skeletal muscle. Proteomics8(9):1885-97.Radke, M., Peng, J., Wu, Y., McNabb, M., Nelson, O.L., Granzier, H., Gotthardt,M. (2007) Targeted deletion of Titin’s N2B region leads to diastolicdysfunction and cardiac atrophy. Proc. Natl. Acad. Sci. USA. 104(9), 3444-3449Peng J., Raddatz, K., Molkentin, J.D., Wu, Y., Labeit, S., Granzier, H., Gotthardt,M. (2007) Cardiac hypertrophy and reduced contractility in titin kinasedeficient hearts. Circulation 13;115(6):743-5Cardiovascular and Metabolic Disease Research 17

Structure of the GroupSalim SeyfriedGroup LeaderDr. Salim SeyfriedScientistsDr. Nana Bit-Avragim*Dr. Nicole Hellwig*Graduate StudentsElena Cibrián-Uhalte*David Hava*Cécile OttenFlorian Priller*Marc RenzStefan Rohr*Sabine Seipold*Justus Veerkamp*Jingjing ZhangTechnical AssistantsNicole Cornitius*Nicole Dittberner*Jana Richter*part of the periodreportedEpithelial Morphogenesis andZebrafish GeneticsVertebrate organs are derived from epithelial sheets that undergo complex morphogenetictransformations. The molecular mechanisms that regulate the polarization of epithelial cells arecrucial in this process. We have studied the early zebrafish heart, a relatively simple organ comparedwith its mammalian counterpart, to better understand the link between cell polarity, epithelialmorphogenesis and signaling events that instruct the assembly of a heart tube. We would like tounderstand: How do the different protein complexes that establish cell polarity interact with eachother? How is cell polarity regulated within epithelial sheets during morphogenesis of tissues andorgans? What are the signals that regulate cell polarity and epithelial morphogenesis duringcardiogenesis? Our long-term interest is to understand how the cellular mechanisms controlling cellpolarity shape our own bodies. Insight from developmental genetics and cell biological approachesdeployed within our research group are being used in collaboration with clinical research teams toidentify genes responsible for human congenital heart disease.Asymmetric behaviors of myocardial cells drivezebrafish heart tube formationStefan Rohr, Cécile OttenMany vertebrate organs are derived from monolayeredepithelia that undergo morphogenetic changes toacquire their final shapes. Little is known about the tissuemovements or cellular dynamics underlying earlycardiac morphogenesis. In particular, the process bywhich the flat heart field is transformed into a lineartube was largely unexplored in vertebrates. In a recentstudy, we described a completely unexpected tissuemorphogenetic process by which the nascent hearttube is generated in the zebrafish embryo. We discoveredthat asymmetric involution of the myocardialepithelium from the right side of the heart field initiatesa complex tissue inversion which creates the ventralfloor of the primary heart tube whereas myocardial cellsderived from the left side of the heart field contributeexclusively to the future dorsal roof of this organ (Fig. 1).Intriguingly, asymmetric left-right gene expression withinthe myocardium correlates with asymmetric tissuemorphogenesis and disruption of left-right gene expressioncauses randomized myocardial tissue involution.Our results demonstrated that asymmetric morphogeneticmovements of the two bilateral myocardial cellpopulations generate different dorso-ventral regions ofthe zebrafish heart tube. Failure to generate a hearttube did not affect the acquisition of atrial versus ventricularcardiac cell shapes. Therefore, establishment ofbasic cardiac cell shapes precedes cardiac function.Together, these results provide a framework for the integrationof single cell behaviors during the formation ofthe zebrafish primary heart tube.Divergent polarization mechanisms duringvertebrate epithelial development mediated bythe Crumbs complex protein Nagie oko/Mpp5Nana Bit-Avragim, Nicole Hellwig, Franziska Rudolph(in collaboration with Chantilly Munson, Didier Y.S.Stainier, University of California, San Francisco)Nagie oko (Nok)/Mpp5 is a member of the conservedStardust/Pals1/Mpp5 family of MAGUK proteins and anintegral component of the apical Crumbs-Patj-Par6-aPKCprotein complex of cell polarity regulators. We performeda functional characterization of its different protein-pro-18 Cardiovascular and Metabolic Disease Research

Transformation of the flat heart field into anelongated heart tube. Myocardial cells (green) ofthe right heart field initiate a tissue involutionprocess which creates the ventral floor of thenascent heart (indicated by white arrow) whereascells on the left side do not change theirmonolayered organization and form the dorsalroof. Various tissues are recognized by aPCK (red)and ALCAM (blue) staining. Inserts indicate theorientation of the section planes shown.tein interaction domains and obtained several surprisingresults that change the understanding of the extensivebiochemical and in vitro protein-protein interaction datathat have previously been described for this protein complex.Therefore, alternative scaffolding interactions mustbe in place, in addition to the canonical model of proteinproteininteractions, to allow association of the largerCrumbs-Nagie oko-Patj-Lin-7-Par6-aPKC protein complexin vivo. Moreover, we provided evidence for differentmechanisms involved in the polarization of the apicalCrumbs complex within different tissues of the zebrafishembryo. These findings suggest that distinct epitheliaemploy divergently composed cell polarity complexesduring tissue polarization.Epithelial functions of aPKCi and Nagieoko/Mpp5 during cardiac morphogenesisStefan Rohr, Nana Bit-AvragimNok/Mpp5 is an essential scaffolding partner for bothCrumbs and for the Par6-aPKC protein complex. In thisstudy, we characterized early cardiogenesis in heart andsoul (has)/aPKCi or nok/mpp5 mutants. Loss of either ofthe two cell polarity regulators disrupted the epithelialorganization of myocardial cells during heart tube formationand blocked the progression of cardiac morphogenesisbeyond the heart cone stage. Has/aPKCi andNok/Mpp5 were shown to function autonomouslywithin the myocardial layer during morphogenesis andthe catalytic kinase activity of Has/aPKCi was essentialfor this process. These findings demonstrated theimportance of correct cell polarity and epithelial morphogenesisduring heart formation.Na + ,K + -ATPase interacts with the apical Crumbscomplex in maintaining myocardial polarityElena Cibrián-Uhalte(in collaboration with Adam Langenbacher, XiaodongShu, Jau-Nian Chen, University of California LosAngeles)In another study, we demonstrated the importance ofcorrect ion balance for junctional maintenance andepithelial character of myocardial cells. The Na + ,K + -ATPase, or Na-pump, is an enzyme primarily involved inthe generation of Na + and K + gradients across membranesthereby regulating ionic concentrations withinepithelial cells. The zebrafish 1B1 subunit of Na + ,K +ATPase is encoded by the heart and mind (had) locusand had mutants show a delayed heart tube elongation.This phenotype is reminiscent of the has/aPKCi ornok/mpp5 mutant phenotypes which are characterizedby a lack of epithelial cell polarity. In genetic interactionstudies, Had/Na + ,K + -ATPase and Nok/Mpp5, a componentof the apical Crumbs protein complex, interactedin the maintenance of apical myocardial junctions raisingthe intriguing possibility that the ion balance producedby the Na-pump is critical for cell polarity. Tofunctionally characterize the role of the ion pump function,we produced a mutant form of Had/Na + ,K + ATPasewhich specifically affects the ATPase activity that isessential for pumping sodium across the plasma membraneand found that it could neither rescue the hearttube elongation phenotype nor myocardial polarity.Our study suggests that the osmotic balance producedby the Na-pump contributes to the maintenance ofapical junction belts, a function that is uncovered uponloss of Nok/Mpp5.Selected PublicationsZhang, J, Piontek, J, Wolburg, H, Piehl, C, Liss, M, Otten, C, Christ, A,Willnow, TE, Blasig, IE, and Abdelilah-Seyfried, S. (2009). Establishment ofa neuroepithelial barrier by Claudin5a is essential for zebrafish brainventricular lumen expansion. Proc. Natl. Acad. Sci USA, Early EditionJanuary 2010.Rohr, S, Otten, C, Abdelilah-Seyfried, S. (2008). Asymmetric involution ofthe myocardial field drives heart tube formation in zebrafish, Circ. Res.102, e12-19.Bit-Avragim, N, Hellwig, N, Rudolph, F, Munson, C, Stainier, D, andAbdelilah-Seyfried, S. (2008). Divergent polarization mechanisms duringvertebrate epithelial development mediated by the Crumbs complexprotein Nagie oko. J. Cell Sci. 121, 2503-2510.Cibrián-Uhalte, E, Langenbacher, A, Shu, X, Chen, JN, Abdelilah-Seyfried,S. (2007). Involvement of Na,K ATPase in myocardial cell junctionmaintenance. J. Cell Biol. 176, 223-230.Rohr, S, Bit-Avragim, N, Abdelilah-Seyfried, S. (2006). Heart and soul/PRKCiand Nagie oko/Mpp5 regulate myocardial coherence and remodelingduring cardiac morphogenesis. Development 133, 107-115.Cardiovascular and Metabolic Disease Research 19

Structure of the GroupFerdinand le NobleGroup LeaderProf. Dr. Ferdinand le NobleScientistsDr. rer. Nat. Christian KleinDr. rer. Nat. Yu ShiDr. rer. Nat. Philipp HillmeisterDr. vet. Med. Andrei DuelsnerGraduate StudentsJanna KruegerDong LiuAnna Maria RohdeTechnical AssistantsJanine MikuttaKatja ScholzAnja ZimmerSecretariatBrunhilde PoppeAngiogenesis and CardiovascularPathologyVascular network remodeling and the formation of new blood vessels (angiogenesis andarteriogenesis) plays an important role in the pathophysiology of ischemic cardiovasculardisease, hypertension, and cancer, which are the most common causes of mortality in westernsociety. Our goal is to generate novel genetic insights in the regulation of vascular developmentthat can translate into therapeutic strategies. Our research projects aim at understanding themolecular regulation of angiogenesis and arteriogenesis. We focus on three crucial aspects:differentiation and guidance of angiogenic vessel sprouts by endothelial tip cells, imprinting ofarterial-venous identity in blood vessels by neural guidance genes and hemodynamic factors,and the formation and adaptation of native collaterals in the context of ischemic diseases.Using an integrative molecular and physiological approach in zebrafish, xenopus, and mouse,we want to investigate in which way manipulation of guidance molecules can betherapeutically relevant.Tip Cell biology and Vessel GuidanceRecent studies of vascular network development in theembryo identified several novel aspects of angiogenesiscrucial to generate a functional and stable branchedvascular network. These aspects include: a) the specificationof arterial and venous identity in vessels, b) thedifferentiation and guidance of endothelial tip cells inangiogenic vessels, c) the formation of branches andnetwork patterning.We discovered that neural guidance genes expressed inthe vascular system control vessel branching morphogenesisby regulating the movement of endothelial tipcells at the leading edge of angiogenic vessel sprouts.We demonstrated that delta-like 4 Notch signaling controlsthe differentiation of endothelial cells into tip cellsin response to VEGF gradients. The recognition thatbranching morphogenesis is controlled by a single celltype, the tip cell, that can sense attractant and repulsivesignaling cues opens novel therapeutic avenues. Ourcurrent research endeavours therefore focus at elucidatingthe transcriptome and proteome of endothelialtip cell differentiation and function. We identified severalnovel candidate molecules involved in endothelial tipcell differentiation and vessel guidance events. We performedloss and gain of function experiments inzebrafish and mouse models to elucidate the mechanismof action of these molecules. Our candidatesappear to directly affect tip cell differentiation fromendothelial cells and coordination of their movement inthe extracellular matrix. Interestingly, the guidancefunction of several of these navigation molecules alsoappears to play a pivotal function in organogenesis ofheart, and liver. In this context we provided the proof ofconcept that in pathological conditions interferencewith guidance molecules maybe beneficial to preventthe development of dilated cardiomyopathy in hypoxichearts.20 Cardiovascular and Metabolic Disease Research

Left panel: in vivo imaging of blood vesselsand organ development in transgeniczebrafish expressing GFP in developingblood vessels. Right panel: detail ofdeveloping hepatic vasculature.(Klein&LeNoble, 2009, unpublished)Mechanosensing, Arteriogenesis and IschemicDiseasesArteriogenesis, the outward remodeling of pre-existingsmall collateral arterial networks, occurs as a responseto vascular occlusion or stenosis and importantly determinesthe clinical outcome of ischemic cardiovasculardisease. Release of vasodilators and activation ofinflammatory pathways allowing influx of monocytesmay result in revascularization and restoration of bloodflow into the hypoperfused ischemic area. Therapeuticarteriogenesis is considered of major clinical importanceto treat the increasing population with complexocclusive artery diseases. Distinct differences existbetween animal strains and patients with regard to collateraldevelopment and response to angiogenicgrowth factors. We aim at understanding the molecularmechanism accounting for such differences. In particularwe focus on the formation of native collateralsand efficiency of collateral recruitment and maintenance.Hemodynamic forces exerted by flowing bloodand neural guidance molecules play a critical role in initiationand maintenance of the arteriogenesisresponse. We are interested in how biophysical signalsexerted by flowing blood activate specific genetic programsessential for arterial differentiation. The role ofneural guidance molecules in arteriogenesis is studiedin conditional mutant mice exposed to ischemic vascularstress (femoral artery occlusion, cardiac infarct,stroke models). We examined adaptive recovery fromsuch pathological insults using laser-doppler flow, perfusion-microspheres,imaging including standardangiography, MRI and micro CT. In addition, we performedextensive molecular and histological analysis oftarget organs. Based on a comprehensive analysis ofhemodynamic parameters in experimental models, andin vitro analysis we identified a specific component ofthe shear stress signal relevant for induction of arterialmarker genes and arteriogenesis. At present we arecharacterizing the promoter elements triggered byshear stress relevant for arterial mechanosensing. Ourdata furthermore show that in mice native collateralsare already present at time of birth, but are progressivelypruned during the course of postnatal life. Both thecollateral numbers at birth, as well as their maintenance,are influenced by neural guidance gene function.The outstanding question is to what extent neuralguidance gene function may account for the differencesbetween “good” and “bad” collateralizationresponses as observed in clinical practice. In collaborationwith clinical research partners we are evaluatingnovel strategies and compounds to stimulate arteriogenesisin patients suffering from vascular occlusivedisease. In this context we now provided the proof ofconcept that, in humans suffering for ischemic heartdisease, stimulation of shear stress using non-invasiveexternal counter pulsation therapy, improves functionalrevascularization towards the ischemic regions.Selected PublicationsBuschmann EE, Utz W, Pagonas N, Schulz-Menger J, Busjahn A, Monti J,Maerz W, le Noble F, Thierfelder L, Dietz R, Klauss V, Gross M, BuschmannIR; On behalf of the Arteriogenesis Network (2009). Improvement offractional flow reserve and collateral flow by treatment with externalcounterpulsation. Eur J Clin Invest. 2009 Jul 2. [Epub ahead of print]Tintu A, Rouwet E, Verlohren S, Brinkmann J, Ahmad S, Crispi F, van BilsenM, Carmeliet P, Staff AC, Tjwa M, Cetin I, Gratacos E, Hernandez-AndradeE, Hofstra L, Jacobs M, Lamers WH, Morano I, Safak E, Ahmed A, le Noble F(2009). Hypoxia induces dilated cardiomyopathy in the chick embryo:mechanism, intervention, and long-term consequences. PLoS One. 2009;4(4): e5155.Buschmann IR, Lehmann K, Le Noble F (2008). Physics meets molecules: ismodulation of shear stress the link to vascular prevention? Circ Res.102(5): 510-2.Suchting S, Freitas C, le Noble F, Benedito R, Bréant C, Duarte A, EichmannA (2007). The Notch ligand Delta-like 4 negatively regulates endothelialtip cell formation and vessel branching. Proc Natl Acad Sci U S A. 104(9):3225-30.Lu X*, Le Noble F*, Yuan L*, Jiang Q, De Lafarge B, Sugiyama D, Bréant C,Claes F, De Smet F, Thomas JL, Autiero M, Carmeliet P, Tessier-Lavigne M,Eichmann A (2004). The netrin receptor UNC5B mediates guidanceevents controlling morphogenesis of the vascular system. Nature432(7014): 179-86. *equal contributionCardiovascular and Metabolic Disease Research 21

Francesca M. SpagnoliStructure of the GroupGroup LeaderDr. Francesca M. Spagnoli**ScientistsDr. Elisa Rodríguez Seguel**part of the period reported** start of the group: July 2008Graduate StudentsNuria Cerdá-Esteban*Kristin Petzold*Igor Pongrac*Technical AssistantsHeike Naumann*Molecular and Cellular Basis ofEmbryonic DevelopmentAfundamental question in developmental biology is how a specialized tissue originatesfrom a pluripotent precursor cell in the embryo. The endoderm germ layer gives rise to anumber of vital organs in our body, including the lungs, liver, pancreas and intestine. Thisremarkable diversity derives from a homogenous and multipotent precursor cell population.The central aim of our research is to understand the mechanisms that pattern and establishcompetence within anterior embryonic endoderm in order to progressively specify thepancreatic organ domain. In addition, we focus on spatio-temporal mechanisms that restrictspecification of the pancreas versus neighboring tissues, such as the liver. A completeunderstanding of these early events will provide insights into the development of theseorgans. Finally, this information might be crucial for advances in regenerative medicinestrategies for the treatment of incurable diseases, such as diabetes.ProjectsIn vivo lineage analysis of pancreatic and hepaticprecursor cellsElisa Rodríguez Seguel, Igor PongracDuring embryogenesis, the pancreas originates fromdistinct outgrowths of the dorsal and ventral foregutendoderm. Both outgrowths give rise to endocrine andexocrine cells and, subsequently, fuse to form one singleorgan. The ventral pancreas arises next to thehepatic endoderm and they possibly originate from acommon bipotent precursor. However, a single cell havingthe dual potential to differentiate along the hepaticand pancreatic lineages has never been isolated neitherin vivo nor in vitro. One main focus of my laboratoryis to investigate how pancreatic versus hepatic fatedecision occurs in the endoderm at both the cellularand molecular level.To conduct a comprehensive in vivo analysis of thehepatic-pancreatic lineage in the mouse embryo, weuse transgenic reporter models that express EGFP orphotoconvertible fluorescent proteins under the controlof lineage-specific promoters. We are using thesenew genetic tools to: i. address in vivo and in vitro if theliver and pancreas arise from a common bipotent precursor;and ii. to trace and molecularly profile the presumptiveprecursor cell and its descendants in themouse embryo. All together, these experiments willdetermine how the hepatic-pancreatic lineage is establishedin vivo, whether a bipotent endodermal precursorexists, and provide us with its molecular signature.Molecular mechanisms controlling pancreas versusliver fate decisionNuria Cerdá-Esteban, Heike NaumannHepatic and pancreatic endoderm share a common setof intrinsic regulatory factors, such as the FoxA andGATA transcription factors, and are exposed to the sameextrinsic signals, FGF and BMP. However, it remainsunclear how the same factors can activate pancreaticgenes, such as Pdx1 and Ptf1a, in the future pancreas,without inducing them in hepatic progenitors; andanalogously, they enable liver development, but notpancreatic development in the hepatic endoderm. Both22 Cardiovascular and Metabolic Disease Research

Stepwise specification of pancreatic progenitor cells within the endoderm. (A) Schematic modelof the emergence of the pancreatic and hepatic lineage from a common endodermal precursor.(B-D) Prox1-EGFP transgenic (Prox1-EGFP Tg) mouse strains reproduce the endogenous pattern ofProx1 expression in the mouse embryos between embryonic stage (E) 7.5 and 9.5. Arrows indicateProx1 expression in the anterior definitive endoderm (B, B’) and foregut endoderm (C) at E7.5 andE8.5, respectively. (E) Whole-mount in situ hybridization analysis of Pdx1 marks both dorsal andpancreatic buds at E9.5. Abbreviations: Alb, albumin; BP, bipotent precursor; DE, definitive endoderm;Lv, liver; Pa, pancreas; vp, ventral pancreas; dp, dorsal pancreas.intrinsic and extrinsic regulators of this cell fate decisionare under study in my laboratory.Our previous studies have elucidated the molecularevents downstream of the GATA factors within theanterior endoderm in Xenopus laevis. We identifiedGATA downstream targets, such as TGIF2, acting asdevelopmental regulators of the pancreatic versushepatic fate decision. Interestingly, TGIF2 promotes pancreaticfate within the endoderm at the expense ofhepatic markers. Conversely, in TGIF2-depleted embryoswe observed expansion of the hepatic domain at theexpense of pancreatic specification. We are currentlyinvestigating whether TGIF2 controls pancreatic versushepatic fate decision and converts liver to pancreasupon over-expression in mammalian systems.In parallel, we study how and when extrinsic factors,such as BMP, control the emergence of liver versus pancreasfrom the same embryonic region.All together, these experiments will define developmentalregulators that are able not only to specify onefate, but also antagonize the other (eg. pancreatic versushepatic). This knowledge will be crucial for defininglineage reprogramming strategies of liver to pancreastoward a new cure for diabetes.Novel signals guiding endodermal progenitorstoward pancreatic fateKristin Petzold, Heike NaumannIn another line of research, we are investigating molecularplayers that act as instructive factors during earlypancreatic development. In previous studies using theXenopus system, we identified a novel pancreatic factorthat we called Shirin. Very little is known about the biologicalfunction of this protein and, in particular, noembryological function has been assigned to it inmammalian species. We showed that Shirin is specificallyexpressed in the endoderm and pancreatic rudimentsfrom gastrulation onwards, representing one theearliest marker of pancreatic endoderm. Gain-of-functionexperiments in Xenopus indicated that Shirin aloneis sufficient to induce pancreatic identity, acting as apancreatic instructive factor. Similarly, upon conditionalablation of Shirin gene expression in the mouse weobserve defects in pancreas formation. Taken together,our observations suggest a conserved role for Shirin atvery early stages of pancreatic specification.At later developmental stages, Shirin is also expressedin neural crest territory and muscle progenitor cells. Weare currently investigating a potential role of Shirin inthese tissues in collaboration with the group of Prof. C.Birchmeier at the MDC.Selected PublicationsSpagnoli*, FM, Rosa*, A, Brivanlou, AH. (2009). Mesendodermal lineagespecification in embryonic stem cells is under the control of a conservedmicroRNA family. Developmental Cell 16, 517-27. *co-first authorsSpagnoli, FM and Brivanlou, AH. (2008). The Gata5 target, TGIF2, definesthe pancreatic region by modulating BMP signals within the endoderm.Development 135, 451-461.Spagnoli, FM. (2007). From endoderm to pancreas: a multistep journey.The CMLS Journal 64, 2378-2390.Sapkota, G, Alarcón, G, Spagnoli, FM, Brivanlou, AH., Massagué J. (2007).Balancing BMP signaling through integrated inputs into the Smad1linker. Molecular Cell. 25, 441–454.Spagnoli, FM and Brivanlou, AH. (2006). The RNA Binding Protein, Vg1RBP,is Required for Pancreatic Fate Specification. Developmental Biology. 292,442-456.Cardiovascular and Metabolic Disease Research 23

Structure of the GroupKai M. Schmidt-Ott(Emmy NoetherResearch Group)Group LeaderProf. Dr. Kai M. Schmidt-OttScientistsDr. Max WerthGraduate StudentsAnnekatrin AueAntje ElgerEugenia SingerKatharina WalentinAnne-Katharina WübkenTechnical AssistantsTatjana LuganskajaAntje SommerManager of SponsoredProgramsSusanne WisslerDevelopmental Biology andPathophysiology of the KidneyThe kidney is a central organ in cardiovascular diseases. It excretes toxins into the urine,regulates volume and solute homeostasis in the body, and produces hormones. The kidney isnot only itself a target in cardiovascular disease and but also is centrally involved in cardiovascularhomeostasis. Moreover, kidney failure constitutes one of the most important risk factors for othercardiovascular diseases. The kidney is composed of structural units called nephrons, which consistof several different types of renal epithelial cells that facilitate directional transport. Our groupstudies the molecular mechanisms of nephron morphogenesis and the maintenance of epithelialintegrity later in life. We focus on transcription factors and their regulation of aspects of epithelialdifferentiation. We use mouse models and epithelial cell culture systems and employ a widespectrum of techniques, including genome-wide gene expression analysis, DNA-protein inter -action analysis as well as organ culture techniques of the developing kidney.Kidney DevelopmentIn the mammalian embryo, formation of the definitivekidney is initiated during mid-embryogenesis, when theureteric bud, an epithelial tubule extending from theposterior Wolffian duct, interacts with an adjacent progenitorcell population, the metanephric mesenchyme.The ureteric bud undergoes branching morphogenesisto give rise to the ureter, renal pelvis and collecting ductsystem, while the metanephric mesenchyme convertsinto epithelial cells that subsequently get patternedalong the proximal-distal axis to form the different celltypes of the nephron. As these cells differentiate, theyobtain epithelial characteristics, including the establishmentof apico-basal polarity and the formation ofepithelial-specific junctions. Our laboratory investigatesthe molecular mechanisms underlying theseevents, which are intriguing for several reasons.First, mammalian embryonic kidney development constitutesa classical model system in developmental biology,in which branching morphogenesis and tubulogenesisoccur in parallel. The sequence of events can beclosely monitored in organ cultures, in which key in vivoaspects of nephrogenesis are recapitulated. Exogenousor genetic perturbations of kidney development resultin congenital kidney diseases. Furthermore, adult kidneyepithelia preserve the ability to reactivate molecularpathways from earlier developmental stages in certaindisease states, including tumors, kidney fibrosis,and acute tubular injury.Wnt signaling in Kidney Development andDiseaseWnt proteins are a family of secreted molecules thatare important for various aspect of embryonic development.Several WNT molecules, including Wnt4, Wnt7b,and Wnt9b, are centrally involved in kidney development.We previously found that Wnt signaling via the β-catenin/TCF/Lef pathway mediates aspects of epithelialdifferentiation in metanephric mesenchymal progenitorcells. In addition, this pathway provides proliferativeand antiapoptotic signals that regulate the size of theprogenitor pool. As part of a DFG funded project (Schm1730/2-1) we characterize the target gene program of24 Cardiovascular and Metabolic Disease Research

TCF/Lef signaling in the kidney. One crucial aspect ofour future studies will be to elucidate the role of Wntsignaling in kidney disease, with an emphasis on thecanonical TCF/Lef-dependent pathway. For this purposewe are using different genetic and experimental mousemodels, which we analyze using genome-wide analysisof expression and transcription factor binding. We areworking in close collaboration with Thomas Willnow(MDC), Friedrich C. Luft (ECRC), and Jonathan Barasch(Columbia University, New York). We believe that adetailed elucidation of the transcriptional network controlledby TCF/Lef will yield fundamental insights intogrowth, remodeling, and regeneration in the kidney.CABTranscription factors involved in terminal renalepithelial differentiationWhile TCF/Lef-dependent signaling accounts for earlydevelopmental programs in renal epithelial progenitors,it may in fact be inhibitory to terminal differentiationof tubular epithelia. Therefore, we are seeking complementarytranscriptional regulators that induce theestablishment of epithelial polarity and the expressionof segment-specific markers. Using expression profiling,we identified two candidate transcriptional regulatorsof terminal differentiation, which belong to theCP2 group of transcription factors (Tcfcpl1 and Grhl2),which are highly and specifically expressed in the distalnephron. Characterization of Grhl2 is part of a DFGfundedproject (Schm 1730/3-1) within FG “Epithelialmechanisms of renal volume regulation” (Speaker:Sebastian Bachmann, Charité Berlin). The projects benefitfrom our close collaborations with JonathanBarasch (Columbia University, New York), ThomasWillnow (MDC), and Michael Bader (MDC).Biomarkers of Renal InjuryCharacterization of transcription factors in nephron epithelia.A. β-Galactosidase expressed from the Grhl2 locus displays an epithelialspecificpattern in the early postnatal kidney. B. Nuclear localization ofthe transcription factor Tcfcp2l1 (green) in a subset of nephron epithelia(red) as detected by confocal microscopy. C. Transcription factor bindingto genomic DNA in a renal epithelial cell line as determined by chromatinimmunoprecipitation followed by massively parallel sequencing.The frequency plot reveals strongly enriched transcription factor bindingto genomic DNA around transcriptional start sites (TSS).Re-expression of embryonic marker molecules is a commonfeature in disease states and is believed to participatein compensation and regeneration. Neutrophilgelatinase-associated lipocalin (NGAL) is a protein inthe developing kidney that is sufficient to induce differentiationin embryonic renal epithelial progenitors.NGAL is also markedly reactivated in tubular injury ofthe kidney and its urinary excretion is closely correlatedwith the temporal onset and severity of tubular injury.In collaboration with Jonathan Barasch (ColumbiaUniversity, New York), Friedrich C. Luft (ECRC) and RalphKettritz (ECRC), we are conducting clinical studies totest the performance of NGAL as a urinary and plasmabiomarker of acute kidney injury. We are aiming to optimizediagnostic algorithms that utilize NGAL measurementsto predict renal injury or to differentiate renalinjury from related clinical entities. This project servesas a prime example to illustrate the importance of anunderstanding of basic molecular mechanisms in theembryo to address the diagnosis of renal disease in theadult. Translational aspects of our research are markedlyenhanced by our close ties to the ‘Experimental andClinical Research Center’ (see ECRC section).Selected publicationsLi JY, Paragas N, Ned RM, Qiu A, Viltard M, Leete T, Drexler IR, Chen X,Sanna-Cherchi S, Mohammed F, Williams D, Lin CS, Schmidt-Ott KM,Andrews NC, Barasch J. (2009) Scara5 is a ferritin receptor mediatingnon-transferrin iron delivery. Dev Cell. 16:35-46.Schmidt-Ott KM, Barasch J. (2008) WNT/beta-catenin signaling innephron progenitors and their epithelial progeny. Kidney Int. 74:1004-8.Schmidt-Ott K.M., Masckauchan T.N., Chen X., Hirsh B.J., Sarkar A., Yang J.,Paragas N., Wallace V.A., Dufort D., Pavlidis P., Jagla B., Kitajewski J.,Barasch J. (2007) -Catenin/TCF/Lef Controls A Differentiation-AssociatedTranscriptional Program In Renal Epithelial Progenitors. Development.134(17):3177-90.Schmidt-Ott, K.M., Mori, K., Li, J.Y., Kalandadze, A., Cohen, D.J., Devarajan, P.,Barasch, J. (2007) Dual action of neutrophil gelatinase-associatedlipocalin. J Am Soc Nephrol. 18, 407-13.Schmidt-Ott, K.M., Chen, X., Paragas, N., Levinson, R.S., Mendelsohn, C.L.,Barasch,, J. (2006) c-kit delineates a distinct domain of progenitors in thedeveloping kidney. Dev Biol. 299, 238-49.Cardiovascular and Metabolic Disease Research 25

Genetics and Pathophysiology of Cardiovascular DiseasesStructure of the GroupNikolaus RajewskyGroup LeaderProf. Dr. Nikolaus RajewskyScientistsCatherine AdamidiDr Kevin ChenDr Minnie FangGraduate StudentsMarc FriedlaenderMarvin JensAzra Krek*Svetlana LebedevaJonas MaaskolaSebastian Mackowiak*Systems Biology of Gene RegulatoryElementsMy lab uses experimental (molecular biology, biochemistry) together with computationalmethods (bioinformatics, computational biology, etc) to dissect, systems-wide, functionand evolution of gene regulation in metazoans. One major focus is to understand moreabout post transcriptional gene regulation exerted by small RNAs, in particular microRNAs.We are developing predictive models for targets of microRNAs. We also investigate generalmechanisms of gene regulation by microRNAs and RNA binding proteins in cell lines and invivo. For example, we are studying the function of small RNAs during early development inC. elegans (Fig. 1). Furthermore, we have established planaria (Fig. 2) as a model system in ourlab. These freshwater flatworms are famous for their almost unlimited ability to regenerateany tissue via pluripotent, adult stem cells. We investigate the role of small RNAs inplanarian stem cell biology and regeneration.IntroductionA major lesson from recent genomics is that metazoansshare to a large degree the same repertoire of proteinencodinggenes. It is thought that differences betweencells within a species, between species, or betweenhealthy and diseased animals are in many cases due todifferences in when, where and how genes are turnedon or off. Gene regulatory information is to a largedegree hardwired into the non-coding parts of thegenome. Our lab focuses on decoding transcriptionalregulation (identification and characterization of targetsof transcription factors in non-coding DNA) andpost-transcriptional control mediated by small, noncodingRNAs, in particular microRNAs. microRNAs are arecently discovered large class of regulatory genes, presentin virtually all metazoans. They have been shown tobind to specific cis-regulatory sites in 3’ untranslatedregions (3’ UTRs) of protein-encoding mRNAs and, byunknown mechanisms, to repress protein production oftheir target mRNAs. Our understanding of the biologicalfunction of animal microRNAs is just beginning toemerge, but it is clear that microRNAs are regulating orinvolved in a large variety of biological processes andhuman diseases, such as developmental timing, differentiation,long-term memory, signaling, homeostasis ofkey metabolic gene products such as cholesterol, apoptosis,onset of cancer, Tourette’s syndrome, and others.Overall, however, it is clear that miRNAs are only a smallpart of the entire post transcriptional gene regulationapparatus used by cells, and we are beginning to alsoexplore the role of RNA binding proteins.Systems Biology of Gene RegulationCatherine Adamidi, Kevin Chen, Minnie Fang, MarcFriedlaender, Signe Knespel, Azra Krek, AndreasKuntzagk, Svetlana Lebedeva, Jonas Maaskola, MarlonStoeckius, Nadine Thierfelder26 Cardiovascular and Metabolic Disease Research

Pinar Oenal*Marlon StoeckiusNadine ThierfelderTechnical AssistantsSalah Ayoub*Signe Knespel (Lab Manager)Tatjana Luganskaja*SecretariatAlex Tschernycheff*part of the period reportedFigure 1. C. elegansIt is clear that a better understanding of gene regulationand in particular of the just emerging universe ofnon-coding RNAs can only come by integrating variousdata sources (comparative sequence analysis, mRNAexpression data, protein-protein interactions, mutantphenotypes from RNAi screens, polymorphism data,experimentally defined gene regulatory networks,ChIP-chip data, etc) since each data source alone is onlya partial description of how cells function. For example,to understand microRNA function, we not only need toidentify their targets but also to decode howmicroRNAs are transcriptionally regulated. A majorfocus of the lab is therefore in developing methods thatintegrate different data sources and methods to produceglobal and yet specific predictions about how,when, and where gene are regulated. This will ultimatelylead to the identification and functional descriptionof gene regulatory networks. We will continue to test,develop and “translate” these methods and their predictionsusing specific biological systems, such asmetabolism in mammals, regeneration in planarians,early embryogenesis in C. elegans, and within collaborationswith other experimental groups.Function of microRNAs(Azra Krek, Marc Friedlaender, Minnie Fang, MarlonStoeckius, Jonas Maaskola, Nadine Thierfelder,Svetlana Lebedeva, Sebastian Mackowiak, MarvinJens).We have developed one of the first microRNA targetfinding algorithms and could later on show thatmicroRNAs very likely regulate thousands of geneswithin vertebrates, flies, and nematodes. We have furtherhelped to elucidate the function of microRNAs inpancreatic beta cells (insulin secretion), in liver (cholesterollevel), and other systems. More recently, we haveshown that microRNAs can leave cell type specificmRNA expression signatures on hundreds of genes,and that human genotyped SNP data can be used toexplicitly demonstrate and quantify the contribution ofmicroRNA targets to human fitness. All microRNA targetpredictions of our algorithm PicTar can be accessedat our searchable PicTar website. We have further developedcomputational methods to predict miRNAs fromhigh throughput sequencing data (see miRDeep webpage).We have also pioneered approaches that allowedCardiovascular and Metabolic Disease Research 27

Figure 2. Planariato experimentally assay, genome-wide, the impact ofmiRNAs on protein synthesis (see pSILAC website). Amajor ongoing effort is currently to use and developseveral key high throughput technologies for in vivostudies in C. elegans and planaria: high-throughputproteomics (SILAC), RNA sequencing, and new methodsthat allow the genome wide identification of bindingsites if RNA binding proteins.These projects involved or involve, in part, collaborationswith the following labs: Markus Stoffel lab (ETHZurich), Fabio Piano and Kris Gunsalus (NYU), MatthiasSelbach (MDC), Markus Landthaler (MDC).Early embryogenesis in C. elegansMarlon Stoeckius, Jonas Maaskola, Nadine ThierfelderAlthough C. elegans is one of the most famous modelsystems for developmental biology, it has been impossibleto use most high-throughput technologies to studydifferential gene expression and networks during veryearly embryogenesis (for example the oocyte to onecellembryo transition upon fertilization). However,high-throughput technologies are needed to solve severalfundamental problems in embryogenesis, forexample how post-transcriptional and later transcriptionalregulatory networks drive development. One keyproblem is that the state of the art method to obtainprecisely staged early embryos consists of sortingembryos via mouth pipetting, thus making it impracticalto obtain large samples. To overcome this problem,we have developed a novel method (“eFACS”) thatallows us to sort embryos at precise stages duringembryogenesis via FACS sorting (Stoeckius, Maaskola etal, Nature Methods 2009). For example, we can nowroutinely obtain ~60,000 one-cell stage embryos (at apurity of >98%) in one FACS run, enough to apply virtuallyany high throughput method of interest (Figure 3).We have used eFACS to assay the dynamics of smallRNA expression during embryogenesis. We discovered awealth of orchestrated, specific changes between andwithin virtually all classes of small RNAs. These findingsopen the door for many computational and functionalfollow up studies. For example, we are proceeding todevelop and use in vivo SILAC to study the functionalimpact of certain small RNAs on the post-transcriptionallevel. These projects involve collaborations with the F.Piano lab (NYU), M. Selbach (MDC), W. Chen (MDC) andothers.Small RNAs in planarian stem cell biologyCatherine Adamidi, Marc Friedlaender, Pinar Oenal,Sebastian MackowiakWe used massive next generation sequencing to identifymiRNAs and piRNAs in S. mediterranea. We also identifiedmiRNAs that seem specifically linked to stem cellbiology. A number of these miRNAs are conserved inhumans (Friedlaender & Adamidi et al.PNAS 2009). Weare currently starting to functionally follow up some ofthese results. We are also starting to systematicallyidentify the proteome of planarian stem cells and areinterested to use these data to identify and characterizekey genes involved in regeneration mediated by28 Cardiovascular and Metabolic Disease Research

Figure 3. Proof of principle: FACS sorting of a mixed C. elegans embryo population (eFACS) routinely yields tens of thousands of almost perfectlyclean one-cell embryo population (Stoeckius & Maaskola et al, Nature Methods 2009). A transgenic strain that expresses GFP mosthighly in the one cell embryo is used. Embryos are fixed with Methanol, and the GFP positive population marked in green (a) is resorted (b). Arandom sample of 96 embryos from the resorted population consists to at least 98% of one cell embryos, as shown by microscopic analysis(c) and also confirmed by PCR markers (d).stem cells. These projects involve, in part, collaborationswith the Sanchez lab (U. of Utah), S. Kempa (MDC), W.Chen (MDC) and others.Selected PublicationsKrek, A, Gruen, D, Poy, MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P,da Piedade I, Gunsalus KC, Stoffel M, and Rajewsky N (2005).Combinatorial microRNA target predictions. Nature Genetics 37, 495-500Chen, K and Rajewsky, N. (2006). Natural selection on human microRNAbinding sites inferred from SNP data. Nature Genetics 38, 1452-1456.Chen, K and Rajewsky, N. (2007). The evolution of gene regulation bytranscription factors and microRNAs.. Nature Reviews Genetics 8, 93-103.Selbach M; Schwanhaeusser B; Thierfelder N; Fang Z; Khanin R; andRajewsky N (2008). Widespread changes in protein synthesis induced bymicroRNAs. Nature 455, 58-63Friedlaender MR; Adamidi C; Han T; Lebedeva S; Isenbarger TA; Hirst M;Marra M; Nusbaum C; Lee WL; Jenkin JC; Alvarado AS; Kim JK; andRajewsky N. (2009). High-resolution profiling and discovery of planariansmall RNAs. PNAS 28, 11546-11551Cardiovascular and Metabolic Disease Research 29

Norbert HübnerStructure of the GroupGroup LeaderProf. Norbert HübnerScientistsDr. Anja BauerfeindDr. Claudia GöseleOliver HummelDr. Svetlana PaskasDr. Giannino PatoneDr. Carola RintischDr. Franz RüschendorfDr. Klaus RohdeDr. Kathrin SaarDr. Herbert SchulzMedical Genomics and Genetics ofComplex Cardiovascular DiseasesMy group is investigating the molecular genetic basis of common cardiovascular riskfactors and disorders in experimental rodent and human populations. We are workingwith inbred rat strains since they provide one of the most relevant models of commonmultifactorial cardiovascular human disease. It has been the major model for physiologicalinvestigation, providing a body of data on patho-physiology, including detailed mechanistic,biochemical and metabolic characterisation that cannot easily be replaced by other models.The work of our group focuses on the development of genomic tools and to utilize these forfunctional genetic and genomic approaches for complex disease gene identification.Additionally my group has an increasing interest in translating findings from modelorganisms to humans by comparative genome analysis.Establishment of functional genetic and genomicresources for the ratWe have developed a large number of genomicresources to facilitate functional genomic studies in therat. These efforts included long-range physical mapsand characterization of single nucleotide variationwithin the rat genome. We contributed to the annotationand assembly of the rat genome sequence. Therecent availability of the rat genome sequence andassociated genomic tools has raised the profile andpace of research into genetic analysis of rat traits anddramatically accelerated prospects for gene identification.Decades of exquisite phenotyping and detailedanalysis of crosses of inbred rats have resulted in initiallocalization of hundreds of loci involved in complex diseaseand quantitative phenotypes, but with very feweventual gene identifications to date. A clear understandingof the origin and structure of genetic variationin the rat will provide a key-missing piece of this puzzle.To fully realize the power of the recent rat genomesequence, we are currently initiating the completegenetic dissection of the ancestral segments makingup the most commonly used inbred lines.Haplotype mapping for genetic analysis in the ratGenetic variation in genomes is organized in haplotypeblocks and species-specific block structure is defined bydifferential contribution of population history effects incombination with mutation and recombination events.Haplotype maps characterize the common patterns oflinkage disequilibrium in populations and have importantapplications in the design and interpretation ofgenetic experiments. Although evolutionary processesare known to drive the selection of individual polymorphisms,their effect on haplotype block structuredynamics has not been shown.We have led an international consortium (STAR) andreported a survey of genetic variation based on almost3 million newly identified SNPs. We constructed highdensitygenetic maps, creating a large dataset of fullycharacterized SNPs for disease gene mapping. Our datacharacterize the population structure and illustrate thedegree of linkage disequilibrium. We provide a detailedSNP map and demonstrate its utility for mapping quantitativetrait loci. This community resource is openlyavailable and augments the genetic tools for studyingthis model of human disease.30 Cardiovascular and Metabolic Disease Research

Graduate StudentsSamreen FalakJudith FischerKatharina GrunzMatthias HeinigHenrike MaatzTechnical AssistantsSusanne BlachutGabriele BornMathias GerhardHeide KistelAnita MüllerSabine SchmidtMadeleine Skorna-NußbeckSecretariatKornelia DokupGenome approaches to dissecting cardiovascularand metabolic diseaseThe rat genome sequence together with other genomicresources, including genetic and structural variationmaps, provide exceptional opportunities for identifyinggenes and pathways underlying disease phenotypes.Our collaborators and we have devised a systemsapproach for dissecting genetic networks systematicallyacross the biological scale, from molecular to physiological,using segregating rat populations We combinedlinkage analyses with genome-wide expression profilingand identified independent genes contributing toheart failure susceptibility, increased left ventricularmass, and hypertension in spontaneously hypertensiverats (SHR) and SHR derived substrains. Moreover, we areinterested in the genetic regulation of gene expressionin a range of insulin sensitive tissues including fat, kidney,adrenal, heart, skeletal muscle, the vasculature, andliver in rat RI strains. Using the data collected acrossmultiple tissues we can now detect the genotypedependent co-expression of gene networks and suggesttheir possible biological implications for commoncardiometabolic disorders. By identifying several ofrobustly mapped cis- and trans-acting expression QTLsin a model with large number of existing physiologicalQTLs we generated a permanent resource to test thehypothesis that genetic variation in gene expressionhas a key role in the molecular evolution of complexphysiological and pathophysiological phenotypes thatmay be shared in common with similar disorders inhumans.Next generation sequencing of thespontaneously hypertensive rat genomeThe genome sequence of the normotensive BN strain isavailable but only limited knowledge exists of genomicsequence variability between SHR and BN. Within aninternational collaboration we started to sequence theSHR genome using paired-end sequencing techniqueon the Solexa platform. Nearly 816 million reads (33.8GB) were sequenced of which 87% of reads weremapped to the reference BN genome giving 10.7x coverageof the SHR genome. We identified 3.6 million highquality SNPs and 343,243 short indels (1 to 15 bp)between SHR and BN with a very low false positive rate(

Structure of the GroupGroup LeaderFriedrich C. LuftMouse physiologyVolkmar GrossGene labSylvia BähringEicosanoid metabolismWolf-Hagen SchunckFriedrich C. LuftHypertension, Vascular Disease,Genetics, and NephrologyThe Department of Nephrology/Hypertension/Clinical Pharmacology, headed by Friedrich C.Luft (since 1992) encompassed numerous groups that are now represented independentlyelsewhere (Clinical Research Center, Ralph Kettritz, Dominik N. Müller, Maik Gollasch, KaiSchmidt-Ott). The core group has focused on genetic rearrangements as a cause of somaticalterations and cardiovascular disease. The group has also directed its attention to elucidatingmechanisms of hypertension by means of novel mouse models. In addition, the group has alsofocused on alternative mechanisms of mineralocorticoid-induced vasculopathy. The group haselucidated the role of eicosanoids in arrhythmia generation and gap junction protein function.Finally, the group has collaborated in efforts directed at salt-sensitive hypertension and noveldriving forces.Molecular geneticsSylvia Bähring leads “gene lab” a laboratory dedicated toclarifying complex genetic issues. She is not focusing onstraight-forward mutations. The primary projectinvolves autosomal-dominant hypertension withbrachydactyly type E (BDE). We now know that these(we have 5 families) are complex rearrangement syndromeson Chromosome 12p. The inversion region forhypertension and brachydactyly on chromosome 12pfeatures multiple splicing and noncoding RNA. We havesuccessfully dissected the disease-carrying chromosomalbands on the short arm of 12p with help from collaboratorsin Jena, and will now subject this material to”next-generation” sequencing. Atakan Aydin is focusingon this important issue supported by the groups of WeiChen and Norbert Hübner at the Genome Center. Weshowed earlier that our primary suspect (a gene withno open reading frames or Kozac sequences) codes for amicroRNA that is responsible for the syndrome. We areexpending most of our energies to elucidate this importantproject. Our efforts include a collaboration withMatthias Selbach and Nikolaus Rajewsky to express ourputative microRNA in HELA cells in order to find out thetarget genes on RNA and protein level and to elucidatethe functional pathways leading to the phenotypes.Philipp Maass pursues another important project of ourgroup. We studied a family BDE, albeit without hypertension.We found a t(8;12)(q13;p11.2) translocation withbreakpoints upstream of PTHLH on chromosome 12p11.2and disrupted KCNB2 on 8q13. The PTHLH translatedPTHrP protein regulates chondrocyte differentiation viaa negative-feedback loop implicating the signaling factorIHH. We determined a PTHLH down- and IHH upregulationin chondrogenic induced fibroblasts fromaffected BDE patients. We sequenced the translocationbreakpoints and found a conserved AP1 transcriptionfactor binding site on chromosome 12p11.2 and an ETS1binding site from 8q13, which resides near the AP1 sitedue to the translocation. We observed binding of bothtranscription factors at the breakpoint sequence. SinceAP1 and ETS1 interact, we tested if these factors regulatePTHLH in murine and human chondrocyte cell lines.Furthermore chondrocytes and BDE fibroblasts showeddifferent epigenetic modifications between the wildtypeand breakpoint allele with an enrichment of histonemodifications H3K4me1 and H3K4m3, which are32 Cardiovascular and Metabolic Disease Research

Mineralocorticoid receptorAnette FiebelerScientists and studentsAtakan AydinCarsten LindschauPhilipp MaassCosima SchmidtAnne KonkelMichael OechsnerAndrey da Costa-GoncalvesTechnical AssistantsAstrid MühlEireen KleinIrene HolfingerYvette NeuenfeldChristel AndréeRamona ZummachIlona KamerFigure 1. Type E brachydactyly, balancedtranslocation t(8;12)(q13;p11.2)in Metaphase-FISH, fibroblasts transformedto chondrogenic cells, upregulationof Indian hedgehog (IHH) anddownregulation of parathyroid-likehormone (PTHLH).generally associated with regulatory activity. Our findingshave broader implications for understanding howchromosomal aberrations affect epigenetic modificationsand long-range gene regulation. The results indicatethat dysregulation of chondrogenesis due to anovel site for interaction of AP1 and ETS1 upstream ofPTHLH, is the underlying cause for the BDE.That is all very nice but it’s miceVolkmar Gross and Friedrich C. Luft have been collaboratorsfor almost 20 years. They have maintained thestandard of conservative cardiovascular physiology asopposed to descriptive RT-PCR, RNase protectionassays, and Western blotting (where physiology ends bythe others), although we have incorporated all thesetechniques to test our hypotheses. Our goal is to showit at the experimental level, gene expression can be nosubstitute. Gross and Luft have attempted to bring allof cardiovascular physiology to mice and particularly tounique mice. Recently, we investigated synophilin (acollaboration with Paul Greengard, Nobel Prize 2000).The spinophilin protein controls intensity/duration of Gprotein-coupled receptor signaling and thereby influencessynaptic activity. We hypothesized thatspinophilin affects blood pressure through centralmechanisms. We measured blood pressure and heartrate in SPL-deficient -/-, SPL+/-, and SPL +/+ mice bytelemetry combined with fast Fourier transformation.We found that an increase in central sympathetic outflowparticipates in blood pressure and heart rateincreases in SPL -/- mice. The elevated blood pressure inSPL(-/-) mice was associated with attenuated baroreflexsensitivity and decreased parasympathetic activity.Our study is the first to show a role for the spinophilingene in blood pressure regulation. These novel resultshave a bearing on centrally (brain) mediated control ofblood pressure. The group has pursued additionalmouse-related projects. These include work on db/dbleptin receptor deficient mice and mice deficient in thesolulble epoxide hydrolase.Mineralocortcoid receptor signalingAnnete Fiebeler has led a group of investigators specificallyinterested in mineralocorticodi-reseceptor signaling.In a recent study, she and friends showed that corticosteroneinduces rapid mineralocorticoid receptorCardiovascular and Metabolic Disease Research 33

Figure 2. Omega-3 fatty acids protect against connexin 43 dyslocalization and sudden cardiac death. Connexin 43 (Cx43), a mediator of intermyocyteconduction, is redistributed to the lateral cell membranes in the hearts of dTGR making them highly susceptible to fatal arrhythmias.Dietary omega-3 fatty acids (Omacor) and aliskiren normalized Cx43 localization to the gap junctions within the intercalated disks and protectedthe animals against sudden cardiac death.signaling in vascular smooth muscle cells that involvesmitogen-activated protein kinase kinase/extracellularsignal-regulated kinase-dependent pathways. Thesenew mineralocorticoid receptor-dependent signalingpathways suggest that glucocorticoids may contributeto vascular disease via mineralocorticoid receptor signaling,independent of circulating aldosterone. Morerecently, the group showed that the growth arrest-specificprotein 6 (GAS6) participates in desoxycorticosterone-induced(DOCA) hypertension and target organdamage. They found that Gas 6 was upregulated in ratswith high aldosterone levels. Mineralocorticoid receptorblockade prevented target organ damage anddecreased the elevated Gas 6 expression. Vascularsmooth muscle cells given aldosterone increased theirGas 6 expression in vitro. Cardiac expression of interleukin6 and collagen IV was blunted in Gas 6(-/-) mice,indicating reduced inflammation and fibrosis. Gas 6(-/-)mice also had an improved renal function with reducedalbuminuria, compared to wild-type mice. Gas 6appears to play a role in mineralocorticoid receptormediatedtarget organ damage. Furthermore, becausewarfarin interferes with Gas 6 protein expression, thefindings could be of clinical relevance for anticoagulantchoices.EETs, HETEs and alternativesRole of enhanced EET-degradation in heart failureWolf-Hagen SchunckA major route of EET-degradation is catalyzed by thesoluble epoxide hydrolase (sEH), an enzyme that is upregulatedby angiotensin II (Ang II) in the heart and vasculature.We collaborated with Jan Monti (ECRC),Norbert Hübner (MDC), and numerous others andshowed that spontaneously hypertensive rats developingheart failure (SHR-HF) map to the gene locus forsEH (EPHX2). Monti and Hübner found a mutation inthe EPHX2-promoter. We used sEH -/- mice and materialfrom the rats and showed that this mutation directlyaffects sEH-expression and cardiac EET-levels. We furthershowed that sEH -/- mice were relatively resistantto Ang II- and pressure overload-induced heart failureand cardiac arrhythmia. This project involving collaborationbetween MDC basic science and ECRC cliniciansis an excellent example of translational medicine.Role of omega-3 fatty acid derivedCYP-eicosanoids in cardiac arrhythmiaOur team collaborated with Robert Fischer and DominikMüller and demonstrated that eicosapentaenoic (EPA)and docosahexaenoic acid (DHA) protect against34 Cardiovascular and Metabolic Disease Research

arrhythmia and sudden cardiac death. Dietary EPA/DHAsupplementation shifted the cardiac CYP-eicosanoidprofile in rats from AA- to EPA- and DHA-derivedmetabolites. One of these novel metabolites (17,18-epoxyeicosatetraenoic acid; 17,18-EETeTr) exerted a negativechronotropic effect, reduced the ß-adrenergicresponse and protected against Ca 2+ -overload in neonatalrat cardiomyocytes (in collaboration with GerdWallukat). 17,18-EETeTr was effective with an EC 50 of 1-2nM. We concluded that this metabolite may mediatethe antiarrhythmic effect of omega-3 fatty acids. Wethen identified the 11,12-double bond and the17(R),18(S)-epoxy group as the structural elementsessential for the biological activity of 17,18-EETeTr (incollaboration with John R. Falck, UT Southwestern,Dallas). Moreover, we have developed and patented syntheticagonists with improved chemical and biologicalstabilities as a basis for novel antiarrhythmic drugs.Role of catechol-o-methyltransferase (COMT) and20-HETE in acute kidney injuryAcute kidney injury (AKI) is a severe problem in variousclinical settings ranging from renal transplantation tocardiac surgery. Collaborating with Duska Dragun(Charité, Berlin), we found that carriers of the low activityCOMT allele are at significantly higher risk to developAKI after cardiac surgery. In a recent joint study, wetested the hypothesis that overproduction of 20-HETEduring ischemia/reperfusion (I/R) contributes to AKIusing an experimental rat model. We found that compoundsthat inhibit 20-HETE synthesis or 20-HETEaction significantly protected against I/R-induced vascularinflammation, tubular injury and loss of renalfunction (compare figure). These findings may offernovel therapeutic opportunities for preventing AKI andpreserving kidney function during transplantation.Selected publicationsBähring, S, Kann, M, Neuenfeld, Y, Gong, M, Chitayat, D, Toka, HR, Toka, O,Plessis, G, Maass, P, Rauch, A, Aydin, A, Luft, FC (2008) Inversion region forhypertension and brachydactyly on chromosome 12p features multiplesplicing and noncoding RNA. Hypertension. 51, 426-431.da Costa-Goncalves, AC, Tank, J, Plehm, R, Diedrich, A, Todiras, M, Gollasch,M, Heuser,, A, Wellner, M, Bader, M, Jordan, J, Luft, FC, Gross, V. (2008) Roleof the multidomain protein spinophilin in blood pressure and cardiacfunction regulation. Hypertension. 52, 702-707.Park, JK, Theuer, S, Kirsch, T, Lindschau, C, Klinge, U, Heuser, A, Plehm, R,Todiras, M, Carmeliet, P, Haller, H, Luft, FC, Muller, DN, Fiebeler, A. (2009)Growth Arrest Specific Protein 6 Participates in DOCA-Induced Target-Organ Damage. Hypertension. 54, 359-364.Monti, J, Fischer, J, Paskas, S, Heinig, M, Schulz, H, Gösele, C, Heuser, A,Fischer, R, Schmidt, C, Schirdewan, A, Gross, V, Hummel, O, Maatz, H,Patone, G, Saar, K, Vingron, M, Weldon, SM, Lindpaintner, K, Hammock, BD,Rohde, K, Dietz, R, Cook, SA, Schunck, WH, Luft, FC, Hubner, N. (2008)Soluble epoxide hydrolase is a susceptibility factor for heart failure in arat model of human disease. Nat Genet. 40, 529-537.Machnik, A, Neuhofer, W, Jantsch, J, Dahlmann, A, Tammela, T, Machura, K,Park, JK, Beck, FX, Müller DN, Derer, W, Goss, J, Ziomber, A, Dietsch, P,Wagner, H, van Rooijen, N, Kurtz, A, Hilgers, KF, Alitalo, K, Eckardt, KU, Luft,FC, Kerjaschki, D, (2009) Titze J. Macrophages regulate salt-dependentvolume and blood pressure by a vascular endothelial growth factor-Cdependentbuffering mechanism. Nat Med. 5, 545-552.Collaborations Jens TitzeOur group has collaborated closely with Jens Titze(University of Erlangen) for the past half-decade. Wedetermined that salt has a non-osmotic storage compartmentand that salt elucidates signals regardingangiogenesis (lymph vessels). The findings imply theimportance of an osmosensitive transcription factor(TonEBP) and suggest mechanisms to test the saltresistantor salt-sensitive hypertension hypothesis. Wewill rely on sodium-directed magnetic resonance imagingand spectroscopy to test our findings in humans.Cardiovascular and Metabolic Disease Research 35

Structure of the GroupDominik N. Müller(Delbrück Fellow)Group LeaderDr. Dominik N. MüllerScientistsDr. Petra GratzeDr. Heda KvakanDr. Verena FokuhlDr. Norbert HenkeDr. Fatimunnisa QadriAssociated ScientistsDr. Ralf DechendDr. Wolf-Hagen SchunckDr. Robert FischerGraduate StudentsUlrike MaschkeAnne KonkelTechnical AssistantsMay-Britt KöhlerJutta MeiselGabi N’diayeJuliane AndersManager of sponsored programsSusanne WisslerMechanisms of Hypertension-InducedTarget Organ DamageDominik N. Müller leads a group of young investigators pursuing the question of how hypertensioninduces target-organ damage. In a translational approach funded by an ECRC grant, Ralf Dechend(Helios Clinic) and Dominik Müller focus primarily on the placenta, heart and kidneys. The primarymediator that has captured their attention is the renin-angiotensin system. The group also cooperatesclosely with MDC scientists. The group has also been a resource for young clinicians and doctoralstudents beginning their careers in experimental cardiovascular research.The group collaborated with Michael Bader and OliverDaumke on the (pro)renin receptor, with Wolf-HagenSchunck and Robert Fischer (Charité) on target organprotective role of polyunsaturated fatty acid and theidentification eicosanoid receptors, with Ruth Schmidt-Ullrich and Claus Scheidereich on the role of NF-κB intarget organ damage, with Martin Zenke (Aachen) toexplore the role of the “inhibitor of differentiation 2”(ID2) in hypertension-induced kidney injury, and withMarkus Kleinewietfeld to elucidate the protective role ofregulatory T cells (Tregs) in hypertension-induced cardiacinjury. Finally, the group made the novel observationthat the human renin promulgates obesity in transgenicrats by inducing changes in energy metabolism.Immune mechanisms in hypertensionMice deficient for Id2(-/-) lack Langerhans and splenicCD8a+ dendritic cells, have reduced natural killer cells,and have altered CD8 T-cell memory. Petra Gratze andthe group tested the hypothesis that an alteration inthe number and quality of circulating blood cellscaused by Id2 deletion would ameliorate angiotensin(Ang) II-induced target-organ damage. Id2-/- micefailed to develop an increase in blood pressure wheninfused with Ang II and had no target-organ damage.The group conducted kidney (figure 1) and bone marrowtransplants that did not restore the sensitivity toAng II. They also found that vascular smooth musclecells from Id2-/- mice showed an antisenescence phenotype.The study identified a previously undefined rolefor Id2 in the pathogenesis of Ang II-induced hypertension.This role is being further explored.Heda Kvakan led the project studying the role ofimmunosuppressive CD4+CD25+ regulatory T (Treg)cells in the pathogenesis of hypertensive target organdamage. The group conducted adoptive transfer of Tregcells into Ang II-infused hypertensive mice. Treg cellrecipients exhibited improved cardiac hypertrophy andless cardiac fibrosis despite sustained hypertension.Amelioration of cardiac morphology was accompaniedby an improvement in arrhythmogenic electric remodeling,indicating the functional significance of theenhanced cardiac morphology. Pronounced connexin43 immunoreactivity was found at the lateral bordersof cardiomyocytes in Ang II-treated mice, implicatingthis gap-junction protein in the arrhythmias. In contrast,connexin 43 was restricted to the intercalateddisk regions in sham controls. Surprisingly, Ang II+Tregtreatedmice showed normal connexin 43 gap junctionprotein localization. Thus, Treg cells ameliorated cardiacdamage and accounted for the improved electricremodeling independently of blood pressure-loweringeffects. The results provide new insights into the pathogenesisof hypertensive cardiac damage and couldtherefore lead to new therapeutic approaches thatinvolve manipulation of the immune system.The (pro)renin receptorThe Müller laboratory collaborates with GenevieveNguyen (INSERM Paris), Michael Bader and Oliver36 Cardiovascular and Metabolic Disease Research

Daumke (both MDC) on studying the (pro)renin receptor,a transmembrane protein that binds either renin orprorenin. Prorenin is activated by this binding to allowthe cleavage of angiotensinogen to Ang I (and then toAng II through the angiotensin converting enzyme).Independent of that process, the (pro)renin receptoralso signals via extracellular-regulated kinase (ERK1/2)and can activate the transforming growth factor (TGF)pathway. Others claim that by blocking the (pro)reninreceptor, by means of a decoy peptide sequence termedthe handle-region peptide (HRP), the vascular damagecaused by diabetes mellitus can be completely ameliorated.The Müller laboratory has worked intensively onthis important problem. Sandra Feldt was primarilyresponsible for this project. The group tested whetherhuman prorenin and renin induce ERK1/2 activationand whether the direct renin inhibitor aliskiren or theHRP inhibits the receptor. The (pro)renin receptormRNA and protein was detected in isolated humanmonocytes and in U937 monocytes. In U937 cells, thegroup found that both human renin and prorenininduced a long-lasting ERK1/2 phosphorylation despiteAng II type 1 and 2 receptor blockade. A mitogen-activatedprotein kinase kinase 1/2 inhibitor inhibited bothrenin and prorenin-induced ERK 1/2 phosphorylation.Neither aliskiren nor HRP inhibited binding of (125)Ireninor (125)I-prorenin to the (pro)renin receptor.Fluorescence-activated cell sorter analysis showed that,although fluorescein isothiocyanate-labeled HRPbound to U937 cells, HRP did not inhibit renin orprorenin-induced ERK1/2 activation. Thus, prorenin andrenin-induced ERK 1/2 activation are independent ofAng II. The signal transduction is different from thatevoked by Ang II. Aliskiren has no (pro)renin receptorblocking effect and did not inhibit ERK1/2 phosphorylationor kinase activity. There was no evidence that HRPaffects renin or prorenin binding and signaling. TheCouncil for High Blood Pressure Research of theAmerican Heart Association voted this work as the“best basic paper of the year”, published in the journalHypertension.Renin and metabolismRenin initiates Ang II formation and (aside from the(pro)renin receptor) has no other known functions.Petra Gratze and the Muller group observed that transgenicrats (TGR) overexpressing the human renin gene(hREN) developed moderate obesity with increasedbody fat mass and glucose intolerance compared withnontransgenic Sprague-Dawley (SD) rats. The metabolicchanges were not reversed by an angiotensin-convertingenzyme inhibitor, a direct renin inhibitor, or by(pro)renin receptor blocker treatment. The obese phenotypein TGR(hREN) originated from higher foodintake, which was partly compensated by increases inRenal Id2 deficiency does not the genesis of hypertension andrenal damage.resting energy expenditure, total thermogenesis (postprandialand exercise activity), and lipid oxidation duringthe first 8 weeks of life. Once established, the differencein body weight between TGR(hREN) and SD ratsremained constant over time. The group observed nochanges in the cocaine and amphetamine-regulatedtranscript, pro-opiomelanocortin, both anorexigenic, orneuropeptide Y, orexigenic, mRNA levels in TGR(hREN)versus SD controls. However, the mRNA level of theagouti-related peptide, orexigenic, was significantlyreduced in TGR(hREN) versus SD controls at the end ofthe study, which indicates a compensatory mechanism.The group suggested that the human renin transgeneinitiates a process leading to increased and earlyappetite, obesity, and metabolic changes not related toAng II. The mechanisms are independent of any currentlyknown renin-related effects. The novelty here is theintroduction of renin as an obesity-related enzyme. Thegroup is busy exploring mechanisms further.Selected PublicationsGratze, P, Dechend, R, Stocker, C, Park, J-K, Feldt, S, Shagdarsuren, E, Wellner,M, Gueler, F, Rong, S, Gross, V, Obst, M, Plehm, R, Alenina, N, Zenclussen, A,Titze, J, Small, K, Yokota, Y, Zenke, M, Luft, FC, Muller, DN. (2008) Novel rolefor inhibitor of differentiation 2 in the genesis of angiotensin II-inducedhypertension. Circulation. 117, 2645-2656.Kvakan, H, Kleinewietfeld, M, Qadri, F, Park, JK, Fischer, R, Schwarz, I, Rahn,HP, Plehm, R, Wellner, M, Elitok, S, Gratze, P, Dechend, R, Luft, FC, Muller, DN.(2009) Regulatory T cells ameliorate angiotensin II-induced cardiacdamage. Circulation. 119, 2904-2912Feldt, S, Maschke, U, Dechend, R, Luft, FC, Muller, DN. (2008) The putative(pro)renin receptor blocker HRP fails to prevent (pro)renin signaling. J AmSoc Nephrol. 19, 743-748.Feldt, S, Batenburg, WW, Mazak, I, Maschke, U, Wellner, M, Kvakan, H,Dechend, R, Fiebeler, A, Burckle, C, Contrepas, A, Danser, JAH, Bader, M,Nguyen, G, Luft, FC, Muller, DN. (2008) Prorenin and renin-inducedextracellular signal-regulated kinase 1/2 activation in monocytes is notblocked by aliskiren or the handle-region peptide. Hypertension. 51,682-688.Gratze, P, Boschmann, M, Dechend, R, Qadri, F, Malchow, J, Graeske, S, Engeli,S, Janke, J, Springer, J, Contrepas, A, Plehm, R, Klaus, S, Nguyen, G, Luft, FC,Muller, DN. (2009) Energy metabolism in human renin-gene transgenicrats: does renin contribute to obesity? Hypertension. 53, 516-523.Cardiovascular and Metabolic Disease Research 37

Rainer DietzStructure of the GroupGroup LeaderProf. Dr. Rainer DietzScientistsDr. Florian BlaschkePD Dr. Ralf DechendDr. Stefan DonathDr. Jens FielitzDr. Robert FischerDr. Jan MontiPD Dr. Cemil ÖzcelikDr. Laura ZelarayánClinical CardiologyThe main topic of the group is to address the molecular signaling pathways leading to heartfailure. Heart failure, a highly heterogenous disease, is characterized by a symptomcomplex of congestion and exertional fatigue. Heart failure is mainly caused by coronaryartery disease/myocardial infarction, arterial hypertension, dilated and hypertrophiccardiomyopathies, and inflammatory heart disease. In a translational approach new animalmodels have been established mimicking those human cardiovascular disorders and theireffects. Detailed phenotyping of those heart failure models has revealed new insights intodisease development and adaptive compensatory pathways prior to the onset of heart failurewhich may lead to new preventive and therapeutic strategies.BackgroundAlthough great progress has been achieved in the treatmentof heart failure, it remains a leading cause ofdeath and disability throughout the world and prevalenceand incidence increase year-by-year. Heart homeostasisis regulated by apoptosis, cardiomyocyte hypertrophyand myocardial regeneration determining thehearts ability to response to pathologic loads. The balancebetween cardiomyoycte survival and apoptoticpathways as well as heart regeneration versus deleteriouscardiac remodelling appear to be key determinantsof the transition from heart hypertrophy to ventriculardilatation.Complex Genetics of Hypertension andHeart FailureJan Monti, Jens ButtgereitIn humans, hypertension associated with sustained cardiachypertrophy represents one of the most commoncauses of heart failure. Starting from high blood pressure,the pathophysiological cardiac remodelling cascadeproceeds to left ventricular hypertrophy as a primarilyadaptive process. Later on, left ventricular dilatation,decreased systolic function, and cardiac arrhythmiascan occur in some patients, whereas others retainstable systolic function without clinical signs of heartfailure.Heart failure is thought to result from complex interactionsbetween genetic susceptibility and lifestyle/environmental factors. In close collaboration withNorbert Hübner, Jan Monti aimed to identify gene variantsunderlying heart failure in the spontaneouslyhypertensive heart failure (SHHF) rat. They generatedexperimental evidence that genetic variance in theEphx2 (soluble epoxide hydrolase) gene facilitates progressionfrom hypertension and cardiac hypertrophy toheart failure in that rat model. The demonstratedcausative impact for Ephx2 in the complex initiation ofheart failure in rats and mice inaugurate Ephx2 as anew heart failure treatment avenue.Hypertension and Heart FailureRalf Dechend, Florian Herse, Katrin WenzelIn addition to genetic predisposition the subgroup ofRalf Dechend examined the relevance of “agonistic”autoantibodies directed against the α1-adrenergicreceptor in patients with refractory hypertension.Almost half of them displayed α1-autoantibodies.Interestingly, the removal of the α1-autoantibodies byimmunoabsorption significantly reduced blood pressureduring the observed follow-up phase of 180 days.By this innovative therapeutic approach Ralf Dechend38 Cardiovascular and Metabolic Disease Research

Graduate StudentsPhilipp DuBoisSanthosh Kumar GhadgeJida HamatiLydia HeringDörte LodkaClaudia NoackMarcel NowakAnke RengerBastian SpallekAnnett SpitzlTimm ZörgiebelTechnical AssistantsMarlies DudaMarlies GriebenRené KolbBeatrice LeipAnika LindnerJutta MeiselJeanette MothesBärbel PohlTanja SchalowAstrid SchicheFigure 1. αMHC-dependent β-catenin depletion attenuates post-infarctLV remodeling. β-catenin depletion (β-cat ∆ex3–6 ) results in improved fractionalshortening 4 weeks after infarct associated with a prominentsub-endocardial and sub-epicardial layer of cardiomyocytes as identifiedby Troponin T (cTnT) staining.and his colleagues provide evidence that α1-autoantibodiesare of potential pathophysiological relevanceand could represent a factor contributing to the developmentof refractory hypertension.Experimental Electrophysiology in Heart FailureRobert FischerIn endstage heart failure a significant portion of heartfailure patients die suddenly due to malignant arrhythmias.Robert Fischer and his subgroup are performingelectrophysiological examinations in murine models toevaluate the risk of sustained ventricular arrhythmias.Recently, they were able to substantiate the beneficialeffects of omega-3-polyunsaturated fatty acids in preventingarrhythmias in a heart failure model.Patent Application 01/2009: EP18174 “Novel eicosanoidderivatives”.Genetics of Hypertrophy and Heart FailureCemil Özcelik, Christian Geier, Andreas Perrot,Maximilian PoschHypertrophic cardiomyopathy (HCM) is the most commongenetic myocardial disease with a prevalence of0.2 % in adults. Left ventricular hypertrophy in theabsence of other causes is the clinical hallmark. In addition,the clinical phenotype is characterized by suddencardiac death. Mutations in a number of sarcomericcontractile-protein genes are causative in approximately60 % of individuals with HCM. Christian Geier fromCemil Özcelik´s subgroup has identified a missenseCardiovascular and Metabolic Disease Research 39

Figure 2. Cardiac-restricted ablation of PKD1. Upon the onset of pressureoverload PKD1 mutants showed less hypertrophy and a significantreduction in cardiac fibrosis. TAC: Thoracic aortic banding.mutation in the “muscle LIM protein” (MLP) in patientswith HCM. Surprisingly, immunohistochemical analysisrevealed that MLP is mainly localized in the cytosoliccomponent, rather than in the sarcomere, indicatingthat HCM is not exclusively a sarcomeric disease.Furthermore, their data suggested that impairedmechano-sensory stress signaling might be involved inthe pathogenesis of HCM.Nuclear Receptors in Cardiac MetabolismFlorian BlaschkeThe cardiac phenotype of left ventricular hypertrophycaused by genes that are involved in metabolism andenergy production underscores the impact of energeticpathways on cardiac phenotype and function. A betterunderstanding of the role of cardiac energy metabolismand hypertrophic gene expression may consequentlylead to more effective therapeutic strategies for40 Cardiovascular and Metabolic Disease Research

the prevention and treatment. The subgroup of FlorianBlaschke is elucidating the role of those nuclear receptorsin cardiac metabolism and hypertrophy both invitro and in vivo and characterize the molecular mechanismsutilized to regulate gene expression involved incardiomyocyte growth and energy metabolism.Apoptosis and Regeneration in Heart FailureStefan Donath, Junfeng AnAnother focus of the group has addressed the issue ofapoptosis in heart failure progression. ARC (apoptosisrepressor with caspase recruitment domain) is a masterregulator of cardiac death signaling, as it is the onlyknown factor that specifically inhibits both deathreceptor and mitochondrial apoptotic death pathways.By generating ARC deficient mice the subgroup ofStefan Donath attempted to elucidate the physiologicalrole of ARC in the heart. Upon biomechanical stressinduced by aortic banding, ARC null mice developedaccelerated cardiomyopathy compared with littermatecontrols that was characterized by reduced contractilefunction, cardiac enlargement, and myocardial fibrosis.Likewise, ischemia-reperfusion injury of ARC null miceresulted in markedly increased myocardial infarct sizes.The pathophysiological relevance was underscored byspecimens from failing human hearts showing markedlyreduced ARC protein levels. Thus, they identified a tissue-specificanti-apoptotic factor which is downregulatedin human failing myocardium and which isrequired for cardioprotection in pressure overload andischemia. Inhibition of apoptosis might have a similareffect on restoration of the balance between loss of cardiomyocytesand heart renewal by strengthening theself-renewal properties of the heart.Transcription Factors in Myocardial StressRemodellingLaura Zelarayán, Martin Bergmann (guest scientist),Maria-Patapia ZafiriouThe subgroup of Laura Zelarayán and Martin Bergmannelucidated the role of the Wnt/β-catenin pathway inactivation of endogenous cardiac progenitor cells.Therefore, they deleted the β-catenin gene in themyocardium by using the Cre/loxP technology. The conditionalknockout of β-catenin resulted in cardiachypertrophy, suggesting that β-catenin downregulationmight be part of the adaptive cardiac hypertrophyresponse. Additionally, the depletion of β-catenin positivelyaffected LV-function and survival after experimentalmyocardial infarction (figure 1). Furthermore,conditional β-catenin deficient mice displayed betterremodelling upon cardiac stress. Isolated Sca-1 pos cellsfrom mutant mice showed significantly increased differentiationcapacity.Pathological Cardiac Remodelling in Heart FailureJens FielitzA further gene family that suppresses stress-dependentremodelling of the heart via association with the MEF2transcription factor are class II histone deacetylases(HDACs). Protein kinase D (PKD) is a stress-responsivekinase that phosphorylates class II HDACs, resulting intheir dissociation from MEF2 with consequent activationof MEF2 target genes. The subgroup of Jens Fielitzwas able to show that the cardiac ablation of PKD1showed less hypertrophy and a dramatic reduction incardiac fibrosis compared to wild type mice when subjectedto aortic banding (figure 2). In addition, PKD1mutant mice were resistant to left ventricular dilatationand a decrease in contractility. This study indicatesthat PKD1 functions as a key transducer of stress stimuliinvolved in pathological cardiac remodelling in vivo.These findings provide the first evidence that deletionof a class II HDAC kinase in vivo diminishes stressinducedhypertrophy.Selected PublicationsAn, J, Li, P, Li, J, Dietz, R, Donath, S. (2009). ARC is a critical cardiomyocytesurvival switch in doxorubicin cardiotoxicity. J Mol Med. 87, 401-10.Fielitz, J, Kim, MS, Shelton, JM, Qi, X, Hill, JA, Richardson, JA, Bassel-Duby, R,Olson, EN. (2008). Requirement of protein kinase D1 for pathologicalcardiac remodeling. Proc Natl Acad Sci U S A. 105, 3059-3063.Geier, C, Gehmlich, K, Ehler, E, Hassfeld, S, Perrot, A, Hayess, K, Cardim, N,Wenzel, K, Erdmann, B, Krackhardt, F, Posch, MG, Osterziel, KJ, Bublak, A,Nagele, H, Scheffold, T, Dietz, R, Chien, KR, Spuler, S, Furst, DO, Nurnberg, P,Ozcelik, C. (2008). Beyond the sarcomere: CSRP3 mutations causehypertrophic cardiomyopathy. Hum Mol Genet. 17, 2753-2765.Monti, J, Fischer, J, Paskas, S, Heinig, M, Schulz, H, Gosele, C, Heuser, A,Fischer, R, Schmidt, C, Schirdewan, A, Gross, V, Hummel, O, Maatz, H,Patone, G, Saar, K, Vingron, M, Weldon, SM, Lindpaintner, K, Hammock, BD,Rohde, K, Dietz, R, Cook, SA, Schunck, WH, Luft, FC, Hubner, N. (2008).Soluble epoxide hydrolase is a susceptibility factor for heart failure in arat model of human disease. Nat Genet. 40, 529-537.Wenzel, K, Haase, H, Wallukat, G, Derer, W, Bartel, S, Homuth, V, Herse, F,Hubner, N, Schulz, H, Janczikowski, M, Lindschau, C, Schroeder, C,Verlohren, S, Morano, I, Muller, DN, Luft, FC, Dietz, R, Dechend, R,Karczewski, P. (2008). Potential relevance of alpha(1)-adrenergic receptorautoantibodies in refractory hypertension. PLoS One. 3, e3742.Zelarayán, LC, Noack, C, Sekkali, B, Kmecova, J, Gehrke, C, Renger, A, Zafiriou,MP, van der Nagel, R, Dietz, R, de Windt, LJ, Balligand, JL, Bergmann, MW.(2008). Beta-Catenin downregulation attenuates ischemic cardiacremodeling through enhanced resident precursor cell differentiation.Proc Natl Acad Sci U S A. 105, 19762-19767.Cardiovascular and Metabolic Disease Research 41

Ludwig ThierfelderStructure of the GroupGroup LeaderProf. Dr. Ludwig ThierfelderScientistsDr. Florian BlaschkeDr. Jörg DrenckhahnDr. Brenda Gerull*Dr. Michael Gramlich*Dr. Arnd HeuserDr. Susanne Probst*Dr. Sabine KlaassenDr. Bertold StrukCardiovascular Molecular GeneticsMolecular genetics of cardiomyopathies, regeneration of the embryonic heart andelucidation of the molecular pathology/genetics of vascular lesions are the majortopics of our research group. A number of cardiomyopathy causing mutations have beenidentified by our research group and the molecular pathology of those mutations iscurrently under investigation. The capacity of the embryonic heart to regenerate fromgenetic damage is based on a complex mechanism of compensatory proliferation, inhibitionof apoptosis, and adaptation of various signaling pathways. Liver X receptor agonists arepotential targets for the treatment of metabolic, inflammatory and cardiovascular diseasesand negatively interfere with cytokine-induced nuclear receptor corepressor dissociationfrom the C-reactive protein promoter, thus maintaining this gene in a repressed state.Genetics of cardiomyopathiesBrenda Gerull, Sabine Klaassen, Arnd Heuser,Michael GramlichArrhythmogenic right ventricular cardiomyopathy(ARVC) is an inherited heart disease predominantlyaffecting the right ventricle and a prevalent cause ofventricular arrhythmias and sudden death, especially inyoung adults. Genetically, heterozygous loss-of-functionmutations in desmosomal proteins (desmocollin2,desmoglein2, plakoglobin, desmoplakin, andplakophilin2) have been mainly associated with ARVC.We have described mutations in plakophilin2 (PKP2)and desmocollin2 (DSC2) as a cause of autosomal dominantARVC. PKP2 mutations account for a significantproportion of ARVC cases (10-45%). PKP2 and DSC2 arecomponents of the desmosomal intercellular junctioncomplex (see figure 1) known to be essential for maintainingtissue integrity and increasingly implicated incell signaling. While the involvement of multipledesmosomal protein genes has led to speculationregarding the sensitivity of myocardium to mechanicaldisruption, the pathogenic mechanisms leading toARVC in humans are largely unknown. We currently tryto elucidate the genetic and molecular mechanisms ofvarious human PKP2 mutations and their consequencesin the pathology of the intercellular junction complexusing cell culture experiments and transgenic mousemodels. Furthermore, we investigate different knockoutmodels of desmosomal components to define theadhesive and signaling contributions of thesel proteinsin the maintenance of cardiac tissue.Left ventricular noncompaction of the myocardium(LVNC) has recently been recognized as a distinct primarycardiomyopathy with a genetic etiology. LVNC ischaracterized by a unique congenital cardiac morphology,consisting of numerous, excessively prominentventricular trabeculations and deep intertrabecularrecesses. The heterogeneity of the clinical featuresincludes progressive deterioration in cardiac functionresulting in congestive heart failure, arrhythmias,thrombembolic events, and sudden cardiac death. Thedisorder is assumed to result from an intrauterinearrest in the process of compaction of the developingmyocardium. We found mutations in genes encodingsarcomere proteins in a significant proportion of LVNCpatients. Heterozygous mutations in genes encoding –myosin heavy chain (MYH7), -cardiac actin (ACTC), andcardiac troponin T (TNNT2) account for 17% of cases ofisolated LVNC in adult patients. As hypertrophic cardiomyopathy(HCM) and dilated cardiomyopathy42 Cardiovascular and Metabolic Disease Research

Graduate StudentsFlorian KirchnerManuela MagarinUte Rimpler*Robert ZinkeTimm Zörgiebel*Technical AssistantsIska LiebnerSigrid MilanElke, Nickel*Martin TaubeJana Treutler**part of the period reported(DCM) are also caused by mutations in sarcomere proteingenes our analyses support the concept of a sharedmolecular etiology of these different cardiomyopathicphenotypes.Mutations in the giant sarcomeric protein TTN areresponsible for inherited dilated cardiomyopathy (DCM)in humans. To dissect affected pathways leading totitin-based DCM and to investigate the role of titin incardiac development, we generated a mouse model thatmimics a TTN truncation mutation (c.43628insAT) previouslyidentified in a large family with autosomal dominantDCM. Heterozygous mice chronically exposed tocardiac stress displayed features of DCM, thereby recapitulatingthe human phenotype. The mutant embryoshomozygous for the Ttn knock-in mutation showedsevere defects in sarcomere formation and died beforeE9.5 due to non-beating hearts. Our data demonstratethat the removal of titin’s M-line region leads tounformed sarcomere structures and provide a chronologicalrelationship between titin expression, sarcomereformation, and embryonic lethality due to absent cardiaccontractile function.Regeneration of the embryonic heartJörg DrenckhahnRegeneration of functional myocardium after cardiacinjury is currently one of the most rapidly developingfields in cardiovascular research. Several differentapproaches based on injection of various cell types aswell as mobilisation or stimulation of endogenous cellsto repair the damaged heart have been reported.Despite lots of encouraging findings some controversialresults have highlighted the need for a better under-Figure 1. The structural organization of desmosomes. (A) The schematic drawingshows desmosomal cadherins (DSG, desmoglein; DSC, desmocollin); the armadillofamilymembers plakoglobin (PG) and the plakophilins (PKP); and the intermediatefilament (IF)-binding protein desmoplakin (DSP). (B) Electron micrograph ofdesmosomes from murine heart.Figure 2. Immunolocalisation of desmosomal proteinsPlakophilin2 (green) and Plakoglobin (red) inneonatal rat cardiomyocytes ; Nkx2.5 (magenta).Cardiovascular and Metabolic Disease Research 43

standing of the molecular and cellular mechanismsunderlying a solid myocardial regeneration. In thisregard, the embryonic heart is a perfect model to studythese processes as key events like cardiomyocyte proliferation,differentiation as well as regional and functionalspecification occur physiologically in the developingheart.Our recent findings have shown that the embryonicmurine heart has a remarkable regenerative capacity.We have inactivated the X-linked gene encodingHolocytochrome c synthase (Hccs), an enzyme essentialfor normal function of the mitochondrial electrontransport chain, specifically in the developing mouseheart. Loss of Hccs activity results in cellular energystarvation causing disturbed cardiomyocyte differentiationand ultimately cellular degeneration. In contrast tothe observed mid-gestational lethality of hemizygousHccs knock-out (KO) males, heterozygous femalesappeared normal during the first months of life withsurprisingly few clusters of affected cardiomyocytes,considering an expected mosaic of affected and normalcardiomyocytes as a result of random X chromosomalinactivation. However, analyses of heterozygous femaleembryos revealed the expected 50:50 ratio of Hccs deficientto normal cardiac cells at mid-gestation with aprogressive reduction in disease tissue to 10% prior tobirth. We could show that this significant change isaccounted for by increased proliferation of remaininghealthy cardiac cells. These data reveal a previouslyunrecognised but impressive regenerative capacity ofthe mid-gestational heart that can compensate for aneffective loss of at least 50% of cardiac tissue to enableformation of a functional heart at birth. Yet despite thisregeneration, hearts of neonatal heterozygous Hccs KOfemales do not appear completely normal but showmorphological, cellular as well as molecular signs ofimmaturity. These changes, however, normalize untiladulthood suggesting activation of compensatory cardiacgrowth mechanisms in the postnatal heart afterdisturbed heart development.The detailed characterisation of molecular signalingpathways as well as cell types involved in embryonicheart regeneration is currently underway and shouldprovide major new insights into heart developmentand cardiac organ size control. Furthermore, the identificationof regenerative factors and stimuli within theembryonic heart might potentially enable the developmentof new therapeutic strategies for cardiac repair inthe adult. Finally, this model might provide a useful toolto study the impact of disturbed heart development onthe incidence of postnatal cardiac disease in the contextof fetal programming.Nuclear Receptors as Potential Target for theTreatment and Prevention of Metabolic,Inflammatory and Cardiovascular DiseaseFlorian BlaschkeMembers of the nuclear receptor superfamily of liganddependenttranscription factors play essential roles indevelopment, homeostasis, reproduction and immunefunction. Several members of this family, including theestrogen receptor (ER) and peroxisome proliferator-acti-Figure 3. β-Galactosidase staining of 19.5 dpc (days postcoitum) fetal hearts prior to birth (counterstained with Eosin).The control heart (♀ +lacZ /+, heterozygous for an X-linked lacZreporter gene) shows large patches of β-Gal positive and negativecells due to random X chromosome inactivation. In theheterozygous Hccs-knock-out heart (♀ -lacZ /+) the lacZ reportergene is linked to the X chromosome carrying the defective Hccsgene allowing the detection of Hccs deficient cells by β-Galstaining. Due to cardiac regeneration during embryonic developmentthe proportion of Hccs deficient cells is minimizeduntil birth. Very few isolated Hccs deficient cardiomyocytes canbe detected within the ventricular and atrial myocardium withonly the basal region of the interventricular septum showing alarger cluster of β-Gal positive cells (see arrow). RA = right atrium,LA = left atrium, RV = right ventricle, LV = left ventricle.44 Cardiovascular and Metabolic Disease Research

vated receptors (PPARs) are target of drugs that areused in a variety of clinical settings. The ability ofnuclear receptors to switch from a transcriptionalrepressor to a transcriptional activator by binding ofsynthetic or natural ligands provided important insightinto the mechanism(s) of gene regulation. Given theirpleiotropic effects and their activation by specific ligands,new drugs targeting nuclear receptors are emergingas promising therapeutics for the treatment of cardiovascular,metabolic and inflammatory disease.Activation and proliferation of vascular smooth musclecells (VSMCs) are recognized to play a decisive role invascular proliferative diseases such as primary atherosclerosis,postangioplasty restenosis, vein graft disease,and transplant vasculopathy. Due to the crucial role ofVSMCs in the pathobiology of vascular occlusive disease,a pharmacologic blockade of VSMC activation andthe cell cycle machinery is a promising therapeuticapproach to prevent and treat vascular proliferative disease.Using both in vitro and in vivo approaches, we areinvestigating the role of various nuclear receptors andtheir activation by synthetic ligands in VSMC activationand cell cycle progression and are characterizing themolecular pathways utilized to regulate gene expression.Cardiac hypertrophy leading to heart failure is a majorcause of morbidity and mortality worldwide. The reasonswhy cardiac dilatation and failure eventually occurare unknown although alterations in energy status andmetabolism are proposed to play an important role. Weare elucidating the role of nuclear receptors in cardiacmetabolism and hypertrophy both in vitro and in vivoand characterize the molecular mechanism(s) utilizedto regulate gene expression involved in cardiomyocytegrowth and energy metabolism.known as MRP-6), cause PXE. Recently, an extensivemutation screen of 81 PXE families revealed 59 distinctABC-C6 mutations. The mode of inheritance in thiscohort is solely recessive or pseudo-dominant if a diseasedindividual married a carrier. Our data do not supportany evidence for a dominant mode of inheritanceof PXE. Current research focuses on the identification ofmolecular mechanisms for disease development in thedifferent tissues involved.Selected PublicationsBlaschke F, Takata Y, Caglayan E, Collins A, Tontonoz P, Hsueh WA, TangiralaRK. (2206). A nuclear receptor corepressor-dependent pathway mediatessuppression of cytokine induced C-reactive protein gene expression byliver X receptor. Circ Res. 99(12):e88-99Heuser A, Plovie ER, Ellinor PT, Grossmann KS, Shin JT, Wichter T, BassonCT, Lerman BB, Sasse-Klaassen S, Thierfelder L, MacRae CA, Gerull B.(2006). Mutant desmocollin-2 causes arrhythmogenic right ventricularcardiomyopathy. Am J Hum Genet. 79(6):1081-8Klaassen S, Probst S, Oechslin E, Gerull B, Krings G, Schuler P, GreutmannM, Hürlimann D, Yegitbasi M, Pons L, Gramlich M, Drenckhahn JD, HeuserA, Berger F, Jenni R, Thierfelder L. (2008). Mutations in sarcomere proteingenes in left ventricular noncompaction. Circulation.117(22):2893-901Drenckhahn JD, Schwarz QP, Gray S, Laskowski A, Kiriazis H, Ming Z,Harvey RP, Du XJ, Thorburn DR, Cox TC. (2008). Compensatory growth ofhealthy cardiac cells in the presence of diseased cells restores tissuehomeostasis during heart development. Dev Cell.15(4):521-33.Gramlich M, Michely B, Krohne C, Heuser A, Erdmann B, Klaassen S,Hudson B, Magarin M, Kirchner F, Todiras M, Granzier H, Labeit S,Thierfelder L, Gerull B. (2009). Stress-induced dilated cardiomyopathy in aknock-in mouse model mimicking human titin-based disease.J Mol CellCardiol.. [Epub ahead of print]Molecular Genetics of Pseudoxanthomaelasticum (PXE)Berthold StrukPseudoxanthoma elasticum is a heritable systemic disorderof the elastic tissue characterized by degenerativecalcification with subsequent disintegration anddestruction of the elastic tissue of several organs.Cardiovascular disease encompasses a wide clinicalspectrum from mental fatigue syndrome to early cardiovasculardeath due to myocardial infarction or, veryrarely, gastrointestinal hemorrhage. We have previouslymapped the PXE locus to a 500 kb interval on chromosome16p13.1. and have shown that mutations in atransmembrane transporter protein, ABC-C6 (alsoCardiovascular and Metabolic Disease Research 45

Structure of the GroupThoralf NiendorfGroup LeaderProf. Dr. rer.nat Thoralf NiendorfScientistsTobias FrauenrathFabian HezelAndreas Pohlmann (as of 11/09)Davide SantoroLukas Winter (as of 01/10)Graduate StudentsThibaut de Geyer d’Orth (as of 10/09)Nishant PatelJan RiegerMagnetic ResonanceThe group’s research concentrates on the development of MR-methodology and MR techno -logy with a focus on new ways of mapping and probing morphology, function, physiology andmetabolism together with explorations of the benefits and challenges of ultrahigh-field imagingto advance cardiovascular, neurovascular, molecular and other MRI applications. These efforts aredesigned to spatially resolve and characterize (patho) physio logical processes and biophysicalmechanisms to promote a transfer from basic research to (pre)clinical studies and vice versa.However, signal-to-noise ratio (SNR) and imaging speed have become an increasingly stringentlimit in new MRI applications. Promising in this regard is the increase in magnetic field strengthsavailable for both animal (9.4 T) and whole-body MR (7.0 T) scanners, though ultrahigh-field MRIhas earned the moniker of being among the most challenging MRI applications.Myocardial T 2 * MappingEmerging cardiovascular MRI applications include T 2 *relaxation sensitized techniques which are increasinglyused in basic research and (pre)-clinical imaging. Forthese reasons, an imaging strategy that avoids imagedistortions and has the advantage that susceptibilityweighting can be adjusted from zero upwards is anappealing strategy in anatomically accurate T 2 * mappingof the heart. Members of our group patented andestablished a free-breathing, cardiac-gated, susceptibilityweighted fast spin-echo technique in conjunctionwith black blood preparation and navigator gated respiratorymotion compensation. The applicability of thisapproach has been demonstrated in myocardial T 2 *imaging/mapping (Figure 1) at 3.0 T and is now beingtransferred to the 7.0 T whole body scanner and the 9.4T animal system.Development of Novel SynchronizationTechniquesObtaining MRI images of moving organs requires speedand efficiency due to physiological motion and flowconstraints, which dictate the viable window for dataacquisition. Cardiac motion is commonly dealt withusing electrocardiographic (ECG) gating techniques tosynchronize data acquisition with the cardiac cycle. Asultrahigh-field cardiac MRI becomes more widespread,the sensitivity of ECG recordings to interference fromelectromagnetic fields and to magneto-hydrodynamiceffects increases, requiring a practical gating/triggeringalternative. For these reasons, an MR-stethoscope hasbeen proposed and clinically evaluated by members ofour group to successfully meet the demands of cardiactriggered MRI at 7.0 T (Figure 2).Rapid Functional Brain Mapping Free of ImageDistortionThe potential of higher magnetic field strengths forfunctional brain mapping in clinical practice and basicresearch has yet to be fully realized. Here, one importantquestion is the choice of pulse sequence for optimalimage quality in functional brain imaging. EchoPlanar Imaging (EPI) is the most frequently appliedtechnique for fMRI because images may be acquiredrapidly and they are inherently sensitive to BloodOxygen Level Dependent contrast. However, a significantdrawback of EPI is its sensitivity to inhomogeneitiesin B 0 , leading to signal loss and image distortionin regions of spatially varying magnetic susceptibility.These effects are more apparent at high and ultra-46 Cardiovascular and Metabolic Disease Research

Christof Thalhammer (as of 11/09)SecretariatSonja Eising (as of 10/09)Start of Group: August 2009high magnetic field strengths B 0 . Rapid spin echo basedpulse sequences successfully developed by members ofour group offer a distortion-free alternative to EPI forfMRI. Consequently, activation may be mappedthroughout the brain using spin-echo based methodswith greater confidence than when using EPI. The challengesto spin echo techniques are adequate temporalresolution for fMRI and avoiding excessive RF powerdeposition which will be a focus in the anticipatedresearch program.Figure 1. QuantitativeT 2 *-relaxation map of thehuman heart superimposedto a short axisanatomical view of thehuman heart. Themyocardial T 2 *-mappingapproach proposed by ourgroup is of value for theassessment of myocardialiron content and myocardialtissue oxygenation.RF Coil DevelopmentsThe sensitivity advantage afforded by ultrahigh-fieldimaging is the driving force behind several technologicaldevelopments. One ongoing development pioneeredby our group is a broad move towards MR systemswith 32 or more receiver channels. This includesthe development and clinical evaluation of many elementRF coil arrays designed for RF transmission andsignal reception. Here, the group’s research activitiesaim to go beyond conventional proton imaging to fosterexplorations into imaging of sodium, fluor, andother nuclei to gain a better insight into metabolic andmolecular processes.Selected PublicationsT. Frauenrath, F. Hezel, U. Heinrichs, S. Kozerke, J. F. Utting, M. Kob, C.Butenweg, P. Boesiger, T. Niendorf (2009) Feasibility of Cardiac GatingFree of Interference with Electro-Magnetic Fields at 1.5 Tesla, 3.0 Teslaand 7.0 Tesla Using an MR-Stethoscope. Invest. Radiol., [Epub ahead ofprint].Figure 2. Short axis views of the heart together with whole R-Rinterval time series of one-dimensional projections along the dottedline profile marked in the short axis view. CINE images wereobtained at 7.0 T using conventional ECG (left) the ACousticTriggering (ACT) (right) approach proposed by our group. ECG triggeredCINE imaging was prone to severe cardiac motion artifactsdue to R-wave misregistration. In comparison, acoustically triggeredCINE imaging at 7.0 T produces images of the human heart free ofmotion artifacts.J.F. Utting, S. Kozerke, R. Luechinger, R. Schnitker, R. Vohn, T. Niendorf(2009)Feasibility of k-t BLAST for BOLD fMRI with a Spin-Echo BasedAcquisition at 3.0 T and 7.0 T. Invest. Radiol., [Epub ahead of print].U. Heinrichs, J. F. Utting, T. Frauenrath, F. Hezel, G. A. Krombach, M. A. J.Hodenius, S. Kozerke, T. Niendorf (2009) Myocardial T2* Mapping Free ofDistortion Using Susceptibility Weighted Fast Spin-Echo Imaging: AFeasibility Study at 1.5 T and 3.0 T. Magn. Reson. Med., 62:822-828.T. Niendorf, C.J. Hardy, R.O. Giaquinto, P. Gross, H. Cline, Y. Zhu, G. Kenwood,S. Cohen, A. Grant, S. Joshi, N.M. Rofsky, D.K. Sodickson (2006) TowardsSingle Breath-Hold Whole Heart Coverage Coronary MRA using HighlyAccelerated Parallel Imaging with a 32-Channel MR System. Magn. Reson.Med., 56:167-176, IF 3.427.T. Niendorf, D.K. Sodickson. (2006) Parallel Imaging in Cardiovascular MRI:Methods and Applications. NMR Biomed. 19:325-41, IF 3.626.Patent applicationsT. Niendorf, U. HeinrichsMethod for Susceptibility Weighted, Distortion Free Magnetic ResonanceImaging of the Cardiovascular SystemPatent DE 10 2007 045 172.7 (2008)T. Niendorf, F. HezelMethod for Automatic Motion Analysis and Quality Assurance inCardiovascular Magnetic Resonance ImagingPatent application 10 2009 011 382.7 (2009)Cardiovascular and Metabolic Disease Research 47

Michael BaderStructure of the GroupGroup LeaderProf. Dr. Michael BaderScientistsDr. Natalia AleninaDr. Carlos Barros*Dr. Saleh BashammakhDr. Philipp Boyé*Dr. Jens ButtgereitDr. Cibele Cardoso*Dr. Ilya ChuykinDr. Johan Duchene*Dr. Robert Fischer*Dr. Alessander Guimaraes*Cardiovascular HormonesThe group focuses on the molecular biology and function of hormone systems involved incardiovascular regulation. The physiological functions of these systems are analyzed by theproduction and analysis of transgenic and gene-targeted animal models. Among the hormonesstudied, serotonin is of special interest, since it is not only involved in vascular homeostasis andother peripheral functions, but also serves as potent and multifunctional neurotransmitter inthe brain. In addition, the group is interested in embryology and stem cell research, aiming toapply these fields to the rat and to establish gene targeting in this species.Renin-angiotensin systemNatalia Alenina, Brit Rentzsch, Mihail Todiras, Ping Xu,Aline Hilzendeger, Luiza Rabelo, Sergio Santos, PhilipBoyé, Gabin Sihn, Cibele CardosoThe renin-angiotensin system (RAS) is of central importancein blood pressure regulation and in the initiationof target organ damage. In particular, local angiotensin-II generating systems in tissues such as brain, heart,vessels, and kidney are involved in these processes.Therefore, transgenic rats with local up- or downregulationof RAS components in these organs, e.g. by the localexpression of antisense-RNA or of a peptide-liberatingprotein, were produced and analyzed to clarify the localfunctions of angiotensin II. Other genetically alteredmouse and rat models for non-classical RAS componentssuch as ACE2, the renin receptor, angiotensin(1-7)and its receptor Mas, have elucidated the physiologicalfunction of these factors. Together with transgenic ratsoverexpressing this peptide, Mas-knockout mice characterizedthe angiotensin(1-7)/Mas system as a cardioprotectiveaxis that counteracts the classical RASeffects in particular improving endothelial function.Furthermore, these animals showed that angiotensin(1-7) and Mas are important for insulin sensitivity andthe pathogenesis of metabolic syndrome.Kallikrein-kinin systemInes Schadock, Robert Fischer, Marcelo Mori, CarlosBarros, Alessander Guimaraes, Fatimunnisa Qadri,Anthony Rousselle, Johan Duchene, Vanessa MerinoThe kallikrein-kinin system (KKS) is an important hormonesystem for cardiovascular regulation also mostlycounteracting the effects of the RAS. As models for thefunctional analysis of the KKS in intact animals, transgenicrats were generated expressing different componentsof the system, such as tissue kallikrein, the kininB1 or the B2 receptor either ubiquitously or specificallyin cardiovascular organs. These animals supported theprotective role of the KKS in kidney and heart againstischemic, diabetic, and hypertrophic injury. Knockoutmice for the kinin B1 receptor were generated andrevealed important functions of this protein in painperception and inflammation. Moreover, the B1 receptorturned out to be involved in sepsis, stroke, multiple sclerosisand high-fat diet induced obesity. Mice lackingboth kinin receptors and thereby being devoid of afunctional KKS were also generated and shown to becompletely normal at baseline suggesting that the KKSis irrelevant for development and basic regulation ofthe cardiovascular system but is involved in the pathogenesisof multiple diseases and, thus, a relevant drugtarget.48 Cardiovascular and Metabolic Disease Research

Dr. AlexanderKrivokharchenkoDr. Irina Lapidus*Dr. Vanessa Merino*Dr. Marcelo Mori*Dr. Elena PopovaDr. Fatimunnisa QadriDr. Luiza Rabelo*Dr. Franziska RotherDr. Tanja ShmidtDr. Gabin SihnDr. Katja Tenner*Dr. Mihail Todiras*Graduate StudentsAline Hilzendeger*Stefanie HügelDana Kikic*Katarina Kotnik*Susann MatthesValentina MosienkoSilke MühlstedtBrit Rentzsch*Anthony Rousselle*Sergio Santos*Ines SchadockLarissa VilianovichPing Xu*Technical AssistantsAdelheid Böttger*Cathrin GerhardSusanne GoncalvesSabine GrügerAnne Koehn*Andrea MüllerIrene StraussLieselotte Winkler*SecretariatIris Apostel-Krause*part of the period reportedABGrowth retardation in Tph2-deficient mice lackingserotonin in the brain. (A) Absence of Tph2-mRNAand serotonin (5-HT) immunoreactivity in the brainstem of Tph2-deficient mice. (B) A 15 days old Tph2-deficient mouse (below) is much smaller than awild-type littermate (above).Cardiovascular and Metabolic Disease Research 49

Natriuretic peptide systemJens ButtgereitThere are 3 natriuretic peptides (NP), ANP, BNP, and CNP,which interact with two natriuretic peptide receptors,NPR-A and NPR-B, to induce a multitude of actions inheart, kidney, vessels, brain and other tissues. The receptorsare dimeric molecules, which after activation synthesizecyclic GMP. We have shown that dimerization isessential for the activation of the receptors and havedesigned dominant negative mutants to downregulatethe activity of the receptors in cells and transgenic animals.Transgenic rat models expressing a dominantnegative mutant for NPR-B exhibit sympathetic activationand develop cardiac hypertrophy supporting a cardioprotectiveaction of this receptor and its ligand CNP.Moreover, these animals show an impaired bonegrowth in accordance with the phenotype of knockoutmice for NPR-B and CNP and humans with mutations inthe NPR-B gene.Serotonin systemNatalia Alenina, Dana Kikic, Katja Tenner, KatarinaKotnik, Saleh Bashammakh, Valentina Mosienko,Susann MatthesSerotonin is a monoamine which functions as animportant neurotransmitter in the central nervous systemand as a major peripheral mediator produced byenterochromaffin cells of the gut and transported andreleased by platelets in the circulation. We discoveredthat vertebrates have two tryptophan hydroxylases, therate limiting enzymes in serotonin synthesis, TPH1 andTPH2. Mice deficient in TPH1, the isoform responsible forthe synthesis of serotonin in the gut, showed thatperipheral serotonin is involved in thrombosis,pulmonary hypertension, remodelling of mammaryglands, tumor angiogenesis, liver regeneration, andhepatitis. Mice deficient in TPH2, the isoformresponsible for the synthesis of serotonin in the brain,were surprisingly viable and fertile, despite a near completelack of serotonin in the brain, and showed growthretardation and altered autonomic control leading toimpairment of sleep, respiration, and cardiovascularparameters. In addition, these mice exhibit increasedaggression and maternal neglect. Furthermore,searching for molecules, which are crucial for thedevelopment of serotonergic neurons, we developedprotocols for the differentiation of embryonic stem(ES) cells into serotonin-producing neurons. Basedon genetic modifications of the ES cells, the methodallows to select this neuronal population and tomonitor their development in in-vitro and in-vivostudies.Androgen receptorGabin Sihn, Silke MühlstedtIn a collaborative work with the Charité studying gendereffects in cardiac hypertrophy and failure, the groupgenerates and characterizes animal models withaltered androgen receptor expression in distinct celltypes of the heart.ImportinsFranziska Rother, Tanja Shmidt, Stefanie HügelImportins are essential components of the machinerythat transports proteins into the nucleus of eukaryoticcells. In an approach to study the physiological functionof alpha importins we have generated knockout micefor all five known paralogs in the mouse. The most obviousphenotype was discovered in mice lacking importinalpha7: Both sexes of these animals are infertile. Themolecular basis of this phenotype is currently analyzed.In addition, we could show that the absence ofimportin alpha5 during mouse development does notsignificantly interfere with neuronal differentiation andproper brain development, in contrast to the predictionbased on a study in cell culture. Comparative studies onalpha-importin deficient mice and cells derived fromthem will allow to discover novel redundancies andspecificities in nuclear transport.Transgenic and stem cell technologyAlexander Krivokharchenko, Elena Popova, IrinaLapidus, Natalia Alenina, Katarina Kotnik, LarissaVilianovitch, Ilya ChuykinThe group has also a strong emphasis in the field of ratembryology and stem cell research. The rat is the preferredanimal in physiological and behavioral studies.However so far, there is no reliable method of generatingtargeted genetic alterations in these species. Inorder to obtain rat pluripotent stem cells two methodologieswere applied in our group: isolation of ES(embryonic stem) from rat preimplantation embryosand generation of induced pluripotent stem (iPS) cellsfrom fibroblasts upon infection with lentiviruses carryingpluripotency genes. These cells are used to explorethe signaling cascades underlying mechanisms ofpluripotency in the rat, to develop protocols in regenerativetherapy, and to establish homologous recombinationand thereby allowing gene targeting in the rat.Furthermore, transgenic rats have been produced carry-50 Cardiovascular and Metabolic Disease Research

ing constructs, which express small interference RNAssuited to downregulate specific genes. The first targetgene was the insulin receptor yielding a uniqueinducible rat model for diabetes mellitus type 2.Selected PublicationsAlenina, N., Kikic, D., Todiras, M., Mosienko, V., Qadri, F., Plehm, R., Boye, P.,Vilianovich, L., Sohr, R., Tenner, K., Hörtnagl, H., Bader, M. (2009) Growthretardation and altered autonomic control in mice lacking brainserotonin. Proc. Natl. Acad. Sci. USA. 106, 10332-10337.Kotnik, K., Popova, E., Todiras, M., Mori, M.A., Alenina, N., Seibler, J., Bader, M.(2009) Inducible transgenic rat model for diabetes mellitus based onshRNA-mediated gene knockdown. PLoS ONE. 4, e5124Schulze-Topphoff, U., Prat, A., Prozorovski, T., Siffrin, V., Paterka, M., Herz, J.,Bendix, I., Ifergan, I., Schadock, I., Mori, M.A., van Horssen, J., Schröter, F.,Han, M.H., Bader, M., Steinman, L., Zipp, F. (2009) Activation of kininreceptor B1 limits encephalitogenic T lymphocyte recruitment to the CNS.Nat. Med., 15, 788-793.Shmidt, T., Hampich, F., Ridders, M., Schultrich, S., Hans, V.H., Tenner, K.,Vilianovich, L., Qadri, F., Alenina, N., Hartmann, E., Köhler, M., Bader, M.(2007) Normal brain development in importin alpha 5 deficient mice.Nat. Cell. Biol. 9, 1337-1338.Langenickel, TH, Buttgereit, J, Pagel-Langenickel, I, Lindner, M, Monti, J,Beuerlein, K, Al-Saadi, N, Plehm, R, Popova, E, Tank, J, Dietz, R, Willenbrock,R, Bader, M. (2006). Cardiac hypertrophy in transgenic rats expressing adominant-negative mutant of the natriuretic peptide receptor B. Proc.Natl. Acad. Sci. USA 103, 4735-4740.Cardiovascular and Metabolic Disease Research 51

Structure of the GroupZsuzsanna IzsvákEuropean YoungInvestigator(EURYI) AwardCo-head: Zoltán IvicsGroup LeadersProf. ZsuzsannaIzsvák/Dr. Zoltán IvicsScientistsDr. Lajos MátésDr. Csaba MiskeyGraduate StudentsAndrea SchmittDianaPryputniewiczYongming WangDawid GrzelaAnantharamDeverajSanum BashirJichang WangKatrin VoigtAndrea SchornIvana GrabundzijaTobias JurschMarta SwierczekFrank SchulzeTechnical AssistantsVerena SladekAnna DaldaSecretariatKornelia DokupMobile DNATransposons (“jumping genes”) are discrete segments of DNA that have the distinctive abilityto move and replicate within genomes across the tree of life. Transposons offer a new modelto study DNA recombination in higher organism, as well as host-parasite interaction.Transposons are also natural gene delivery vehicles that are being developed as genetic tools.Our laboratory is following the strategy of understanding the mechanism of transposition andits regulation and translate this knowledge to derive transposon-based genetic tools forgenome manipulation or for gene therapy.Quality controls in Sleeping Beauty element (SB)transpositionDiana Pryputniewicz, Tobias Jursch, Andrea SchornOur understanding the way of how eukaryotic recombinasesare working is mostly based on assuming analogiesto V(D)J recombination. Besides the basic chemicalreaction, the different elements have a variety of “builtin”regulatory mechanisms, often involving host factors,to provide specificity to the transposition reaction. Onemain function of such regulation is to impose “qualitycontrol” on transposition in the form of regulatorycheckpoints, at which certain molecular requirementshave to be fulfilled for the transpositional reaction toproceed. The role of these regulatory checkpoints is toavoid accumulation of incorrect reaction products ingenomes, possessing a threat of genome instabilityassociated by transposition. We also investigate thepossible involvement of small, germline-specific RNAsin transposon regulation in zebrafish, as well as theeffect of chromatin structure of both donor and targetsites on transposition.Transposon-host interactionsYongming Wang, David Grzela, Anantharam Deveraj,Andrea SchmittTransposons occupy a significant portion of ourgenomes. However, the vast majority of transposonsremain silent due to accumulated mutations in theirgenomes. The transposition of the few, active copies isstrongly regulated, but this control is sensitive to environmentalstress. Our results show that transposonsmight exist in a “latent” form in the genome and areable to sense developmental and environmentalchanges and manipulate stress signaling. Cellularmechanisms that are directly involved in repairingtransposition-inflicted DNA lesions or can attenuateDNA damage should have crucial role in establishingstable host-transposon co-existence. Our results suggestthat SB transposon takes advantage of the cellularrepair machinery and/or during DNA replication toamplify their own genome.Domesticated, transposon-derived cellular genesCsaba Miskey, Marta SwierczekOne particular copy of the transposase gene of theancient Hsmar1 human transposon has been underselection. This transposase coding region is part of theSETMAR gene, in which a histone methylatransferaseSET domain is fused to an Hsmar1 transposase domain.SETMAR retains ist ability to bind to transposonsequences in vitro, and we are currently investigatingthe cellular function of this gene by integrated genomicand transcriptomic approaches.The active copy of a rat endogenous retrovirusYongming WangEndogenous retroviruses were repeatedly demonstratedto influence the expression of rat genes, withoutknowing the active copy. A phenotype of hypodactyly inrat was associated with recent retroviral activity. We52 Cardiovascular and Metabolic Disease Research

have identified an “active” copy of novel endogenousretrovirus in the rat genome, and showed that the elementis active in retrotransposition. The transpositionallyactive copy has an intact env gene, so elementmight be capable of infection.Genome manipulation – Transposon mutagenesisin rat spermatogonial stem cellsLajos Mátés, Ivana GrabundzijaTransposons can be harnessed as vehicles for introducingmutations into genes. Our goal is to establish toolsbased on SB as well as on the piggyBac and Tol2 transposonsystems to manipulate vertebrate genomes(transgenesis, genomic screens) in organisms wherethis technology was not available before. One particularapplication relates to loss-of-function insertionalmutagenesis in rats with the goal of generating knockoutanimals. The genes inactivated by transposoninsertion are “tagged” by the transposable element,which can be used for subsequent cloning of themutated allele. With the goal of knocking out genesimplicated in disease, we carried out a pilot screen inrat spermatogonial stem cells. The project has enormouspotential to develop powerful genomic tools forrat that is the preferred model organism of cardiovascular,as well as toxicology and behavioral studies.Deciphering the genetic background of neuroblastomaand hormone-induced breast cancerSanum BashirThe SB transposon is suitable for somatic mutagenesisand emerged as a new tool in cancer research as analternative to retroviral mutagenesis. Transposonbasedinsertional mutagenesis screens are able to identifiyboth oncogenes and tumor-suppressor genes. Weare engaged in two projects aiming at the discovery ofnovel driver mutations that are associated with neuroblastoma(in mice) and estrogen-induced mammarycancer (in rats). The transposon mutagenesis approachis expected to be a powerful tool to decipher gene regulatorynetworks cooperating in cancer development,progression and metastasis.Transposons as non-viral vectors for genetherapeutic approachesIsmahen Ammar, Csaba Miskey, Katrin Voigt,Jichang Wang, Frank SchulzeDNA-based transposons are natural gene delivery vehicles,and molecular reconstruction of SB represents acornerstone in applying transposition-mediated genedelivery in vertebrate species, including humans. Ourrecently developed 100-fold hyperactive SB systemStable gene transfer and expression of green fluorescent protein (GFP)in differentiating, human hematopoietic stem cell lineages (A-C). Thecells only appear green if the GFP marker gene stably integrated intheir chromosomes by transposition. The hyperactive transposase(SB100X) generates far more green cells than an earlier version of theSB transposase (SB32X).opened new avenues for gene therapeutic approaches,and we are currently developing preclinical animalmodels for gene therapy of Wiskott-Aldrich syndrome,chronic granulomatous disease and Goucher disease.SB transposition occurs into chromosomes in a randommanner, which is clearly undesired for human applicationsdue to potential genotoxic effects associatedwith transposon integration. We succeeded in targetingSB transposition into predetermined chromosomalloci. We employed modular targeting fusion proteins, inwhich the module responsible for target binding canbe a natural DNA-binding protein or domain, or an artificialprotein such as a designer zinc finger. Targetedtransposition could be a powerful method for safetransgene integration in human applications.Selected PublicationsIvics, Z, Li, M.A., Mátés, L, Boeke, JD, Nagy, A, Bradley, A, Izsvák, Z. (2009)Transposon-mediated genome manipulations in vertebrates. NatureMethods 6(6), 415-422.Mátés, L, Chuah, MK, Belay, E, Jerchow, B, Manoj, N, Acosta-Sanchez, A,Grzela, DP, Schmitt, A, Becker, K, Matrai, J, Ma, L, Samara-Kuko, E,Gysemans, C, Pryputniewicz, D, Miskey, C, Fletcher, B, VandenDriessche, T,Ivics, Z., Izsvák, Z. (2009). Molecular evolution of a novel hyperactiveSleeping Beauty transposase enables robust stable gene transfer invertebrates. Nature Genet. 41(6), 753-761.Sinzelle, L, Kapitonov, VV, Grzela, DP, Jursch, T, Jurka, J, Izsvák, Z, Ivics, Z.(2008). Transposition of a Reconstructed Harbinger Element in HumanCells and Functional Homology with Two Human Cellular Genes. Proc.Natl. Acad. Sci. USA, 105(12):4715-20Wang, Y, Liska, F, Gosele, C, Sedová, L, Kren, V, Krenová, D, Ivics, Z, Hubner, N,Izsvák, Z. (2009) A novel active endogenous retrovirus family contributesto genome variability in rat inbred strains. Genome Res. [Epub ahead ofprint] PMID: 19887576Ivics, Z, Katzer, A, Stüwe, EE, Fiedler, D, Knespel, S, Izsvák, Z. (2007). TargetedSleeping Beauty transposition in human cells. Mol. Ther. 15, 1137-1144.Cardiovascular and Metabolic Disease Research 53

Young-Ae LeeStructure of the GroupGroup LeaderProf. Dr. Young-Ae LeeScientistsDr. Jorge Esparza-Gordillo(Marie Curie fellow)Dr. Ingo MarenholzDipl.-Biol. Tamara KerscherDipl.-Biol. Florian SchulzDipl.-Biol. Jamina EckardDipl.-Biol. Andrey GiménezTechnical AssistantsChristina FlachmeierSusanne KolbergMonika SchwarzInka SzangoliesSecretariatKornelia DokupGenetics of Allergic DiseaseThe allergic diseases, particularly atopic dermatitis, food allergy, asthma, and hay fever, areamong the most common chronic diseases in man. The prevalence of atopic diseases hasincreased to epidemic dimensions over the past decades. In the industrialized countries, 25-30%of the population are affected. Genetic and environmental factors interact to determine diseasesusceptibility, and family and twin studies indicate that the genetic contribution is substantial.Our group is using genetic and genomic approaches to identify genes and genetic variants thatpredispose to atopic dermatitis and atopy. The identification of the molecular pathwaysunderlying allergic disease will provide novel targets for preclinical diagnosis, diseaseprevention, and therapeutic intervention.Genomewide Association study reveals acommon variant on chromosome 11q13 that isassociated with atopic dermatitisAtopic dermatitis is a chronic inflammatory skin disorderand a major manifestation of allergic disease. Themolecular mechanisms underlying eczema are not fullyunderstood. Skin barrier defect as well as systemic andcutaneous immune dysfunction in response to allergensor bacterial products are thought to play animportant role.To identify genetic variants contributing to atopic dermatitis,we conducted a genome-wide associationstudy in 939 individuals with atopic dermatitis and 975controls as well as 270 complete nuclear families with 2affected siblings on Affymetrix Human mapping 500Kand 5.0 arrays. SNPs consistently associated with atopicdermatitis in both discovery sets were then investigatedin two additional independent replication setstotalling 2637 cases and 3957 controls (Figure 1). Highlysignificant association was found with a commonsequence variant on chromosome 11q13.5 (P combined = 7.6x 10 -10 ) in all 4 study groups. Approximately 13% of individualsof European origin are homozygous for the riskallele, and their risk of developing atopic dermatitis is1.47 times that of noncarriers. Linkage disequilibriumanalysis in Hapmap showed that rs7927894 is locatedin a 200 kb LD block containing a single gene calledC11orf30 (Figure 2).C11orf30 encodes the nuclear proteinEMSY which has been implicated in breast cancer susceptibility,chromatin modification, DNA repair, andtranscriptional regulation. The potential involvement ofC11orf30 in multiple inflammatory and malignantepithelial diseases (atopic dermatitis, Crohn´s disease,and adenocarcinoma) strongly suggests a role forC11orf30 in epithelial immunity, growth, and/or differentiation.Finally, we provide a list of additional candidate genes.Further replication in independent cohorts, fine mappingand functional studies will be required to gain abetter understanding of the physiological mechanismsunderlying this common allergic disorder.Towards the genetic prediction of childhoodasthmaAsthma is a chronic inflammatory lung disease featuringintermittent airway obstruction triggered by environmentalallergens, exercise or viral infections. Theincreasing prevalence of asthma and the lack of curativetherapy underscores the need for effective diseaseprediction and prevention. We have performed the firstgenetic prediction study for asthma using the loss-offunctionmutations in the filaggrin gene (FLG), which54 Cardiovascular and Metabolic Disease Research

Figure 1. Study designFigure 2. Association results and LD structure in the AD associatedregion on 11q13.5. (a) Association results for all GWA markers tested in theregion. Physical positions are based in the NCBI build 36. (b) Genes in theregion from the UniGene database. (c) LD in the CEU Hapmap population.Disequilibrium coefficient values for Hapmap Phase II data (v. 22)were generated with Haploview. The lead SNP is indicated by a green line.were identified to be a strong genetic risk factors foreczema and eczema-associated asthma. We investigatedin the population-based Multicenter Allergy Study(MAS cohort) whether the predictive value of the FLGmutations for asthma was related to infantile eczema.Furthermore, we investigated the role of allergic sensitizationto food allergens which represents the earliestserologic marker for atopy and is a recognized risk factorfor chronic asthma.We found that, in infants with eczema and sensitizationto food allergens within the first three years of life,the presence of a FLG loss of function mutation predictsthe future development of asthma with a specificityand a positive predictive value of 100%. The combinationof FLG mutations and early sensitization to foodallergens, predicted a sizeable proportion (17.2%) of theinfants with eczema who made the transition to asthmalater in childhood. Furthermore, longitudinal pulmonaryfunction measurements demonstrated thatthis subgroup of asthma children identified by the FLGmutations carried a poor prognosis with a steadydecline in pulmonary function until puberty. Hence, thissubgroup might particularly benefit from early predictionof the disease. Our findings indicate that assessmentof the FLG carrier status could improve the predictionof eczema-associated asthma considerably. Themagnitude of the predictive power surpasses the utilityof mutations in the breast cancer susceptibilitygenes BRCA1 and BRCA2 that account for only 2-4% ofall breast cancer cases and for 15-20% among the highrisk group of women with a strong family history andearly onset of the disease in whom targeted genetictesting was recommended. Analogously, in the highrisk group of children with eczema and early food sensitizationthe genotyping of the FLG mutations wouldidentify 35.7% of future asthmatics.In this study, we demonstrate that the determinationof the FLG carrier status in infants with eczema andsensitization to food allergens within the first threeyears of life would allow the early prediction of asthmabefore the onset of symptoms and at a critical time ofimmune development when preventive interventionsare likely to be effective. We suggest our findings couldbe of diagnostic and subsequent therapeutic utility.Selected PublicationsEsparza-Gordillo J, Weidinger S, Folster-Holst R, Bauerfeind A,Ruschendorf F, Patone G, Rohde K, Marenholz I, Schulz F, Kerscher T,Hubner N, Wahn U, Schreiber S, Franke A, Vogler R, Heath S, Baurecht H,Novak N, Rodriguez E, Illig T, Lee-Kirsch MA, Ciechanowicz A, Kurek M,Piskackova T, Macek M, Lee YA *, Ruether A. A common variant onchromosome 11q13 is associated with atopic dermatitis. Nat Genet 41[5],596-601. 2009. * corresponding authorMarenholz I, Kerscher T, Bauerfeind A, Esparza-Gordillo J, Nickel R, Keil T,Lau S, Rohde K, Wahn U, Lee YA. An interaction between filaggrinmutations and early food sensitization improves the prediction ofchildhood asthma. J Allergy Clin Immunol 123[4], 911-916. 2009.Muller S, Marenholz I, Lee YA, Sengler C, Zitnik S, Griffioen RW, Meglio P,Wahn U, Nickel R. Association of Filaggrin loss-of-function mutationswith atopic dermatitis and asthma in the Early Treatment of the AtopicChild (ETAC) population. Pediatr Allergy Immunol . 2009.Pasternack SM, von Kugelgen I, Aboud KA, Lee YA, Ruschendorf F, Voss K,Hillmer AM, Molderings GJ, Franz T, Ramirez A, Nurnberg P, Nothen MM,Betz RC. G protein-coupled receptor P2Y5 and its ligand LPA are involvedin maintenance of human hair growth. Nat Genet 40[3], 329-334. 2008.Schulz F, Marenholz I, Folster-Holst R, Chen C, Sternjak A, Baumgrass R,Esparza-Gordillo J, Gruber C, Nickel R, Schreiber S, Stoll M, Kurek M,Ruschendorf F, Hubner N, Wahn U, Lee YA. A common haplotype of the IL-31 gene influencing gene expression is associated with nonatopiceczema. J Allergy Clin Immunol 120[5], 1097-1102. 2007.Cardiovascular and Metabolic Disease Research 55

Structure of the GroupMatthias SelbachGroup LeaderDr. Matthias SelbachScientistsDr. Marieluise KirchnerDr. Matthias SuryGraduate StudentsOlivia EbnerFabian HospFlorian PaulBjörn SchwanhäusserTechnical AssistantsChristian SommerSecretariatSonja GieringPetra HainkCell Signaling and Mass SpectrometryProteins are the chief actors in almost every biological process. While we know a lot aboutthe function of individual proteins there is little information about the system as a whole.Recent developments in mass spectrometry have dramatically improved the analytical powerof this technology. We are using mass spectrometry-based quantitative proteomics toinvestigate cellular signaling at the protein level on a global scale. Main areas of research arepost transcriptional regulation of gene expression by microRNAs, protein-protein interaction inthe context of neurodegenerative diseases and in vivo quantitative proteomics.Recently developed quantitative methods make it possibleto obtain precise functional information and tomonitor temporal changes in the proteome by massspectrometry. In one approach, named SILAC (for stableisotopelabelling with amino acids in cell culture), cellsare differentially labeled by cultivating them in thepresence of either normal or a heavy isotope–sub -stituted amino acid, such as 13 C-labeled lysine (Figure,left panel). Due to their mass difference, pairs of chemicallyidentical peptides of different stable-isotope compositioncan be distinguished in a mass spectrometer.The ratio of intensities for such peptide pairs accuratelyreflects the abundance ratio for the corresponding proteins.Quantitative proteomics with SILAC has emergedas a very powerful approach to investigate signalingprocesses. We are using this technology as our centraltool to address challenging questions in cell signaling.pSILAC and microRNAsRegulation of gene expression occurs at all stages frommRNA transcription to protein synthesis. It is now clearthat translation itself is a regulated process with a centralrole in cellular physiology and a growing catalogueof human diseases. A prominent example aremicroRNAs: These small non-coding RNAs repress targetgenes by inhibiting translation or by inducingdegradation of mRNAs. In mammals, microRNAs arepredicted to control the activity of ~30% of all proteincodinggenes. They have been shown to be involved inregulation of almost every cellular process investigatedso far. In order to understand microRNA function it iscrucial to investigate how they regulate protein pro -duction.We developed pulsed SILAC (pSILAC) as a novel methodto directly compare protein translation rates betweentwo samples (Figure, right panel). Cells are first cultivatedin standard growth medium with the normal light(L) amino acids. Concomitantly with differential treatment,cells are transferred to culture medium containingheavy (H) or medium-heavy (M) amino acids. Allnewly synthesized proteins will be made in the H or Mform, respectively. Subsequently, both samples are combinedand analyzed together. The abundance ratio of Hversus M peptides reflects differences in translation ofthe corresponding proteins integrated over the pulselabelling incubation time. We employed pSILAC tomeasure changes in synthesis of several thousand proteinsafter misexpression of microRNAs (collaborationwith the group of Nikolaus Rajewsky). Bioinformaticanalysis of the data showed that a single microRNA candirectly repress hundreds of targets and that thisrepression is typically rather mild. By comparing thepSILAC and the microarray data we were able to showthat microRNAs directly repress translation of hundreds56 Cardiovascular and Metabolic Disease Research

The principle standard SILAC (left) and pulsed SILAC (right). Instandard SILAC, cells are completely labeled by cultivating them ingrowth medium containing light (L) or heavy (H) stable (i.e. nonradioactive)amino acids. After several cell generations, all proteinshave uniformly incorporated the isotope label. Differentially labeldcells can be combined and analyzed together by mass spectrometry.The ratio of peak intensities reflects differences in proteinabundance between both samples. In pSILAC, cells are growing innormal (i.e. light) medium. Upon differential treatment, cells aretransferred to medium-heavy (M) of heavy (H) medium for pulselabelling. The ratio of heavy and medium-heavy peaks is a directmeasure of the differences in protein synthesis between bothexperimental conditions.of mRNAs. We are currently extending this analysis tomicroRNAs involved in carcinogenesis. (Olivia Ebner,Björn Schwanhäusser)Protein-protein interaction in neurodegenerativediseasesIdentifying interaction partners is the key to proteinfunction and can provide insight into disease mechanisms.Neurodegenerative diseases like Alzheimer’s arefrequently caused by accumulation of toxic proteinspecies in neuronal cells. We are using quantitativemass spectrometry and network analysis to analyzeprotein-protein interactions (PPIs) involved in diseasepathogenesis. A useful method to study PPIs is affinitypurification using a bait molecule. However, such pulldownassays suffer from the trade-off between sensitivityand specificity: While more stringent purificationconditions can remove some contaminating proteinsthey might also eliminate specific interaction partnerswith weak affinities and/or of low abundance.Quantitative proteomics solves this problem by comparingthe abundance of proteins identified in a pulldownexperiment with a suitable internal control. Thisallows the development of assays that can directly leadto understanding of biological systems. We are employingthis strategy to identify interaction partners of proteinsinvolved in neurodegeneration. Particularly, weare interested in identifying interactions affected bydisease-associated mutations. (Fabian Hosp, FlorianPaul)In vivo quantitative proteomics: The SILAC zooCell culture-based experiments cannot recapitulate allof the complex interactions among different cell typesand tissues that occur in vivo. Small animal modelssuch as worms, fruit flies and zebrafish are attractivealternatives that are extensively used in many areas ofbiomedical research, especially in genetics and developmentalbiology. While SILAC has enormouslyimproved quantitative proteomics in cultured cells, themethod was not yet used in small animal models. Weare currently extending this technology to three of themost important model organisms in biomedicalresearch: Caenorhabditis elegans, Drosophila melano gasterand Danio rerio. (Matthias Sury, Marieluise Kirchner)Selected publicationsSelbach M*, Paul FE, Brandt S, Guye P, Daumke O, Backert S, Dehio C,Mann M.* (2009) Host cell interactome of tyrosine-phosphorylatedbacterial proteins. Cell Host and Microbe 5, 397-403. (*sharedcorresponding authors)Schwanhäusser B, Gossen M, Dittmar G, Selbach M. (2009) Globalanalysis of cellular protein translation by pulsed SILAC. Proteomics 9,205-209.Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, Mann M. (2009) Apractical guide to the MaxQuant computational platform for SILACbasedquantitative proteomics. Nature Protocols 4, 698-705.Selbach M*, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, RajewskyN.* (2008) Widespread changes in protein synthesis induced bymicroRNAs. Nature 455, 58-63. (*shared corresponding authors)Selbach M, Mann M. (2006) Protein interaction screening by quantitativeimmunoprecipitation combined with knockdown (QUICK). NatureMethods 3, 981-983.Cardiovascular and Metabolic Disease Research 57

Matthew PoyStructure of the GroupGroup LeaderDr. Matthew Poy*Graduate StudentsJiaxuan Chen*Maria Dolaptchieva*Dörte Matthäus*Sudhir Gopal Tattikota*microRNAs and Mechanisms ofMetabolic DiseasesType II diabetes has finally become recognized as a major challenge to global health. It isimperative to improve our understanding of the molecular mechanisms behind this disorderand develop new drug therapies. The pathophysiology of diabetes is undoubtedly complex,typically characterized by hyperglycemia resulting from varying states of insulin resistance andimpaired β-cell function. Oftentimes, the failure to regulate circulating blood glucose levels is aconsequence of the inability to produce sufficient amounts of insulin by the pancreatic β-cells.In our group, we focus on fundamental pathways regulating glucose metabolism and howaltered pancreatic islet physiology contributes to metabolic disorders such as type 2 diabetes.The mechanisms governing gene expression patternsintegrate both transcriptional activation, post-transcriptionalgene silencing, and post-translational modifications.In the last decade the complex picture of generegulation has been extended by the discovery ofmicroRNAs. MicroRNAs are short, approximately 22nucleotide long non-coding RNAs which are thought tobe involved in a number of evolutionary conserved regulatorypathways. Many published reports have clearlyillustrated a role for individual microRNA sequences indevelopmental timing, apoptosis, proliferation, differentiation,and organ development. However, in light ofthe many advances in the fields of microRNAs and RNAinterference, many questions remain concerning thefunctional role of microRNAs in tissues like the pancreaticislet. Our work aims to test the hypothesis thatmicroRNAs expressed in the islet play an important rolein the development and function of pancreatic β-cellsthrough their ability to regulate gene expression. Manydirect targets of microRNAs expressed in the pancreaticislet have never been studied with respect to β-cellbiology and it is important to understand how theycontribute to islet function. Using newly developedmouse models, a molecular understanding of the exactnature of microRNAs is necessary to develop therapeuticstrategies for the treatment of metabolic diseaseslike diabetes.Selected PublicationsPoy, MN, Hausser, J, Trajkovski, M, Braun, M, Collins, S, Rorsman, P, Zavolan,M and Stoffel, M. (2009). miR-375 maintains normal pancreatic α- andβ-cell mass. Proc. Natl. Acad. Sci. USA. 106(14):5813-8.Yi, R, Poy, MN, Stoffel, M, and Fuchs, E. (2008) A skin microRNA promotesdifferentiation by repressing ‘stemness’. Nature. 452(7184):225-29.Wolfrum, C, Poy, MN, and Stoffel, M. (2005) Apolipoprotein M is requiredfor pre-HDL formation, cholesterol efflux to HDL and protects againstatherosclerosis. Nature Med. 11, 418-422.Krek, A*, Grun, D*, Poy, MN*, Wolf, R, Rosenberg, L, Epstein, EJ,MacMenamin, P, da Piedade, I, Gunsalus, KC, Stoffel, M, Rajewsky, N. (2005)Combinatorial microRNA target predictions. Nat. Genet. 37(5):495-500.(* equal contribution)Poy, MN, Eliasson, L, Krützfeldt, J, Kuwajima, S, Ma, X, MacDonald, PE,Pfeffer, S, Tuschl, T, Rajewsky, N, Rorsman, P and Stoffel, M. (2004) Apancreatic islet-specific microRNA regulates insulin secretion. Nature.432(7014):226-30.58 Cardiovascular and Metabolic Disease Research

Technical AssistantAndrea Katzer** Start of the group: August 2008Decreased β-cell mass in miR-375KO pancreatic islets. Representativesections of pancreas from 10-week-old a)wild type and b)miR-375KOmice visualized by immunofluorescence after staining with insulin(green) and glucagon (red). Bar represents 25µm.Cardiovascular and Metabolic Disease Research 59

Jana WolfStructure of the GroupGroup LeaderDr. Jana WolfScientistsDr. Antonio Politi*Graduate StudentsKatharina Baum*Uwe Benary*Bente KofahlMathematical Modelling ofCellular SystemsComplex diseases are often characterised by an accumulation of multiple perturbations inrather large and complex cellular networks. The consequences of these perturbations, suchas mutations or over-expression of proteins, can hardly be analysed by pure reasoning. Here,mathematical modelling contributes to a deeper understanding of the regulatory systems andprovides thus a better basis for the interpretation of high-throughput data and identification ofeffective drug targets.Our group develops and analyses mathematical models of signaling pathways and generegulatorynetworks in normal and disease states. For our investigations we use tools suchas simulations, bifurcation analyses and sensitivity analyses. These give insights into thedynamical properties of the systems and help to identify most sensitive processes and criticalregulations. Another important aspect is the investigation of cell specific differences insignaling and gene-regulatory networks since these are critically involved in the predictionof the efficiency and possible side-effects of drugs.Modelling of IKK/ NF-κB signalingBente KofahlIn this collaborative project with the group of ClausScheidereit at the MDC we aim for a systems levelunderstanding of the IKK/NF-κB signaling pathway.This pathway consists of a canonical and a non-canonicalbranch. Both have a distinct timing and distinct biologicalfunctions but are interconnected. On the onehand substrates and inducers of the non-canonicalbranch are produced in the canonical branch, on theother hand a control of the canonical part by the noncanonicalbranch was reported. We are interested in theregulation of the long-time behaviour of the pathwayand its malfunction in diseases, e.g. Hodgkin-lymphoma.In particular, we want to dissect the contributionof canonical and non-canonical modules underthese conditions. To that end we are investigating thekinetic properties, feedback regulations and interactingmodules of both signaling branches. In a first step wehave developed a mathematical model of the noncanonicalIKK/NF-κB pathway, which is currentlyrefined in an iterative cycle of experiments and modellingand extended towards canonical signaling.Wnt/ β-catenin signalingUwe Benary, Bente KofahlWnt/β-catenin signaling plays an important role indevelopment and tissue homeostasis. Its deregulationis associated with various diseases and cancer. We use amathematical model of the Wnt/β-catenin pathway tostudy the effect of β-catenin mutations criticallyinvolved in hepatocellular carcinoma. In collaborationwith Rolf Gebhardts group (Leipzig) we are interested inthe impact of the mutations on the dynamics of thesignaling pathway and the expression of target genesin hepatocytes.60 Cardiovascular and Metabolic Disease Research

SecretariatSonja GieringPetra Haink*part of the period reportedIn other projects we investigate cell-type specific differencesin Wnt/β-catenin signaling, analyse the effect oftranscriptional feedbacks (e.g. via Axin, β-TrCP/ HOS)and that of cross-talks of Wnt/β-catenin signaling toother pathways, most importantly NF-κB.Robustness of cellular rhythmsKatharina Baum, Antonio PolitiRhythmic phenomena are widespread in biology andmany examples can be found on the cellular level, e.g.circadian rhythms, the cell cycle, calcium or metabolicoscillations. These rhythms are involved in various functionsand react specifically to environmental changes.Our project addresses the question of what determinesthe balance between sensitivity and robustness of acellular rhythm. To this end we analyse specific examplesof oscillations. By comparing various well-establishedmathematical models for calcium, glycolytic andcircadian rhythms we could already show that the sensitivityof the oscillatory period against perturbationsstrongly depends on the underlying oscillatory mechanism.We now ask the question to what extent robustnessis determined by local kinetic parameters or thetopology of the systems. In our comparison, we findmodels for calcium oscillations to be very sensitive,those for circadian rhythms very robust. These findingscorrespond well to the biological functions of therhythms since for calcium oscillations a period-encodedsignal-transduction was shown, whereas circadianoscillations generate an internal time information. Inaddition, the effect of the feedback type on the sensitivityis studied in core models.Comparison of the period sensitivity of cellular oscillators.Shown is the overall period sensitivity of ten mathematicalmodels describing three cellular rhythms. Models for calciumoscillations are very sensitive, those for circadian rhythm arevery robust. Models for glycolytic oscillations show an intermediatesensitivity.Selected publicationsJ. Wolf, S. Dronov, F. Tobin & I. Goryanin (2007), The impact of theregulatory design on the response of EGFR-mediated signal transductiontowards oncogenic mutations. FEBS Journal 274, 5505-5517.J. Wolf, S. Becker-Weimann & R. Heinrich (2005), Analysing the robustnessof cellular rhythms, IEE Syst. Biol. 2(1), 35-41.S. Becker-Weimann, J. Wolf, H. Herzel & A. Kramer (2004), Modelingfeedback loops of the mammalian circadian oscillator. BiophysicalJournal 87, 3023-3034.Cardiovascular and Metabolic Disease Research 61

Structure of the GroupGroup LeaderDr. Markus LandthalerTechnical AssistantsJulia KretschmerMarkus LandthalerScientistsAlexander BaltzLea GregersenKerstin BaethgeRNA Biology and Post-transcriptionalregulationOur main interest is the understanding of post-transcriptional regulatory networkscontrolling gene expression in humans. In cells RNA stably associates with RNA-bindingproteins, RNA helicases and nucleases to form ribonucleoprotein complexes. These complexesplay a key role in the regulation of spatial and temporal changes in protein synthesis bycontrolling transport, storage and translation of mRNAs. Deregulation and failed coordinationof these mechanisms contribute to pathophysiologicial development and conditions. Aprerequisite for a systems level understanding of post-transcriptional regulation is atranscriptome-wide high-resolution map of the RNA-protein contacts that allows us to studyhow these interactions control the fate of cytoplasmic RNA. To achieve this goal we use anovel crosslinking-immunoprecipitation approach (PAR-CLIP) in combination with massivelyparallel sequencing to identify functional RNA-protein interactions at single-nucleotideresolution.Post-transcriptional regulation by RNA-bindingproteinsMammalian genomes encode several hundred RNAbindingproteins, each containing one or multipledomains able to recognize target mRNA in a sequenceand/orstructure-dependent manner. The association ofthese proteins with RNA regulates the biogenesis andtranslation of RNA. For a large number of RNA-bindingproteins the target mRNAs and their function in RNAmetabolism are unknown, limiting our understandingof post-transcriptional regulatory processes.In particular, we are interested in RNA-binding proteinsthat positively and negatively modulate the activity ofmicroRNAs. By combining maps of functional RNA-proteininteractions with cell-based and biochemicalassays, we determine the dynamic assembly of RNAbindingproteins and microRNAs on their target mRNAsas well as the elements and mechanisms guidingmRNA maturation, localization, turnover and proteinsynthesis.Specificity and function of RNA helicasesRNA helicases are a family of highly conserved proteinsthat utilize NTP hydrolysis to unwind RNA structuresand/or remodeling of ribonucleoprotein complexes.RNA helicases participate in all biological processesthat involve RNA metabolism, including transcription,splicing and translation and have been implicated indisease states such as tumorgenesis and the replicationof viruses. We are using our crosslinking-immunoprecipitationapproach to define functional interactions ofhelicases and RNA that are typically transient in nature.The identification of RNA target sites provides a foundationfor biochemical and reverse genetic approachesto investigate the remodeling mechanism of ribonucleoproteincomplexes by helicases. These studies will provideinsights into the determinants of target RNA selection,functional interactions with other RNA-interactingproteins and the physiological role of RNA helicases.62 Cardiovascular and Metabolic Disease Research

PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation)Photoactivatable 4-Thiouridine (4SU), when providedto cultured cells, is incorporated into nascentRNAs and crosslinked to RNA-interacting proteins bylong-wave UV irradiation. The protein of interest isimmunoprecipitated and the covalently bound RNAreduced to a short protected segment by ribonucleasetreatment. In contrast to other methods, thesites of crosslinking are identified by mapping T to Ctransitions residing in the cDNA of libraries preparedfrom RNA crosslinked to a specific RNA-interactingprotein.Regulation of gene expression by non-codingRNAsNon-coding RNA (ncRNA) are functional RNAs that arenot translated into proteins. Recent evidence indicatesthat sizable regions of mammalian genomes are transcribedinto ncRNAs, which are alternatively splicedand/or processed into smaller RNAs. microRNAs, themost prominent class of small ncRNAs, mediate destabilizationand inhibition of translation target mRNAs.We are using next generation sequence technology todiscover large ncRNAs in human cells lines. To obtaininsights into the function of identified ncRNAs wedeplete these RNAs by RNA interference and characterizeassociated proteins by photo-crosslinking and massspectrometry.Selected PublicationsHausser J*, Landthaler M*, Jaskiewicz L, Gaidatzis D, Zavolan M. (2009)Relative contribution of sequence and structure features to the mRNAbindingof Argonaute/miRNA complexes and the degradation of miRNAtargets. Genome Res 19. 2009-2020.Zhang Y, Landthaler M, Schlussman SD, Yuferov V, Ho A, Tuschl T, KreekMJ.(2009) Mu opioid receptor knockdown in the substantia nigra/ventraltegmental area by synthetic small interfering RNA blocks the rewardingand locomotor effects of heroin. Neuroscience 158, 474-483Yi R, Pasolli HA, Landthaler M, Hafner M, Ojo T, Sheridan R, Sander C,O’Carroll D, Stoffel M, Tuschl T, Fuchs E. (2009) DGCR8-dependentmicroRNA biogenesis is essential for skin development. PNAS 106,498-502.Landthaler M, Gaidatzis D, Rothballer A, Chen PY, Soll SJ, Dinic L, Ojo T,Hafner M, Zavolan M, Tuschl T. (2008) Molecular characterization ofhuman Argonaute-containing ribonucleoprotein complexes and theirbound target mRNAs. RNA 14, 2580-2596.Landthaler M, Yalcin A, Tuschl T. (2004) The human DiGeorge syndromecritical region gene 8 and Its D. melanogaster homolog are required formiRNA biogenesis. Curr. Biol.14, 2162-7.Cardiovascular and Metabolic Disease Research 63

Cancer Research ProgramKrebsforschungCoordinator: Claus ScheidereitSignaling Pathways, Cell and Tumor BiologyCoordinator: Achim LeutzStructural and Functional GenomicsCoordinator: Udo HeinemannTumor ImmunologyCoordinator: Martin Lipp

Cancer Research ProgramKrebsforschungsprogramm Claus Scheidereit, Achim Leutz, Martin Lipp, Udo HeinemannAs a collective term, cancer denotes a variety ofmalignant neoplastic diseases that originatefrom different organs through the accumulationof multiple mutations in the genome. Thesealterations affect the functioning of sets of geneswhich control the normal behavior of cells in their tissuecontext. It is a shared feature of the differenttypes of cancer cells that they escape the natural surveillanceof growth control by their environment: cancercells proliferate in an uncontrolled manner, theyevade their elimination by natural cell death, escapethe control by the immune system and finally dispersein the body by migrating through the blood andlymph system (metastasis) to form secondarytumors.The objective of the Cancer Research Programis to understand at the molecular and cellular levelhow cancer develops and progresses in order to providethe basis for improved diagnosis and, ultimately,treatment of cancer.The human genome contains around 23,000 proteinencoding genes, a number of which are of particularimportance for the regulation of cellular behavior. Incancer cells, many of these crucial genes are repeatedlyfound to have acquired genetic mutations. Theseaffected genes are categorized as either “oncogenes”or “tumor suppressor genes”, depending on theirmode of action.They code for regulatory and structuralproteins that control critical processes, such as cellproliferation, differentiation, apoptosis, cell migrationor angiogenesis. Often, cancer cells have accumulatedsets of mutated genes for these different functions.Many of these genes are also active during embryogenesisor during the differentiation of distinct celltypes. This is why tumor cells appear to share characteristicsof embryonic cells.The research groups of the MDC Cancer ResearchProgram work in the fields signal transduction andgrowth control, structural genomics and tumorimmunology. The broad expertise in basic biomedicalas well as clinical disciplines allows to investigate thecauses and the emergence of cancer and to findrational treatments. Cancer studies are conducted inclose collaboration with clinical research groups ofthe Charité/ Medical Faculty Berlin. The aim of theProgram is to discover and to characterize genes thatAls Sammelbegriff bezeichnet Krebs eine Bandbreiteunterschiedlicher, bösartiger Tumor-Erkrankungen,die in verschiedenen Organen des Körpers durchAnhäufungen von Mutationen im Genom entstehen können.Diese Mutationen verändern die Funktion von Genen,die das normale Verhalten der Zellen in ihremGewebsverband steuern. Eine gemeinsame Eigenschaftder Zellen verschiedener Krebstypen ist, dass sie sich dernatürlichen Wachstumskontrolle durch ihre Umgebungentziehen. Krebszellen teilen sich spontan und unreguliert,entgehen dem natürlichen Zelltodprogramm und derAbwehr durch das Immunsystem und können denZellverband über Blut- und Lymphkreislauf verlassen, umsich an anderer Stelle als Metastasen anzusiedeln. ImForschungsprogramm des MDC soll auf molekularer undzellulärer Ebene verstanden werden, wie Krebs entsteht,um die Grundlage für eine verbesserte Diagnose undTherapie von Krebserkrankungen zu schaffen.Das menschliche Genom enthält etwa 23000 ProteinkodierendeGene, von denen eine Anzahl für die Steuerungdes Verhaltens der Zelle im Gewebeverband verantwortlichist. Viele dieser Gene finden sich regelmäßig in mutierter,also genetisch veränderter Form, in Krebszellen wieder.Diese Klasse von Genen wird begrifflich, entsprechend ihrerWirkungsweise, in „Onkogene“ (potentiell krebsfördernd)und „Tumor-Suppressor-Gene“ (potentiell krebshemmend)eingeteilt. Alle kodieren für Proteine, die eine Funktion inder Steuerung von Prozessen wie Zellproliferation, Zelldifferenzierung,Apoptose, Zellmigration oder Angiogenesehaben. Krebszellen weisen häufig Mutationen mehrerersolcher Gene auf. Viele dieser Proteine sind normalerweisewährend der Embryogenese und ebenso später bei derRegeneration bestimmter Zelltypen aktiv. Deshalb habenTumorzellen auch zahlreiche Eigenschaften mit embryonalenZellen gemeinsam.Die Forschergruppen des Krebs-Forschungsprogramms desMDC arbeiten auf den Gebieten der Signalübertragung,Wachstumskontrolle, Strukturbiologie des Genoms undder Tumor-Immunologie. Experimentell-theoretische undklinische Expertisen werden systematisch eingesetzt, umdie Ursachen und den Entwicklungsverlauf von Krebs zuverstehen und zielgerichtete Therapien zu entwickeln. DieStudien werden in enger Zusammenarbeit mit klinisch orientiertenForschergruppen der Medizinischen Fakultät Berlin(Charité) durchgeführt. Das Ziel des Krebs-Forschungsprogrammsdes MDC besteht darin, Gene zu identifizierenund zu untersuchen, die für die Entstehung von Krebs verantwortlichsind und zu ermitteln, welche Rolle deren Gen-66 Cancer Research

are responsible for the emergence of cancer and todetermine the function of these gene products in cellularprocesses and disease progression. This understandingwill be crucial for the development of newcancer treatments.Signaling pathways, Cell Biology, and CancerThe research in the program “Signaling pathways, CellBiology and Cancer” aims to unravel the molecularbasis underlying tumor formation. A focus is made onthe study of de-regulated and normal function ofoncogenic genes and on the cellular pathways andsignals that regulate their activity. Thus, the researchfield covers the normal function of crucial regulatorysystems during development or cell differentiation aswell as the role in epithelial cancer and metastasis, inleukemia or lymphoma cells.The program is further strengthened by investigationsof important fundamental cell biologicalprocesses, including embryonic stem cell biology, cellcycle regulation, protein degradation pathways andregulation of chromatin by epigenetic mechanisms.Cancer and metastasisThe process of metastasis is the major risk factor incancer progression. Tumors can be classified into differentcategories according to how they infiltrate surroundingtissues. However, these parameters do notcurrently provide a method to predict the likelihoodof tumor relapse. Currently, one of the best signs of anunfavorable disease course is lymph node infiltration.Patients with this diagnosis may be treated by adjuvantchemotherapy or radiation, yet more than 70 %of them suffer from a loss of quality of life rather thanexperiencing any benefits from adjuvant treatment.This situation could be greatly improved by a comprehensiveunderstanding of the molecular mechanismsof tumor progression and metastasis. That would gohand-in-hand with the identification of reliableprospective markers that can be used to predict thelikelihood of metastasis.The group of Peter M. Schlag, in collaboration withWalter Birchmeier, has identified a previously uncharacterizedgene that encodes the protein MACC1produkte bei zellulären Vorgängen und im Krankheitsverlaufspielen. Ein solches Wissen ist eine entscheidende Voraussetzungfür die Entwicklung zukünftiger Behandlungsverfahren.Signalübertragungsketten, Zellbiologie undKrebserkrankungenDie Forschung in diesem Bereich versucht, die molekularenGrundlagen der Tumorentstehung zu entschlüsseln.Ein Schwerpunkt ist die Untersuchung der dereguliertenund normalen Wirkungsweise von Krebs-assoziiertenGenen und der zellulären Signalwege, die deren Aktivitätsteuern. Es wird dabei die normale Funktion der entscheidendenRegulationssysteme während der Embryonalentwicklungoder Zelldifferenzierung als auch deren Rolle inKarzinomen und bei Metastasenbildung, in Leukämienoder Lymphomen untersucht. Das Programm wird durchdie Erforschung wichtiger grundlegender zellbiologischerThemen, wie embryonale Stammzellbiologie, Steuerungdes Zellzyklus, Kontrolle der Proteinstabilität und epigenetischeRegulation der Chromatinstruktur verstärkt.Krebs und MetastasierungDie Bildung von Metastasen ist der dominierende Risikofaktorim Fortschreiten einer Krebserkrankung. Man kannTumore danach klassifizieren, in welcher Weise sie umgebendesgesundes Gewebe infiltrieren. Leider kann mandaraus noch keine zuverlässigen Vorhersagen für dieWahrscheinlichkeit eines Rückfalls nach einer Krebstherapieableiten. Die beste Vorhersage eines ungünstigenKrankheitsverlaufes resultiert gegenwärtig aus demNachweis von Infiltrationen der Lymphknoten durchKrebszellen. Bei diesen Patienten kann Chemotherapieoder Bestrahlungsbehandlung eingesetzt werden, jedochführt dies bei 70% der Behandelten eher zu einer erheblichenVerschlechterung der Lebensqualität als zu wirklichenBehandlungserfolgen. Diese Situation könnte erheblichdurch eine umfassende Kenntnis der molekularenMechanismen der Tumorprogression und Metastasierungverbessert werden. Dies würde es ermöglichen, prognostischzuverlässige Tumormarker zu identifizieren, mitdenen die Tendenz zur Metastasierung nachgewiesenwerden kann.Die Gruppe von Peter M. Schlag hat in Zusammenarbeitmit Walter Birchmeier ein zuvor noch nicht charakterisiertesGen identifiziert, welches das Protein MACC1 (Metastasis-Associatedin Colon Cancer, publiziert in Nature Medi-Cancer Research 67

(metastasis-associated in colon cancer-1; published inNature Medicine) as a key regulator for HGF and Metmediated metastasis of colon cancer. MACC1 promotesproliferation, invasion, and HGF-induced scatteringof colon cancer cells in culture and tumorgrowth and experimental metastasis in mouse models.Currently, MACC1 is clinically evaluated as a promisingpredictive factor for colon cancer. The group ofWalter Birchmeier, in collaboration with Peter M.Schlag, has also identified genes whose expressioncorrelates with the occurrence of metastasis in coloncancer. Using microarray transcript profiling, a signatureof 115 genes was found whose expression can distinguishnon-metastatic and metastatic primary colorectalcarcinomas. The expression of one of the signaturegenes BAMBI, promotes experimental metastasisin mice and correlates with the survival ofpatients. BAMBI is a TGFβ inhibitor whose expressionis controlled by the β-catenin/Bcl9-2 signaling pathway.A high proportion of bone tumors metastasize. Thegroup of Achim Leutz identified genes that are dysregulatedin human osteosarcomas and are involvedin osteosarcomagenesis in experimental animals.E2F3 is essential for cellular division. Strong expressionof this transcription factor leads to the developmentof tumors in humans.Through interaction studiesnew co-activators were identified in the lab ofUlrike Ziebold. In the future these co-activators maybe used for targeted tumor therapies. In addition,genetically modified mouse strains harbouringpRB/E2F3 mutations are used since these mice developmetastatic medullary thyroid cancers. With the aidof these tumors 123 transcripts were identified, manyof which are also upregulated in human metastasis.The clinical relevance of these markers is currentlyunder investigation.The molecular and functional analysis of cell adhesionand signaling is of fundamental importance indevelopment and tumor progression and is a majorfocus of the laboratory of Walter Birchmeier.Previously, the lab has defined functions of E-cadherin/β-cateninat the cell periphery which mediateadhesion between epithelial cells and prevent invasionand metastasis. However, β-catenin is also a cencine)kodiert. MACC1 ist ein Hauptregulator der durch HGFund Met-vermittelten Metastasierung bei Dickdarmkrebs.Es fördert Wachstum, Invasion und HGF-vermittelte Streuungvon kultivierten Darmkrebszellen, als auch Tumorwachstumund experimentelle Metastasierung in Mausmodellen.Zur Zeit wird MACC1 als vielversprechenderTumormarker für Dickdarmkrebs klinisch evaluiert.In Kooperation mit Peter M. Schlag hat die Gruppe vonWalter Birchmeier ebenfalls eine Reihe von Genen gefunden,deren Expressionshöhe mit dem Auftreten von Metastasenbei Dickdarmkrebs korreliert. Durch Ermittlung vonGenexpressionsprofilen anhand von Mikroarrays wurdeeine Gruppe von 115 Genen identifiziert, mit deren Expressionsverhaltennicht-metastasierende von metastasierendenkolorektalen Karzinomen unterschieden werden können.Die Expression eines dieser Gene, BAMBI, erhöht dieexperimentelle Metastase im Mausmodell und korreliertmit einer schlechten Prognose bei Darmkrebspatienten.BAMBI hemmt TGF und seine Expression wird durch den β-Catenin/Bcl9-2 Signalweg gesteuert.Knochentumoren weisen eine hohe Metastasierungstendenzauf. Die Gruppe um Achim Leutz hat Gene identifiziert,die in menschlichen Osteosarkomen dereguliert sindund an der Entstehung von Osteosarkomen in Versuchstierenbeteiligt sind.E2F3 ist für die Teilung von Zellen wichtig. Eine hoheExpression dieses Transkriptionsfaktors in Geweben korreliertmit der Entstehung von Tumoren beim Menschen.Durch Interaktionsanalysen konnten im Labor von UlrikeZiebold neue Koaktivatoren von E2F3 identifiziert werden.Diese Koaktivatoren könnten künftig für gezielte Tumortherapienbenutzt werden. Zudem werden genetischmanipulierte Mäuse mit pRB/E2F3-Mutationen verwendet.In diesen Mäusen entstehen Metastasen des medullärenSchilddrüsenkarzinoms. Mit Hilfe dieser Tumoren konnten123 Transkripte nachgewiesen werden, von denen vieleauch in menschlichen Metastasen hochreguliert werden.Die klinische Bedeutung dieser Marker wird gegenwärtigweiter untersucht.Die molekulare und funktionelle Analyse der Zelladhäsionund der daran beteiligten Signalübertragungsketten istvon grundlegender Bedeutung für die Entstehung undWeiterentwicklung von Tumoren; sie ist ein wesentlicherSchwerpunkt der Forschungsgruppe von Walter Birchmeier.Durch das Labor wurden bestimmte Eigenschaften desE-Cadherin/β-Catenin-Systems an der Zellperipherie identifiziert,durch die eine Adhäsion der Epithelzellen bewirkt68 Cancer Research

tral component of the canonical Wnt signaling pathway.β-Catenin may translocate from the periphery tothe nucleus, bind to the transcription factors LEF/TCFand activate gene expression. In the present researchperiod, cell type and tissue specific conditional geneablation of β-catenin revealed intricate connectionsbetween the Wnt and BMP signaling pathways in thedevelopment of limbs, the central nervous system,the heart, and in tumor development. In addition toWnt signaling, there is a long-standing interest in thebiochemistry of scatter factor/hepatocyte growthfactor (SF/HGF) and Met receptor signaling. Met signalingwas found to regulate wound healing, and itsdownstream signal transducer Gab1 is required tomediate cell-context specific effects of Met.Hematopoiesis, leukemia, and lymphomaStem cells in the bone marrow continuously replenishhuge numbers of exhausted blood cells, includingerythrocytes, granulocytes, macrophages, and lymphoidcells. In experimental hematology, cells of differentblood cell lineages and maturation stages canbe distinguished by cell surface markers and can beisolated by cell sorting for prospective studies.Differentiation of hematopoietic cells from progenitorsmay then be recapitulated in tissue culture.Moreover, murine hematopoietic stem cell transplantationcan be combined with experimental genetics,two powerful tools to explore somatic stem cell biologyand cell differentiation control.Deregulation of hematopoietic stem cell biology andcell differentiation pathways may cause diseases.Transcription factors are at the core of pathways thatdetermine self-renewal versus differentiation andmaintain cell function. The common view is that keytranscription factors navigate hematopoiesis alonghierarchical cell differentiation routes and that mutationsthat disrupt their normal functions are centralto disease development. Unraveling the genetics andepigenetics of somatic stem cell biology and differentiationare prerequisites for the design of novel therapies.MDC research groups have accomplished majorachievements towards understanding basic mechanismsin hematopoietic stem cell and precursor cellbiology in the last years.und so das Eindringen von Zellen und Metastasierung verhindertwird. β-Catenin ist jedoch auch ein zentraler Faktorin der kanonischen Wnt-Signalübertragungskette. DasProtein kann von der Zellperipherie in den Zellkern wandern,dort an die Transkriptionsfaktoren LEF/TCF bindenund damit die Expression von Genen auslösen. ImBerichtszeitraum gelang es, durch zell- bzw. gewebespezifischeAusschaltung des β-Catenin Gens komplexe Wechselbeziehungenzwischen den Wnt- und BMP-Signalübertragungskettenbei der embryonalen Entwicklung derExtremitäten, des zentralen Nervensystems, des Herzensund bei der Tumorentstehung nachzuweisen. Neben derWnt-Signalkette ist die Forschungsgruppe von WalterBirchmeier seit langem an der Biochemie des Scatter Factor/HepatocyteGrowth Factor (SF/HGF)-Systems und derMet-Rezeptor Signalübertragung interessiert. Das letztereSystem wurde als Regulator der Wundheilung identifiziertund es wurde gefunden, dass ein in der Signalkette weiterabwärts gelegener Signalüberträger, Gab1, für die VermittlungKontext-spezifischer Effekte von Met notwendig ist.Hämatopoese, Leukämie und LymphomeStammzellen des Knochenmarks müssen permanentgroße Mengen verbrauchter Blutzellen, wie Erythrozyten,Granulozyten, Makrophagen und lymphatische Zellen,neu bilden. In der experimentellen Hämatologie könnendie verschiedenen Zelltypen und deren Reifungsgradeunterschieden und im Zellsortierer isoliert werden,wodurch prospektive Studien ermöglicht werden. Die Differenzierungder hämatopoetischen Zellen aus deren Vorläuferzellenkann dann in der Gewebskultur nachvollzogenwerden. Weiterhin kann die Transplantation hämatopoetischerStammzellen in der Maus mit experimentellerGenetik kombiniert werden, zwei wirkungsvolle Werkzeuge,um die Biologie der Stammzellen und die Kontrolle derZelldifferenzierung zu erkunden.Eine Störung der hämatopoetischen Stammzellbiologieund der Signalwege, die zellulären Differenzierung steuern,kann Erkrankungen zur Folge haben. Transkriptionsfaktorensind für die Signalwege, die über Selbsterneuerungoder Differenzierung entscheiden und zelluläreFunktionen aufrecht erhalten, von zentraler Bedeutung.Es wird allgemein angenommen, dass bestimmte Transkriptionsfaktorendie Hämatopoese entlang hierarchischerZelldifferenzierungs-Routen navigieren und dassMutationen, die deren normale Aktivität unterbrechen,zur Krankheitsentwicklung entscheidend beitragen. EineEntschlüsselung der Genetik und Epigenetik der somati-Cancer Research 69

In the lab of Achim Leutz, the transcription factorC/EBPβ was found to be regulated at the level oftranslation initiation. Growth signals and nutritionalconditions determine whether long or short C/EBPβisoforms are generated from its messenger RNA.Genetic manipulation of mice now demonstrated thephysiological importance of translational switchingin C/EBPβ expression. Mice that express too much ofthe truncated C/EBPβ isoform develop osteoporosisdue to hyperactive osteoclasts, a cell type thatdegrades bone and that is derived from myeloidblood cells. Mice that can not switch to the truncatedisoform at all and that express only long C/EBPβ aredefective for liver regeneration (published in EMBOJournal and in Genes & Development). Thus, translationalregulation of C/EBPβ isoforms is important inhomeostasis and regeneration and the pathways andmechanisms uncovered in these studies may providenovel pharmacological targets.DNA methylation is a dynamic epigenetic mark thatundergoes extensive changes during differentiationof self-renewing stem cells. Moreover, altered DNAmethylation is a hallmark of cancer, and drugs targetingmethylating enzymes are used in cancer therapy.Frank Rosenbauer’s research group revealed veryrecently a crucial role of DNA methylation in the controlof both normal and cancerous hematopoieticstem cells (published in Nature Genetics). Theirresults identify DNA methylation as an epigeneticmechanism to block premature activation of predominantdifferentiation programs in stem cells as a keyprerequisite to maintain multipotent developmentaloptions and self-renewal. These data suggest thatcompeting stem cell programs require differentmethylation dosage-dependent control mechanisms,and identify CpG methylation as a shared epigeneticprogram in the control of normal and neoplastic stemcells.Numerous physiological and pathological processesare regulated by transcription factors of the NF-κBfamily and by IκB kinases (IKK), which control NF-κBactivity. In normal physiology, transiently activatedNF-κB transcription factors are central regulators ofthe adaptive and innate immune response and ofinflammation. However, pathological conditions canschen Stammzellbiologie und Differenzierung ist eine Voraussetzungfür die Entwicklung neuer Therapien. Forschergruppendes MDC haben in den letzten Jahren bedeutendeBeiträge zum Verständnis grundlegender Mechanismen inder Biologie hämatopoetischer Stamm- und Vorläuferzellengeleistet.Im Laboratorium von Achim Leutz wurde gefunden, dassder Transkriptionsfaktor C/EPBβ auf Ebene der Translationsinitiationreguliert wird. Wachstumssignale und Nährstoffbedingungenbestimmen, ob lange oder kurze C/EBPβIsoformen von der Boten-RNA gebildet werden. Durchgenetische Manipulationen in der Maus konnte jetzt diephysiologische Bedeutung der translationalen Umschaltungin der Expression von C/EBPβ Isoformen nachgewiesenwerden. Mäuse, die zu viel der verkürzten Isoformexprimieren, entwickeln Osteoporose. Der Grund sindhyperaktive Osteoklasten, ein Zelltyp, der Knochen abbautund von myeloiden Blutzellen abstammt. Mäuse, die nichtzu der verkürzten Isoform umschalten können und ausschließlichlanges C/EBPβ bilden, weisen einen Defekt inder Regeneration der Leber auf (publiziert in EMBO J. undGenes & Development). Die translationale Regulation derC/EBPβ Isoformen spielt also für Homeostase und Regenerierungeine wichtige Rolle und die in diesen Studien entdecktenMechanismen könnten zu neuen pharmakologischeZielstrukturen führen.DNA Methylierung ist eine dynamische epigenetische Markierung,die während der Differenzierung und Selbsterneuerungvon Stammzellen ausgeprägten Veränderungenunterliegt. Weiterhin ist veränderte DNA Methylierung einKennzeichen von Krebszellen. Substanzen, die gegenmethylierende Enzyme gerichtet sind, werden in Krebstherapienverwendet. Die Forschungsgruppe von Frank Rosenbauerhat kürzlich eine entscheidende Rolle der DNA-Methylierung bei der Kontrolle sowohl normaler als auchkrebsartiger hämatopoetischer Stammzellen aufgedeckt(publiziert in Nature Genetics). Durch diese Ergebnisse wirdDNA Methylierung als ein epigenetischer Mechanismusbestimmt, der eine verfrühte Aktivierung von Differenzierungsprogrammenin Stammzellen blockiert, um die Voraussetzungfür multipotente Entwicklungsoptionen undSelbsterneuerung aufrecht zu erhalten.Zahlreiche physiologische und pathologische Vorgängewerden durch Transkriptionsfaktoren der NF-κB-Familieund durch IκB Kinasen (IKK), die NF-κB regulieren, kontrolliert.Normalerweise sind kurzzeitig aktivierte NF-κB-Transkriptionsfaktorenzentrale Regulatoren der angeborenenund der adaptiven Immunreaktion. Viele pathologische70 Cancer Research

lead to an aberrant constitutive activation, refractoryto the normal homeostatic control. Previously, thegroup of Claus Scheidereit in collaboration withBernd Dörken has shown that constitutive NF-κBplays a key role in tumor cell survival in Hodgkin’slymphoma, where it promotes cell cycle progressionand blocks apoptosis (programmed cell death). Theactivation of NF-κB is also thought to be one of thepotential causes for tumor cell resistance to chemoand radiation therapy. However, it was not understoodhow NF-kappaB is switched on by DNA damage,which is caused by these treatments in order toablate tumor cells by apoptosis. The laboratory ofClaus Scheidereit has now succeeded in identifyingthe start signal for an NF-κB dependent cell survivalprogram (published in Molecular Cell). PARP-1, whichdetects DNA strand breaks within seconds, formspoly(ADP-ribose), which acts as a scaffold moleculeand rapidly sequesters IKKγ, the kinase ATM as well asother signaling molecules in the nucleus. Subsequentchemical modifications imposed on IKK then triggerNF-κB activation and protection against apoptosis.These findings have implications for clinical studiesusing PARP-1 inhibitors in tumor therapy.In embryonic development, NF-κB is required for theformation of epidermal structures, including hair follicles,eccrine glands or teeth. Using murine models itcould now been shown how NF-κB and Wnt/βcateninsignaling pathways are integrated to controlin a reciprocal manner the formation and growth offollicle placodes in early embryonic development(published in Development Cell). A cross-talk of NF-κBand Wnt/-β-catenin may also be of importance inepithelial tumors, where these factors are activated.In addition to apoptotic cell death, cellular senescencehas been recognized as another cellular safeguardprogram that responds to cellular insults suchas oncogenic activation or DNA damage by lockingthe cell into a terminal arrest at the G1 phase of thecell-cycle. The group of Clemens Schmitt has made alandmark discovery by showing that the anti-cancersenescence program prevents the Ras oncoproteinfrom induction of lymphoma. The mechanism theydiscovered involves an enzyme that was alreadyknown to shut down genes by histone methylation ofBedingungen können jedoch zu einer abweichenden permanentenAktivierung von NF-κB führen, die der normalenhomöostatischen Kontrolle entgeht. Die Gruppe vonClaus Scheidereit, in Zusammenarbeit mit Bernd Dörken,hatte bereits nachgewiesen, dass die konstitutive Aktivierungvon NF-κB eine kritische Rolle bei dem Überleben vonTumorzellen des Hodgkin-Lymphoms spielt, indem es denZellzyklus antreibt und Apoptose (programmierter Zelltod)blockiert. Es wird angenommen, dass die NF-κB Aktivierungeine der möglichen Ursachen der Resistenz vonTumorzellen gegen Chemotherapie oder Bestrahlung seinkönnte. Man hat jedoch nicht verstanden, wie NF-κBdurch DNA Schäden, die durch die Therapie erzeugt werden,um Tumorzellen durch Apoptose zu beseitigen, aktiviertwird. Im Laboratorium von Claus Scheidereit ist esjetzt gelungen, das Startsignal für das NF-κB abhängigeÜberlebensprogramm zu identifizieren (publiziert inMolecular Cell): PARP-1, das DNA Strangbrüche innerhalbvon Sekunden erkennt, bildet Poly(ADP-Ribose), die alsGerüstmolekül wirkt und IKKγ, die Kinase ATM, sowie weitereSignalmoleküle im Zellkern bindet. Nachfolgende inIKK eingefügte chemische Veränderungen lösen dann NFκBAktivierung und damit Schutz gegen Apoptose aus.Diese Ergebnisse sind für klinische Studien wichtig, beidenen PARP-1 Inhibitoren in der Tumortherapie getestetwerden.In der Embryonalentwicklung wird NF-κB für die kompletteBildung epidermaler Strukturen, einschließlich Haarfollikel,exokriner Drüsen oder von Zähnen benötigt. DurchVerwendung von Mausmodellen konnte jetzt gezeigt werden,wie NF-κB und Wnt/β-Catenin Signalketten bei derBildung und dem Wachstum der frühen Anlagen dieserStrukturen miteinander interagieren (publiziert in DevelopmentalCell). Eine Wechselwirkung zwischen den NF-κBund Wnt/β-Catenin Signalketten kann auch in epithelialenTumoren wichtig sein, bei denen beide aktiviert sind.Neben dem apoptotischen Zelltod ist Seneszenz als einweiteres Schutzprogramm erkannt worden, mit dem Zellenauf die Aktivierung von Onkogenen oder Schädigungder DNA antworten können, indem ein irreversiblerWachstumsarrest in der G1 Phase des Zellzyklus ausgelöstwird. Die Gruppe von Clemens Schmitt hat eine bahnbrechendenEntdeckung gemacht, indem sie zeigen konnte,dass das Seneszenz-Programm der Tumorenstehung entgegenwirktund die Induktion von Lymphomen durch dasRas-Onkoprotein verhindern kann. Zu dem zugrundeliegendenMechanismus wurde herausgefunden, dass einEnzym beteiligt ist, das bereits dafür bekannt war, GeneCancer Research 71

chromatin. These results identify senescence as anovel tumor suppressor mechanism whose inactivationpermits the formation of aggressive but apoptosis-competentlymphomas.Basic mechanisms in cell biology: from proteinquality control to gene boundariesThe controlled degradation of proteins is an importantstep in cellular regulation processes and homeostasis.Proteins that are damaged by heat, oxidation,or other events become toxic to cells because of theirtendency to form aggregates. Protein quality control(PQC) pathways decide whether these faulty polypeptidesare channeled into a re-folding pathway orwhether they are removed by the ubiquitin-proteasomepathway. PQC pathways are found in many cellularcompartments and are essential for properstress response, development, and cancer prevention.A major PQC pathway is found in the EndoplasmicReticulum (ER). Dysfunctions in this system lead tosevere diseases and in addition, some viruses highjackthis system to establish themselves in the infectedcell. Thomas Sommer’s lab has unraveled the ERassociatedprotein degradation (ERAD) pathwaywhich is a basic cell biological process that exists inall eukaryotic cells in a highly conserved manner. Inthe last years, the group could identify the crucial signalthat distinguishes a terminally misfolded proteinfrom an unfolded newly synthesized protein. This signalis a specifically processed glycan structureattached to the protein, a Man7-GlcNAc2. This signalis generated by Htm1 and it is decoded by two componentsof the ERAD specific HRD ubiquitin ligase. Thelectin Yos9 binds the oligosaccharide while Hrd3binds directly to unfolded polypeptides. This dualrecognition provides the selectivity in the ERAD pathway.Investigation of signaling by the Wnt, BMP, and TGFβcascades in human embryonic stem cells is the focusof research of Daniel Besser’s lab. It was found thatFGF treated murine fibroblasts produce secreted factorsthat maintain pluripotency in human stem cells.Identification of these factors by expression profileanalysis may help to close the knowledge gapbetween murine and human embryonal stem cells.durch Methylierung von Histonen im Chromatin abzuschalten.Durch diese Ergebnisse wurde Seneszenz als einneuartiger Tumorsuppressions-Mechanismus identifiziert,dessen Inaktivierung die Bildung eines aggressiven, aberzur Apoptose befähigten Lymphoms erlaubt.Grundlegende zellbiologische Funktionen: Vonder Qualitätskontrolle von Proteinen bis zurAbgrenzung von Gen-OrtenDer kontrollierte Abbau von Proteinen ist ein wichtigerSchritt in zellulären Regulationsprozessen und bei derHomeostase. Proteine, die durch Hitze, Oxidation oderandere Einflüsse geschädigt wurden, haben toxische Auswirkungen,da sie die Tendenz haben, intrazelluläre Aggregatezu bilden. Um mit diesen schädigenden Eigenschaftenumzugehen, entscheiden so genannte Protein-Qualitäts-Kontroll (PQC) Systeme, ob die fehlerhaften Proteine repariertwerden können oder ob sie der Proteolyse zugeführtwerden müssen. Um diese Kontrollfunktion ausführen zukönnen, kooperiert das Ubiquitin-Proteasom-System mitder zellulären Protein-Faltungsmaschinerie. Solche PQCSysteme kommen in allen zellulären Kompartimenten vorund sind essentiell für störungsfreie Entwicklungsvorgängeund für die Verhinderung von Krebswachstum. Einer derwichtigsten PQC-Pathways befindet sich im EndoplasmatischenRetikulum (ER). Fehlfunktionen dieses Systems könnendie Ursache schwerer Krankheiten werden. Überdiesbenutzen einige Viren dieses System, um sich in der infiziertenZelle zu etablieren. Das Laboratorium von ThomasSommer hat die Wirkungsweise des ER-assoziierten Protein-Degradationswegs(ERAD) aufgeklärt, ein grundlegenderzellbiologischer Prozess, der in allen eukaryotischen Zellenin evolutionär hoch konservierter Weise vorhanden ist.Im Laufe der letzten Jahre gelang es, das entscheidendeSignal zu identifizieren, das ein entgültig fehlgefaltetesProtein von einem ungefalteten neu synthetisierten Proteinunterscheidet. Dieses Signal ist eine spezifisch prozessierteGlycan Struktur, die an das Protein geheftet wird, einMan7-GlcNAc2. Dieses Signal wird durch Htm1 erzeugt undvon zwei Komponenten der ERAD-spezifischen Ubiquitinligaseerkannt. Das Lectin Yos9 bindet das Oligosaccharid,während Hrd3 direkt an das ungefaltete Polypeptid bindet.Diese duale Erkennung ist für die hohe Spezifität des Prozessesentscheidend.Die Erforschung der Wnt, BMP und TGF-β Signalketten inmenschlichen embryonalen Stammzellen steht im Mittelpunktder Arbeiten von Daniel Bessers Gruppe. Es wurdeherausgefunden, dass FGF-behandelte Mausfibroblasten72 Cancer Research

How Notch- and TGFβ signaling cascades are interconnectedis studied by the group of HaraldSaumweber using the fruit fly as a model organism.Activated Notch signaling targets the CBF1 complexin the nucleus. CBF1 is the core component of a generegulatory complex that may change from a repressorto an activator. TGFβ signaling activates Smad transcriptionfactors. The Bx42/SKIP protein was found tobe involved in both Notch and TGF -dependent generegulation. Circumstantial evidence suggests thatBx42/SKIP recruits chromatin modifiers and altersgene expression in response to Notch and to TGFβ inan epigenetic fashion. In another project, geneboundaries are studied on Drosophila giant chromosomes.Proper boundary formation between chromosomalgenes shield against adjacent genes and heterochromaticgene silencing. Examination of definedloci in different cell types revealed the presence of aprotein complex responsible for targeting of severalhistone modifying enzymes (Jil-1, H3K4-HMT) that arerequired for local chromatin decondensation andboundary formation between chromatin domainsthat differ in their degree of condensation.Structural and Functional GenomicsStructural biologists aim at describing basic aspectsof physiological and pathophysiological processes atthe level of individual molecules and their interactions.At MDC, two groups employ X-ray crystallographyto gain knowledge of the three-dimensionalstructures of proteins, nucleic acids, membranes, carbohydratesand small metabolites that can provideunique insights into cellular processes and may permitthe design of small molecules that specificallymodify protein and cellular function.In the laboratory of Udo Heinemann, protein-nucleicacidinteractions, vesicular transport at the Golgimembrane, and cellular processes related to humandiseases are being characterized at the atomic level.In a series of crystallographic and biochemical studies,the co-operation of transcriptional regulators incontrolling gene expression in the conjugative plasmidRP4 was elucidated, leading to a model accordingto which a flexible N-terminal domain of the repressorprotein KorB binds to a conserved surface ofbestimmte Faktoren sezernieren, die Pluripotenz beimenschlichen Stammzellen erhalten können. Diese Faktorenwerden gegenwärtig durch Analyse von Expressionsprofilenidentifiziert und sollten später helfen, die Wissenslückehinsichtlich der Eigenschaften von murinen undhumanen embryonalen Stammzellen zu schließen.Wie die Notch- und TGFβ-Signalketten miteinander vernetztsind, wird im Labor von Harald Saumweber in derFruchtfliege als Modellorganismus untersucht. Die aktivierteNotch Signalübertragung ist auf den CBF1-Komplexim Zellkern ausgerichtet. Dieses CBF1 ist die Kernkomponenteeines regulatorischen Komplexes, der von einer Gen-Repressorfunktion auf eine Aktivatorfunktion umschaltenkann. Durch TGFβ vermittelte Signalübertragungen werdenSmad Transkriptionsfaktoren aktiviert. Es wurde herausgefunden,dass das Bx42/SKIP Protein dabei sowohl anNotch- als auch TGFβ-vermittelter Genexpression beteiligtist. Es liegen Hinweise vor, dass Bx42/SKIP Chromatin-Modifikatoren rekrutiert und dadurch die durch Notchund TGFβ ausgelöste Genexpression auf der epigenetischenEbene verändert. In einem weiteren Projekt werdendie Grenzen zwischen den in Riesenchromosomen in Drosophilavorhandenen Genen untersucht. Eine genaueGrenzziehung zwischen chromosomalen Genen schirmtgegen benachbarte Gene und gegen heterochromatinbedingteStilllegung von Genen, „gene silencing“, ab. Anden untersuchten genetischen Orten wurde in allen bisheruntersuchten Zelltypen die Anreicherung eines Proteinkomplexesgefunden, der verschiedene Histon-modifizierendeAktivitäten hinzuziehen kann (Jil-1, H3K4-HMT);diese sind für eine lokale Öffnung des Chromatins sowiefür eine Abgrenzung benachbarter Bereiche unterschiedlichkondensierten Chromatins voneinander notwendig.Strukturgenomik und Funktionelle GenomikStrukturbiologen sind bestrebt, grundlegende Aspektephysiologischer und pathophysiologischer Prozesse aufder Ebene einzelner Moleküle und molekularer Wechselwirkungenzu beschreiben. Zwei Forschungsgruppen desMax-Delbrück-Centrums nutzen die Methode der Röntgenkristallographie,um die dreidimensionalen Strukturenvon Proteinen, Nukleinsäuren, Membranen, Kohlenhydratenund Metaboliten zu untersuchen. Diese Strukturenkönnen sehr detaillierte Einsichten in zelluläre Prozessevermitteln und bei der Identifizierung kleiner Molekülehelfen, welche die Funktion von Proteinen oder Zellenmodulieren.Cancer Research 73

another transcription factor, KorA, at a fixed distance,but with variable geometry. The crystal structure ofthe Golgi marker protein p115 revealed a dimericarmadillo-repeat structure of its globular headregion. This structure could be extended into a modelthat contains the extended coiled-coil region of p115and highlights the interactions of this golgin withRab GTPases and other factors involved in vesiculartransport at the Golgi.Guanine nucleotide binding proteins (G-proteins) areinvolved in various cellular processes including signaltransduction, protein synthesis, sensual perception,and vesicular transport. Large G-proteins of thedynamin superfamily are mechano-chemicalenzymes that use the energy of GTP hydrolysis toactively remodel membranes. The group of OliverDaumke aims to elucidate the interaction and reciprocalmodulation of membranes and G-proteinsusing structural, biochemical and cell-biologicalmethods. Their work is focussed on the membraneremodelling EHD family of G-proteins, the GIMAPGTPases which are proposed to regulate apoptosis incells of the immune system, and the interferoninducedMx (myxovirus-resistance) proteins, keyeffector molecules in the innate immune systemmediating cellular resistance against pathogensincluding influenza virus.Tumor ImmunologyHomeostatic chemokines and their receptors areresponsible for lymphocyte trafficking between andwithin secondary lymphoid tissues and thus requiredfor immune surveillance and the establishment ofself-tolerance. The group of Martin Lipp is particularlyinterested in the role of homeostatic chemokinereceptors in the development and progression ofchronic inflammatory diseases associated with lymphoidneogenesis, i. e. the formation of lymphoid tissue-likestructures at sites of inflammation. Thegroup demonstrated that the homeostaticchemokine receptors CXCR5 and CCR7 are critical signalingmolecules in lymphoid neo-genesis duringchronic inflammation. To this end, they have establisheda novel combined mouse model of collagenandantigen-induced arthritis showing specific fea-Im Labor von Udo Heinemann werden Protein-Nukleinsäure-Wechselwirkungen,vesikulärer Transport an der Golgi-Membran und verschiedene krankheitsbezogene zelluläreProzesse bei atomarer Auflösung untersucht. In einer Reihekristallographischer und biochemischer Studien wurde dieKooperation von Regulatorproteinen der Transkription deskonjugativen Plasmids RP4 untersucht. Diese Arbeitenführten zu einem Modell, nach dem die flexible N-terminaleDomäne des Repressorproteins KorB an eine konservierteOberfläche eines zweiten Transkriptionsfaktors, KorA,über einen festgelegte Abstand, aber mit variabler Geometriebindet. Die Kristallstruktur des Golgi-Markerproteinsp115 zeigte eine dimere aus Armadillo-Motiven aufgebauteStruktur seiner globulären Kopfdomäne. Diese Strukturkonnte zu einem Modell komplettiert werden, das die langgestrecktesuperhelikale Region des p115 enthält und molekulareWechselwirkungen dieses Golgins mit Rab-GTPasenund anderen Faktoren des vesikulären Transports am Golgiaufzeigt.Guaninnukleotid-bindende Proteine (G-Proteine) sind anverschiedenen zellulären Prozessen einschließlich der intrazellulärenSignalübertragung, Proteinsynthese, Sinneswahrnehmungund dem vesikulären Transport beteiligt.Große G-Proteine der Dynamin-Superfamilie sind mechano-chemischeEnzyme, die die Energie der GTP-Hydrolysenutzen, um Membranen aktiv zu remodellieren. Die Gruppevon Oliver Daumke studiert die Wechselwirkung undwechselseitige Modulierung von Membranen und G-Proteinenmit strukturellen, biochemischen und zellbiologischenMethoden. Ihre Arbeiten sind auf die EHD-Familievon Membran-remodellierenden G-Proteinen, die GIMAP-GTPasen, denen eine Funktion bei der Regulierung derApoptose in Zellen des Immunsystems zugeschrieben wird,sowie auf die Interferon-induzierten Mx- (Myxovirus-resistenten)Proteine, die als wichtige Effektoren des angeborenenImmunsystems die zelluläre Abwehr von Pathogenenwie dem Grippevirus vermitteln, fokussiert.TumorimmunologieDie Migration von Lymphozyten zwischen und innerhalbvon lymphoiden Geweben hängt von homöostatischenChemokinen und deren Rezeptoren ab, die deshalb für dieImmunüberwachung und die Ausbildung von Autoimmuntoleranznotwendig sind. Die Gruppe von Martin Lippist in besonderem Maße an der Rolle der homöostatischenChemokine bei der Entstehung und Progression chronischerEntzündungen interessiert, die mit lymphoider Neogeneseeinhergehen, d.h. mit der Bildung lymphknoten-74 Cancer Research

tures of the chronic phase in human disease. In addition,the model is characterized by formation of antibodiesdirected against collagen type II and citrullinatedpeptides (CCP). CCP-specific antibodies have ahigh prognostic value in humans and have so far notbeen described in mouse models of arthritis. Anotherrecent accomplishment of the group was to showthat CCR7 is also a key regulator of homeostatic lymphocyterecirculation through body cavities and nonlymphoidperipheral tissues.The adoptive transfer of regulatory Foxp3-positive Tcells (Treg) has been shown in various animal modelsto prevent inflammatory immune and autoimmunediseases. Translation into therapeutic applications,however, is hindered by the lack of suitable techniquesand markers. CD25, commonly used to isolateTreg cells from mice, has only limited value in humansas it is also present on proinflammatory CD4-positiveeffector cells. The group of Olaf Rötzschke and KirstenFalk has shown that pure populations of human Tregcells can be obtained with antibodies directedagainst the integrin alpha subunit CD49d. The markeris present on proinflammatory peripheral bloodmononuclear cells but is absent on immune-suppressiveTreg cells. Importantly, in combination with themarker alpha-CD127 it allows the isolation of“untouched” Treg cells, that have not been targetedby an antibody during purification, and that are virtuallyfree of contaminating CD25-positive effectorcells. The cells can be expanded in vitro and are effectivesuppressors both in vitro and in vivo. Thus, CD49dprovides access to highly pure populations ofuntouched Treg cells conferring maximal safety forfuture clinical applications.The immune system usually fails to reject establishedtumors. In order to develop effective cancer therapies,it is therefore essential to understand the molecularinteraction of tumor cells, immune cells, and thetumor microenvironment. The group of ThomasBlankenstein has shown in a model of sporadicimmunogenic cancer, that tumor-specific toleranceclosely coincides with the first tumor antigen recognitionby B cells. During the subsequent latency perioduntil tumors progress, the mice acquire generalcytotoxic T lymphocyte (CTL) unresponsiveness. Inähnlicher Gewebsstrukturen in der Umgebung von Entzündungsherden.Die Gruppe konnte nachweisen, dass dieChemokin-Rezeptoren CXCR5 und CCR7 für die Signalübertragungbei der lymphoiden Neogenese im Verlaufechronischer Entzündungen kritische Moleküle darstellen.Für diese Studien wurde ein neuartiges kombiniertesMausmodell Kollagen-und Antigen-induzierter Arthritisetabliert, das spezifische Merkmale der chronischen Phaseder Erkrankung beim Menschen aufweist. Dieses Modellist weiterhin dadurch ausgezeichnet, dass Antikörpergegen Kollagen Typ II und citrullinierte Peptide (CCP)gebildet werden. CCP-spezifische Antikörper haben einehohe prognostische Bedeutung beim Menschen und sindvorher noch nicht für Arthritismodelle der Maus beschriebenworden. Eine weiterer kürzlicher Erfolg der Arbeitsgruppewar der Nachweis, dass CCR7 auch einen Schlüsselregulatorder homöostatischen Lymphozyten-Rezirkulationdurch Körperhöhlen und nicht-lymphoide periphereGewebe darstellt.Es ist in verschiedenen Tiermodellen gezeigt worden, dassder adoptive Transfer von regulatorischen FoxP3-positivenT Zellen (Treg) entzündliche Immun- und Autoimmunerkrankungenverhindern kann. Die Umsetzung in therapeutischeAnwendungen ist jedoch wegen fehlender Technikenund Marker erschwert worden. CD25, welches häufigverwendet wird, um Treg aus Mäusen zu isolieren, hatbeim Menschen einen nur geringen Nutzen, da es dortebenfalls auf pro-entzündlichen CD4-positiven Effektor TZellen vorhanden ist. Die Arbeitsgruppe von OlafRötzschke und Kirsten Falk hat herausgefunden, dassreine Populationen humaner Treg mit Antikörpern erhaltenwerden können, die gegen die Integrin α UntereinheitCD49d gerichtet sind. Dieser Marker ist auf pro-entzündlichenperipheren mononukleären Blutzellen vorhanden,aber nicht auf immun-suppressiven Trg Zellen. In Kombinationmit dem Marker α-CD127 können damit „unberührte“Treg Zellen isoliert werden, die während der Reinigungnoch nicht mit einem Antikörper in Berührunggekommen sind und die vollkommen frei von kontaminierendenCD25-positiven Zellen sind. Die Zellen können invitro vermehrt werden und sind wirkungsvolle Suppressorensowohl in vitro als auch in vivo. Damit liefert CD49dZugang zu hochgradig reinen Populationen unberührterTreg Zellen, womit maximale Sicherheit für künftige klinischeAnwendungen gewährleistet wird.Eine Abstoßung etablierter Tumoren wird durch dasImmunsystem meist nicht zustande gebracht. Für die Entwicklungwirksamer Behandlungsverfahren gegen KrebsCancer Research 75

mice with large non-immunogenic tumors, unrelatedCTL responses are undiminished. These results suggestthat (a) tolerance to the tumor antigen occurs atthe premalignant stage, (b) tumor latency is unlikelycaused by CTL control, and (c) a persistent immunogenictumor antigen causes general CTL unresponsivenessbut tumor burden per se does not.Cytotoxic T cells destroy virus-infected and tumorigeniccells through release (exocytosis) of cytotoxicsubstances from lytic granules. In recent years scientistshave identified molecules within CTLs that helpsecrete the toxins. But little is known about the contraryprocess, which keeps levels of the substanceslow and prevents their release at the wrong time. Thelaboratories of Armin Rehm, Uta Höpken, and BerndDörken at the MDC and Charité University Hospital,along with several collaborating groups, haveexplored a new mechanism that helps cells controlthe toxins. By generating and analyzing Ebag9 (estrogenreceptor-binding fragment-associated antigen9)-deficient mice they showed that loss of EBAG9confers CTLs with enhanced cytolytic capacity in vitroand in vivo. Although loss of EBAG9 did not affectlymphocyte development, it led to an increase in CTLsecretion of granzyme A, a marker of lytic granules.This resulted in increased cytotoxicity in vitro and anenhanced cytolytic primary and memory T cellresponse in vivo. While EBAG9 deficiency did not disruptthe formation of the immunological synapse,lytic granules in Ebag9-deficient CTLs were smallerthan in wild-type CTLs. These results identify a potentiallink between the hormone estrogen and the waythe immune system responds to cancer and suggestthat EBAG9, which seems to be abnormally expressedin some human tumors, is a tunable inhibitor of CTLmediatedadaptive immune response functions.The groups of Thomas Blankenstein, WolfgangUckert and Antonio Pezzutto have a strong interest incancer immunotherapy based on adoptive T celltransfer. In a collaborative effort they have developednovel methods to generate T cells expressing arecombinant T cell receptor (TCR) with defined antigenspecificity. TCR gene therapy bears the risk ofauto-reactive side-effects when the TCR recognizesantigens on self-tissue. The groups have now develistes daher von Bedeutung, die molekularen Interaktionenvon Tumorzellen mit Immunzellen und mit ihrer Mikroumgebungzu verstehen. Die Gruppe von Thomas Blankensteinhat mit einem sporadischen immunogenen Krebsmodellzeigen können, dass eine Tumor-spezifische Toleranzdicht mit der ersten Tumorantigen-Erkennung durchB Zellen zusammenfällt. Während der nachfolgendenLatenzperiode bis sich die Tumoren weiterentwickeln,erwerben die Mäuse eine allgemeine Unansprechbarkeitder zytotoxischen T Lymphozyten (CTL). In Mäusen mitgroßen, nicht-immunogenen Tumoren sind hierauf nichtbezogene CTL Antworten unbeeinträchtigt. Diese Ergebnisselegen nahe, dass (a) eine Toleranz gegen das Tumorantigenim prämalignen Stadium auftritt, (b) die Tumorlatenzwohl kaum durch CTL verursacht wird und (c), dassein fortbestehendes immunogenes Tumorantigen eine allgemeineCTL Unansprechbarkeit verursacht, während diesdurch die Tumorlast allein nicht erreicht wird.Zytotoxische T Zellen zerstören Virus-infizierte und tumorigeneZellen durch Freisetzung (Exozytose) von zytotoxischenSubstanzen aus lytischen Körnern (lytic granules). Inden vergangenen Jahren haben Wissenschaftler Moleküleinnerhalb der CTL identifiziert, die bei der Ausschüttungvon Toxinen behilflich sind. Es war aber wenig über dengegenläufigen Prozess bekannt, der die Menge dieser Substanzenniedrig hält und verhindert, dass deren Freisetzungzum falschen Zeitpunkt stattfindet. Die Laboratorienvon Armin Rehm, Uta Höpken und Bernd Dörken am MDCund an der Charité Universitätsklinik haben, zusammenmit mehreren kollaborierenden Arbeitsgruppen, einenneuen Mechanismus ergründet, mit dem Zellen Toxineunter Kontrolle halten. Durch die Generierung and AnalyseEbag9 (estrogen receptor-binding fragment associatedantigen 9)-defizienter Mäuse konnten sie zeigen, dass CTLdurch den EBAG9 Verlust eine verstärkte zytolytische Aktivität,sowohl in vitro als auch in vivo, erworben haben.Obgleich der EBAG9 Verlust keinen Einfluss auf die Entwicklungder Lymphozyten hatte, führte er zu einer Verstärkungder Auschüttung von Granzym A, einem Markerder lytischen Körner. Dies führte in vitro zu einer erhöhtenZytotoxizität und in vivo zu einer verstärkten Antwort derzytolytischen primären und Gedächtnis (memory) T Zellen.Während der EBAG9 Verlust nicht zu einem Zerfall derimmunologischen Synapse führte, waren die lytischen Körnerin EBAG9-defizienten CTL kleiner als in normalen CTL.Durch diese Ergebnisse konnte eine mögliche Verbindungzwischen dem Hormon Östrogen und der Weise, wie dasImmunsystem auf Krebs reagiert, hergestellt werden und76 Cancer Research

oped a new safeguard that is based on a TCR-intrinsicdepletion mechanism to eliminate auto-reactive TCRredirectedT cells. By modifying TCRs with a 10 aminoacid tag of the c-myc protein, which does not interferewith TCR-mediated effector function, T cells harbouringtag-modified TCRs can be efficiently depletedin vivo with a tag-specific antibody. More important,in vivo depletion of adoptively transferred T cells rescuedmice from lethal autoimmune diabetes. Thisnew safeguard will allow the termination of adoptivetherapy in case of severe side-effects.es wird nahegelegt, dass EBAG9, welches in einigen Tumorenin abweichender Weise exprimiert wird, ein veränderbarerHemmstoff CTL-vermittelter adaptiver Immunfunktionenist.Die Arbeitsgruppen von Thomas Blankenstein, WolfgangUckert und Antonio Pezzutto haben ein starkes Interessean Krebs-Immunotherapie, die auf adoptiven Transfer vonT Zellen basiert ist. In einer kollaborativen Initiative wurdenneue Methoden entwickelt, um T Zellen zu generieren,die einen rekombinanten T Zellrezeptor (TCR) mit definierterAntigen-Spezifität exprimieren. TCR-Gentherapie trägtdas Risiko von auto-reaktiven Nebeneffekten, wenn derTCR Antigene auf Eigengewebe erkennt. Die Arbeitsgruppenhaben jetzt eine neues Sicherungssystem entwickelt,das auf einem TCR-gerichteten Depletierungsmechanismusberuht, um auto-reaktive TCR-modifizierte T Zellen zueliminieren. Durch Anheftung einer 10 Aminosäuren langenSequenz des c-Myc Proteins an den TCR, wodurch dieTCR-Funktion nicht gestört wird, können T Zellen, die dieseSequenz tragen, in vivo effizient mit einem spezifischenAntikörper depletiert werden. Wichtig ist, dass beobachtetwerden konnte, dass eine in vivo Depletierung übertragenerT Zellen Mäuse vor tödlicher Autoimmun-Diabetesretten konnte. Dieses neue Sicherungssystem wird eserlauben, eine adoptive Therapie im Falle schwerer Nebeneffekteabzubrechen.Cancer Research 77

Signaling Pathways, Cell Biology, and CancerStructure of the GroupClaus ScheidereitGroup LeaderProf. Dr. Claus ScheidereitScientistsDr. Annette AhlersDr. Michael HinzDr. Eva KärgelDr. Giulietta RoelDr. Ruth Schmidt-UllrichDr. Buket YilmazSignal Transduction in Tumor CellsThe major interest of our laboratory is the regulation of gene expression by cellular signaltransduction processes. ”Nuclear factor kappaB” (NF-κB) denotes a family of transcriptionfactors whose activities are controlled by inhibitory IκB proteins and IκB kinases (IKK). NF-κB/IKK signaling cascades have wide physiological and medical relevance. Major efforts are todecipher the mechanisms and structures that underlie gene regulation by IKK and NF-κB, thecrosstalk with other gene regulatory systems and to dissect both, the role in development andin the pathogenesis of diseases.Molecules and mechanisms that control IKK andNF-κB activityMost if not all cell types of the body utilize NF-κB transcriptionfactors to regulate under various conditionsthe expression of numerous genes. These encodecytokines, surface receptors, adhesion molecules, transcriptionfactors and other functional classes of proteins.Biological processes which involve NF-κB activationinclude the innate and adaptive immune responses,inflammation, cellular reaction to environmentalstress as well as selective aspects of early embryonicdevelopment. In non-stimulated cells, NF-κB is associatedwith IκB molecules, which inhibit nuclear translocationand DNA binding activity of NF-κB. Cellular exposureto a variety of agents or conditions, includingmicrobial pathogens, cytokines, mitogens, genotoxicstress or morphogens triggers the activation of an IκBkinase (IKK) complex, which consists of catalytic (IKKα,IKKβ) and regulatory (IKKγ/NEMO) components. Thiscomplex phosphorylates IκB molecules, resulting intheir ubiquitination by βTrCP/SCF ubiquitin ligases andproteasomal destruction, followed by liberation ofactive NF-κB.In mammals, the NF-κB family comprises five relatedmembers, p50, p52, p65, c-Rel and RelB. These proteinsform distinct hetero- and homodimers and bind toinhibitory cytoplasmic IκB molecules, IκB α,β or ε, or tothe nuclear IκB homologues Bcl-3 and MAIL. It is a characteristicfeature of NF-κB that two of its subunits, p50and p52, are formed by proteolytic proteasomal processingof their precursor proteins, p105 and p100,respectively. Unprocessed p105 and p100 bind to otherNF-κB subunits and so act as cytoplasmic inhibitors.There are two distinct IKK pathways, which respond toextracellular stimuli and either trigger degradation ofIκBs and liberation of prototypic p50-p65 (canonicalpathway) or proteolytic processing of p100 and productionof p52-RelB complexes (non-canonical pathway)(Figure 1). The two pathways depend on IKKβ andIKKγ/NEMO or on IKKα, respectively. For the canonicalpathway, a critical role for non-degradative, K63-linkedpoly-ubiquitination has been recognized. These ubiquitinmodifications are generated by ubiquitin ligases,such as TRAF2 or TRAF6, and enforce a co-recruitment ofthe IKK complex and other components, which haveubiquitin binding motifs, to receptor-proximal complexes.This results in T-loop phosphorylation and activationof IKKs. A third IKK pathway is activated by DNAdamage-inducing agents or conditions, such as γ-irradiationor chemotherapeutic drugs in the nucleus and78 Cancer Research

Graduate StudentsSeda Cöl ArslanCan Demiroglu*Jan EbertAndrea Oeckinghaus*Kivia A. Pontes de Oliveira*Michael StilmannPhilip Tomann*Technical AssistantsRudolf Dettmer*Sabine JungmannInge KrahnSarah UgowskiSecretariatDaniela Keyner*part of the period reportedFigure 1. Distinct NF-κB dimers are activated by canonical and non-canonical IKK/NF-κB signaling pathways and depend on IKKβ and IKKα,respectively. Both pathways are constitutively activated in Hodgkin lymphoma cells. A nucleus-to-cytoplasm IKK/NF-κB pathway is induced by theDNA damage response and may play a role in therapy resistance of tumor cells. Homeostasis and activity of the IKK complex is regulated by theHsp90-Cdc37 chaperone complex.depends on nuclear shuttling and SUMO-modificationof IKKγ, as well as on the kinase ATM.IKK and NF-κB signal transduction in lymphoidtumor cellsA dysregulation of the IKK/NF-κB system has now beenrecognized as an oncogenic hallmark of an increasingnumber of lymphoma and leukemia entities. We haveinvestigated the origins and the biological functions ofconstitutively activated IKKs and NF-κB and the downstreameffector networks in Hodgkin lymphoma (HL)tumor cells. The IKK/NF-κB system is aberrantly activatedin a cell-autonomous manner, generally involving apersistent activation of the IKK complex, which resultsin constitutive release of NF-κB complexes. In HL cells,both, canonical and non-canonical p100 pathways areactivated (Figure 1). The determination of the global NFκBtarget gene signature in HL cells by microarraysCancer Research 79

Figure 2. Mutual requirements for Eda-A1/Edar/NF-κB and Wnt/β-catenin in early hair follicle development. (A) Wnt activity is maintained in theabsence of Eda-A1/Edar/NF-κB activity. Wnt reporter mice (cond-lacZ) were mated with mice deficient in Eda-A1 (cond-lacZ;Ta/Ta) or Edar expression(cond-lacZ;dl/dl), or with mice lacking NF-κB activity (cond-lacZ;∆N). X-Gal stained E14.5 embryos are depicted. Arrows point to Wnt activity inplacodes and other ectodermal appendages, such as whiskers and eyelids. Note that the placode pattern in embryos lacking epidermal Eda-A1/Edar/NF-κB activity is irregular when compared to controls. Thus, placode pattern refinement requires NF-κB activity. We and others haveshown that Wnt inhibitor Dkk4 is an NF-κB target gene. Dkk4 is known to be involved in placode patterning and spacing. (B) X-Gal stained condlacZ;dl/dland cond-lacZ;∆N embryos at E15.5, when hair placodes have already started to grow. Focal Wnt activity in hair placodes has disappearedin the absence of Eda-A1/Edar/NF-κB activity. We have found that Wnt10b is a direct target gene of Eda-A1/Edar/NF-κB, providing a possible explanationfor this observation. Panel (C) illustrates a model for the molecular interplay of Wnt/β-catenin and Edar/NF-κB signaling pathways. Notethat Wnt10a and Lef-1 have previously been described as potential NF-κB target genes.revealed that constitutive NF-κB drives the expressionof genes with important functions in cell cycle progression,programmed cell death and tropism or migrationof tumor cells. Thus, a central pathogenic role of theIKK/NF-κB pathways is very likely and the distinct contributionsof canonical and non-canonical pathways forthe biology of the tumor cells are now under investigation.In addition to IKK/NF-κB, Hodgkin lymphoma cellsreveal aberrant activation of transcription factor AP-1(activating protein 1), composed of the c-Jun and JunBsubunits. AP-1 cooperates with NF-κB to superactivate asubset of NF-κB target genes in HL. Furthermore, Stat5ais activated by NF-κB in HL cells and may synergize withNF-κB at the level of common target genes.By a genome-wide determination of genes regulated byIKK and NF-κB in activated B cell lineage cells, we couldshow that induced AP-1 activity is entirely dependenton IKK and NF-κB, which regulate expression of Jun, ATFand Maf members. This cross-talk is under further80 Cancer Research

investigation, as is the cause of constitutive IKK/NF-κBand c-Jun activation in Hodgkin lymphoma.Functional control of IKK by transient interactionwith Hsp90 and Cdc37The IKK complex undergoes interactions with a numberof regulatory proteins, including the chaperones Cdc37and Hsp90. We had previously shown that pharmacologicalinhibition of Hsp90 abrogates constitutive IKKactivation in Hodgkin lymphoma cells, resulting instrongly enhanced apoptosis. Using an RNAi approach,we found that Cdc37 recruits Hsp90 to the IKK complexin a transitory manner, preferentially via IKKα. Bindingis conferred by N-terminal as well as C-terminalresidues of Cdc37 and results in the phosphorylation ofCdc37. Cdc37 is essential for the maturation of de novosynthesized IKKs into enzymatically competent kinases,but not for assembly of an IKK holocomplex. MatureIKKs, T-loop phosphorylated after stimulation either byreceptor-mediated signaling or upon DNA damage, furtherrequire Hsp90-Cdc37 to generate an enzymaticallyactivated state. Thus, the Hsp90-Cdc37 chaperone-cochaperonecomplex is an essential regulatory componentin IKK signaling cascades and a potential drug targetin tumor therapy.Mutual requirements of Eda-A1/Edar/NF-κB andWnt/β-catenin signaling in early ectodermalorganogenesisTo allow a functional analysis of NF-κB in early embryonicdevelopment and in disease models, we have generatedNF-κB repressor and reporter mice. The repressormice carry a dominant negative IκBα mutant (IκBα∆N)as a conditional knock-in allele, while the reporter miceexpress an NF-κB-driven β-gal transgene. Using thesemice, we could previously demonstrate novel morphogenicfunctions for NF-κB, including an early role inthe development of epidermal organs, such as hair follicles(HF). In early HF placodes, NF-κB is specifically activatedby TNF family members Eda-A1 and its receptorEdar. Furthermore we have identified an intense molecularcross-talk of Eda-A1/Edar/NF-κB with other signalingpathways required for HF development, such as Wntand Sonic Hedgehog (SHH).According to our current knowledge, Wnt/β-cateninsignaling specifies the initial hair fate decision of epidermalkeratinocytes, while NF-κB is required for hairplacode growth by activating SHH signaling andrepressing Bone Morphogenic Protein (BMP) activity. Incollaboration with the laboratory of Dr. Sarah E. Millar(U. Pennsylvania, Philadeplhia, USA) we have nowdemonstrated that Wnt/β-catenin is initially activatedindependently of Eda-A1/Edar/NF-κB, but depends onNF-κB activity for focal hair placode patterning. In contrast,initial Eda-A1/Edar/NF-κB signaling is not activatedin skin of mice expressing the secreted Wnt inhibitorDKK1, or lacking epithelial β-catenin (Figure 2). In thiscontext we have shown that Wnt/β-catenin isabsolutely essential for NF-κB activation and have providedevidence that Edar is a direct Wnt/β-catenin targetgene. However, at later time points of hair follicledevelopment, localized Wnt activity disappears fromEda-A1/Edar/NF-κB mutant skin, implying that Eda-A1/Edar/NF-κB is required for the maintenance of Wntsignaling (Figure 2).We found that NF-κB is essential forplacodal up-regulation of Wnt10a and Lef-1, and wecould identify Wnt10b as a direct target gene of NF-κB.Our data reveal a complex interplay and interdependenceof Wnt/β-catenin and Eda-A1/Edar/NF-κB signalingpathways, which may not only have importantimplications in organ development and morphogenesis,but also in cancer and inflammatory diseases.Our current and future studies will continue to focus onthe Wnt/NF-κB cross-talk in development/morpho -genesis and disease. Furthermore, in collaboration withDr. Nathaniel Heintz (Rockefeller I., New York, NY, USA)and Ines Ibañez-Tallon (MDC) we have developed amouse model (bacTRAP: translating ribosome affinitypurification (TRAP)) that allows the specific isolation ofHF keratinocytes. This mouse model will be used toidentify novel NF-κB target genes in early HF developmentand to perform detailed gene profiling studies onthe molecular controls of HF induction.Selected publicationsSchmidt-Ullrich, R, Tobin, DJ, Lenhard, D, Schneider, P, Paus, R, Scheidereit,C. (2006). NF-κB transmits Eda A1/EdaR signaling to activate Shh andcyclin D1 expression, and controls post-initiation hair placode downgrowth.Development 133, 1045-1057.Oeckinghaus, A, Wegener, E, Welteke, V, Ferch, U, Çöl Arslan, S, Ruland, J,Scheidereit, C, Krappmann, D. (2007). TRAF6 mediated Malt1 ubiquitinationtriggers IKK/NF-κB signaling upon T cell activation. EMBO J. 26,4634-4645.Hinz, M, Broemer, M, Çöl Arslan, S, Dettmer, R, Scheidereit, C. (2007).Signal-responsiveness of IκB kinases is determined by Cdc37-assistedtransient interaction with Hsp90. J. Biol. Chem. 282, 32311-32319.Zhang, Y, Tomann, P, Andl, T, Gallant, N, Huelsken, J, Jerchow, B, Birchmeier,W, Paus, R, Piccolo, S, Mikkola, ML, Morrisey, EE, Overbeek, PA, Scheidereit,C, Millar, SE, Schmidt-Ullrich, R. (2009). Reciprocal requirements forEda/Edar/NF-κB and Wnt/β-catenin signaling pathways in hair follicleinduction. Dev. Cell, 17, 49-61.Stilmann, M, Hinz, M, Cöl Arslan, S, Zimmer, A, Schreiber, V, Scheidereit, C,(2009). A nuclear poly(ADP-ribose) dependent signalosome confers DNAdamage induced IκB kinase activation. Mol Cell, in press.Cancer Research 81

Walter BirchmeierStructure of the GroupGroup LeaderProf. Dr. Walter BirchmeierScientistsDr. Felix Brembeck*Dr. Marta De Rocha RosárioDr. Johannes FritzmannDr. Katja GroßmannDr. Klaus Hellmuth*Dr. Jane HollandDr. Alexandra KlausSignals Provided by Wnt/β-cateninand Met/Gab1/Shp2 in Developmentand CancerThe molecular and functional analysis of cell adhesion and signaling in development andtumor progression has been the major focus of my laboratory. We previously definedfunctions of E-cadherin/β-catenin, which mediate cell-cell adhesion of epithelial cells andprevent invasion and metastasis. Moreover, we discovered that β-catenin, which is a centralcomponent of canonical Wnt signaling (armadillo in Drosophila), binds to the transcriptionfactors LEF/TCF. This interaction allows the nuclear translocation of β-catenin, and theregulation of gene expression. Signals provided by Wnt/β-catenin are essential in manydevelopmental processes and in tumor progression. We have used conditional loss-of-andgain-of-functionmutations to study the role of β-catenin in the skin, and in development oflimbs and brain, and observed essential functions of β-catenin signaling in these organs.In the skin, specification of stem cells to the hair, but not the epidermal lineage requiresβ-catenin signals. In the dorsal spinal cord, Wnt/β-catenin signaling controls the transcriptionfactor Olig3, which is required for the specification of two types of neurons, dI2 and dI2.Moreover, our genetic analysis of two other members of the armadillo gene family,plakoglobin (γ-catenin) and plakophilin2, established a role of theses molecules in the stabilityof cell junctions in the heart, and implicate these molecules in heart disease. We recentlydiscovered that the β-catenin-binding protein BCL9-2, a homologue of legless in Drosophila,induces epithelial-mesenchymal transition and activation of canonical Wnt signaling.In addition, we have a long standing interest in the biochemistryof scatter factor/hepatocyte growth factor(SF/HGF) and Met receptor signaling, and have analyzedfunctions of these molecules in development andtumor progression. We characterized the downstreameffectors of Met, Gab1 and Shp2. We were able to show,by genetic means, that signaling of Gab1 through thetyrosine phosphatase Shp2 controls migration of muscleprecursor cells to the limbs. Shp2 acts also downstreamof many other receptor and non-receptorkinases.In the present report period, we have performed the followinginvestigations on Wnt/β-catenin and receptortyrosine kinase signaling:A colorectal cancer expression profile thatincludes the β-catenin target and TGFβ inhibitorBAMBI predicts metastatic potentialJohannes Fritzmann, Daniel Besser, Frauke Kosel, Felix H.Brembeck, Ulrike Stein, Iduna Fichtner, Peter M. Schlag,Walter Birchmeier, Markus Morkel (MPI for MolecularGenetics, Berlin) and Jan Budczies (Charité Berlin)82 Cancer Research

Graduate StudentsÖzlem Akilli ÖztürkLiang Fang*Tamara GrigoryanRitesh Gupta*Julian HeubergerJingjing Qi*Peter WendRegina WilleckeTechnical AssistantsChris Eckert*Lysann Hinz*Frauke KoselMarion MüllerSandra Rohrmoser*Simone SteinRegina VogelSecretariatIrmgard Wiznerowicz*part of the period reportedA colorectal cancer expression profile that includes transforminggrowthfactor beta inhibitor BAMBI predicts metastatic potential(A) Mean expression levels of 115 signature genes that distinguishmetastatic from non-metastatic primary tumors. Hierarchical clusteringdendrogram is shown on top: average gene expression levels inmetastatic (mPT) and non-metastatic (nPT) primary tumors (insideblack box), lymph node (LN), liver (Hep) and lung (Pul) metastases andnormal tissue (N). Selected genes that are involved in signal transduction,gene transcription or metastasis are indicated. (B) Kaplan-Meiersurvival analysis of patients from an independent cohort (50 primarytumors), as determined by quantitative RT-PCR. All patients were freeof distant metastasis at the time of sample collection.Much is known about the genes and mutations thatcause colorectal cancer (CRC), yet only a few have beenassociated with CRC metastasis. Mutations of genes ofthe Wnt/β-catenin pathway are responsible for thegeneration of over 90 % of CRC. We performed expressionprofiling experiments to identify genetic markersof risk and to elucidate the molecular mechanisms ofCRC metastasis. We compared gene expression patternsbetween metastatic and non-metastatic, stagematchedhuman colorectal carcinomas. We establisheda signature of 115 genes that differentiated metastaticfrom non-metastatic primary tumors. Among these,the TGFβ inhibitor BAMBI was highly expressed in abouthalf of metastatic primary tumors and metastases butnot in non-metastatic tumors. We have identifiedBAMBI as a target of canonical Wnt signaling thatinvolves the β-catenin co-activator BCL9-2. We observedan inverse correlation between level of BAMBI expres-Cancer Research 83

The tyrosine phosphatase Shp2 directs Neuregulin-1/ErbB signaling throughout Schwann cell developmentIn vivo analysis of Schwann cells associated with peripheral nerves in control and Wnt1-cre Shp2 fl/fl mice at the indicated developmental stagesusing (A,B) immunohistochemistry for BFABP (red) and neurofilament (NF, green), (C,D) in situ hybridization with a Sox10-specific probe, and (E,F)histological sections stained with hematoxylin and eosin. Note the reduced numbers of Schwann cells at Shp2 mutant peripheral nerves.In vitro analysis of the role of Shp2 during Schwann cell proliferation and migration. (G) Southern blot and (H) Western blot analyses after HTNcreinduced recombination in cultured Shp2 fl/fl Schwann cells. (I,J) Quantification of the Nrg1-induced proliferation and migration indicates strongreduction of both cellular responses in the absence of Shp2. Shown are the percentage of Schwann cells that incorporated BrdU, or the percentageof Schwann cells that migrated into the scratched area, using control and Shp2 ∆/∆ Schwann cells (HTN-cre-treated Shp2 fl/fl Schwann cells) in thepresence and absence of Nrg1. (K-L´) Migration of Schwann cells into a scratched area.sion and metastasis-free survival time of patients.BAMBI inhibits TGFβ signaling and increases migrationin colon cancer cells. In mice, overexpression of BAMBIcaused colon cancer cells to form tumors that metastasizedmore frequently to liver and lymph nodes thancontrol cancer cells. Thus BAMBI regulates colorectalcancer metastasis by connecting the Wnt/β-cateninand TGFβ signaling pathways. The metastatic expressionsignature we describe, along with BAMBI levels,may be used in prognosis in the future. Our data alsoshow that developmental signaling pathways act inhierarchies and cooperate in tumor cell migration, invasionand metastasis.Skin cancer stem cell maintenance is dependanton catenin signaling throughout Schwann celldevelopmentIlaria Malanchi, Deepika Kassen, Thomas Hussenet, JörgHuelsken (EPFL/ISREC Lausanne), Amparo Cano (CSIC-UAMMadrid) and Walter Birchmeier.Proper canonical Wnt signaling guides healthy developmentin many tissues, but defects play a role in tumors,as revealed by the analysis of loss- and gain-of functionmutations in β-catenin. The recently identified cancerstem cells and normal stem cells share many characteristics,like the capacity for self-renewal and differentiationand their dependence on a particular microenvi-84 Cancer Research

onment, the (cancer) stem cell niche. We have identifieda population of cancer stem cells in mouse epidermaltumors, i.e. early squamous cell carcinomas, whichare dependent on β-catenin and that are phenotypicallyand functionally similar to normal bulge skin stem cells.These cancer stem cells can be isolated by cell sortingbased on the presence of the stem cell marker CD34 andabsence of other markers. In normal mouse skin, CD34 +bulge stem cells account for approximately 1.8% of keratinocytes.However, cutaneous tumors, induced bychemical (DMBA/TPA) carcinogenesis or by expression ofthe Ras oncogene, contain a 9-fold increase of the CD34 +cell population. The tumorigenic capacity of the CD34 +cells was over 100-fold greater than that of unsortedcells, when these were transplanted into the back skin ofNOD/SCID mice. Secondary tumors that formed aftertransplantation resembled the parental tumors; theycontained a small population of CD34 + stem cells.Remarkably, the deletion of β-catenin in DMBA/TPA orRas-induced tumors using an inducible system thatallows conditional mutagenesis after the addition oftamoxifen (K14-cre ERT2 ; β-catenin flox ) resulted in completetumor regression. Thus, canonical Wnt signals arerequired for the maintenance of skin cancer stem cells,whereas in normal skin they instruct bulge stem cellstowards the hair cell fate.The tyrosine phosphatase Shp2 directsNeuregulin-1/ErbB signaling throughoutSchwann cell developmentKatja Grossmann, Hagen Wende, Florian Paul, Cyril Cheret,Alistair Garratt, Daniel Besser, Herbert Schulz, MatthiasSelbach, Walter Birchmeier and Carmen Birchmeier.The non-receptor tyrosine phosphatase Shp2 has beenimplicated in tyrosine kinase, chemokine and integrinreceptor signaling. We have produced a floxed allele ofthe Shp2 gene in mice in order to study the biologicalfunction of this tyrosine phosphatase. We showed thatconditional mutation of Shp2 in neural crest cells and inmyelinating Schwann cells resulted in deficits in glialdevelopment that are remarkably similar to thoseobserved in mice mutant for Neuregulin-1 (Nrg1) or theNrg1 receptors, ErbB2 and ErbB3. In cultured Shp2mutant Schwann cells, Nrg1-evoked cellular responseslike proliferation and migration were virtually abolished,and Nrg1-dependent intracellular signaling wasaltered. Pharmacological inhibition of Src family andMAP kinases mimicked all cellular and biochemicaleffects of the Shp2 mutation, implicating Src as a primaryShp2 target during Nrg1 signaling. Our phenotypicand biochemical analyses thus demonstrate thatShp2 is an essential component in the transduction ofNrg1/ErbB signals.Specific inhibitors of the protein tyrosinephosphatase Shp2 identified by high-throughputdockingKlaus Hellmuth, Ching Tung Lum, Martin Würtele, MartaRosario, Walter Birchmeier, Stefanie Grosskopf (FMP),Jörg Rademann (FMP) and Jens von Kries (FMP).The protein tyrosine phosphatase Shp2 is a positive regulatorof growth factor signaling. Gain-of-functionmutations in several types of leukemia define Shp2 as abona fide oncogene. We performed a high-throughputin silico screen for small-molecular-weight compoundsthat bind the catalytic site of Shp2. We have identifiedthe phenylhydrazonopyrazolone sulfonate PHPS1 as apotent and cell-permeable inhibitor, which is specificfor Shp2 over the closely related tyrosine phosphatasesShp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellularevents such as hepatocyte growth factor/scatter factor(HGF/SF)-induced epithelial cell scattering and branchingmorphogenesis. PHPS1 also blocks Shp2-dependentdownstream signaling, namely HGF/SF-induced sustainedphosphorylation of the Erk1/2 MAP kinases anddephosphorylation of paxillin. Furthermore, PHPS1 efficientlyinhibits activation of Erk1/2 by the leukemiaassociatedShp2 mutant, Shp2-E76K, and blocks theanchorage-independent growth of a variety of humantumor cell lines. The PHPS compound class is thereforesuitable for further development of therapeutics for thetreatment of Shp2-dependent diseases.Selected PublicationsKlaus, A., and Birchmeier, W. (2008): Wnt signalling and its impact ondevelopment and cancer. Nature Rev. Cancer 8, 387-398Grigoryan, T., Wend, P., Klaus, A., and Birchmeier, W. (2008): Decipheringthe function of canonical Wnt signals in development and disease: conditionalloss- and gain-of-function mutations of beta-catenin in mice.Genes & Development 22, 2308-2341Malanchi, I., Peinado, H., Kassen, D., Hussenet, T., Metzger, D., Chambon,P., Huber, M., Hohl, D., Cano, A., Birchmeier, W., and Huelsken, J. (2008):Cutaneous cancer stem cell maintenance is dependent on beta-cateninsignalling. Nature 452, 650-654Hellmuth, K., Grosskopf, S., Tung Lum, C., Wuertele, M., Roeder, N., Kries v.,JP, Rosário, M., Rademann, J., and Birchmeier, W. (2008): Specific inhibitorsof the protein tyrosine phosphatase Shp2 identified by high-throughputdocking. Proc. Natl. Acad. Sci. USA. 105, 7275-7280Fritzmann, J., Morkel, M., Besser, D., Budczies, J., Kosel, F., Brembeck, F.H.,Stein, U., Fichtner, I., Schlag, P.M., and Birchmeier, W. (2009): A colorectalcancer expression profile that includes transforming growth factor betainhibitor BAMBI predicts metastatic potential. Gastroenterology 137,165-175PatentMDC 0501: Shp-2 inhibitors, pharmaceutical compositions comprisingthem and their use for treating phosphatase-mediated diseases.W. Birchmeier, K. Hellmuth. EP 05 090 160.2.Cancer Research 85

Daniel Besser(Delbrück Fellow)Structure of the GroupGroup LeaderDr. Daniel BesserGraduate StudentsSebastian DieckeAngel Quiroga-NegreiraTorben RedmerSignaling Mechanisms in EmbryonicStem CellsEmbryonic stem cells (ESC) are pluripotent cells, which can proliferate indefinitely andparticipate in the formation of most cell types. Studies on human ESCs (hESC) provide aninsight into human embryogenesis and allow the development of tools for pharmacology andregenerative medicine. The focus of our studies is the maintenance of the pluripotent state inmurine ESCs (mESCs) and hESCs. We found that the Activin/Nodal pathway is activated inpluripotent hESCs and blocked upon differentiation. Pluripotent cells require these signalingregulating a specific subset of target genes. In addition, the BMP pathway is in the off-state inpluripotent cells and activated upon differentiation. This pathway counteracts the effects ofActivin signaling. Moreover, it has been shown that somatic cells for various sources can bereprogrammed by transfection of four defined transcription factors, i.e. Oct4, Sox2, Klf4, andc-Myc, to a pluripotent state. These induced pluripotent stem cells (iPS) are very exciting newcell populations for stem cell research.Role of TGFβ signaling in pluripotency ofembryonic stem cellsAngel Quiroga-Negreira, funded by the DFG, SPP1109Embryonal and Tissue-specific Stem Cells and HGF-systemsbiology NW1 SB Cancer DKFZ.I.4Our previous observations show that Activin/Nodal signalingis required for maintenance of the undifferentiatedstate in hESCs while BMP signaling induces differentiation.This raises important questions regarding thedownstream target genes that are important for theregulation of pluripotency as well as differentiation.Another important question is how cross talk betweenthese two pathways is established. In this project wehave found that intrinsic Activin/Nodal and BMP signalingin hESCs controls the molecular events and theregulation of a whole set of downstream target genes.In a recent analysis of global expression profiles usingAffymetrix microarrays in a dense time course in hESCsand mESCs upon differentiation, activation and inhibitionof Activin/Nodal signaling and activation of BMPsignaling we gained deeper insight into the regulatoryevents underlying these processes. Moreover, by mathematicalmodeling we are in the process of using thisdataset for a detailed analysis of gene regulation inpluripotent ESCs and during early differentiation. Wefound that in hESCs, Activin A treatment under differentiatingconditions leads to establishment of thepluripotency program, while inhibition of Activin signalingand BMP4 treatment strongly induces differentiationinto specific cell lineages. The data also suggestthat molecules that regulate cell-cell interaction andcell motility, especially molecules of the Cadherin familyand molecules regulating the small GTP-binding proteinRho are regulated very early after induction of differentiation.Regulation of Oct4: How the regulator isregulatedSebastian Diecke and Angel Quiroga-NegreiraIt is well established that three transcription factors, i.e.Oct4 (Oct3/4, Pou5F1), Nanog, and Sox2 that interactwith each other, are at the core of the molecular events86 Cancer Research

The pluripotent state of murine andhuman embryonic stem cells (m/hESC).mESCs can be maintained undifferentiatedin LIF, while hESCs require conditionedmedium (CM) from mouse embryo fibroblasts(MEF) leading to activation of Activinsignaling and block of BMP4 signaling.Upon differentiation, i.e. by withdrawal ofLIF or cultivation in non-conditioned medium(nCM) the pluripotency transcriptionfactor Oct4 is downregulated and weobserve changes in cell morphology reminiscentto epithelial to mesenchymal transition(EMT).maintaining the pluripotent state. However, we observea significant decrease of Oct4 levels only several daysafter induction of differentiation, although molecularchanges related to the differentiation can be observedduring the first 24 hr. In addition, we observed in chromatinprecipitation experiments that Oct4 levels bindingin specific promoters decreases before a decrease intotal protein level is detected. Thus, additional signalingevents such as phosphorylation may have an impact onthe activity of the Oct4 transcription factor complex.Interestingly, we observed a change in the charge ofOct4 protein in differentiating versus undifferentiatedcells, suggesting differential phosphorylation of Oct4upon differentiation. Comparison of Oct4 orthologesequences from different species show a highly conservedhomology surrounding the nuclear localizationsignal (NLS) and in the C-terminal domain containingthreonine and serine residues, which are potentialphosphorylation sites. We are currently testing whetherphosphorylation of these sites influence the cellulardistribution of Oct4 during the early differentiation andcontribute to its inactivation. The regulation of Oct4 byphosphorylation may also explain why signaling pathways,such as Activin/Nodal have an impact on the regulationof pluripotency.Deciphering mechanisms during reprogrammingof mouse embryonic fibroblasts to pluripotentstem cellsTorben Redmer and Sebastian Diecke, funded bySTART-MSC Consortium, BMBF joint project grantThe reprogramming of murine fibroblasts by transductionof the transcription factors Oct4, Sox2, Klf4 andc-Myc has become an established method to generatepluripotent cells, but mechanisms that govern thesesteps are still poorly understood. We identified a populationof cells that seems to be partially reprogrammedand display features of fibroblasts, although viral transcriptsof the four factors are present. Using FACSortingwe separated cells positive and negative for the cell surfacemarker SSEA-1 (stage-specific embryonic antigen-1). These two populations differ in their morphology, differentiationcapacity like formation of embryoid bodiesand in gene expression levels of endogenouslyexpressed markers of pluripotent stem cells like Nanog.These findings led us conclude that expression of thefour critical factors is necessary to induce reprogrammingbut its not sufficient for fully generation of stableiPS cells. Therefore additional mechanisms and signalingpathways play a role during this process.Selected PublicationsBesser, D. (2004) Expression of Nodal, Lefty-A, and Lefty-B in undifferentiatedhuman embryonic stem cells requires activation of Smad2/3. J. Biol.Chem. 279, 45076-45084.James, D., Levine, A.J., Besser, D. & Brivanlou, A.H. (2005)TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotencyin human embryonic stem cells. Development 132, 1273-1282.Diecke, S., Quiroga Negreira A., Redmer, T., and Besser, D. (2008) FGF2 signalingin mouse embryonic fibroblasts is crucial for self-renewal ofembryonic stem cells. Cells Tissues Organs 188, 52-61.Kaufman, W.L., Kocman, I., Agrawal, V., Rahn, H.P., Besser, D., and Gossen, M.(2008) Homogeneity and persistence of transgene expression by omittingantibiotic selection in cell line isolation. Nucleic Acid Res. 36, e111.Fritzmann, J.*, Morkel, M.*, Besser, D. *, Budczies, J., Kosel, F., Brembeck, F.H.,Stein, U., Fichtner, I., Schlag, P.M., and Birchmeier, W. (2009) A ColorectalCancer Expression Profile that Includes Transforming Growth Factor βInhibitor BAMBI Predicts Metastatic Potential. Gastroenterology, 137, 165-175 (* equal contribution).Cancer Research 87

Structure of the GroupUlrike ZieboldGroup LeaderDr. Ulrike ZieboldGraduate StudentsChristna ChapKirsten VormbrockBjörn von EyßSebastian MemczakKatharina MöllmannTechnical AssistantsGitta BlendingerCynthia VossSecretariatPetra HainkSonja GieringGenetics of Tumor Progression andMetastasisThe mammal cell cycle has been extensively studied nevertheless its intricacies remainelusive. Furthermore, the knowledge of cancer stem cells is at its beginnings. To tackle thesecomplex questions, we modify murine ES-cells to characterize both transcriptional and signalingmodules in stemness, proliferation or differentiation. Similarly, we use mouse models to definethe molecular circuitry underlying cancer causing stem cells. Central to our work is theunderstanding of the molecular consequences of deregulation of the retinoblastoma protein(RB) and E2Fs, since in most human tumors the RB pathway is mutated leading to unfetteredactivity of the E2F transcription factors. To reveal how E2Fs control growth, we search forinteraction partners and chromatin modifiers. We are also interested in the specific contributionof E2F3 to tumor progression by identifying novel metastasis specific targets. In summary, ourgoal is to understand how specific biological processes as cellular stemness, growth control andsuppression of tumorigenesis are coupled.Interaction partners and possible co-activatorsof E2F3Björn von Eyss and Katharina MöllmannThe inactivation of the RB tumor suppressor pathwaydepends on the activity of the E2F-transcription factors,since in pRB mutant mice E2F3 contributes extensivelyto the ectopic S-phase and apoptosis. Depleting E2F3alone leads to cellular growth defects but the molecularmechanism of E2F3 is not understood. Searching forspecific interaction partners of E2F3, we identified anumber of novel molecules, among them putative chromatinregulators. For some of these regulators we wereable to show that they can be detected on E2F-regulatedpromoters, that they are essential for the progressionthrough cell cycle and are co-expressed with E2F3in human tumors. Lastly, we are using ChIP coupled todeep sequencing to reveal the whole scope of co-regulatedtarget genes. So far, our genome wide searchrevealed unanticipated E2F-target genes, which will befocus of our future research.E2F target genes in proliferation and metastasisKirsten Vormbrock and Sebastian MemczakE2f3 is the only E2F amplified in human tumors, wherebyhigh E2F3 expression marks invasiveness predictingpoor patient survival. This led to the hypothesis thatE2F3 regulates unique sets of target genes. To search forsuch targets we use a mouse strain deficient for bothpRB/E2F3. In this model, merely few pRB-deficient micedevelop small medullary thyroid carcinomas (MTCs),while most pRB/E2F3 deficient mutants developmetastatic MTCs. Using micro-array gene-chips weestablished the transcriptional profile of these MTCs.The resulting sets of transcripts are putative metastaticmarkers. Importantly, for four of these markers anidentical pattern in human metastatic thyroid malignancieswas found, a first proof that these genes areprognostic markers. Judged by chromatin immune precipitationassays, all of the genes are direct targets ofE2F. Thus, we link individual E2F-action to specific E2Ftargetgenes and the onset of metastasis.88 Cancer Research

In the morula of the early mouse embryo the Spry4 protein is expressed uniformly in all blastomeres. Upon differentiation, both Spry4 and Nanogare limited to the inner cell mass of the blastocyst. Cdx2 is a marker of the extra-embryonic lineage, showing that cells of this lineage are devoidof Spry4 or Nanog.Proliferation and pluripotency in stem cellsChristna Chap and Gitta BlendingerThe ability to derive multiple differentiated lineages is ahallmark of embryonic stem cells (ES cells). Since ES-celltransplanted into nude mice develop primitive tumors,ES cells are viewed as an ideal in vitro model for earlymammalian development and tumorigenesis. UsingES-cells differentiation, we identified Sprouty4 (Spry4)as a novel player of the pluripotency network. Also, in EScells Spry4 acts as an inhibitor of the ERK pathway toregulate pluripotency. Using ChIP assays, we establishedthat Spry4 is transcriptionally repressed byNanog. This repression is essential, since over-expressionof Spry4 renders ES-cells insensitive to differentiation.Consistent with a role for Spry4 in pluripotency, wefind its expression restricted to the inner cell mass ofmouse embryos (see Figure). Our results show thatSpry4 tightly regulates several signal pathways tomaintain the pluripotent state of ES cells. In the future,we will characterize specific mutants of Spry4 that likelyinfer commitment to differentiation lineages. Sincewe believe, that Spry4 impinges upon more pathwaysthan it is currently recognized, we intend to establishnew Spry4 mouse models allowing us to dissect cooperatingsignaling events in cancer and stem cells.Determinants of cancer stem cells activityIn collaboration with Peter Wend and Walter BirchmeierRecent evidence suggests that organ as well as cancerstem cells share core transcriptional and signalingmodules. This points towards the fact that both celltypes share a common ancestor. To understand if this istrue, we deployed genetic mouse models and studiedWnt/β-catenin and Bmp signaling in salivary glandstem cells. Our results imply that both Bmp and properWnt/β-catenin signals are required to properly controlcell division, apoptosis and differentiation of salivarygland stem cells. Significantly, if mutations in bothpathways are joined, a tenfold increase of stem cells isobserved. These stem cells are activated upon regenerationand are highly tumorigenic. In the future, we willdissect the transcriptional profiles of these stem cellsto understand their specific contribution to tumorigenesis.Selected PublicationsLee, E.Y., Cam, H., Ziebold, U., Rayman, J.B., Lees, J.A., and Dynlacht, B.D.(2002). E2F4 Loss suppresses Tumorigenesis in RB Mutant Mice Through aNovel Mechanism. Cancer Cell 2, 463-472.Cloud, J.E., Rogers, C., Reza, T.L., Ziebold, U., Stone, J.R., Picard, M.H., Caron,A.M., Bronson, R.T., and Lees, JA. (2002). Mutant mouse models reveal therelative roles of E2F1 and E2F3 in vivo. Mol. Cell. Biol. 22, 2663-2672.Ziebold, U., Caron, A., Bronson, R., and Lees, J.A. (2003). E2F3-loss has opposingeffects on different pRb-deficient tumors, resulting in suppression ofpituitary tumors but metastasis of medullary thyroid carcinomas. Mol.Cell. Biol. 18, 6542-6552.Boden, C., and Ziebold, U. (2004). Cell division – an Overview, inEncyclopedic Reference of Genomics and Proteomics in MolecularMedicine K. Ruckpaul and D. Ganten, Eds. Springer Verlag, Heidelberg.Tapon, N. and Ziebold, U. (2008). Invasion and metastasis: stem cells,screens and survival. EMBO Rep. 9, 1078-1083.PatentsMIT Case No. 8756 “ANTIBODY TO P10”.Cancer Research 89

Achim LeutzStructure of the GroupGroup LeaderProf. Dr. Achim LeutzScientistsDr. Valérie BégayDr. Elisabeth Kowenz-LeutzDr. Jörn Lausen*Dr. Ole PlessDr. Goncalo Regalo*Dr. Marina SchellerDr. Jeske SminkDr. Klaus WethmarDr. Katrin Zaragoza-DoerrCell Differentiation and TumorigenesisStem cells, transcription factors, & differentiationSomatic stem cells undergo self-renewal to maintainthe stem cell pool and give rise to transit amplifyingprogenitor cells that terminally differentiate intomature cells. Self-renewal, proliferation, and cell differentiationare interlaced genetic programs that aretightly controlled by gene regulatory proteins (transcriptionfactors). Transcription factors downstream ofsignaling cascades dynamically adjust the expressionof developmental genes. Some transcription factorsrepresent nodal points in such regulatory networks ofgrowth and differentiation. These key transcription factorsmay mutate and become tumorigenic by blockingdifferentiation or deregulating self-renewal.We focus on the dynamic regulation of CCAAT EnhancerBinding Protein (C/EBP) α and β transcription factorsand their interaction with co-factors of the epigeneticmachinery and of cell division control. C/EBPs regulategenes involved in stem cell functions, proliferation, differentiation,metabolism, immunity, senes cence, andtumorigenesis and determine lineage commitment inthe hematopoietic system and myeloid trans-differentiation.Of particular interest is how extracellular signalsinstruct C/EBPs to orchestrate “stemness”, cell multiplication,or lineage restricted terminal differentiation.Lineage specific gene expression patterns are establishedduring cell differentiation while alternative differentiationoptions and self-renewal are extinguished.The choreography of all these steps is essential to properdifferentiation, as many malignant diseases, in particularleukemic diseases, display conflicting geneexpression patterns and “multi-lineage competence”,known as lineage infidelity. How “multi-lineage competence”is maintained in stem cells, how it is extinguishedduring lineage commitment and why it resurfacesduring tumorigenic conversion are major topics ofour research.C/EBP: Structure & FunctionC/EBPs comprise a family of 6 genes in vertebrates. Fourmembers, C/EBPα,β,δ,ε, are highly related whereas twoothers, γ and ζ, are more divergent and display homologyonly in their C-terminal ~100 amino acid long basicDNA binding leucine zipper domain (bZip) that forms abifurcated coiled-coil domain which interacts with specificcis-regulatory sites on DNA. The N-terminal part ofC/EBPα,β (first ~100 amino acids) represent strongtrans-activation domains (TAD) whereas the centersequences (aa ~100-200) contain regulatory domains(RD).Knockout analysis of individual C/EBPs in mice and phylogenetictree construction suggested that C/EBPα andC/EBPβ are the most ancient C/EBPs. Combined loss ofC/EBPαβ, causes placental defects in trophoblast cellsand early embryonic lethality, suggesting that they arethe most important C/EBPs.The activity of C/EBα,β is regulated by translationalcontrol and by post-translational modifications thatdetermine combinatorial co-factor interactions withthe gene regulatory and the epigenetic machineries.Multiplicity of signaling regulated modifications permitmany combinatorial interactions and plasticity ofdevelopmental processes to be accomplished with alimited set of regulators.Translational regulation of C/EBPsValérie Bégay, Jeske J. Smink, Klaus WethmarC/EBPα and C/EBPβ transcripts harbor small upstreamopen reading frames (uORF) in their mRNAs that sensethe activity of translation initiation factors and relay initiationto alternative in-frame start sites. As a result,truncated proteins that lack part of the N-terminalsequences are generated. Besides C/EBPs, many transcriptsof key regulatory genes involved in growth, differentiation,and proliferation harbor uORFs, suggesting90 Cancer Research

Graduate StudentsRami Hamscho*Qingbin LiuBilyana StoilovaMagdalena Schmude*Undergraduate StudentsSusanne Eiglmeier*Julien Karriche*Francisca Leonard*Julia Tornack*Lina Zschockelt*Technical AssistantsCorinna BeckerKarolin FriedrichNatalie HaritonowMaria KnoblichSarah Schmidt*Ruth Zarmstorff*SecretariatSylvia Sibilak*part of the period reportedan important role of uORF-mediated translational controlin mammalian development and physiology.Nevertheless, genetic models in mammals are lackingto explore the physiological relevance of uORF-regulatedtranslation initiation.Truncated C/EBP isoforms sustain proliferation, whereasfull-length forms are inhibitors of cell division.Previously, we showed that anaplastic large cell lymphomaand Hodgkin Lymphoma express predominantlytruncated C/EBPβ. Rapamycin, an antibiotic thatinhibits mTOR signaling, shuts down the truncatedC/EBP isoform and concomitantly inhibits growth inboth types of lymphomas. Ectopic expression of truncatedC/EBPβ restored proliferation, suggesting thattruncated C/EBPβ represents a translationally controlledoncogene.By targeted recombination we have now generatedmouse C/EBPβ mutants that express distinct C/EBPβisoforms. The murine mutants revealed that C/EBPβisoform switching is important during liver regeneration,bone homeostasis and tumorigenesis. Mice deficientfor the C/EBPβ uORF initiation codon(C/EBPβ∆uORF) fail to initiate translation of the autoantagonistic,truncated “LIP” C/EBPβ isoform. Livers ofC/EBPβ∆uORF mice displayed defective regeneration.After partial hepatectomy, delayed and blunted entry ofhepatocytes into S-phase, persistent repression of E2Fregulatedgenes, and hyperactivation of acute phaseresponse genes became evident. Thus, switching to thetruncated isoform is important during liver regenerationand in shutting off acute phase response.C/EBPβ deficient mice and mice expressing only thetruncated “LIP” isoform (LIP knock-in mice, L/L) both displayincreased bone resorption. This is due to enhanceddifferentiation of the bone resorbing cell, the osteoclast.Failure to switch back to the long C/EBPβ isoformaugmented osteoclastogenesis. Indeed, ectopic expressionof the long isoform “LAP” in monocytes inhibitedformation of multi-nucleated osteoclasts. Rapamycin,an inhibitor of mTOR signaling that increases the “LAP”over “LIP” ratio, also inhibited osteoclastogenesis in wildModel of integrated control of osteoclastogenesis by C/EBPβ.Top to Bottom: Long (LAP) and truncated (LIP) C/EBPβ isoforms aregenerated from a single mRNA. The ratio between LAP and LIP is determinedby the activity of translation initiation factors, which are downstreamof growth factor, hormone, nutrient, and stress signaling pathways.The mammalian Target Of Rapamycin (mTOR) kinase integratesthese signals and plays an important role in adjusting the LAP/LIPratio (blue: low activity; red: high activity). The LAP isoform inducesMafB, an inhibitor of osteoclastogenesis that suppresses the activity ofseveral osteoclastic regulators (Mitf, Fos, NFATc1). Osteoclast precursorsthat lack C/EBP or that express LIP only, display strongly augmentedformation of giant osteoclasts that readily destroy bone substance.Cancer Research 91

type but not in C/EBP deficient or L/L osteoclasts thatboth can not switch to “LAP” expression. Profiling analysisof the transcriptome showed that rapamycin treatmentor ectopic expression of “LAP” activated expressionof MafB, a negative regulator of osteoclastogenesis.This suggested that restriction of osteoclastogenesisby “LAP” or rapamycin is dependent on MafB. Inaccordance, knock-down of MafB induced osteoclastogenesis,regardless of rapamycin treatment or C/EBPisoform expression. Altogether, the data showed thatdifferential regulation of MafB gene expression byC/EBPβ isoforms determines the balance of boneturnover and that the control of C/EBPβ isoform translationrepresents a target for osteoporosis treatment.Post-translational C/EBP modifications andepigenetic functionsElisabeth Kowenz-Leutz and Ole PlessSeveral years ago, we found that C/EBPα,β may instructeven non-hematopoietic cells, such as skin fibroblasts,to express myeloid genes. Others have shown thatC/EBPs may reprogram lymphocytes into myeloid cells.Accordingly, C/EBPs entail epigenetic competence andgene regulatory functions to determine cell fate. Wehad also shown that cellular signaling cascades regulatethe activity of C/EBPβ to convert extracellular informationinto gene regulation. C/EBPβ is a ras/ MAP kinasesignal sensitive transcription factor that regulatesgenes involved in metabolism, proliferation, differentiation,immunity, senes cence, and tumorigenesis. Thefunctional capacity of C/EBPβ is governed by proteininteractions that depend on post-translational C/EBPβmodifications. In a proteome-wide interaction screen,the histone-lysine N-methyl trans ferase, H3 lysine-9specific 3 (G9a) was found to directly inter act with theC/EBPβ transactivation domain (TAD). G9a, but not adefective G9a mutant abrogated the transactivationpotential of wild type C/EBPβ. Metabolic labelingshowed that C/EBPβ is post-translationally modified bymethylation of its TAD. A conserved lysine residue (K39)in the C/EBPβ-TAD served as a substrate for G9a mediatedmethylation. A C/EBPβ K39 alanine exchangemutant was resistant to G9a mediated inhibition andconferred super-activation of myeloid genes. These dataidentified C/EBPβ as a direct substrate of G9a thatalters the functional properties of C/EBPβ by posttranslational lysine methylation.Mass spectrometry of cell derived C/EBPβ (in collaborationwith Gunnar Dittmar, MDC) revealed extensivemethylation of N-terminal arginine residues in C/EBPβ.The protein arginine methyl-transferase 4 (PRMT4) wasfound to interact with C/EBPβ and to di-methylate theconserved arginine residue (R3) in the C/EBPβ TAD.Phosphorylation of the regulatory domain of C/EBPβ byras/ MAP kinase signaling abrogated the inter actionbetween C/EBPβ and PRMT4. Differential proteomicscreening with R3-methylated and un-methylatedC/EBPβ peptides, protein interaction studies, and mutationalanalysis revealed that methylation of R3 constrainedthe interaction between C/EBPβ with SWI/SNFand Mediator complexes. Both complexes play essentialroles in chromatin remodeling and transcription initiationby polymerase II and were previously implicated inC/EBPβ functions. Mutation of the R3-C/EBPβ methylationsite alters the ability of C/EBPβ to induce myeloidand adipogenic differentiation. Thus, phospho ry lationof the transcription factor C/EBPβ couples ras signalingto arginine methylation and regulates the interactionof C/EBPβ with epigenetic gene regulatory protein complexesduring cell differentiation.A number of implications and conceptual advances arecontained in these results. An “indexing code” of posttranslationaltranscription factor modifications hasrecently been suggested (Sims & Reinberg, 2008),although experimental evidence is currently still scarce.Covalent modifications by “writers” (here, R-methylationby PRMT4, and K-methylation by G9a) determinemodification dependent docking of “readers” (SWI/SNF;Mediator; G9a). Our results imply that writing/readingsuch an indexing code is downstream of receptor tyrosinekinase signaling, relaying signals to epigeneticevents that finally determine cell fate. This concept is animportant extension of the Histone Code hypothesis tonon-histone transcription factor proteins, which comefirst in gene regulation and before chromatin modificationsoccur. Many mechanistic (gene regulation indevelopment and disease) and medical issues (pharmacology)are implied.Functional interactions between C/EBPalpha andE2F-DP complexesKatrin Zaragoza and Qingbin LiuC/EBPα coordinates proliferation arrest and differentiationin many cell types. C/EBPα transactivates lineagespecific differentiation genes and inhibits proliferationby repressing E2F-regulated genes. The myeloproliferativeC/EBPαBRM2 mutant serves as a paradigm forrecurrent human C-terminal bZip C/EBPα mutationsthat are involved in acute myeloid leukemogenesis.BRM2 fails to repress E2F and fails to induce adipogen-92 Cancer Research

esis and granulopoiesis. We showed that C/EBPα orBRM2 interact with the dimerization partner (DP) of E2Fand that BRM2 displays enhanced interaction with theE2F-DP complex. Augmented interaction with E2F-DPprevents binding of C/EBP to its cognate sites on DNAand thus prevents transactivation of C/EBP targetgenes. Repression of C/EBPα by E2F-DP occurs independentlyof pocket proteins, including the retinoblastomaprotein Rb and the related p107,and p130.Although BRM2 is more susceptible to E2F-DP repressionit retains transactivation potential and differentiationcompetence, as disclosed by knock-down of E2F orDP expression. These data suggested that a tripartidebalance between C/EBPα, E2F/DP, and pocket proteinscontrol proliferation and differentiation and that E2F-DP mediated block of C/EBPα functions may play a rolein tumorigenesis.Selected PublicationsMo X., Kowenz-Leutz E., Xu H., and Leutz A. (2004) Ras Induces MediatorComplex Exchange on C/EBPbeta. Molecular Cell, 13:241-250Mo, X., Kowenz-Leutz, E., Laumonnier, Y., Xu, H., and Leutz, A. (2005)Histone H3-tail positioning and acetylation by c-Myb but not the v-MybDNA binding SANT Domain. Genes & Development, 19: 2447-2457Scheller, M.; Huelsken, J.; Rosenbauer, F.; Taketo, M.M.; Birchmeier, W.;Tenen, G.D. and Leutz, A. (2006). Hematopoietic stem cell and multi lineagedefects by β-catenin activation. Nature Immunology, 7:1037-1047Smink, J.J, Bégay,V., Schoenmaker, T., Sterneck, E., de Vries, T.J., and Leutz, A.(2009) Transcription factor C/EBPβ isoform ratio regulates osteoclastogenesisthrough MafB. The EMBO J., 28: 1769-1781Klaus Wethmar, Valérie Bégay, Jeske J. Smink, et al. (2010) C/EBPβ ∆uORFmice−−a genetic model for uORF-mediated translational control inmammals Genes Dev. 24: 15-20Cancer Research 93

Structure of the GroupFrank RosenbauerGroup LeaderDr. Frank RosenbauerScientistsDr. Andreas EnnsDr. Saeed GhaniDr. Christiane Kuhl*Graduate StudentsAnn-Marie BröskeLena VockentanzJörg SchönheitChiara PerrodMathias Leddin* part of the period reportedTechnical AssistantsVictoria MalchinChristiane Graubmann*Nancy Endruhn*SecretariatSonja GieringPetra HainkCancer, Stem Cells, andTranscription FactorsStem cells are capable of life-long self-renewal and multi-lineage differentiation. Theformation of early progenitors of the myeloid lineage from stem cells is orchestrated bya relatively small number of transcription factors. Among them are PU.1, CCAAT/enhancerbinding protein α (C/EBPα), growth factor independent 1 (GFI1), interferon-regulatory factor8 (IRF8), Runt-related transcription factor 1 (RUNX1) and stem-cell leukemia factor (SCL).Mice in which these genes have been knocked out displayed profound hematopoieticdefects. Moreover, these transcription factors were shown to regulate a broad range ofpivotal target genes, thereby directly programming precursors to differentiate along acomplex developmental pathway. A block in normal differentiation is a major contributingfactor towards the development of solid tumors and leukemias and cells from leukemiapatients frequently harbor mutated or dysregulated transcription factor genes. Thissuggests that altered transcription factor activity is a major driving force behind thepathology of transformation and the development of cancer stem cells.Dynamic PU.1 expression in hematopoiesis andleukemiaOne of the main interests of our laboratory is to understandhow transcription factors direct normal stem cellfunctions, such as self-renewal and differentiation, howthey program precursors to adopt a certain lineagechoice and how disruption of transcription factor activityleads to cancer (stem) cell transformation. Usingboth transgenic and knockout mouse models, we areparticularly interested in discovering crucial molecularup- and downstream mechanisms that regulate theexpression and function of transcription factors. A currentresearch focus in our laboratory is on PU.1. The Etsfamilymember PU.1 is essential for both myeloid andlymphoid lineages. PU.1 knockout mice exhibit earlylethality and lack of B-lymphocytes and mature myeloidcells in fetal livers. In addition, PU.1 is important for HSCself-renewal and differentiation into the earliestmyeloid and lymphoid progenitors. Furthermore, PU.1must be properly downregulated in early thymocytes toallow normal T cell development. It was shown thatgraded changes in PU.1 concentrations have drasticeffects on lineage fate decisions. Therefore, a greaterunderstanding of PU.1 gene regulation is the key todeciphering its role in normal hematopoiesis andmalignant transformation.We identified a novel distal DNA element (termedupstream regulatory element, URE) which is pivotal forproper PU.1 gene expression in vivo. We generated UREdeficient mice (URE ∆/∆ ) using targeted recombination inES cells. Remarkably, URE deletion led to a markeddecrease in PU.1 expression in HSCs, macrophages and Bcells, but an increase in PU.1 expression in early thymocytes.This demonstrated that the URE has an essentialcell context specific regulator function, and directs PU.1expression as an enhancer in myeloid and B-lymphoidcells but functions as a repressor in T cells. Due to theseprofound effects of URE deletion on PU.1 expression,94 Cancer Research

URE ∆/∆ mice regularly developed aggressive hematopoieticmalignancies, such as acute myeloid leukemia, Tcell lymphoma and B1 cell chronic lymphoid leukemia.Results from the URE ∆/∆ animal model provided the firstdemonstration that interference with the fine-tunedregulation of a single transcription factor, through disruptionof a key cis-regulatory element, can be sufficientto initiate the formation of cancer stem cells andsubsequent tumor development.Epigenetic control of hematopoietic stem cellfunctionMethylation of CpG dinucleotides within the DNA is amajor epigenetic modification, which in mammals iscontrolled by at least 3 different DNA-methyltranferases(DNMTs): DNMT3a and -b for de novo methylationand DNMT1 for methylation maintenance. The impactof methylation on stem cell features has been studiedin embryonic stem (ES) cells, but little is known aboutits function in somatic stem cells. Recent advances inthe genome-wide mapping of DNA methylationdemonstrated that methylation of CpGs are dynamicepigenetic marks that undergo extensive changes duringcellular differentiation. However, whether and howthese changes are required for cell fate choice, in particularfor that of stem cells, remained unknown.Moreover, altered methylation is a hallmark of cancer,and drugs targeting methylating enzymes are used incancer therapy. The relationship between tumor-associatedalterations in methylation and cancer stem cellproperties was still elusive.We could show that alternative functional programs ofHSCs are governed by gradual differences in the methylationlevel (Bröske et al. in press). Constitutive methylationis essential for HSC self-renewal, but dispensablefor homing, cell cycle control and suppression of apoptosis.Remarkably, HSCs from mice with reduced DNMT1 activity fail to suppress key myeloerythroid regulatorsand as a consequence can differentiate into myeloerythroidbut not into lymphoid progeny. We revealed thata similar methylation dosage effect controls stem cellfunction in leukemia. Thus, our data identified DNAmethylation as an essential epigenetic mechanism toprotect stem cells from premature activation of predominantdifferentiation programs and suggest thatmethylation dynamics determines stem cell functionsin tissue homeostasis and cancer (Figure 1).Consequently, these results provide the hope thatdemethylating drugs may be instrumental to impairthe function of cancer stem cells in cancer therapy.Model of DNA methylation dosage effects on stem cell multipotency.In the presence of physiological DNMT1 concentrations (top panel),stem cells are competent of a diverse functional repertoire whichincludes self-renew, differentiation into myeloid or lymphoid progeny(MP, LP) and suppression of apoptosis. The molecular basis of thisdiversity lies in a tightly balanced expression of genes that control selfrenewalwith genes that prime for certain developmental fates.Reduction in DNMT1 levels (middle panel) narrows the functionaloptions of stem cells by lifting the suppression of a predominantmyeloerythroid gene program via insufficient promoter methylation.As a consequence, expression of self-renewal and lymphoid genesbecomes blocked. Finally, complete loss of DNMT1 (bottom panel)blocks the entire functional stem cell repertoire and causes the rapidelimination of the stem cell pool by cell-autonomous induction ofapoptosis. Methyl +++: high methylation level required, Methyl +: lowmethylation level required.Selected publicationsSteidl U, Rosenbauer F, Verhaak RGW, Gu X, Out HH, Bruns I, Steidl C,Costa DB, Klippel S, Wagner K, Aivado M, Kobbe G, Valk PJ, Passegué E,Libermann TA, Delwel R, and Tenen DG. (2006) Essential role of Jun familytranscription factors in PU.1-induced leukemic stem cells. Nature Genet38, 1269-1277.Scheller M, Huelsken J, Rosenbauer F, Taketo MM, Birchmeier W, Tenen DG,and Leutz A. (2006) Hematopoietic stem cell and multi lineage defects byβ-catenin activation. Nature Immunol 7, 1037-1047.Rosenbauer F, Owens BM, Yu L, Tumang JR, Steidl U, Kutok JL, Clayton LK,Wagner K, Scheller M, Iwasaki H, Liu C, Hackanson B, Akashi K, Leutz A,Rothstein TL, Plass C, and Tenen DG. (2006) Lymphoid cell growth andtransformation are suppressed by a key regulatory element of the geneencoding PU.1. Nature Genet 38, 27-37.Rosenbauer F and Tenen DG. (2007) Transcription factors in myeloid development:Balancing differentiation with transformation. Nature RevImmunol 7,105-117.Bröske AM, Vockentanz L, Kharazi S, Huska M, Mancini E, Scheller M, EnnsA, Prinz M, Jaenisch R, Nerlov C, Leutz A, Andrade-Navarro MA, JacobsenSEW, Rosenbauer F. (2009). DNA methylation protects hematopoieticstem cell multipotency from myeloerythroid restriction. Nature Geneticsin press.Cancer Research 95

Peter M. SchlagStructure of the GroupGroup LeaderProf. Dr. Peter M. SchlagScientistsDr. Mathias DahlmannDr. Marion FehlkerDr. Johannes FritzmannDr. Wolfgang HaenschPD Dr. Wolfgang Kemmner(Group Leader Gene Profiling)Prof. Dr. Ulrike Stein (GroupLeader Tumor Metastasis)Surgical OncologyOur group has been focusing on the establishment of predictive and prognostic gene signaturesand expression profiles for analysis of metastases and drug resistance in colorectal cancer. Thedata provide the basis for novel therapeutic concepts in tumor treatment that are applicable andvalidated in the clinic. We were successful in identifying new metastases-associated genes, theirimportance for cancer metastases and their function in signaling pathways. We isolated andcharacterized the new gene MACC1, revealed S100A4/metastasin as novel target gene of theβ-catenin pathway and analyzed the interplay of BMP-4 and Bambi in the context of β-cateninsignaling. This provided insights into the tight association between early and late events of themetastasis process particularly in colorectal cancer, and opens opportunities for targetedtherapies. In the identification of patient-individualized gene expression profiles for improveddiagnosis and prediction regarding metastases formation and patient survival, the achievementswere made in close collaboration with the groups of W. Birchmeier and M. Lipp.In parallel to our achievements in understanding of new key regulators and molecularmechanisms in metastasis, we significantly advanced in the clinical development of local nonviralgene transfer for effective and applicable cancer gene therapy. We successfully performed a clinical"proof of principle“ phase I gene transfer trial at the ECRC using jet-injection for transfer of nakedDNA. In conclusion of this trial we made great efforts to further improve safety and efficiency ofnonviral vector systems and tested their therapeutic potential for clinical application.Classification of gastrointestinal carcinomas bygene expression profilingW. Kemmner, W. Qing, S. Förster, Q. Wang, P. M Schlag. Incooperation with M. Yashiro (Department of SurgicalOncology, Osaka City University Graduate School ofMedicine,Osaka, Japan), M. Vieth (Institute of Pathology,Klinikum Bayreuth, 95445 Bayreuth), P. Malfertheiner(Department of Gastroenterology, Hepatology andInfectious Diseases, Otto-von-Guericke UniversityMagdeburg, 39120 Magdeburg)The aim of this project was to find gene signatures thatallow a more precise differentiation of prognostic subtypesof gastrointestinal carcinomas. RNA was extractedfrom 60 colorectal, 72 gastric and 130 esophagealcancers and precancerous lesions respectively (e.g.GERD). After quality controls of the microarray data,ensuring the comparability of the different arrays, normalizationand data analysis was performed. Microarrayresults were validated by qRT-PCR analysis (TaqMan)and by immunohistochemistry.In each of the three cancer entities differentiallyexpressed genes were found which allow e.g. to discriminatebetween carcinoma subtypes. In the case ofesophageal neoplasias we were able to describe a newpotentially "high risk" group of Barrett's esophagus, ingastric carcinomas we described new candidate geneswith prognostic relevance and in colorectal cancer tissuewe identified a set of 42 genes which are significantlyupregulated in patients who are highly at risk forrecurrence after radical surgery. Most of the identified96 Cancer Research

PD Dr. Wolfgang Walther (GroupLeader Gene Therapy)Dr. Kayiu WanDr. Qing WangGraduate and undergraduatestudentsFranziska ArltOlga BöllingPia ForberichSusann FörsterInken KelchDennis KobeltStoyan PetkovAndreas PichornerUlrike SackCeline SchäferFelicitas SchmidTechnical AssistantsJutta AumannLisa BauerSabine GrigullPia HerrmannGudrun KochBianca KochnowskyClaudia RoefzaadJanice SmithSecretariatCornelia GroßeTime interval after treatment:t=120min t=180min t=240min t=300minPpIX-fluorescence in tumor xenografts. Human breast carcinoma cells MDA-MB-435 were injected subcutaneously (s.c.) into the fat pad of nudemice. Treatment of xenografts with liposomes, ALA or FECH siRNA alone showed little nonspecific fluorescence. Dramatic enhancement of PpIXfluorescent signals from the areas of xenografted tumors were detected in mice which were treated with folate-PEG cationic lipoplexes containingFECH-1140 siRNA followed by a single dose of 15mg/kg ALA. Localized fluorescent hotspots were detected at the sites of the millimeter-sizedtumors. Circles indicate the xenografted tumorsgenes are known to have a tumor biologic functionand/or are related to pathways.Silencing of human ferrochelatase (FECH) leadsto abundant accumulation of fluorescent protoporphyrin-IX(PpIX) in colorectal carcinoma cells -a new molecular amplification approach for earlyimaging of tumorsW. Kemmner, K. Wan, P. M. Schlag. In cooperation withBernd Ebert (Department of Biomedical Optics, Physi -kalisch-Technische Bundesanstalt Berlin) and R. Haag(Institute for Chemistry and Biochemistry, Free UniversityBerlin)Silencing of FECH leads to an endogenous fluorescenceby affecting the cellular heme metabolism. Applicationof siRNA may provide a general means for cellular imaging.Any nucleated human cell requires heme for hemecontainingenzymes essential for the cellular energymetabolism. The last step of heme synthesis is incorporationof iron into Protoporphyrin IX (PpIX) that takesplace in the mitochondria catalyzed by the enzymeFECH. PpIX is a fluorescing and photodynamically activechromophore which can be used for detection andtreatment of neoplasias. Quantitative RT-PCR of humantissue samples revealed a significant down-regulationof FECH expression in various gastrointestinal carcinomas.Experimentally induced knock down of FECHexpression in carcinoma cells by RNA-interference(siRNA) led to an accumulation of fluorescent PpIX ofmore than 50-fold. Moreover, PpIX fluorescence wasdramatically enhanced in xenografted tumors of nudemice treated with lipoplexes containing FECH-siRNA orwith siRNA bound to Polyglycerol cationic polymers.FECH-siRNA silencing might be a versatile tool formolecular imaging and cancer treatment. We reportedfor the first time the functional use of siRNA for cellularimaging. The presented approach exhibits no relevanttoxicity, because siRNA-silencing of FECH led to anendogenous and non-toxic fluorescence by affectingthe cellular heme metabolism.MACC1, a newly identified gene regulatesHGF/Met signaling and predicts colon cancermetastasisU. Stein, W. Walther, F. Arlt, J. Smith, P.M. Schlag. Incooperation with W. Birchmeier, I. FichtnerWe identified the novel gene MACC1 (Metastasis-Associatedin Colon Cancer 1) in subjects with colon cancerby genome-wide expression analysis in primary andmetastatic carcinomas, adenomas and normal tissues.MACC1 is located on chromosome 7. It encodes a proteinof 852 amino acids with domains that enable MACC1 forCancer Research 97

protein-protein interactions. We found several sites forserine/threonine and tyrosine phosphorylation; featuresthat predestine MACC1 as a signal transducer.We identified the gene Met as transcriptional target ofMACC1. Following application the hepatocyte growthfactor (HGF), MACC1 translocates from the cytoplasminto the nucleus, binds to the Met promoter, andinduces its transcription. The HGF/Met pathway is particularlyinvolved in colon cancer metastasis. Thus, weidentified MACC1 as a major regulator of this importantcellular metastasis pathway.MACC1 induces migration, invasion, proliferation, colonyformation, and HGF-mediated scattering in cell culture.These functional MACC1 effects were reverted byMACC1- or Met-specific siRNA. Cells with mutantMACC1 protein interaction domains did neither showMACC1-induced cell motility nor proliferation. Subcutaneous,orthotopic or intrasplenic transplantation ofMACC1-expressing colon cancer cells in mice resulted inelevated tumor growth and increased numbers of distantmetastases. MACC1- or Met-specific shRNA led tolowered numbers of metastases in vivo.MACC1 was overexpressed in human colon cancer comparedto normal tissues. Importantly, MACC1 expressionswere significantly higher in those tumors, thatmetachronously metastasized compared to those,which did not metastasize within up to 10 years. MACC1levels predict the probability of metastasis linked tometastasis-free survival. The correct positive or negativeprediction for metastasis was 74% and 80%, respectively.The 5-year-survival was 80% for patients with lowMACC1, compared to 15% for patients with high MACC1expressions. MACC1 in tumor specimens is an independentprognostic indicator for metastasis andmetastasis-free survival. For clinical practice, MACC1 isan important prognostic gene for early identification ofhigh-risk subjects with colorectal cancer, and a promisingnew target for intervention of metastasis formation.Inhibition of colon cancer metastasis by smallmolecules targeting the metastasis progressorS100A4U. Stein, U. Sack, W. Walther, P.M. Schlag. In cooperationwith W. Birchmeier, I. Fichtner (MDC), N. Scudiero, M. Selby(SAIC, Frederick, MD), Robert H. Shoemaker (NationalCancer Institute-Frederick, MD)The calcium-binding protein S100A4 is involved inmetastasis formation and aggressive tumor growth inmany types of cancer. We identified S100A4 as a Wnt/βcatenintarget gene which induces migration, invasionHGF-induced translocation of MACC1 from the cytoplasm into thenucleus in human colon cancer cells, detected by immunofluorescence(bar 50 µm). Stein U, Walther W, Arlt F, Schwabe H, Smith J, Fichtner I,Birchmeier W, Schlag PM. MACC1, a newly identified key regulator ofHGF/Met signaling, predicts colon cancer metastasis. Nature Med15:59-67, 2009and angiogenesis. Hence, its suppression bears potentialfor therapeutic intervention of metastasis.Here we report the identification of small moleculeinhibitors which significantly reduce S100A4 expressionin colorectal carcinoma cells. These inhibitors wereidentified from a high throughput screening of 1,280compounds, employing the S100A4 gene promoterreporter system, and by targeting key molecules of theWnt/β-catenin signaling pathway.Functional assays with selected and validated compoundsrevealed that proliferation rates were diminishedupon treatment while cell viability was only slightlyaffected. More strikingly, migration and invasion ratesof treated cells were significantly decreased by theseinhibitors, but could be rescued by overexpressingS100A4 cDNA. The impact of these small molecules onmetastasis formation in vivo was demonstrated withsmaller and fewer metastases per animal. In summary,our findings present a new strategy to restrict S100A4induced metastasis formation in colon cancer.Clinical application of nonviral gene therapy forlocal treatment of solid tumorsW. Walther, D. Kobelt, R. Siegel, J. Aumann, S. Burock,U. Stein, P. M. Schlag. In cooperation with M. Dietel (Instituteof Pathology, Charité), M. Schleef (PlasmidFactory,Bielefeld), A. Menne (EMS Medical, Nyon, Switzerland)98 Cancer Research

For the delivery of naked DNA into cells or tissues agreat variety of procedures is employed in vitro and invivo. Among other physical delivery systems jet-injectionhas developed to an applicable gene transfer technology,which allows gene transfer of small amounts ofnaked DNA into different tissue types with deeper penetrationand improved dispersion.At the Clinic for Surgical Oncology, Charité, Berlin aphase I clinical trial (DeReGe 62) was conducted to evaluatesafety, feasibility and efficiency of intratumoral jetinjectiongene transfer of β-galactosidase (LacZ)-reporter expressing plasmid-DNA into skin metastasesfrom breast cancer and melanoma. Seventeen patientswere enrolled and treated with jet-injection into a singlecutaneous lesion. In the study the safety and efficiencyof the jet-injection gene transfer was shown. Thestudy revealed efficient LacZ mRNA- and proteinexpressionin all treated lesions and rapid clearance ofplasmid-DNA from patient’s blood. The treatment waswell tolerated by all patients and no side effects wereexperienced, indicating clinical applicability of this nonviralapproach for local tumor gene therapy.Use of the minimalistic MIDGE vector forimproved safety, gene transfer and expression inclinical application of gene therapyW. Walther, S. Burock, D. Kobelt, J. Aumann, U. Stein,P. M. Schlag. In cooperation with B. Wittig and M. Schmidt(MOLOGEN AG, Berlin), U. Trefzer (Charité) and I. FichtnerIn result of our clinical gene transfer trial we currentlymake great efforts for further optimization of the nonviralvector by removing unnecessary sequences for theimprovement of transfer- and expression efficiency. Theminimalistic immunologically defined gene expression(MIDGE, Mologen AG, Berlin) vector system provides anonviral linear DNA molecule that is depleted of anybacterial origin, antibiotic resistance sequences or replicationbackbone sequences. This makes MIDGE of highvalue with respect to regulatory requirements for productsafety in clinical applications. Thus, MIDGE vectorsare substantially reduced in size, resulting in highertransfer efficiencies and improved transgene expression.Our in vitro analyses in different human tumor celllines revealed pronounced increase in transfer efficiencyand in transgene expression of the MIDGE vector systemcompared to plasmid-based vectors. The use of thissystem for expression of therapeutic genes, such ashuman TNF-α, mediated high-level expression in vitroand more importantly in vivo and resulted in effectiveantitumoral effects. These expression characteristicsmake MIDGE a good candidate for use in clinical genetherapy applications.Based on our pre-clinical and clinical experiences onnonviral gene transfer a new phase I trial will be initiatedto evaluate safety and efficiency of nonviral jet-injectionapplication of the TNF-α expressing MIDGE-basedvector in patients with skin metastasis from mela -noma. This dose-escalation study will reveal, whetherthe MIDGE-vector is efficiently expressing TNF-α afterthe intratumoral application by jet-injection. It will beimportant to correlate the applied vector dose and theamount of TNF-α expressed in the tumor lesion. Thesafety of the expressed TNF-α and the vector-DNA doseapplied will be examined and will be tested in a clinicalphase II trial, to evaluate the safety and efficacy ofthis approach for the combined modality treatment ofpatients with malignant melanoma.Selected PublicationsStein, U., Walther, W., Arlt, F., Schwabe, H., Smith, J., Fichtner, I., Birchmeier,W., Schlag, P.M. (2009) MACC1, a newly identified key regulator of HGF-MET signaling, predicts colon cancer metastasis. Nat Med. 15, 59-67Fritzmann J, Morkel M, Besser D, Budczies J, Kosel F, Brembeck FH, Stein U,Fichtner I, Schlag PM, Birchmeier W. (2009) A colorectal cancer expressionprofile that includes transforming growth factor beta inhibitor BAMBIpredicts metastatic potential. Gastroenterology 137; 165-175Kemmner W, Wan K, Rüttinger S, Ebert B, Macdonald R, Klamm U, MoestaKT (2008) Silencing of human ferrochelatase causes abundant protoporphyrin-IXaccumulation in colon cancer – a new tool for molecular imaging.FASEB J 22, 500-509Walther W, Siegel R, Kobelt D, Knösel T, Dietel M, Bembenek A, Aumann J,Schleef M, Baier R, Stein U, Schlag PM. (2008) Novel jet-injection technologyfor nonviral intratumoral gene transfer in patients with melanomaand breast cancer. Clin Cancer Res 14, 7545-53.Stein, U., Arlt, F., Walther, W, Smith, J., Waldman, T., Harris, E.D., Mertins,S.D., Heizmann, C.W., Allard, D., Birchmeier, W., Schlag, P.M., and Shoemaker,R.H (2006). The metastasis-associated gene S100A4 is a novel target ofβ-catenin/T-cell factor signalling in colon cancer. Gastroenterology 131,1486-1500.PatentsKemmner W, Schlag PMMicroarray für das Expression Profiling der kompletten zellulärenGlykosylierungsmaschineriePatent application: 9.10.2008Stein U, Lange C, Walther W, Schlag PM.Use of the human LRP/MVP promoter for a vector that can be induced bytherapy.Patent EP 1332219 10.5.2006US 7,297,535 20.11.2007Stein U, Schwabe H, Walther W, Schlag PM.Verwendung des neu-identifizierten Gens 7a5/Prognostin fürTumordiagnostik und Tumortherapie.7a5/Prognostin and its use for tumor diagnostics and therapy.Patent application US 10/564,823 2006Stein U, Walther W, Arlt F, Schlag PM.Methods for diagnosing metastasis by analysing mutations in β-catenin.Patent application US 60/847,547 2006Patent application EP 008333 2007Cancer Research 99

Structure of the GroupClemens A. SchmittGroup LeaderProf. Dr. Clemens Schmitt, M.DScientistsDr. Soyoung Lee, Ph.D.Dr. Maurice Reimann, Ph.D.Dr. Mathias Rosenfeld, M.D.Dr. Eva Wönne, Ph.D.Bianca Teichmann, M.Sc.Graduate StudentsHenry DäbritzJan DörrJing DuAnja HaugstetterYong YuTechnical AssistantsNadine BurbachCarmen JudisSven MaßwigSandra WegenerCancer Genetics and Cellular StressResponses in Pathogenesis andTreatment of Lymphatic MalignanciesOur research program is driven by our interest in cellular stress responses (so called ‘failsafemechanisms’) that may serve as anti-tumor barriers when challenged by transformingoncogenes, and, in turn, must be bypassed or inactivated before a full-blown malignancy canactually form. Importantly, ultimate stress responses such as apoptosis or cellular senescence –both terminal ‘cell-cycle exit’ programs – do not only counter tumorigenesis, but are utilized aschemotherapy-induced stress responses as well. Hence, principles of oncogenesis andmechanisms of treatment sensitivity seem to critically overlap and impinge on each other duringtumor formation, cancer therapy and relapsed or progressive disease conditions. Moreover, recentevidence points towards interferences between cell-autonomous failsafe programs and the tumorenvironment, which, in turn, evoke feedback mechanisms that may significantly alter tumorbiology. To test the impact of genetic lesions in cellular stress response programs on tumordevelopment and treatment outcome under most physiological conditions in vivo, we generatemouse models harboring lymphomas (and other tumor entities) with defined genetic lesions.FoxO transcription factors suppress Myc-drivenlymphomagenesis via direct activation of ARFSoyoung Lee and collaboration partnersFoxO transcription factors regulate numerous cellularfunctions including proliferation, stress sensitivity, andcellular survival. Conditional co-deletion of FoxO familiymembers 1, 3 and 4 in the absence of a defined oncogenicstimulus unveiled a context-dependent cancerpronephenotype after long latencies. We investigatedin an oncogene-driven setting, namely the Eµ-myctransgenic mouse model, whether ablation of FoxOfunction via transduction of hematopoietic stem cellswith a dominant-negative FoxO moiety would give riseto accelerated tumor onset. We not only demonstrateda tumor-suppressive role of FoxO transcription factorsin Myc-induced lymphomagenesis but showed geneticallyan interference of FoxO function with the p53pathway. Precisely, we found FoxOs to bind to theINK4a/ARF locus and to induce ARF expression whenMyc is constitutively expressed. Thus, FoxO transcriptionfactors have tumor-suppressive potential and actby promoting ARF expression in response to oncogenicactivation – a pivotal pathway with FoxOs now identifiedto mediate the missing link between Myc activationand ARF induction.Oncogenic signaling evokes a DNA damageresponse that is selected against in manifestlymphomasMaurice Reimann, Ines Schildhauer, Bianca Teichmann,Bernd Dörken and collaboration partnersIn addition to the ARF/p53 pathway, the DNA damageresponse (DDR) has been recognized as another oncogene-provokedanti-cancer barrier in early humantumorigenesis leading to apoptosis or cellular senescence.DDR mutations may promote tumor formation,100 Cancer Research

ACBDMyc and FoxO transcription factors co-operate in tumor-suppressiveARF induction. We tested the questions whether FoxO transcriptionfactors possess tumor-suppressive potential in Myc-driven tumordevelopment and whether a dominant-negative FoxO moiety(dnFoxO) may act via interference with the ARF/p53 pathway (A). Eµmyctransgenic, p53 +/+ or p53 +/- hematopoietic stem cells were stablytransduced with dnFoxO or mock, and lymphoma onset was monitored.Note that p53 +/+ ,dnFoxO lymphomas formed much faster thancontrols (i.e. p53 +/+ lymphomas without dnFoxO), and arose virtuallyindistinguishable from the p53 +/- group were lymphomas typically losethe remaining p53 allele (‘p53null’) (B). ARF expression is detectable inlymphoma sections independent of their p53 status, but only in theabsence of the dnFoxO moiety (C). FoxO transcription factors bind tothe FBS2 consensus site in the INK4a/ARF promoter, but only inductionof Myc, not activation of FoxOs by the PI3 kinase inhibitor LY294002(both resulting in equal binding of FoxOs to FBS2 [data not shown]),promote ARF expression (D).but their impact on treatment outcome remainsunclear. In this study, we generated Atm (ataxia telangiectasiamutated)-proficient and -deficient B-cell lymphomasin Eµ-myc transgenic mice to examine the roleof DDR defects in lymphomagenesis and treatmentsensitivity. Atm inactivation accelerated developmentof lymphomas, and their DNA damage checkpointdefects were virtually indistinguishable from thoseobserved in Atm +/+ -derived lymphomas that spontaneouslyinactivated the pro-apoptotic Atm/p53 cascadein response to Myc-evoked reactive oxygen species(ROS). Importantly, acquisition of DDR defects, but notselection against the ARF pathway, could be preventedby lifelong exposure to the ROS scavenger N-acetyl-cysteine(NAC) in vivo. Following anticancer therapy, DDRcompromisedlymphomas displayed apoptotic, but, surprisingly,no senescence defects, and achieved a muchpoorer long-term outcome when compared to DDRcompetentlymphomas treated in vivo. Hence, Atmeliminates pre-neoplastic lesions by converting oncogenicsignaling into apoptosis, and selection against anAtm-dependent response promotes formation of lymphomaswith predetermined treatment insensitivity.Apoptotic lymphoma cells trigger TGF-β secretionof host macrophages that results in tumorsuppressivelymphoma cell senescenceMaurice Reimann, Soyoung Lee, Jan Dörr, Bernd Dörkenand collaboration partnersActivated RAS/BRAF oncogenes induce cellular senescenceas a tumor suppressive barrier in early cancerdevelopment – as recently demonstrated by our group(Braig et al., Nature, 2005) – at least in part, via an oncogene-evokedDNA damage response (DDR). In contrast,Myc activation – although producing a DDR as well – isknown to primarily elicit an apoptotic countermeasure.Using the Eµ-myc transgenic mouse lymphoma model,we found that both a cell-autonomous DDR-dependentand a non-cell-autonomous transforming growth factor–(TGF-β)-mediated induction of cellular senescencelimits Myc-driven tumor development. While acuteinduction of Myc signaling is sufficient to produce lymphomacell senescence in vitro, we identified TGF-β asthe pivotal senescence trigger in vivo. We demonstratethat TGF-β is not secreted by lymphoma cells but isdelivered by macrophages upon their activation byapoptotic lymphoma cells. These findings, detectable inhuman aggressive B-cell lymphomas as well, establisha novel network of heterotypic cell-cell interactions inwhich apoptotic tumor cells launch a paracrineresponse in non-malignant bystanders that limitstumorigenesis by cellular senescence.Selected PublicationsBouchard*, C., S. Lee*, V. Paulus-Hock, C. Loddenkemper, M. Eilers and C.A.Schmitt. 2007. FoxO transcription factors suppress Myc-driven lymphomagenesisvia direct activation of ARF. Genes Dev. 21: 2775-2787. *) equalcontributionKilic, M. and C.A. Schmitt. 2008. Exploiting drug-induced senescence intransgenic mouse models. In Beyond apoptosis – cellular outcomes ofcancer therapy; edited by Roninson, I.B., J. M. Brown and D.E. Bredesen.Informa Health Care USA, Inc. – New York, London: 273-294.Reimann, M., C. Loddenkemper, C. Rudolph, I. Schildhauer, B. Teichmann, H.Stein, B. Schlegelberger, B. Dörken and C.A. Schmitt. 2007. The Myc-evokedDNA damage response accounts for treatment resistance in primary lymphomasin vivo. Blood 110: 2996-3004.Reimann, M., C. Loddenkemper, S. Lee, Dörr, J., Tabor, P. Aichele, V., H. Stein,B. Dörken, T. Jenuwein and C.A. Schmitt. 2009. Tumor-stroma-derived TGF-βlimits Myc-driven lymphomagenesis via Suv39h1-dependent senescence.In press.Schmitt, C.A. 2007. Cellular senescence and cancer treatment. Biochim.Biophys. Acta 1775: 5-20.Cancer Research 101

Structure of the GroupGroup LeaderDr. Thomas SommerScientistsDr. Christian HirschDr. Ernst JaroschDr. Birgit MeusserThomas SommerMechanisms of Protein Quality ControlThe endoplasmic reticulum (ER) is a cellular organelle through which a significant proportionof proteins pass on their way to their functional sites in membranes, exocytic and endocyticcompartments, or the cell exterior. Far from being a passive traffic way, the ER is home to anarray of molecular chaperones, which help proteins to fold and guide their maturation. Despitethis support, protein biogenesis is an error-prone process. A considerable fraction of all newlysynthesized polypeptides fail to attain their native conformation due to mutations,transcriptional and translational errors, folding defects, or imbalanced subunit synthesis.Mature proteins can be damaged by environmental stress conditions, such as high-energyradiation, chemical insults, or metabolic by-products. Malfunction or aggregation of defectiveproteins challenges the homeostasis of the ER and the cell as a whole. As a consequence,evolution has produced a protein quality control (PQC) network that operates on several levelsto maintain the integrity of the ER.The work of this group focuses on how the ER proteinquality control system selectively disposes aberrantproteins without jeopardizing nascent polypeptidesthat also populate this compartment. Current data suggestthat polypeptides are initially protected fromdegradation by a specific N-linked glycan structure toallow their maturation. Later, ER mannosidases generatea unique glycan code that flags potentially misfoldedsubstrates. This signal is decoded by an ubiquitin ligaseanchored in the ER membrane. Proteins committedfor degradation are transported across the ER membranein a process termed protein dislocation.Subsequently, substrate molecules are ubiquitylatedand degraded by the 26S proteasome. This process isreferred to as ER associated degradation or ERAD. Butmisfolded proteins are not the only substrates of thissystem. It also regulates sterol synthesis by eliminatingthe pathway’s rate-limiting enzyme when sterols areabundantly available. This example shows that ERADalso has a regulatory component that may not be limitedto this anabolic pathway.Since the ERAD pathway appears to be conserved fromyeast to mammals, we use the model organismSaccharomyces cerevisiae to investigate the fundamentalmechanisms and to identify the key components ofthis important pathway. These are the HRD ubiquitinligase and the Doa10 ubiquitin ligase. The HRD-ligase iscrucial for turnover of membrane-bound (ERAD-M) andER-luminal substrates (ERAD-L). Doa10 targets membraneproteins for degradation that carry lesions intheir cytoplasmic domains (ERAD-C). Both yeast ubiquitinligases and their co-factors that have been identifiedso far are summarized in Fig. 1. The mammaliancounterparts of the yeast components are mentionedas well.Usa1 Functions as a Scaffold of the HRD-ligaseSabine Horn, Ernst Jarosch, Christian Hirsch in collaborationwith Jennifer Hanna, Anja Schütz, and Udo HeinemannThe multiprotein HRD-ligase (HMG-CoA ReductaseDegradation) singles out terminally misfolded proteinsof the ER and routes them for degradation to cytoplas-102 Cancer Research

Graduate StudentsKatrin BagolaHolger BrendebachSabine HornLaura JänickeMartin MehnertMarcel NowakFranziska ZimmermannTechnical AssistantsCorinna VolkweinAngelika WittstruckSecretariatSylvia KlahnFigure 1. ER-associated protein degradation in yeast and mammalian cells.Molecular chaperones and proteins of the glycosylhydrolase-47 family (Mns1 andHtm1) detect misfolded polypeptides and direct them to membrane bound ligases(Doa10, RMA1, HRD). After dislocation to the cytosolic face of the ER membrane,substrates are ubiquitylated by an ubiquitin ligase. All ligase complexes comprise acentral, catalytic active RING finger protein (E3), ubiquitin-conjugating enzymes(E2), and additional factors. The AAA ATPase Cdc48 releases ubiquitylated moleculesfrom the ER membrane. The adapter proteins Rad23 and Dsk2 escort the ubiquitylatedsubstrates to the 26S proteasome for degradation. Concurrently Png1 deglycosylatesglycoproteins through its association with Rad23. Proteins containing a glycan-interactionmotif or ubiquitin-binding domains are depicted in red and blue,respectively. Proteins are labeled with their yeast names in blue and green lettersindicate the mammalian counterpart.Cancer Research 103

Figure 2. Processing of N-linked glycans in the yeast and mammalian ER. (a) A glucose 3 -mannose9-N-acetylglucosamine 2 oligosaccharide is initiallyattached to asparagine in NXS/T motifs by the oliglsyccharyltransferase. Gls1 (glucosidase 1) and Gls2 remove the two outmost glucoseresidues and generate a glucose 1 -mannose 9 -N-acetylglucosamine 2 sugar (step 1). Further processing by Gls2 results in a mannose 9 -N-acetylglucosamine2 glycan that protects the glycoprotein from disposal (step 2). Mns1 trims the outmost mannose moiety of the B branch, yielding a mannose8 -N-actelyglucosamine 2 oligosaccharide, which indicates a protein that is retained in the ER for a prolonged period of time (step 3).Subsequently Htm1 processes the C-branch yielding a mannose7-N-actelyglucosamine2 oligosaccharide (step 4). (b) In contrast to yeast, in mammaliancells removal of the residual glucose residue from the A branch by glucosidase-II (GII, step 2) is reversible. GT reglucosylates glycoproteinsthat have not attained their native fold (step 3). Extensive demannosylation by ERManI and probably the EDEM proteins yield a mannose 5 -Nacetylglucosamine2 glycan that is a signal for disposal (step 4). Light blue mannose residues protect the glycoprotein from degradation until theyare removed.mic 26S-proteasomes. Specific functions, like substrateselection and ubiquitylation, are assigned to distinctsubunits of the HRD-ligase. For example substratebinding involves the ER-luminal domain of Hrd3 andthe associated lectin Yos9. Ubiquitylation depends onthe RING-finger protein Hrd1 and the ubiquitin-conjugatingenzyme Ubc7 and its co-factor Cue1. The drivingforce for dislocation is provided by ATP hydrolysis at thehexameric Cdc48 (in mammals p97 or VCP) complex. Itconsists of Cdc48 itself and the associated co-factorsUfd1 and Npl4. This ATPase binds to Ubx2 and in additionto poly-ubiquitylated substrates at the ligases. Twoadditional subunits of the ligase complex have beenidentified, Der1 and Usa1, but their precise function isstill under debate. It is furthermore speculated that theligase forms a channel in the membrane through whichsubstrates are dislocated. However, other channels havebeen discussed as well and direct evidence for thechannel hypothesis is still missing.By biochemical analyses we were now able to showthat Usa1 plays a dual role within the HRD ligase complex:First, it recruits the ancillary factor Der1 and secondit mediates oligomerization of the HRD ubiquitinligase.These separate activities mirror different requirementsfor the processing of malfolded proteins withdistinct topology. On one hand, Usa1 promotedoligomerization of Hrd1 is predominantly required forthe breakdown of membrane proteins while it is dispensablefor the turnover of soluble polypeptides. Onthe other hand, Der1 recruitment, in turn, is a prerequisiteonly for the degradation of soluble substrates. Inthis collaborative work with the group of Udo104 Cancer Research

Heinemann, we could also identify the relevantdomains of Usa1, which are involved in the relevant proteininteractions at the ligase complex. The N-terminusof Usa1 binds the very C-terminal 40amino acids ofHrd1. Binding of Der1 involves the C-terminal region ofUsa1. Thus, Usa1 functions as a scaffold that assemblesthe different activities of the HRD-ligase for processingof different classes substrates that differ in their topology.In a broader sense, our data strengthen the viewthat scaffold proteins modulate ubiquitin ligase activitiesrather then being passive devices.Htm1 protein generates the N-glycan signal forglycoprotein degradation in the endoplasmicreticulumChristian Hirsch in collaboration with Simone Clerc,Daniela Maria Oggier, Paola Deprez, Claude Jakob, andMarkus AebiN-linked glycans are essential for the breakdown of glycoproteins.The covalently attached oligosaccharidestructure is used as a signal to display the folding statusof the protein. Newly synthesized proteins receive aGlc3Man9GlcNAc2 modification. Such a glycan structureprotects a newly synthesized protein from degradation.Subsequently it is trimmed by glucosidases andmannosidases until a specific signal is generated, whichis recognized by the quality control ubiquitin ligase.Since trimming of glycans is slow, these processingsteps provides a time window in which a newly synthesizedprotein can adopt its cognate conformation.In a collaborative effort we were able to define thefunction of Htm1 as an α1,2-specific exo-mannosidasethat generates the Man7GlcNAc2 oligosaccharide witha terminal α1,6-linked mannosyl residue on degradationsubstrates. This oligosaccharide signal is decodedby the ER-localized lectin Yos9 that in conjunction withHrd3 triggers the ubiquitin-proteasome dependenthydrolysis of these glycoproteins. The Htm1 exo-mannosidaseactivity requires processing of the N-glycan byglucosidase I, II and mannosidase I, resulting in asequential order of specific N-glycan structures thatreflect the folding status of the glycoprotein (Fig. 2).Since Htm1 generates the crucial signal that flags a proteinfor degradation, its activity must be tightly controlled.Thus, we searched for associated factors thatcould be involved in controlling Htm1 activity.Surprisingly, we co-purified the protein disulfide isomerasePdi1 together with Htm1. Binding of Pdi1 occursat the Htm1 C-terminus whose function is unknown.Our results raise the sepculation that the activity ofHtm1 is linked to incorrect disulfide bridge formation.Selected PublicationsNeuber, O., Jarosch,E., Volkwein, C., Walter, J., and Sommer, T. (2005)Ubx2/Sel1p links the Cdc48p/p97-Complex to Endoplasmic ReticulumAssociated Protein Degradation. Nature Cell Biol., 7, 993-998.Horn, S., Hanna, J., Hirsch, C., Volkwein, V., Schütz, A., Heinemann, U.,Sommer, T., and Jarosch, E. (2009) Usa1 functions as a scaffold of theHRD-ubiquitin ligase. Mol Cell 36, 782-793Clerc, S., Hirsch, C., Oggier, D. M., Deprez, P., Jakob, C., Sommer, T., and Aebi,M. (2009) HTM1 protein generates the N-glycan signal for glycoproteindegradation in the endoplasmic reticulum. J. Cell. Biol. 184, 159-172Hirsch, C., Gauss, R., Horn, S.C., Neuber, O., and Sommer, T. (2009) The ubiquitylationmachinery of the endoplasmic reticulum. Nature 458, 453-460.Gauss, R., Jarosch, E., Sommer, T., and Hirsch, C. (2006) A complex of Yos9pand the HRD ligase integrates endoplasmic reticulum quality control intothe degradation machinery. Nature Cell Biol., 8,849-854Cancer Research 105

Structure of the GroupHarald SaumweberGroup LeaderProf. Dr. HaraldSaumweberScientistsDr. Dereje NegeriPhD studentsDr. Miao GanShaza Dehne*UndergraduatesAndrea Kiep*Nadine Schweizer*Alexander Glahs**part of the period reportedNuclear Signaling andChromosomal DomainsUsing Drosophila as a model our group investigates chromatin switches that arecrucial for Notch and TGF-β signal transduction and mechanisms involved incompartmentalization of chromatin.Skip/Bx42 dependent target gene and cell cycleregulation related to Notch signalingOn binding transmembrane ligands of adjacent cellsthe Notch receptor splits off its intracellular domain(Notch-IC) which migrates into the nucleus and transientlybinds to its target genes. On binding Notchrecruits an activator complex to genes that previouslywere repressed by a silencing complex bound to a chromatinplatform provided by the conserved CSL proteinsCBF1/Su(H)/Lag3. By exchanging the repressor complexfor the Notch activator complex target genes becomeactivated. However, neither the exact composition ofthe activator and the repressor complexes nor themode of the switching mechanism are as yet known.We previously characterized Bx42/SKIP a conservedchromatin co-regulator protein. As in Drosophila themammalian homologue interacts with nuclear componentsof the Notch pathway like Notch-IC andSu(H)/CBF1. By induced Bx42-RNAi we demonstratedthat these interactions are biologically important sincethe expression of several Notch target genes is dependentof the presence of Bx42/SKIP and tissue specificknock down of Bx42/SKIP results in Notch-like phenotypes.As a precondition for crystallographic studies wecurrently map the interaction sites of Bx42/SKIP withNotch-IC, Su(H) and the Hairless protein. For instance, incontrast to previous work by others we mapped theSkip interaction side on the C-term of Notch-IC downstreamof the ankyrin repeats and the RAM domain.Work is in progress to investigate how the Skip/Notch-IC interaction is translated into the activation of targetgenes Expression of the most conserved part of Skip,the central SNW region, results in dominant negativephenotypes that are suppressed by over-expression ofthe FL Skip protein, by Notch gain of function mutationsor putative Skip interacting proteins and enhanced byNotch loss of function alleles. Over-expression of SNWregion in late larval eye discs results in a small eye phenotypeby specific suppression of the Notch-dependentdivision of retinal precursor cells at the G2-M transitionthat is cyclin A dependent. Comparing the expressionprofile of the Bx42 dominant negative to wild type cellson microarrays we find that a restricted number ofgenes is affected by SNW over-expression, amongstthem Dp, a dimerization partner of E2F and the chromatinrepressor protein Sina. Interestingly, in mammaliancells Bx42/SKIP interacts with and counteractsthe repressive effect of the Rb protein and shows aninteraction with the E2F family of cell cycle regulators.The role of Skip and interacting proteins in cell cycleregulation will be investigated in more detail inDrosophila cell culturesChromatin domains and boundariesTransgenes inserted into the mammalian genome regularlybecome silenced and occasionally hyperactivatedat their site of insertion reflecting the impact of localchromatin structure. Interestingly, elements found atchromosomal domain boundaries shield for sucheffects by providing enhancer blockers or barrier ele-106 Cancer Research

Above: Tip of chromosom3L showing the Z4 in interbands red (IIF) andthe DNA in blue. Inserts indicate the chromatid organisation in bands(30 nm fiber) and interbands (10 nm fiber) respectively. Below: Schemeof the Z4-complex with Chriz- (C), Z4- (Z), MBD-R2- (M) and other yetuncharacterized proteins that results in recruitment of the H3S10kinase Jil-1 and H3S10 phosphorylation (yellow flag) followed by otherchromatin modifications like H3K4 trimethylation (red flag).ments that insulate nearby regulatory elements or thespreading of adjacent heterochromatin. Thus, besidesfor scientific curiosity, the knowledge of chromosomaldomain structure and their boundaries is of immediatemedical importance, in particular in gene therapy.Formation of boundaries is part of a concept, thateukaryotic genomes are organized into functionallyindependent chromosomal domains. A well knownchromosomal domain is the globin domain in man,mouse and chicken whose boundary function essentiallyrequires the CTCF protein. The Drosophila homologuedCTCF is found at a restricted number of interbandson polytene chromosomes and is required forboundary formation in homeotic gene clusters. It interactswith the protein CP190 present at band interbandboundaries At some sites CP190 is essential for dCTCFchromosomal binding. dCTCF and CP190 binding wasmapped genome-wide by ChIP on Chip experimentsand found to be correlated to active promoters andinsulators.On Drosophila polytene chromosomes chromosomaldomains differing in their degree of condensationbecome apparent as a conserved pattern of dark bands(chromatids as >30 nm fibers) and light interbands(chromatids as ~10 nm fibers). The pattern is conservedbetween tissues, suggesting a constitutive chromosomaldomain organisation established and maintainedby chromatin boundaries. By localization, isolating andcharacterizing DNA sequences from band/interbandunits, their associated proteins and histone modificationswe try to understand the process of chromosomaldomain formation. Mutation of the interband specificzinc finger protein Z4 results in a dramatic loss ofband/interband structure, presumably by affecting themaintenance of chromosomal boundaries. The Z4 proteinis complexed with other interband proteins likethose organizing dosage compensation on the X chromosomesand the histone kinase Jil-1 [Gan PhD thesisHumboldt University 2009]. Tethering Jil-1 kinase inchromatin results in local decondensation and Z4-mutations affect interband specific histone H3S10phosphorylation and H3K4 trimethylation. Z4 interactsin a protein complex with the novel chromodomainproteins Chriz and MBD-R2. Chriz is required for Jil-1 andZ4 binding, H3S10 interphase phosphorylation and themaintenance of chromosome structure [Gan PhD thesisHumboldt University 2009]. The DNA regionrequired for Z4/Chriz complex binding was mapped onpolytene chromosomes by high resolution in situhybridization and by ChIP on chromatin of diploid cellswe could show that the complex binds to the sameregion in these cells too, suggesting its general role in aconstituting a chromatin structure that is sharedbetween cells of different tissues.Selected PublicationsBartkuhn M, Straub T, Herold M, Herrmann M, Rathke C, Saumweber H,Gilfillan GD, Becker PB, Renkawitz R. (2009) Active promoters and insulatorsare marked by the centrosomal protein 190. EMBO J. 2009 28, 877-888.Mohan, M., Bartkuhn, M., Herold, M., Philippen, A., Heinl, N., Leers, J., White,R. A. H., Renkawitz-Pohl, R., Saumweber, H., and Renkawitz, R. (2007) EMBOJ 26, 4203-4214-Mendjan, S, Taipale, M, Kind, J, Holz, H, Gebhard, P, Schelder, M, Vermeulen,M, Buscaino, A, Duncan, K, Mueller, J, Wilm, M, Stunnenberg, H,Saumweber, H and Akhtar, A (2006) Nucleoporins are involved in thetranscriptional regulation of dosage compensation in Drosophila. MolCell 21, 1-13.Gortchakov, A., Eggert, H., Gan, M., Mattow, J., Zhimulev, I.F., Saumweber,H. (2005) Chriz, a chromodomain protein specific for the interbands ofDrosophila melanogaster. Chromosoma 114, 54-66.Eggert, H., Gortchakov, A, Saumweber H. (2004) Identification of theDrosophila interband-specific protein Z4 as a DNA binding zinc-fingerprotein determining chromosomal structure. J. Cell Sci. 117, 4253-4264.Cancer Research 107

Structural and Functional GenomicsStructure of the GroupUdo HeinemannGroup LeaderProf. Dr. Udo HeinemannScientistsDr. Ulrich Gohlke*Dr. Ulf LenskiDr. Klaas E.A. MaxDr. Jürgen J. Müller*Dr. Yvette RoskeDr. Jörg Schulze*Dr. Anja SchützMacromolecular Structure andInteractionInside cells the interplay of large and small molecules is responsible for the catalysis ofchemical reactions, the regulation of gene expression and the transport of proteins and othercargo to their sub-cellular destinations. These activities depend on the fine details ofmacromolecular interactions which are amenable to structural characterization at the atomiclevel. We are combining the powerful methodology of X-ray crystallography with biochemicaland biophysical studies of proteins, nucleic acids, carbohydrates, lipids, membranes and smallmetabolites to analyze their structures and interactions. The results of these studies haveyielded insight into the architecture and glycosyl hydrolase activity of bacteriophage tailspikeproteins, the cooperation of transcriptional regulators in controlling gene expression in theconjugative RP4 plasmid, and structural aspects of vesicle tethering to the Golgi apparatus. Ourwork is greatly facilitated by privileged access to the synchrotron storage ring BESSY in Berlinand the Helmholtz Protein Sample Production Facility.Tailspike endoglycosidasesTailed bacteriophages consist of an icosahedral headfilled with double-stranded DNA and a portal channelwith tail machine. In the P22 phage, 12 subunits of thegp1 protein form the portal structure which serves asthe base for the association of 39 additional subunits offour different proteins, gp4, gp9, gp10 and gp26. The sixtailspike proteins (TSP, gp9 of P22) are thought to establishphysical contact with the bacterial host cell afterdegrading the cell-surface glycans.The high-resolution crystal structures of phage Sf6 TSPand phage HK620 TSP show a conserved tripartitearchitecture of these proteins with an α-helical N-terminalregion, a large right-handed β-helix and a β-sheetC-terminal domain. The central β-helix bears distantstructural similarity to cross-β structures known toexist in amyloid deposits. Quite surprisingly, the crystallographicand mutational analysis revealed the locationof the Sf6 endorhamnosidase active site in a cleftbetween the subunits of the trimeric TSP, whereas singlesubunits of both P22 and HK620 are capable of glycanhydrolysis. The C-terminal domain of Sf6 TSP sharesa conserved fold with viral capsid proteins, suggesting acommon evolutionary origin of capsid and tail components.Up until recently, all phage TSP were crystallizedin the absence of their N-terminal domains whichattaches them to the base of the tail machine throughgp10 and gp4. We have now designed a P22 TSP mutantthat yields crystals and will permit the fitting of anintact tailspike protein structure to high-resolutioncryo-electron microscopic images of the tail machine.Nucleic acid-interacting proteinsTranscriptional regulators have to recognize specificsequence motifs in double-stranded DNA. Their activity108 Cancer Research

Graduate StudentsSarbani Bhattacharya*Kerstin BöhmClaudia Maria Haas*Jennifer HannaBettina KönigHarald StrieglChengcheng WangTechnical AssistantsIngrid Berger*Anette FeskeMathias Hoeschen*Silvia Kaminski*Andreas KnespelSilke Kurths*Janett TischerSecretariatBirgit Cloos*part of the period reportedFigure 1. Co-operation of transcriptional regulators in RP4 geneexpression. (A) Crystal structure of the RP4-encoded repressor KorA.The DNA-binding domain including a classical helix-turn-helix motif iscolored yellow, the dimerization region orange. (B) Dimer iza tion andDNA binding of KorA. The two subunits of the dimer are colored in yellowand red. The view is along the DNA double helix. (C) End-to-endstacking of two 18-bp DNA fragments bound to KorA dimers in crystalsof the KorA-DNA complex. The operator sequence is recognizedthrough specific hydrogen bonding of protein sidechains from therecognition helix to base pairs in the major groove of the DNA. Thetwo strands of the double helix are colored green and gray. (D) Modelfor the KorA-KorB interaction in RP4 gene regulation. The flexible N-terminal domains of KorB are predicted to contact the conserveddimerization domain of KorA over a distance which remains nearlythe same for operator midpoints at 32, 33 or 36 bp distance, becauseeach basepair added to the spacer DNA rotates the contact regionstowards each other while increasing the lateral separation.is modulated by binding to small molecules, co-activatoror co-repressor proteins. We are studying transcriptionalrepressor co-operation in a simple model system,the conjugative plasmid RP4. Genes encoded on thisplasmid are under the control of two global repressors,KorA and KorB, that recognize multiple bindings siteseach, O A and O B . At five promoter regions of RP4, an O Asite is near an O B site, and at four of these promotersthe center-to-center distance of these operators isbetween 32 and 36 basepairs. In earlier work, we haddetermined the crystal structure of a central domain ofKorB, KorB-O, bound to double-stranded DNA containingthe O B 1 sequence. KorB was characterized as a proteincontaining natively unfolded regions near its N-terminusand in a segment connecting KorB-O with the C-terminal dimerization domain, KorB-C.Cancer Research 109

Figure 2. Crystal structure of the human golgin p115. The globular head region of p115, p115 GHR , is composed of nine canonical armadillo repeats(ARM1 to ARM9) and an unsual repeat, ARM10, whose additional α-helix (USO) folds into the concave surface of p115 (left). In the crystal, p115 GHRis in a dimeric arrangement with C-termini in close vicinity (center). A model of intact p115 containing the coiled-coil and acidic C-terminalregions (right) closely resembles electron micrographs of human p115 and the homologous yeast Uso1p.The crystal structure of KorA bound to an 18-basepairDNA fragment containing a consensus O A site revealeda classical binding mode in which sidechains from therecognition helix of the KorA helix-turn-helix motifmake specific hydrogen-bonded contacts to basepairsin the DNA major groove. The dimer iza tion domain of ofKorA has a sequence that is conserved in the homolo -gous RP4-encoded proteins TrbA and KlcB. Based on thecrystal structures of DNA-bound KorA and KorB we proposea model for repressor co-operation in RP4 generegulation in which a contact between the flexible N-terminus of KorB with the conserved dimerizationdomain of KorA or TrbA is postulated (Figure 1).According to this model, the flexibility of KorB serves tomeasure the distance between operators along theDNA while allowing for different spatial dispositions ofthe contact sites on KorA or TrbA caused by the doublehelixtwist.Vesicular transport to the Golgi apparatusVesicular transport in eukaryotic cells depends on conservedsets of proteins involved in the sequential stepsof vesicle budding, uncoating, and tethering to the targetmembrane, as well as in membrane fusion andcargo release. Tethering, the process in which a firstphysical contact between vesicle and target membraneis established, is regulated by a small GTPase belongingto the Rab/Ypt family and involves both heteromultimerictethering complexes and factors characterized byextended coiled-coil regions. Vesicle tethering to theGolgi apparatus of human cells thus involves the Rab1GTPase, the transport protein particle (TRAPP) consistingof at least seven different subunits and p115 or othercoiled-coil containing proteins of the golgin family. Weare taking a combined biochemical and structuralapproach to studying the role of these molecules in110 Cancer Research

vesicle transport to the Golgi. Our earlier work hasfocussed on subunits and sub-complexes of TRAPPRecently, the golgin p115 was studied by X-ray diffractionmethods. The crystallized protein fragment comprisedthe globular head region (p115 GHR ) and lacked 50N-terminal residues as well as the coiled-coil and acidictail regions of p115. The crystal structure reveals anarmadillo fold of p115 GHR with nine canonical and oneunusual, C-terminal repeat (Figure 2). This C-terminalrepeat contains an extra α-helix which folds back intothe concave surface of the armadillo region to give theC-terminal half of the armadillo domain a compact,globular appearance. In the crystal, two p115 GHR moleculesare arranged to form a symmetric dimer. A modelfor an intact p115 based on this dimeric arrangement incombination with a coiled-coil region of appropriatelength agrees well with electron microscopic images ofp115 and its yeast homolog, Uso1p. The crystal structureof p115 GHR reveals important determinants of this golgin’sinteraction with components of the COP I vesiclecoat, the conserved oligomeric Golgi (COG) complexand the Rab1a GTPase in vesicular transport at theGolgi.Selected PublicationsBarbirz, S, Müller, JJ, Uetrecht, C, Clark, AJ, Heinemann, U. Seckler, R (2008).Crystal structure of Escherichia coli phage HK620 tailspike: Podoviral tailspikeendo glycosidase modules are evolutionarily related. Mol. Microbiol.69, 303-316.Gräslund, S, Nordlund, P, Weigelt, J, Bray, J, Gileadi, O, Knapp, S,Oppermann, U, Arrowsmith, C, Hui, R, Ming, J, dhe-Paganon, S, Park, H-w,Savchenko, A, Yee, A, Edwards, A, Vincentelli, R, Cambillau, C, Kim, R, Kim, S-H, Rao, Z, Shi, Y, Terwilliger, TC, Kim, C-Y, Hung, L-W, Waldo, GS, Peleg, Y,Albeck, S, Unger, T, Dym, O, Priluski, J, Sussman, JL, Stevens, RC, Lesley, SA,Wilson, IA, Joachimiak, A, Collart, F, Dementieva, I, Donnelly, MJ,Eschenfeldt, WH, Kim, Y, Stols, L, Wu, R, Zhou, M, Burley, SK, Emtage JS,Sauder, JM, Thompson, D, Bain, K, Luz, J, Gheyi, T, Zhang, F, Atwell, S, Almo,SC, Bonanno, JB, Fiser, A, Swaminathan, S, Studier, FW, Chance, MR, Sali, A,Acton, TB, Xiao, R, Zhao, L, Ma, CL, Hunt, JF, Tong, L, Cunningham, K,Inouye, M, Anderson, S, Janjua, H, Shastry, R, Ho, CK, Wang, D, Wang, H,Jiang, M, Montelione, GT, Stuart, DI, Owens, RJ, Daenke, S, Schütz, A,Heinemann, U, Yokoyama, S, Büssow, K, Gunsalus, KC (2008). Protein productionand purification. Nature Methods 5, 135-146.Müller, JJ, Barbirz, S, Heinle, K, Freiberg, A, Seckler, R, Heinemann, U (2008).An inter-subunit active site between supercoiled parallel -helices in thetrimeric tail spike endo rhamnosidase of Shigella flexneri phage Sf6.Structure 16, 766-775.König, B, Müller, JJ, Lanka, E, Heinemann, U (2009). Crystal structure ofKorA bound to operator DNA: Insight into repressor cooperation in RP4gene regulation. Nucleic Acids Res. 37, 1915-1924.Striegl, H, Roske, Y, Kümmel, D, Heinemann, U (2009). Unusual armadillofold in the human general vesicular transport factor p115. PLoS ONE 4,4656.1-4656.8.Protein Sample Production FacilityOur group at MDC closely co-operates with theHelmholtz-Zentrum für Infektionsforschung (HZI) inBraunschweig in the framework of the HelmholtzProtein Sample Production Facility (Helmholtz PSPF).The PSPF offers expertise in protein production forstructural biology using various host systems tailoredto specific experimental requirements. A reliablemethodology for the production of crystallizable proteinsamples in bacterial, insect or mammalian cells hasbeen established since the start of the PSPF, where theBerlin group focusses on parallelized expression cloningand protein production using Escherichia coli whereasthe group at HZI is developing methods that rely oneukaryotic cell culture systems.Recently, structural biologists have increasingly beenlooking past single protein molecules and turned theirattention to structural studies of firm or transient proteininteractions. Various approaches to the productionand characterization of protein complexes for structuralstudies have been evaluated and implemented. Thesetechniques include a set of vectors allowing the coexpressionof an, in principle, unlimited number of protein-codinggenes in E. coli and the analysis of proteinproteinand other protein-ligand interactions in thermofluorassays.Cancer Research 111

Structure of the GroupOliver DaumkeGroup LeaderDr. rer. nat.Oliver DaumkeScientistDr. Katja FälberGraduate StudentsDavid SchwefelSong GaoJanko BrandClaudio ShahChris FröhlichTechnical AssistantsSabine WernerMarion PapstSecretariatBirgit CloosStructure and Mechanism of MembraneremodelingG-proteinsGuanine nucleotide binding proteins (G-proteins) are involved in a diverse range of cellularprocesses including protein synthesis, sensual perception, vesicular transport and signaltransduction cascades. Whereas small G-proteins are molecular switches that cycle between anactive GTP-bound form and an inactive GDP-bound form, large G-proteins of the dynaminsuperfamily are mechano-chemical enzymes that use the energy of GTP hydrolysis to activelyremodel membranes. Members of both groups bind to membranes, and this interaction iscrucial for their function. Our projects aim to elucidate the interaction and reciprocalmodulation of membranes and G-proteins using structural, biochemical and cell-biologicalmethods.1. EHD as a molecular model for membraneremodelling G-ProteinsMembers of the dynamin superfamily are multidomainproteins with an amino-terminal G-domain. Itsfounding member dynamin oligomerises around theneck of clathrin-coated vesicles and induces vesicle scissionin a reaction which is dependent on GTP hydrolysis.The mechanism of vesicle scission is poorly understoodat the molecular level.In this project, we want to establish the less characterisedEHD family as a model system to understandprinciples of membrane remodelling in the dynaminsuperfamily. EHDs comprise a highly conserved eukaryoticprotein family with four members (EHD1-4) inmammals and a single member in C. elegans and D.melanogaster. The proteins are built of an amino-terminalG-domain, followed by a helical domain and a carboxy-terminalEH-domain known to interact with linearpeptide motifs of proteins involved in endocytosis.EHDs can be found at vesicular and tubular structuresin vivo, and EHD family members have been shown toregulate several trafficking pathways including the exitof cargo proteins from the endocytic recycling compartment.We could show that EHD2 binds with low affinity tonucleotides, like other members of the dynamin superfamily.Surprisingly, ATP rather than GTP was bound. Wedemonstrated that EHD2 could also bind to negativelycharged liposomes, and this binding resulted in thedeformation of the liposomes into long tubular structuresaround which EHD2 oligomerised in ring-likeoligomers (Figure 1a). Furthermore, the slow ATPaseactivity of EHD2 was enhanced in the presence of liposomes,which is another typical feature of dynaminrelatedproteins.We solved the crystal structure of an EHD2 dimer in thepresence of a non-hydrolysable ATP analogue (Figure1b). Dimerisation is mediated via a highly conserved surfacepatch in the G-domain. We could show that thelipid-binding sites in each dimer are located at the tip ofthe helical domains and create a highly curved lipidinteraction site, which might contribute to the membraneremodelling activity of EHD2. We further predictedthe architecture of the EHD2 oligomeric ring where20 EHD2 dimers assemble via the G-domain to form atightly packed oligomer (Figure 1c).Having solved the structure, we will continue this projectto understand the structural changes associated112 Cancer Research

acbFigure 1a: EHD2 is tubulatingphosphatidyl-serine (PS)liposomes and oligomerisesin ring-like oligomers asanalysed by negative-stainelectron microscopy.Micrographs of PS liposomesin the absence (top)or presence (bottom) ofEHD2 and 1 mM ATP-γ-S.Figure 1b: Ribbon-typepresentation of the EHD2dimer. The structure ofEHD2 was determined byX-ray crystallography. Thetwo-fold axis is indicted bya dashed line.Figure 1c: Top and side viewof the proposed EHD2oligomer with the lipidbindingsites of EHD2 pointingtowards the liposomesurface. The EH-domainsare omitted for clarity.with ATP binding and hydrolysis. Furthermore, wewould like to obtain structural information of membrane-boundoligomerised EHD2 using electron-paramagneticresonance studies. Finally, we are interestedto study the physiological function of EHD2 using cellularstudies.2. Structure and function of the GIMAP familyThe GIMAP GTPases comprise seven members inhumans which are predominantly expressed in cells ofthe immune system. Some of the members localise tothe mitochondrial membrane and are proposed to regulateapoptosis by regulating the entry of cytochrome cfrom the mitochondria into the cytosol. We will clarifythe exact function of this protein family at the mitochondriaand the interaction with membranes usingstructural, biochemical and cell-biological methods.These results will have implications for several types ofleukaemia in which GIMAP members are overexpressed.3. Structural insights into the antiviral effector MxA.Mx (myxovirus-resistance) proteins are interferoninducedkey effector molecules in the innate immunesystem mediating cellular resistance against a widerange of pathogens including influenza virus. The proteinsbelong to the dynamin superfamily and havebeen shown to tubulate liposomes and oligomerise inrings around the tubulated liposomes. We are interestedto understand the structural details of oligomerisationand to decipher the exact mode of antiviral actionusing a structural approach.Selected PublicationsDaumke O, Lundmark R, Vallis Y, Martens S, Butler PJ, McMahon HM.(2007) Architectural and mechanistic insights into an EHD ATPase involvedin membrane remodelling. Nature 449, 923-927.Henne WM, Kent HM, Ford MG, Hegde BG, Daumke O, Butler PJ, Mittal R,Langen R, Evans PR, McMahon HT. (2007) Structure and analysis of FCHo2F-BAR domain: A dimerizing and membrane recruitment module thateffects membrane curvature. Structure 15, 839-852.Kupzig S, Deaconescu D, Bouyoucef D, Walker SA, Liu Q, Polte CL, DaumkeO, Ishizaki T, Lockyer PJ, Wittinghofer A, Cullen PJ. (2006) GAP1 familymembers constitute bifunctional RAS and RAP GTPase-activating proteins.J Biol Chem 281, 9891-9900.Chakrabarti PP, Daumke O, Suveyzdis Y, Kötting C, Gerwert K, WittinghoferA. (2006) Insight into catalysis of a unique GTPase reaction by a combinedbiochemical and FTIR approach. J Mol Biol 367, 983-985.Daumke O, Weyand M, Chakrabarti PP, Vetter I, Wittinghofer A. (2004). TheGTPase-activating protein Rap1GAP uses a catalytic asparagine. Nature429, 197-201.Cancer Research 113

Structure of the GroupChristof TannertGroup LeaderDr. Christof Tannert(retired, further supporting)Dr. Burkhard Jandrig(ad interim)ScientistsDr. Burkhard JandrigProf. em. Dr. Erhard GeißlerDr. Horst-Dietrich Elvers*Michaela Blankenburg*Lutz Haberland*Student SupportLars KaufmannKatarina Komenska*(University of Presov,Slovakia)*part of the period reportedBioethics and Science CommunicationThe group Bioethics and Science Communication was established in 2002. We analyzeethical, scientific, and social aspects of new health technologies. Areas of conflictconcerning future developments of these technologies are disclosed by the comparison ofscientific expectations with the ongoing social change.Our research focuses on prerequisites and practical questions for communicating scientificchallenges and results to the public in a procedure without preempting results, facilitatingthus an open, informed, and fair discourse.In particular, we are focusing on the evaluation of scientific uncertainties in health technologyassessment by omics technologies, risk perception and risk communication, lay-expertrelationships, and options for decision making.Active participation in health technology assessment by different stakeholders includingexperts and lay people is crucial to our research. By developing and evaluating rationalapproaches, we aim at contributing to new forms of governance and deliberative democracy.Relevant Topics:ethical, legal and social aspects (ELSA) and assessments ofbiomedical technologies, especially of (1) stem cellresearch and (2) radiofrequency electromagnetic fields(RF-EMF) and cancer.IMBA – Implications of Biomedicine for theAssessment of Human Health RisksThe project analyzes how toxicogenomics will trans -form the present risk management framework forbiomedicine. The focus of the research is on the impactof risk characterization, risk perception, and riskcommunication concerning RF-EMF and cancer. IMBA isa project of the “Strategy Group Systems Analysis andTechnology Assessment in the Helmholtz Association”(HGF SO-033 System Analysis). It started in June 2006and will be completed in December 2009. Theproject:· involves research groups and programmes inside theHGF, in close collaboration with German universitiesas well with international centers of excellence andadds critical mass to social science research on theimpacts of toxicogenomic-based risk assessment byinterdisciplinary collaboration between life scienceand social science· contributes to the public debate on the impact oftoxicogenomics on risk assessment throughdialogue with various stakeholders· fosters international collaboration throughinternational networks and conferences.SET-DEV – Science, Ethics and TechnologicalResponsibility in Developing and EmergingCountriesThe project SET-DEV aims in supporting the researchsystems of developing countries and countries withemerging economies, respectively, and especially thosecountries who are interested in developing their ownethical perspective in a more general context of sharing114 Cancer Research

KnowledgeProbabilitiesknownPossible future eventswith known adverseoutcomesWhat can I know?What shall I do?What may I hope?What is the human being?(Immanuel Kant, 1800)ProbabilitiesunknownIgnoranceclosedopenopenclosedI know enoughI knowsomethingI need toknowI do not wantto knowI cannotknow“Certainty” Risk DangerGalileo Effect NescienceThreatacceptedor imposedThreat neitheraccepted orimposedField of uncertaintyAs an outcome of the IMBA project, our group created the so-called “Igloo of Uncertainty”, an epistemic topology for the term in question aswell as a taxonomy of uncertainty (see also http://en.wikipedia.org/wiki/Uncertainty and Tannert et al., 2007).responsibility for scientific and technological researchwith all involved parties. The SET-DEV project uses internationaldialogue, to contribute to capacity building indeveloping countries and emerging economies in thefield of ethics and science. Our group incorporates thespecific aspects of bioethics into this international EUproject.The project is structured in three interconnected stages:· development by a comparison between networks ofEuropean and non-European researchers, includingthose working in emerging and developingcountries (technological responsibility);· implementation of a pilot programme, centred onactions of public dialogue, capacity building andtraining on the themes of ethics and technologicalresponsibility;· definition and dissemination of policy guidelines forstrengthening the ethical orientation of researchsystems.Internet presenceThe website www.bioethik-diskurs.de with interactivediscourse-pages, and the Online-Game GenEthix registersabout 7,000 site visitors per month (May 2009).Selected PublicationsElvers, HD, Jandrig, B, Grummich, K, Tannert, C. (2009). Mobile phones andhealth: Media coverage study of German newspapers on possible healtheffects of mobile phone use. Health Risk Soc. 11, 165-179.Blankenburg, M, Haberland, L, Elvers, HD, Tannert ,C, Jandrig, B. (2009).High-throughput omics technologies: Potential tools for theinvestigation of influences of EMF on biological systems. Curr.Genomics 10, 86-92.Jandrig, B. (2008). The 2008 stem cell research debate in Germany. AICGSGerman-American Issues 9, 37-42.Tannert, C, Elvers, HD, Jandrig, B. (2007). The ethics of uncertainty. In thelight of possible dangers, research becomes a moral duty.EMBO Rep. 8, 892-896.Tannert, C. (2007). The autonomy axiom and the cloning of humans.Hum. Reproduct. Genet. Ethics 13, 4-7.Cancer Research 115

Tumor ImmunologyMartin LippStructure of the GroupGroup LeaderDr. Martin LippScientistsDr. Uta E. HöpkenDr. Gerd MüllerDr. Hossein PanjidehDr. med. Tobias SchulzeDr. Jörg Rossbacher*Dr. Peter Rahn (FACS Operator)Differentiation and Growth Controlin Lymphocyte Development andImmunopathogenesisChemokines are essential regulators of lymphocyte migration throughout the body. Thechemokine system controls lymphocyte recirculation in immune system homeostasis aswell as the activation-dependent and tissue-selective trafficking of lymphocytes and dendriticcells during immune responses. In addition, chemokines are critical factors for the developmentand organization of secondary lymphoid organs. Our main focus is the role of homeostaticchemokine receptors like CXCR5 and CCR7 in lymphoid organ development, systemic immuneresponses, and chronic inflammatory diseases. In addition, we are interested in the immunemodulatory and growth-inducing functions of chemokine receptors encoded by humanherpesviruses, and the function of sphingophospholipid receptors in the immune system.CCR7 mediates homeostatic lymphocyterecirculation and mucosal immunityThe homeostatic chemokine receptor CCR7 controls notonly lymphocyte trafficking to and within secondarylymphoid organs, but also homeostatic migration of Tand B lymphocytes through non-lymphoid peripheraltissues. CCR7 deficiency results in a massive accumulationof T- and B-lymphocytes in the peritoneal cavity.Mechanistically, the increase in the number of peritoneallymphocytes is caused by an impaired egress ofCCR7-deficient lymphocytes from body cavities. Mostinterestingly, a disturbed peripheral recirculation oflymphocytes also resulted in the development ofectopic lymphoid-like follicles and age-dependenthistopathological changes in the gastrointestinal tractof CCR7-deficient mice. Since the formation of ectopicfollicles precedes the development of epithelial histomorphologicalchanges, we suggest that crosstalkbetween lymphoid aggregates and their adjacentepithelial tissue might contribute to the developmentof hypertrophic gastropathy. The underlying molecularmechanisms have been addressed by performingexpression profiling of differentially expressed genesbetween wild-type epithelial tissue and CCR7 -/- epithelialtissue or mucosal lymphoid aggregates. Severaltranscription factors and signaling molecules wereselected on basis of the microarray data and are furthercharacterized as potential mediators involved in thiscellular crosstalk.CXCR5-dependent autoantigen-driven lymphoidneo-genesis in a chronic murine model ofrheumatoid arthritisRheumatoid arthritis (RA) is a common autoimmunedisease with unknown etiology that affects around 1%of the population. In RA, chronic inflammation of the116 Cancer Research

Graduate and undergraduatestudentsUta BaddackSven HartmannSusann WinterMathias KochKristina SchradiKatharina WichnerNedjoua MallemJohannes Neuhaus*Felix Oden*Dennis Stauß*Claudia Krause*Elena Babych*Technical AssistantsAndra EisenmannJenny GrobeKerstin KrügerKatrin RäbelHeike SchwedeThorsten Riepenhausen*Florian Weigend*Susanne Scheu*Jens-Philipp Otto*SecretariatDaniela Keyner*part of the period reportedsynovium of diarthrodial joints leads to irreversiblejoint damage, which results in chronic pain and disability.A characteristic feature for RA is the infiltration ofthe synovial tissue by granulocytes and large numbersof mononuclear cells. Another hallmark is the developmentof self-reactive T and B cells, leading to autoantibodyproduction. We have developed a novel combinedmouse model of collagen- and antigen-induced arthritisshowing specific features of the chronic phase inhuman disease. The model is characterized by a rapidformation of ectopic lymphoid follicles as well as antibodiesdirected against collagen type II and citrullinatedpeptides (CCP). CCP-specific antibodies have a highprognostic value in humans and have so far not beendescribed in mouse models of arthritis. However, theprocesses leading to chronic inflammation and thefunction of complex lymphoid microstructures havenot been characterized so far. In this context, we arecharacterizing the role of cytotoxic T lymphocytes(CTLs) and follicular B helper T (T FH ) cells, which inducethe differentiation of B cells into (auto) antibody-producingplasma cells. The development and organizationof these ectopic structures were severely impaired inCXCR5-deficient mice proving its critical role as a signalingmolecule in lymphoid neo-genesis during chronicinflammatory autoimmune diseases. Our results reinforcethe link between chronic inflammation and thegeneration of tertiary lymphoid tissue at extra-nodalsites, which in turn drives local self-antigen-dependentinteraction of memory/effector B and T lymphocytesresulting in aberrant chronic autoreactive immuneresponses.Functional role of CXCR5 in the pathogenesis ofH. pylori-induced chronic inflammatory gastritisA potential relationship between chronic inflammation,establishment of extranodal tertiary follicles andlymphoma pathogenesis has been inferred from gastricMALT lymphomas in humans. In particular, expressionof CXCL13, the ligand for CXCR5, has been associatedwith the formation of ectopic lymphoid follicles and thedevelopment of MALT-lymphoma in H. pylori-infectedpatients. We have analyzed the role of the homeostaticchemokine receptor CXCR5 in the formation of mucosaltertiary lymphoid tissue after H. pylori infection. CXCR5-deficient mice failed to develop lymphoid aggregates inthe glandular stomach and exhibited lower H. pylorispecificserum IgG responses compared to infectedwild-type mice. Thus, the development of mucosal tertiaryectopic follicles during chronic H. pylori infection isstrongly dependent on the CXCL13/CXCR5 signalingaxis.Transcriptional control of follicular B helper T cell(T FH ) differentiationHumoral immunity relies on B cell differentiation intoantibody-secreting plasma cells. Follicular T helper (T FH )cells, a specialized subset of effector T cells, are indispensablefor the generation of these plasma cells, byproviding B cell help during the germinal center (GC)reaction. T FH cells supply signals for the survival, affinitymaturation, isotype switching and immunoglobulinsecretion of GC B cells and are thus important players inthe generation of long-lived, high-affinity antibodysecretingplasma cells as well as memory B cells. Still,the molecular mechanisms through which naïve CD4 Tcells become T FH cells are largely unknown. The projectaims at the identification of critical checkpoints in thedifferentiation of T FH cells in order to better understandthe development of adaptive immune responses. Largescalegene expression analysis of human and murineT FH cells as well as other CD4 T cell populations revealedunique expression patterns for transcriptional regulatorsand signaling molecules belonging e.g. to the deltanotch signaling pathway. Our data suggests that T FHcells constitute a separate lineage of T helper cells witha discrete developmental program and distinguishableeffector function. As cell specialization is oftenCancer Research 117

A novel animal model to study the role of epigeneticmechanisms in immune escape andtumor progressionA: BALB/c-3T3 fibroblasts have been transformedby retroviral transduction of the humanherpervirus 8-encoded oncogenic chemokinereceptor vGPCR. B: vGPCR-transformed BALB/c-3T3 fibroblasts that are tumorigenic in BALB/cnude mice, but as expected fail to inducetumors in their immunocompetent BALB/ccounterparts. C: Tumor fragments obtainedfrom nude mice, which comprise stroma cells ofthe tumor microenvironment, grow progressivelyin immunocompetent BALB/c mice. D: vGPCRexpressingBALB/c cell lines have been establishedfrom grafted tumor fragments underpuromycin selection to get rid of any hostderivedcells. E: Unexpectedly, the tumor-derivedvGPCR-BALB/c cells gave rise to progressivelygrowing tumors in immunocompetent BALB/cmice (Thirunarayanan et al., Oncogene 14:523,2007). Ongoing experiments support ourhypothesis that the passage of vGPCR-3T3 cellstrough nude and immunocompetent miceleads to the induction of chromatin remodelingand epigenetic changes in the tumor-derivedcell lines resulting in immune escape and progressivetumorigenesis.associated with epigenetic changes in chromatin structurewe are performing genome-wide ChiP-on-Chipanalyses to define the landscape of activating/repressinghistone modifications during T FH cell differentiationin order to identify novel regulatory elementsthat may not be readily discernible throughexpression analysis and comparative genomics.The human herpesvirus 8 encoded chemokinereceptor vGPCR triggers progressive tumor -igenicity of fibroblasts in immuno competentBALB/c mice: A novel animal model to study therole of epigenetic mechanisms in immune escapeand tumor progressionThe human herpes virus 8 (HHV-8)-encoded G proteincoupledchemokine receptor (vGPCR) has been implicatedin the pathogenesis of Kaposi´s sarcoma (KS) particularlybecause of its high constitutive signaling activity.We have used retroviral transduction to generatevGPCR-expressing BALB/c-3T3 fibroblasts that aretumorigenic in nude mice, but as expected fail toinduce tumors in their immunocompetent counterparts.However, tumor fragments obtained from nudemice grow progressively in immunocompetent BALB/cmice. Unexpectedly, vGPCR-expressing cells establishedfrom grafted tumor fragments gave rise to tumors inimmunocompetent mice. These tumors exhibit a strikinghistological resemblance to KS including plumpspindle cell morphology, a high degree of vascularizationand brisk mitotic activity. Short interfering RNAdirected at vGPCR abrogated or significantly delayedtumorigenesis of tumor-derived cells in nude mice,demonstrating that the tumor development is specificallydriven by the vGPCR oncogene, but not by other118 Cancer Research

successive oncogenic mutations. We have now comparedgene expression profiles of vGPCR-inducedtumors in immunocompetent and nude mice and celllines established from tumors in immuncompetentmice to the profiles of parental BALB/c-3T3 and vGPCRtransformedBALB/c-3T3 cells. This approach, in combinationwith with ChIP-on-Chip analyses, led to the identificationof genes regulated by epigenetic modificationsrelated to the successive passage of vGPCR-3T3cells in nude and immunocompetent mice. Hence, thisnovel animal model will contribute to our understandingof the role of the tumor microenvironment for theinduction of chromatin remodeling and epigeneticchanges resulting in immune escape and progressivetumorigenesis.Role of sphingophospholipid receptors in theimmune systemThe group of sphingosine-1-phosphate (S1P) receptorscomprises five G protein-coupled receptors mediating awide variety of biological functions. In order to characterizethe as yet unidentified in vivo function of the S1P 4receptor that was initially described in our laboratory,we have created and analysed two different S1P 4 -deficientmouse models, including a lacZ knock-in reporterstrain. The phenotype of these S1P 4 -deficient animalssuggest a role of S1P 4 in megakaryocyte maturation aswell as in T cell biology. The biological behavior of S1P 4 -deficient lymphocytes suggests furthermore an intricateinteraction of the two S1P receptors predominantlyexpressed on lymphocytes, S1P 1 and S1P 4 . Our resultsSelected PublicationsWengner, AM, Höpken, UE, Petrow, PK, Hartmann, S, Schurigt, U, Bräuer, R,and Lipp, M. (2007) CXCR5- and CCR7-dependent lymphoid neo-genesis ina murine model of chronic antigen-induced arthritis. Arthritis & Rheum.56, 3271-3283Thirunarayanan N, Cifire F, Fichtner I, Posner S, Benga J, Reiterer P,Kremmer E, Kölble K, Lipp M. (2007) Enhanced tumorigenicity of fibroblaststransformed with human herpesvirus 8 chemokine receptorvGPCR by successive passage in nude and immunocompetent miceOncogene. 14, 523-532Achtman AH, Höpken UE, Bernert C, Lipp M. (2009). CCR7-deficient micedevelop atypically persistent germinal centers in response to thymusindependenttype 2 antigens. J. Leucoc. Biol., 85, 409-417. [Epub 2008Dec 12]Buonamici S, Trimarchi T, Ruocco MG, Reavie L, Cathelin S, Mar BG, KlinakisA, Lukyanov Y, Tseng JC, Sen F, Gehrie E, Li M, Newcomb E, Zavadil J,Meruelo D, Lipp M, Ibrahim S, Efstratiadis A, Zagzag D, Bromberg JS,Dustin ML, Aifantis I. (2009) CCR7 signalling as an essential regulator ofCNS infiltration in T-cell leukaemia. Nature, 459: 1000-1004Wolf SA, Steiner B, Wengner A, Lipp M, Kammertoens T, Kempermann G.(2009). Adaptive peripheral immune response increases proliferation ofneural precursor cells in the adult hippocampus. FASEB J., 23, 3121-3128[Epub 2009 May 11]show for the first time an in vivo function of the S1P 4receptor. Our results identify the S1P 4 receptor as apotential therapeutic target in clinical situation wherean increased generation of platelets is required (i.e.massive bleeding) or, at the opposite, reactive thrombocytosisis to be prevented (i.e. thromobogenic clinicalsettings).Cancer Research 119

Structure of the GroupGroup LeaderPD Dr. Uta E. HöpkenKristina SchradiSusann WinterUta E. Höpken(Delbrück Fellow)Graduate StudentsAngela MensenKatharina WichnerTechnical AssistantsKatrin RäbelHeike SchwedeRegulatory Mechanisms of LymphocyteTrafficking in Homeostasis andImmunopathogenesisRegulated lymphocytic recirculation is pivotal in immune system homeostasis andimmunopathogenesis. Our work is focused on the role of the chemokine/chemokinereceptor system in homeostatic lymphocytic recirculation, systemic and mucosal immuneresponses, and lymphoid neo-organogenesis during chronic inflammatory or infectiousdiseases. We further focus on the molecular mechanisms of immune surveillance inpreclinical mouse models for B cell lymphoma.Lymphocytic homeostasis and mucosal immunityChemokine receptors regulate peripheral homeostaticlymphocyte recirculation and mucosal immunityRecently, we showed that the chemokine receptor CCR7controls not only lymphocyte trafficking to and withinsecondary lymphoid organs, but also homeostaticmigration of T and B lymphocytes through non-lymphoidperipheral tissues. CCR7 deficiency results in massiveaccumulation of T- and B-lymphocytes in the peritonealcavity. Mechanistically, an increase in peritoneallymphocyte numbers is caused by impaired egress ofCCR7-deficient lymphocytes from body cavities. Mostinterestingly, disturbed peripheral recirculation of lymphocytesalso resulted in the development of ectopiclymphoid-like follicles and age-dependent histopathologicalchanges in the gastrointestinal tract of CCR7-deficient mice. Since the formation of ectopic folliclesprecedes the development of epithelial histomorphologicalchanges, we suggest that crosstalk betweenlymphoid aggregates and their adjacent epithelial tissuemight contribute to the development of hypertrophicgastropathy.The underlying molecular mechanisms have beenaddressed by performing expression profiling of differentiallyexpressed genes between wild-type epithelialtissue and CCR7 -/- epithelial tissue or mucosal lymphoidaggregates. Several transcription factors and signalingmolecules were selected on basis of the microarraydata and are further characterized as potential mediatorsinvolved in this celluar crosstalk.Chemokine/chemokine receptor function ininfectious gastrointestinal cancer modelsThere is considerable evidence for the involvement ofhomeostatic chemokines in the formation of tertiarylymphoid tissues during chronic inflammatory processessuch as rheumatoid arthritis and Helicobacter pyloriinducedgastritis. A potential relationship betweenchronic inflammation, establishment of extranodal tertiaryfollicles and lymphoma pathogenesis has beeninferred from gastric MALT lymphomas in humans. Wehave analysed the role of the homeostatic chemokinereceptor CXCR5 in the formation of mucosal tertiarylymphoid tissue after H. pylori infection. CXCR5-deficientmice failed to develop lymphoid aggregates in theglandular stomach and exhibited lower H. pylori-specificserum IgG responses compared to infected wild-typemice. Thus, the development of mucosal tertiary ectopicfollicles during chronic H. pylori infection is stronglydependent on the CXCL13/CXCR5 signaling axis.To date, we study the function of additional chemo -kine/chemokine receptors during the pathogenesis of120 Cancer Research

CXCR5 drives the development of ectopic follicles in the gastric mucosa inH. pylori-infected mice. Histological analysis of paraffin embedded stomachsof CXCR5 -/- compared to wild type mice 5-6 month after H. pyloriinfection showed that ectopic follicles do not develop in CXCR5 -/- mice.MALT-lymphoma and gastric cancer in in vivo mousemodels.Immunosurveillance and interactions betweentumor cells and its microenvironmentIdentification of a chemokine receptor profile characteristicfor mediastinal large B-cell lymphoma (MLBCL)in cooperation with A. Rehm, I. Anagnostopoulos, H. Stein,and B. Dörken, MDC, Charité, BerlinWe have shown that the most frequent lymphomaentities involving the mediastinum can be diagnosedbased on the combination of five different homeostaticchemokine receptor stainings. In contrast to diffuselarge B cell lymphoma (DLBCLnos) and classicalHodgkin lymphoma, MLBCLs are largely devoid of thehomeostatic chemokine receptors CXCR5 and CCR7, andalso lack CCR6. These findings suggest that extranodallocalization and lack of nodal dissemination in MLBCLare related to the expression of those lymph nodeaddressins. Conversely, expression of these receptors onessentially all B cell neoplasms with a widespread nodaldissemination provide a rationale for the therapeutictargeting of homeostatic chemokine receptors withantibodies or antagonistic compounds.Cellular interactions between lymphoma cells andtheir accessory or tumor stroma cellsin cooperation with A. Rehm, and B. Dörken, MDC, Charité,BerlinBased on our phenotypical analysis of chemokine/chemokine receptor expression in MLBCL, we now focuson the cellular interactions between lymphoma cellsand their accessory or tumor stroma cells. Investi gat -ions on the relationship between lymphoma cells andits local immune environment includes the analysis ofactive immune escape mechanisms in murine B celllymphoma. We backcrossed the transgenic malignantlymphoma mouse strain, Eµ-Myc, onto diversechemokine receptor KO strains, which will help to decipherthe influence of tumor cell homing within a specificlymph node and splenic niche onto disease onsetand progression. A more profound understanding ofmechanisms that may cause non-Hodgkin lymphomato be addicted to the local microenvironment could providenew targets for therapeutic intervention.Immunoregulatory functions of the tumorassociatedantigen EBAG9/RCAS1in cooperation with A. Rehm, and B. Dörken, MDC, Charité,BerlinThis project studies whether the estrogen-inducibletumor-associated antigen, EBAG9, has a concurrentimpact on T cell-mediated tumor immunosurveillance.Cytotoxic T lymphocytes (CTL) are essential forimmuno surveillance and score cells for the display oftumor-derived peptides. For target cell destruction, CTLsemploy polarized secretion of lytic granules. We generatedEBAG9 knockout mice and characterized the consequencesof its deletion in CTL-mediated immuneresponses. Loss of EBAG9 amplifies the release of lyticgranules and confers CTLs with an enhanced cytolyticactivity, in all likelihood through improved formation offusion- and release-competent secretory lysososomes.With regard to tumor immunosurveillance and tumorimmunotherapy, modulating the cell biological roadblocksin T cell activation and cytolytic capacity on a singlecell level emerges as a strategy to increase avidityand to strengthen anti tumor T cell efficiency. Our identificationof the estrogen tunable repressor of cytotoxicT cell activity, EBAG9, will allow us to suppress its activitythrough pharmacological estrogen receptor blockade.Selected PublicationsHöpken, UE, Winter, S, Achtman, AH, Krüger, K, Lipp, M. (2010). CCR7regulates lymphocyte egress and recirculation through body cavities.J. Leukoc. Biol. In press.van de Pavert SA, Goverse G, Vondenhoff MF, Beke P, Höpken UE, Lipp M,Niederreither K, Blomhoff R, Agace WW, Mebius R (2009): CXCL13 is essentialfor lymph node initiation and is induced by retinoic acid and neuronalstimulation. Nature Immunology 10, 1193-1199.Rüder, C, Höpken, UE*, Wolf, J, Mittrücker, H-W, Engels, B, Erdmann, B,Wollenzin, S, Uckert, W, Dörken, B, Rehm, A*. (2009). The tumor-associatedantigen EBAG9 negatively regulates the cytolytic capacity of mouseCD8+ T cell. J. Clin. Invest. 119: 2184-2203*shared corresponding authorRehm, A, Anagnostopoulos, I, Gerlach, K, Broemer, M, Scheidereit, C,Jöhrens, K, Hübler, M, Stein, H, Lipp, M, Dörken, B, Höpken, UE. (2009).Identification of a chemokine receptor profile characteristic for mediastinallarge B-cell lymphoma. Int. J. Cancer 125, 2367-2374Höpken, UE, Wengner, AM, Loddenkemper, C, Stein, H, Heimesaat, MM,Rehm, A, Lipp, M. (2007). CCR7 Deficiency causes ectopic lymphoidneogenesis and disturbed mucosal tissue integrity. Blood 109, 886-95.Cancer Research 121

Thomas BlankensteinStructure of the GroupGroup LeaderProf. Dr. Thomas BlankensteinScientistsDr. Jehad CharoDr. Thomas KammertönsDr. Ibrahim Kocman*Dr. Catarina Leitao*Dr. Liang-Ping LiDr. Joanna ListopadDr. Jan SchmollingerDr. Gerald WillimskyMolecular Immunology andGene TherapyMost of the current experimental cancer models do not reflect the pathophysiology of reallifecancer. Cancer usually occurs sporadically and is clonal in origin. Between tumorinitiation and progression clinically unapparent pre-malignant cells may persist for years ordecades in humans. More recently, mouse models of sporadic cancer have been developed. Themouse germ-line can be engineered with high precision so that defined genes can be switchedon and off in the adult organism, ideally in a locally and timely controlled fashion. However,analysis of the immune response against sporadic tumors requires the knowledge of a tumorantigen.In the last two years our group focused on several areas in cancer immunology. We analysed atwhich point of cancer development T cells become aware of the cancer cells. We asked whetherthe initial recognition of cancer cells by cytotoxic T-lymphocytes (CTL) results in tumor immunityor tolerance. Additionally, we employed different strategies of cellular therapy.Immunogenicity of premalignant lesions is theprimary cause of general cytotoxic T lymphocyteunresponsivenessCancer is sporadic in nature, characterized by an initialclonal oncogenic event and usually a long latency.When and how it subverts the immune system isunknown. We show, in a model of sporadic immunogeniccancer, that tumor-specific tolerance closely coincideswith the first tumor antigen recognition by B cells.During the subsequent latency period until tumorsprogress, the mice acquire general cytotoxic T lymphocyte(CTL) unresponsiveness, which is associated withhigh transforming growth factor (TGF)β 1 levels andexpansion of immature myeloid cells (iMCs). In micewith large nonimmunogenic tumors, iMCs expand butTGF-β 1 serum levels are normal, and unrelated CTLresponses are undiminished. We conclude that (a) toleranceto the tumor antigen occurs at the premalignantstage, (b) tumor latency is unlikely caused by CTL control,and (c) a persistent immunogenic tumor antigencauses general CTL unresponsiveness but tumor burdenand iMCs per se do not.Engineering antigen-specific primary human NKcells against HER-2 positive carcinomasNK cells are promising effectors for tumor adoptiveimmunotherapy, particularly when considering the targetingof MHC class I low or negative tumors. Yet, NKcells cannot respond to many tumors, which is particularlythe case for nonhematopoietic tumors such as carcinomasor melanoma even when these cells lose MHCclass I surface expression. Therefore, we targeted primaryhuman NK cells by gene transfer of an activatingchimeric receptor specific for HER-2, which is frequentlyoverexpressed on carcinomas. We found that these targetedNK cells were specifically activated upon recognitionof all evaluated HER-2 positive tumor cells, includingautologous targets, as indicated by high levels ofcytokine secretion as well as degranulation. The magnitudeof this specific response correlated with the level122 Cancer Research

Graduate and undergraduatestudentsWisam AlsamahKathleen AndersDana BriesemeisterChristian Buschow*Xiaojing ChenDana HoserAna JukicaAnna KruschinskiCynthia PerezJelena PopovicMichael RotheChristian SchönKarin SchmidtChristian SchönTechnical AssistantsKathrin BorgwaldAngelika GärtnerMarkus HenselSabrina HornStefanie KupschTanja SpecowiakMartin Textor*Christel WestenSecretariatKarin KaramatskosSylvia Klahn*part of the period reportedof HER-2 expression on the tumor cells. Finally, thesereceptor transduced NK cells, but not their mock transducedcounterpart, efficiently eradicated tumor cells inRAG2 knockout mice as visualized by in vivo imaging.Taken together, these results indicate that the expressionof this activating receptor overrides inhibitory signalsin primary human NK cells and directs them specificallytoward HER-2 expressing tumor cells both in vitroand in vivo.A safeguard eliminates T cell receptor genemodifiedautoreactive T cells after adoptivetransferBy transfer of T cell receptor (TCR) genes, antigen specificityof T cells can be redirected to target any antigen.Adoptive transfer of TCR-redirected T cells into patientshas shown promising results. However, thisimmunotherapy bears the risk of autoreactive sideeffects if the TCR recognizes antigens on self-tissue.Here, we introduce a safeguard based on a TCR-intrinsicdepletion mechanism to eliminate autoreactive TCRredirectedT cells in vivo. By the introduction of a 10-aatag of the human c-myc protein into murine (OT-I, P14)and human (gp100) TCR sequences, we were able todeplete T cells that were transduced with these myctaggedTCRs with a tag-specific antibody in vitro. T cellstransduced with the modified TCR maintained equalproperties compared with cells transduced with thewild-type receptor concerning antigen binding andeffector function. More importantly, therapeutic in vivodepletion of adoptively transferred T cells rescued miceshowing severe signs of autoimmune insulitis fromlethal diabetes. This safeguard allows termination ofadoptive therapy in case of severe side effects.Selected PublicationsKieback, E., Charo, J., Sommermeyer, D., Blankenstein, Th. And Uckert, W.(2008). A safeguard eliminates T cell receptor gene-modified autoreactiveT cells after adoptive transfer. Proc. Natl. Acad. Sci. USA 105: 623-628.Li, L.-P., Willimsky, G., Seitz, S., Xu, Y., Li, Y., Schwarz, L., Schlag, P. andBlankenstein, Th. (2008). SV40 large T antigen-transformed human primarynormal and cancerous mammary epithelial cells are phenotypicallysimilar but can be distinguished in 3D culture with selection medium.Int. J. Cancer 123: 1516-1525.Willimsky, G., Czeh, M., Loddenkemper, C., Gellermann, J., Schmidt, K.,Wust, P., Stein, H. and Blankenstein, Th. (2008). Immunogenicity of premalignantlesions is the primary cause of general cytotoxic T lymphocyteunresponsiveness. J. Exp. Med. 205: 1687-1700.Kruschinski, A., Moosmann, A., Poschke, I., Norell, H., Chmielewski, M.,Seliger, B., Kiessling, R., Blankenstein, Th., Abken, H. and Charo, J. (2008).Engineering antigen-specific primary human NK cells against HER-2 positivecarcinomas. Proc. Natl. Acad. Sci. USA 105: 17481-17486.Kammertoens, T. and Blankenstein, T. (2009) Making and circumventingtolerance to cancer. Eur. J. Immunology, in press.Cancer Research 123

Structure of the GroupGroup LeaderProf. Dr. Wolfgang UckertScientistsDr. Haike Gelfort*Dr. Elisa KiebackDr. Lilian StärckWolfgang UckertMolecular Cell Biology andGene TherapyThe introduction of T cell receptor (TCR) genes into T cells has been developed over the past yearsas a strategy to induce defined antigen-specific T cell immunity. The potential value of TCR genetherapy was well established in mouse models and the feasibility of infusion of TCR gene-modifiedautologous T cells was shown in first clinical studies. The next steps will be the translation of TCRgene therapy from an experimental technique into a robust and safe clinical strategy.The focus of our group lies on the therapy of cancer and viral diseases using TCR gene therapy.We address questions related to: generation of T cells with new antigen specificity, modificationof TCR genes to generate T cells with high functional avidity, safety aspects of TCR genemodifiedT cells with respect to the recognition of self-antigens, adoptive transfer of TCR genemodifiedT cells in mice as preclinical models, and optimization of TCR transfer vectors.Redirection of T cell antigen specificity by TCRgene transferMatthias Leisegang, Daniel Sommermeyer, Lilian Stärck,Peter Meyerhuber, Haike Gelfort in collaboration withHelga Bernhard, Antonio Pezzutto, Dolores SchendelWe have molecularly cloned TCRα/β genes recognizingtumor-associated antigens (HER2, Mart-1, Melan-A, NY-ESO-1, RCC-26, RCC-53, Survivin, Tyrosinase), virus-specificantigens (CMV, EBV-EBNA3a, EBV-LMP2a), and modelantigens (LCMV-gp33, ovalbumin). Using retrovirus vectors,TCR genes were transferred into T cell lines and primaryT cells of mouse and human origin. TCR-α andβ-chains were expressed on the cell surface and functionalityof transgenic TCRs was demonstrated by antigenrecognition, cytokine secretion and lysis of tumor cells.Designer T cells by TCR optimizationDaniel SommermeyerTo increase the avidity of TCR-redirected T cells, weintroduced different modifications into the TCR genes(codon optimization, additional disulfide bond,murinization of constant regions of human TCR chains)and demonstrated exemplarily for the NY-ESO-1 TCRthat functional T cell avidity can be considerablyimproved (Figure).One of these modifications – “murinization” – whichreplaces the human TCR and TCR constant regions bytheir murine counterparts was analyzed in detail. Usinga series of mouse-human hybrid constructs, we identifiednine amino acids responsible for the improvedexpression of murinized TCRs. Five critical amino acidexchanges were identified in the TCR constant region,with exchange of an acidic glutamic acid (human) for abasic lysine (mouse) at position 18 of the constantregion, being most important. For the TCR constantregion, a domain of four amino acids was sufficient forimproved expression. The minimal murinized TCR variantsenhanced expression of human TCRs by supportingpreferential pairing of transferred TCR chains.Furthermore, usage of minimal murinized TCR chainsimproved the function of transduced primary human Tcells when compared to cells transduced with wild-typeTCRs. For TCR gene therapy, the utilization of minimal124 Cancer Research

Graduate andUndergraduate StudentsMario BunseFlorian Helm*Matthias LeisegangPeter Meyerhuber*Simone ReußDaniel SommermeyerTechnical AssistantsUta FischerJanina HauchwitzKordelia HummelMartina KalupaIrmgard KüttnerMatthias Richter*SecretariatAnette Madel*part of the period reportedFigure Modifications increase the avidity of TCR genemodifiedT cells. A NY-ESO-1-reactive wild-type (wt) TCRwas modified by murinization (mu; substitution of humanTCR constant regions by mouse counterparts), introductionof a cysteine bond (cys), codon optimization (co) orcombinations of modifications. Transduction efficiency(%), TCR expression level (MFI) and fuctionality of TCRgene-modified human PBL after co-culture with antigenpresenting tumor cells (IFN-γ release) were determined.instead of completely murinized constant regions dramaticallyreduces the number of foreign residues andthereby the risk for immunogenicity of therapeutic TCRs.Designer T cells by vector optimizationMatthias Leisegang, Peter Meyerhuber, Elisa Kieback,Daniel Sommermeyer, Simone Reuß in collaboration withHans Stauss,For clinical application of TCR-redirected T cells, efficientfunctional expression of the transgenic TCR is a key prerequisite.We compared the influence of the transgenecassette on the expression and function of the murineTCR P14 (recognizing a LCMV gp33 epitope) and thehuman TCR WT-1 (recognizing an epitope of the tumorassociatedantigen WT-1). We constructed different vectors,in which TCR-α and β-chain genes were (i) linkedby an IRES element, (ii) combined by a 2A peptide or (iii)introduced into two individual retroviral constructs. Wefound that IRES-, but not 2A-employing TCR expressionis hampered in primary T cells, where the transgenicTCR has to compete with endogenous TCR chains.Differences in expression were independent of copynumber integration as shown by quantitative PCR.Thus, linking TCR-α and β-chain genes by a 2A peptideseems superior to an IRES element and to single genevectors for TCR expression and T cell function.Construction of TCR-retrovirus packaging cell linesSimone Reuß, Matthias LeisegangWe constructed suspension cell line-based packagingcells derived from human lymphoblastoid -Jurkat cellsto produce TCR-retroviruses. These cells lack endogenousTCRβ-chains and are unable to present CD3 moleculeson the cell surface. Packaging functions weretransferred into ∆β-Jurkat cells by electroporation ofplasmids encoding the gag-pol gene of murine leukemiavirus (MLV) and the GALV or MLV-10A1 env gene.Cell clones expressing high amounts of gag-pol and envgene products were determined by RT-PCR and Westernblot analysis. Upon introduction of a TCR-encodingretroviral vector, ∆β-Jurkat packaging cells shiftedthrough the expression of the transgenic TCR-chainfrom CD3-negative to CD3-positive cells. CD3 highexpressingpackaging cells were enriched by FACS sortingand produced high-titer TCR-retrovirus containingsupernatant.Selected PublicationsSommermeyer, D, Neudorfer, J, Weinhold, M, Leisegang, M, Charo, J, Engels,B, Nößner, E. Heemskerk, M, Schendel, DJ, Blankenstein, T, Bernhard, H,Uckert, W. (2006). Designer T cells by T cell receptor replacement. Eur. J.Immunol. 36, 3052-3059.Reuss, S, Biese, P, Cosset, FL, Takeuchi, Y, Uckert, W. (2007). Suspensionpackaging cell lines for the simplified generation of T cell receptor encodingretrovirus vector particles. Gene Therapy 14, 595-603.Leisegang, M, Engels, B, Meyerhuber, P, Kieback, E, Sommermeyer, D, Xue,S-A, Reuß, S, Stauss, H, Uckert, W.(2008). Enhanced functionality of T cellreceptor-redirected T cells is defined by the transgene cassette. J. Mol.Med. 86: 573-583.Kieback, E, Charo, J, Sommermeyer, D, Blankenstein, T, Uckert, W. (2008). Asafeguard eliminates T cell receptor gene-modified autoreactive T cellsafter adoptive transfer. Proc. Natl. Acad. Sci. USA, 105: 623-628.Uckert, W, Schumacher, T. (2009). TCR transgenes and transgene cassettesfor TCR gene therapy: status in 2008. Cancer Immunol. Immunother. 58:809-822.Cancer Research 125

Bernd DörkenStructure of the GroupGroup LeaderProf. Dr. Bernd DörkenScientistsDr. Armin RehmDr. Stephan MathasDr. Martin JanzDr. Stephan KreherDr. Friderike Blumenthal-BarbyDr. Constantin Rüder*Graduate StudentsJana WolfBiology and Targeted Therapyof LymphomaStudying the molecular basis underlying lymphoid cell development and differentiation isone of the key approaches to understand the pathways leading to disease. We areinterested in the mechanisms that give rise to the development of human hematologicmalignancies, in particular to that of classical Hodgkin lymphoma (cHL) and anaplastic largecell lymphoma (ALCL). Our work focuses on the characterization of the molecular basis forthe dedifferentiation program of these lymphoid cell-derived malignancies and furthermoreaims to characterize molecular defects that are responsible for tumor cell transformation,proliferation and apoptosis protection. It is the ultimate aim of our work to identify targetsfor the development of new treatment strategies.Characterization of deregulated transcriptionfactor networks in Hodgkin lymphoma andanaplastic large cell lymphomaS. Mathas, M. Janz, B. Dörken in cooperation with H. Stein(Charité)Using classical Hodgkin lymphoma (cHL) as a modelsystem, we are investigating the role of transcriptionfactors in lymphoma development. Malignant transformationof hematopoietic cells is associated with profoundalterations in the transcriptional program resultingin deregulated proliferation, differentiation, andapoptosis. It is our aim to identify genes and transcriptionfactor networks specifically deregulated in malignantlymphoma cells. Our work revealed in HRS cells afunctional disruption of the B cell-specific transcriptionfactor network, which is composed of the transcriptionfactors E2A, EBF, and Pax5. In particular, the B cell-determiningtranscription factor E2A is inhibited by its overexpressedantagonists activated B cell factor 1 (ABF-1)and inhibitor of differentiation 2 (Id2). Importantly,these factors are able to down-regulate the expressionof B cell-specific genes and to allow up-regulation of Blineage-inappropriate genes. We could also show that Blineage-inappropriate genes like IL-21 contribute to theattraction of immune cells supporting the growth ofHRS cells. In addition, the loss of the B cell phenotype inprimary effusion lymphoma (PEL) is based on similarmolecular mechanisms, and in these cells reconstitutionof B cell specific E2A activity resulted in inductionof apoptosis. These data support the concept that theloss of lineage-specific transcription factors in lymphoidcells might be linked to the process of malignanttransformation. Thus, further understanding of thededifferentiation process in lymphoid cells provides abasis for the development of novel targeted therapeuticsfor lymphoma therapy.Identification of survival pathways of lymphomacells for the development of new therapeuticstrategiesM. Janz, S. Mathas, B. DörkenIn continuation of earlier work of our group, we focuson the analysis of the NF-κB and AP-1 transcription factorsystem with respect to apoptosis resistance andproliferation. These pathways are investigated not onlyin HRS of cHL, but also in lymphoma entities such asanaplastic large cell lymphoma (ALCL) and multiplemyeloma. Recent results show that overexpression ofthe NF-κB/IκB family member Bcl-3 constitutes a novelmolecular defect of the NF-κB system in cHL and ALCL.126 Cancer Research

Angela MensenStefanie WittstockBjörn LamprechtKarl KöchertShuang LiKatrin UllrichKathrin D. WursterTechnical AssistantsKerstin GerlachSimone KressmannFranziska HummelBrigitte Wollert-WulfUte Nitschke*part of the period reportedBcl-3 might be involved in apoptosis protection of thesecells. Furthermore, we have shown that AP-1 is involvedin the dedifferentiation process of HRS cells by maintaininghigh expression of the E2A antagonist inhibitorof differentiation 2 (Id2), and the overexpression of theAP-1 family member Fra2 in ALCL cells contributes totheir malignant transformation. The AP-1 activity mightfurther be enhanced by a specific overexpression of theCREB family member ATF3. ATF3 is specifically overexpressedin HRS and ALCL tumor cells and protects atleast HRS cells from apoptosis. In addition, we showedthat p53-dependent apoptosis can be induced in HRScells by the MDM2-antagonist nutlin-3, and thus theactivation of the p53 pathway might represent a noveltreatment strategy for cHL. These data provide insightsinto the deregulated apoptosis and survival signalingpathways in HRS cells.Analysis of the mechanisms leading to formationof the ALCL-specific translocation t(2;5)(p23;q35)S. Mathas, B. Dörken in cooperation with T. Misteli (NCI,Bethesda)Whereas the identification and characterization oftranslocations rapidly increases, little is known aboutthe mechanisms how translocations occur in vivo.Using anaplastic large cell lymphoma (ALCL) with andwithout the characteristic non-random translocationt(2;5)(p23;q35) as model system, we study the mechanismsof formation of t(2;5)(p23;q35) translocations inthese cells. We could show that several genes surroundingthe ALCL translocation breakpoint are deregulatedregardless of whether the tumor contains the t(2;5). Theaffected genes include the oncogenic transcription factorFra2 (located on 2p23), the HLH protein Id2 (2p25)and the oncogenic tyrosine kinase CSF1-receptor(5q33.1), and their up-regulation promotes cell survivaland repression of T cell-specific gene expression programs,a feature that is characteristic for ALCL. By 3Dinterphase FISH analyses we demonstrated that thederegulated genes are in spatial proximity within thenuclear space of t(2;5)-negative ALCL cells, facilitatingtheir translocation. These data suggest that deregulationof breakpoint proximal genes occurs prior to formationof translocation and that aberrant transcriptionalactivity of genome regions is linked to theirpropensity to undergo chromosomal translocations.How to deliver cytotoxic agents during effector Tcell mediated adaptive immune responsesA. Rehm, B. Dörken in cooperation with U.E. Höpken, T.Willnow, MDCEstrogen plays a critical role in the regulation of growth,development and cell type-specific gene expression invarious tissues, including the immune system.Alterations in the response to estrogen are associatedwith hormone-dependent diseases, such as breast andovarian cancer. Adjuvant therapies with estrogenreceptorinhibitors mounted to a tremendous improvementof tumor-free survival among patients, which wasexplained primarily through the inhibition of the proproliferativeeffect of estrogen in tumor cells itself.However, implications for a concurrent impact on theimmune system remained unclear. We have characterizedan estrogen-inducible tumor-associated antigen,EBAG9, through engineered deletion of the gene inmice.In cytotoxic T lymphocytes from these mice, equippedwith lytic granules that undergo polarized transportand exocytosis in a Ca 2+ -dependent manner, release ofcytolytic mediators was enhanced, an effect that translatedinto the rapid clearance of intracellular bacteria.Furthermore, a tumor-peptide labeled target cell populationwas eliminated in an accelerated fashion. Thus,under physiological conditions EBAG9 tempers lymphocytekilling activity. Upon estrogen-induced enhancedgene expression, EBAG9 could act as an inhibitor offull effector T cell activation with an ensuing lack ofimmunosurveillance.While the function of the neuronal sorting receptorsSorLA/LR11 and Sortilin within the neuronal system hasbeen well established, little is known about their role inthe immune system. In a Sortilin-genetically deletedmouse strain we have elucidated that this sortingreceptor plays a pivotal and non-redundant role duringthe cytotoxic effector function. However, unlike EBAG9-Cancer Research 127

ABGene deregulation and its impact on formation of translocations in anaplastic large cell lymphoma (ALCL). (A) Immunohistchemistry of Fra2and Id2 in ALCL carrying (left) or lacking (right) t(2;5). Cells with positive signals are stained in red. Large neoplastic cells show a strong nuclearstaining for Fra2 and Id2, whereas no (Fra2) or only weak (Id2) signals are detectable in the small non-neoplastic cells. (B) Schematic view of thebreakpoint proximal genes up-regulated in ALCL cells showing the localization of FRA2 (located on 2p23), ID2 (2p25), and CSF1R (5q33.1). In thecase of t(2;5), the fusion of NPM (5q35) and ALK (2p23) results in the NPM-ALK fusion gene. Colored bars indicate positions of probes used forFISH in Mathas et al., PNAS, 2009.Activation-dependent intracellular redistribution of EBAG9 toward the immunological synapseEBAG9 (green) relocalizes toward the immunological synapse when cytotoxic T lymphocytes are conjugated withCD3/CD28 coated microbeads, which act as surrogate antigen-presenting cells (opaque structures).CTLs were mixed with CD3/CD28 coated microbeads, incubated for 5 min at 37°C, plated on coverslips, and incubatedfor another 30 min before fixation and permeabilization. Lytic granules were stained with anti-Cathepsin D antibody(red). A non-conjugated and non-polarized T cell is shown in the top row. Polarized T cells with conjugated beads areshown below. Images were obtained with a Zeiss LSM 510 confocal microscope setup. Scale bars, 5 micron. Insets haveadditional magnifications of 2- to 3-fold.128 Cancer Research

deficient mice, both major effector T cell populations,CD8+ cytotoxic T cells and CD4+ Th1 cells, were found tobe affected in Sortilin KO animals.From both animal models we infer that the modulationof cell biological roadblocks in T cell activation and cytolyticcapacity on a single cell level emerges as a strategyto increase avidity and strengthen anti tumor T cell efficiency.Conversely, augmentation of EBAG9 or Sortilinactivity could turn out to be beneficial in the treatmentof autoimmune disorders.Selected publicationsMathas, S, Kreher, S, Meaburn, KJ, Jöhrens, K, Lamprecht, B, Assaf, C, Sterry,W, Kadin, ME, Daibata, M, Joos, S, Hummel, M, Stein, H, Janz, M,Anagnostopoulos, I, Schrock, E, Misteli, T, Dörken, B. (2009). Gene deregulationand spatial genome reorganization near breakpoints prior to formationof transclocations in anaplastic large cell lymphoma. PNAS. 106,5831-5836.Lamprecht, B, Kreher, S, Anagnostopoulos, I, Jöhrens, K, Monteleone, G,Jundt, F, Stein, H, Janz, M, Dörken, B, Mathas, S. (2008). Aberrant expressionof the Th2 cytokine IL-21 in Hodgkin lymphoma cells regulates STAT3signaling and attracts Treg cells via regulation of MIP-3alpha. Blood. 112,3339-3347.Mathas*, S, Janz*, M, Hummel, F, Hummel, M, Wollert-Wulf, B, Lusatis, S,Anagnostopoulos, I, Lietz, A, Sigvardsson, M, Jundt, F, Jöhrens, K, Bommert,K, Stein, H, Dörken, B. (2006). Intrinsic inhibition of E2A by overexpressedABF-1 and Id2 is involved in reprogramming of the neoplastic B cells inclassical Hodgkin lymphoma. Nature Immunol. 7, 207-215. *contributedequallyJanz, M, Hummel, M, Truss, M, Wollert-Wulf, B, Mathas, S, Jöhrens, K,Hagemeier, C, Bommert, K, Stein, H, Dörken, B, Bargou, RC. (2006).Classical Hodgkin lymphoma is characterized by high constitutiveexpression of activating transcription factor 3 (ATF3) which promotesviability of Hodgkin/Reed-Sternberg cells. Blood. 107, 2536-2539.Rüder C, Höpken UE, Wolf J, Mittrücker HW, Engels B, Erdmann B,Wollenzin S, Uckert W, Dörken B, Rehm A (2009). The tumor-associatedantigen EBAG9 negatively regulates the cytolytic capacity of mouseCD8+ T cells. J. Clin. Invest. 119:2184-203Cancer Research 129

Structure of the GroupAntonio PezzuttoGroup LeaderProf. Dr. med. AntonioPezzuttoScientistsDr. med. Jörg WestermannDr. rer. Nat. Tam Nguyen-HoayDr. rer. Nat Oliver SchmetzerDr. rer. Medic Sonia WaicziesGraduate andundergraduate studentsGerd BaldenhoferNina SchmolkaAndreas WilhelmMona SalehTechnical AssistantsMargarete GriesNicole HellmigPeter TangMolecular ImmunotherapyGoal of our work is the implementation of basic research into preclinical models and clinicaltrials. Our strategies cover passive and active immunotherapy. We have recently concludeda pilot clinical study with a gene-modified tumor cell vaccine for treatment of advancedmetastatic renal cancer. A follow-up study by combining this vaccine with antibody therapy tomodulate regulatory T cells (T-regs) is in preparation in patients who responded to therapy withthyrosine kinase inhibitors such as sorafenib and sunitinib.Clinical vaccination studies using dendritic cellsor modified tumor cellsJörg Westermann and J. Kopp, in cooperation with Th.Blankenstein, W. Uckert and D. Schendel (GSF, Munich).In renal carcinoma (RCC), a gene-modified, tumor cellvaccine that expresses co-stimulatory molecules andsecretes Interleukin-7, generated in cooperation withTh. Blankenstein and W. Uckert (MDC) as well as D.Schendel (GSF, Munich) has been produced according toGMP rules in our laboratory at the Robert-Rössle Building.The pilot trial has been safely concluded. Based onour recent data concerning the numbers of regulatory Tcells in RCC patients undergoing therapy with the multikinaseinhibitors sorafenib and sunitinib we are planninga follow-up trial in conjunction with antibodiesthat increase T-cell stimulation by reducing regulatoryT-cells.In a recently concluded clinical vaccination trial using invitro-generated dendritic cells (DC) in patients withchronic myeloid leukemia (CML) we have seen reductionin the tumor load in 50% of the patients. A follow-upmulticenter national trial is starting in fall 2009 in CMLpatients with clinical but not molecular remission upontreatment with the tyrosine-kinase inhibitor imatinib.DNA Vaccination: preclinical modelsJörg Westermann, Tam Nguyeng Hoay and Sonia Waicziesin cooperation with U. Höpken and M. Lipp andPhysikalische Bundesanstalt, BerlinDNA vaccination offers several advantages over the useof peptides as vaccines (DNA covers several MHC-I andMHC-II epitopes, directly targets the endogenous presentationpathway and contains immunostimulatoryCpG sequences). In cooperation with the group of M.Lipp (Molecular Tumor Genetics) we have explored thepossibility of recruiting immune cells at the vaccine siteby inserting DNA sequences coding for the chemokinesCCL19 and CCL21 as possible adjuvants. Coexpression ofCCL19 with tumor antigens results in enhancement of aTh1-polarized immune response with substantialimprovement of the protective effect of the vaccine.Further improvement of the vaccine potency has beenachieved using a novel gene-gun for vaccine application(Cooperation with the Robert-Koch Institute). We haveset up a preclinical model using the human breast-cancerassociated antigen Her-2 as a target antigen toexplore the potential use of a vaccine for breast cancerpatients. S. Vaiczies is exploring magnetic resonanceimaging to study dendritic cell migration in vivo.Immunity against EpCam (Epithelial Celladhesion molecule) / CD3 kappa as animmunomodulatory polypeptideOliver Schmetzer in cooperation with P. Schlag andG. Moldenhauer (DKFZ)Heteroclitic peptides derived from the Epithelial AdhesionMolecule EpCam appear able to induce a strong T-cell stimulation by upregulating the TCR. A splice vari-130 Cancer Research

ant of CD3 delta coding for a 45-mer polypeptideappears to mediate the increase of the T-cell receptordensity. Characterization of this polypeptide and evaluationof its properties as an immunomodulatory agentis ongoing.Adoptive T-cell therapy for lymphomasTuan Duc Nguyen and Nina Schmolka in cooperation withT. Blankenstein and W. UckertHodgkin, Nasopharyngeal Carcinomas, and Post-TransplantLymphoproliferative Disorders (PTLD) are associatedwith Epstein-Barr Virus (EBV) infection. CD8 and CD4clones have been generated against EBV proteins. Thecorresponding TCRs have been cloned in expression vectorsfor generation of transgenic T cells for adoptivetherapy of EBV-associated diseases. Optimization ofTCR expression led to improved killing efficiency ofgene-modified T cells in vitro.Moreover, the B-cell antigens CD19 and CD22 areappealing targets for a transgenic T cell recognition.Generation of TCR vectors recognizing epitopes of theseantigens is being pursued in cooperation with Th.Blankenstein in a murine system with humanized TCRgene sequences.Development of less toxic immunotoxinstargeting CD22 positive lymphomasTuan Duc Nguyen, Oliver Schmetzer in cooperation withG. Moldenhauer (DKFZ)Immunoconjugates coupled to toxic agents havepotent biological activity but side effect that canseverely limit their use. In a new approach, our groupgenerates immunotoxins which are based on non-toxicsubstances like dithiocarbamate-based compounds orcatechins which has been isolated from green tea. Coupledto an Anti-CD22-antibody, which is rapidly internalizedfrom lymphoma cells, leads to apoptosis inductionin cancer cells.Selected PublicationsSchmetzer, O, Moldenhauer, G, Riesenberg, R, Pires, JR, Schlag, P, Pezzutto,A. (2005). Quality of recombinant protein determines the amount ofautoreactivity detected against the tumor-associated epithelial celladhesion molecule antigen: low frequency of antibodies against the naturalprotein. J Immunol. 174, 942-952.Sebelin-Wulf, K, Nguyen, TD, Oertel, S, Papp-Vary, M, Trappe, RU, Schulzki, A,Pezzutto, A, Riess, H, Subklewe, M. (2007). Quantitative analysis of EBVspecificCD4/CD8 T cell numbers, absolute CD4/CD8 T cell numbers andEBV load in solid organ transplant recipients with PLTD. Transpl Immunol.17, 203-210.Westermann, J, Nguyen-Hoai, T, Baldenhofer, G, Hopken, UE, Lipp, M,Dorken, B, Pezzutto, A. (2007). CCL19 (ELC) as an adjuvant for DNA vaccination:induction of a TH1-type T-cell response and enhancement of antitumorimmunity. Cancer Gene Ther. 14, 523-532.Westermann, J, Kopp, J, van Lessen, A, Hecker, AC, Baskaynak, G, Le Coutre,P, Dohner, K, Dohner, H, Dorken, B, Pezzutto, A. (2007). Vaccination withautologous non-irradiated dendritic cells in patients with bcr/abl+ chronicmyeloid leukaemia. Br J Haematol. 137, 297-306.Westermann J, Hecker AC, Flörcken A, Dörken B, Pezzutto A. Granulocytemacrophage-colony stimulating factor plus interleukin-2 plus alphainterferonplus 5-fluorouracil in the treatment of metastatic renal cellcancer: induction of CD80/86+ T cells indicates adverse outcome. JImmunother. 2009 Jul-Aug;32(6):667-75.Patent applicationsEpCAM MHC-Klasse-II bindende Peptide und davon abgeleitete Mutantenals Verstärker der zellulären tumorreaktiven immunantwort (MDC0506 EP)Peptides regulating the surface expression of the T cell receptorEuropean Patent Application EP1870420Cancer Research 131

Structure of the GroupPeter DanielGroup LeaderProf. Dr. Peter DanielScientistsDr. Bernhard GillissenDr. Philipp HemmatiDr. Christian ScholzDr. Isrid SturmDr. Jana WendtGraduate StudentsCindrilla ChumduriMonika DejewskaNina GebhardtClinical and Molecular OncologyVirtually all medical anticancer therapies rely on the induction of cell cycle arrest or celldeath in the malignant cells. Consequently, the analysis of such genetic events allows forthe identification of patients at risk for an insufficient response to treatment and poorsurvival. Such analyses therefore provide a rational basis for a molecular understanding of theresponse to anticancer therapies and the clinical use of cancer therapeutics. The aim of thegroup is, therefore, to define genetic defects in cancer that result in aggressive disease, poorprognosis, and resistance to clinical cancer therapy. To this end, we have established anextensive genotyping and functional genomics program in solid tumors and leukemias. Recentdata indicate that specific defects in cellualr stress pathways leading to cellular resistancemay be overcome by rationally selected targeted anticancer drugs. In addition, these systemsare exploited to gain insights into novel aspects of cell cycle and cell death regulation andtheir intricate interactions.Understanding resistance to anticancer therapyMany anticancer therapies activate nuclear stressresponses to induce cell cycle arrest and DNA repair.When repair fails, the same stress responses trigger cellularsenescence or death and demise of the affectedcell. The molecular basis of these events has been studiedextensively during recent years and comprehensivemodels are now established for large parts of these signalingevents. We have investigated the consequencesof genetic defects in genes acting as effectors or inducersof p53 that trigger apoptosis and cell cycle arrestprograms upon genotoxic stress. In this context, werecently described selective loss of multiple BH3-onlyproteins, pro-apoptotic homologs of the Bcl-2 family,including Nbk and Bim in renal carcinoma. This is a unifyingfeature of renal carcinoma and appears to belinked to the impressive clinical resistance of this tumorentity to anticancer therapy.Regulation of cell death by pro-apoptotic Bcl-2family membersApoptosis is mediated through at least three majorpathways that are regulated by (1) the death receptors,(2) the mitochondria, and (3) the endoplasmic reticulum(ER). In most cells, these pathways are controlled bythe Bcl-2 family of proteins that can be divided intoantiapoptotic and proapoptotic members. Althoughthe overall amino acid sequence homology betweenthe family members is relatively low, they contain highlyconserved domains, referred to as Bcl-2 homologydomains (BH1 to BH4) that are essential for homo- andheterocomplex formation as well as for their cell deathinducing capacity. Structural and functional analysesrevealed that the proapoptotic homologs can be subdividedinto the Bax subfamily and the growing BH3-onlysubfamily. BH3-only proteins link upstream signalsfrom different cellular or functional compartments tothe mitochondrial apoptosis pathway (see figure).Puma, Noxa, Hrk, and Nbk (Bik) are induced by p53 andmediate cell death originating from the nucleus, e.g.upon DNA damage. Nbk localizes to the ER and activatesBax (but not Bak) indirectly, through a ER-initiateddeath pathway that has been recently elucidated by ourgroup.The aim of our work is to gain structural and functionalinsights into how these subfamilies promote orinhibit cell death signals and how these properties maybe utilized for development of apoptosis-promoting132 Cancer Research

Robert KudernatschLi-Min LiuAna MilojkovicAnnika MüerAnja MüllerTim OverkampThomas PretzschThoma RathjenTechnical AssistantsAnja RichterAntje RichterJosephine RußKerstin Dietze*Jana Roßius**part of the period reportedcancer therapies. Our studies therefore deal with questionssuch as how cell cycle stress responses includinganticancer therapies and oncogene deregulation feedinto the mitochondrial death pathway. We recentlyestablished that Nbk stabilizes the anti-apoptotic multidomainprotein Mcl-1 that acts as an endogenousinhibitor of Bak. This fully explains the entirely Baxdependent induction of apoptosis by Nbk. Ongoingwork addresses the transcriptional control of Nbkexpression and its functional involvement in the regulationof cell death following ER stress responses.Regulation of the Bak pathway by Mcl-1 (but not Bcl-x L )is of general relevance for targeted cancer therapy andwas shown to mediate TRAIL resistance in Bax deficientcarcinoma cells.Cell cycle and apoptosisUsing the apoptosis, cell cycle arrest and senescenceinducing tumor suppressor gene p14 ARF expression as amodel system, we explore the intricate interconnectionsbetween cell cycle stress responses and apoptosisinduction. P14 ARF expression is induced upon cellularstress, especially following deregulation of oncogenes.While physical interaction of p14 ARF with numerous regulatoryproteins, induction of p53-dependent cell cyclephenomena and cellular senescence by p14 ARF are wellestablished, little is known how p14 ARF induces celldeath. Notably, we established that the induction ofmitochondrial apoptosis by p14 ARF is entirely independentfrom p53 and Bax in p53-deficient cells where Bakcan fully complement for Bax function. Apoptosis ismediated, at least in p53-proficient cells, via the BH3-only protein Puma/bbc3 and relies on procaspase-3 forcell death execution. In contrast to apoptosis induction,the triggering of a G1 cell cycle arrest (and presumablypremature cellular senescence) by p14 ARF is entirelydependent on p53 and p21 CIP/WAF-1 , indicating that the signalingpathways for p14 ARF -induced G1 arrest and apoptosisinduction dissociate upstream of p53. Noteworthy,loss of p21 and/or 14-3-3σ strongly enhances apoptosisinduction by p14 ARF . Nonetheless, we recently demonstratedthat, in the absence of functional p53 and/orp21, p14 ARF triggers a G2 cell cycle arrest by downregula-Function of BH3-only proteins as death sensors. A: BH3-only proteinsact as functional interface between death signals and the mitochondrialapoptosis pathway. Anti-apoptotic Bcl-2 proteins put an at least duallayer of protection on activation of Bax/Bak that redistribute upon activationto form pores in the outer mitochondrial membrane for therelease of pro-apoptotic factors such as cytochrome c. B: Conditionaladenoviral expression of Nbk induces redistribution of Bax (EGFP, green)to mitochondria (TOM20, red) and a punctuate formation of Bax clustersdue to oligomerization. Blue colour: DAPI stained nuclei.Mitochondria fragment and cluster around the nucleus in apoptoticcells (on condition) as compared to the control (off).tion of cdc2-kinase activity, protein expression, andcytoplasmic localization in these cells whereas p14 ARF islocalized to the nucleus, i.e. mediates cdc2 sequestrationand induction of mitochondrial apoptosis throughan indirect mechanism. Such p53-independent mechanismsof p14 ARF induced apoptosis and arrest in the celldivision cycle represent fail-safe mechanisms thatallow for efficient growth suppression following inductionof p14 ARF -mediated stress responses in p53 pathwaydeficient cells.Selected PublicationsHossbach J, Michalsky E, Henklein P, Jaeger M, Daniel PT, Preissner R.Inhibiting the inhibitors: Retro-inverso Smac peptides. Peptides. 2009 Aug12. [Epub ahead of print]Gillissen B, Essmann F, Hemmati P, Richter A, Richter A, Öztop I,Chinnadurai G, Dörken B and Daniel PT. Mcl-1 mediates the Bax dependencyof Nbk/Bik-induced apoptosis. J Cell Biol 2007, 179: 701-15.Hemmati PG, Normand G, Gillissen B, Wendt J, Dörken B, Daniel PT.Cooperative effect of p21Cip1/WAF-1 and 14-3-3s on cell cycle arrest andapoptosis induction by p14ARF. Oncogene 2008, 27:6707-19.Daniel PT, Koert U, Schuppan J. Apoptolidin: induction of apoptosis by anatural product. Angew Chem Int Ed Engl. 2006, 45:872-93.Sturm I, Stephan C, Gillissen B, Siebert R, Janz M, Radetzki S, Jung K,Loening S, Dörken B, Daniel PT. Loss of the tissue-specific proapoptoticBH3-only protein Nbk/Bik is a unifying feature of renal cell carcinoma.Cell Death Differ. 2006, 13:619-27.Cancer Research 133

Kirsten Falk andOlaf RötzschkeStructure of the GroupGroup LeaderDr. Kirsten FalkDr. Olaf RötzschkeScientistsDr. Mireille StarkeDr. Markus KleinewietfeldGraduate andundergraduate studentsSabine HöpnerShashank GuptaKatharina DickhautReiner MailerAlexander SternjakCellular Immunology of Autoimmunereactions – Controlling the Balancebetween Effector and Suppressor T cellsDirection and strength of the immune response is largely controlled by the equilibrium betweeneffector and suppressor T cells. While effector T cells drive proinflammatory reactions toeradicate pathogens and transformed cells, suppressor T cells prevent autoimmune reactions aswell as the collateral damage by keeping the effector cells in check. It is now generally acceptedthat regulatory T cells (Treg) are a key suppressor population responsible for the maintenance ofperipheral tolerance. Characteristic marker of Treg cells is the transcription factor Foxp3. Specificaim of the group is to understand the composition and function of Treg subsets and to exploretheir role for future immune interventions in autoimmune diseases and cancer.Antigen-specific Recruitment of Treg cells andTreg-based cell therapiesCooperation with the SFB650, Berlin, the BMBF networkproject ‘BIOTIA’, and the ‘Singapore Immunology Network’(SIgN), SingaporeTreg cells may offer a novel perspective for the treatmentof autoimmune diseases as the antigen-specificrecruitment of these cells could result in dominant protectionfrom the autoimmune attacks. In several experimentalanimal models we have demonstrated thatrepeat antigens consisting of linear copies of a T cellepitope are particularly effective to inducing this effect.This applies for the treatment of multiple sclerosis-likeautoimmune disease (EAE), as well as for experimentalautoimmune neuritis (EAN) and an animal model oftype I diabetes (T1D). Recent studies further suggestthat the strategy is also widely used by parasites suchas plasmodium spec to mediate immune evasion. In a“bionics approach” tolerogenic repeat regions identifiedin the malaria parasite plasmodium falciparum are currentlybeing tested in the autoimmune model of multiplesclerosis.in the context of the BIOTIA network.In another approach the group has developed a methodthat provides access to ‘untouched’ human Treg cells. Ithas been documented numerous times in mice that theadoptive transfer of Treg cells is a very effective celltherapeuticapproach to treat inflammatory diseasesand syndromes. Translation of these approaches intoclinical praxis however was hindered by the lack of suitablecell isolation techniques. The new approach allowsnow obtaining highly pure populations of human Tregcells particularly suitable for therapeutic applications Ina mouse model of ‘graft vs. host disease’ (GvHD), thecells have been proven already to be capable of controllingthe disease. In a joint project with SIgN, a clinicalphase I trial has been launched in Singapore, in which‘untouched’ Treg cells will be used to treat GvHD inleukaemia patients.Extracellular ATP and CD39+ Treg cellsCooperation with Santa Lucia Foundation, Rome, Italy, theGRK 1258, Berlin and SIgN, SingaporeAs indicator of ‘non-natural’ (necrotic) cell death ATP isreleased through the damaged cell membrane into theextracellular space. It triggers various proinflammatoryreactions such as the maturation of dendritic cells (DC)and the regulation the inflammasome-mediatedrelease IL-1. The latter is also known as ‘endogenous134 Cancer Research

Sebastian GüntherTechnical AssistantsSabrina KleißleJörg ContzenSecretariatSonja Gieringpyrogen’ as it acts as key-factor in igniting the immuneresponse. We have recently shown that Treg cells expresswith CD39 a surface ATPase, able to degrade extracellularATP to ADP and AMP. Studies in the animal model ofmultiple sclerosis (MS) as well as in human MS indicatethat CD39+ T REM cells play indeed a major role in controllingthe disease. CD39-/- mice are susceptible to EAEand MS patients have strikingly reduced numbers ofCD39+ Treg cells in the blood (Fig. 1). Thus the control ofextracellular ATP-levels by these cells seems tom be crucialin suppressing the autoimmune disease.Notably, the individual variations in the cell numberseem to have genetic causes. Recent studies inSingapore indicate that the CD39-Treg phenotype ismuch more frequent in the Asian population. Given itsapparent importance of this polymorphism for inflammatoryimmune responses a study has been launchedin Singapore to determine whether a ‘single nucleotidepolymorphism’ (SNP) can be identified that and can beused as a diagnostic indicator explains the underlyingmechanism.The impact of ‘MHC-loading enhancer’ (MLE) onthe immune responseCooperation with the BMBF network project‘MHCenhancer’ and the European MC-RTN ‘Drugs forTherapy’Class II MHC molecules are receptor molecule presentingantigens in the form of short peptides for the surveillanceby CD4+ T cells. Cell surface MHC moleculesthat have lost their ligand rapidly inactivate. Theyacquire a ‘non-receptive state’, presumably to preventthe ‘accidental’ exchange of peptide ligands on the cellsurface. While this safe guard mechanism minimizesthe uncontrolled loading of MHC molecules, it alsoinhibits the effective antigen-loading needed for peptidevaccinations. During the past years the group hadidentified a number of small molecular compoundstermed ‘MHC-loading enhancer’ (MLE) that can bypassthis safety mechanism. By acting directly on cell surfaceMHC molecules they can reconstitute the non-receptiveMHC molecule in a catalytic fashion.In cooperation with partners from the ‘Leibnitz Instituteof Molecular Pharmacology’ (FMP) and other membersof the ‘MHCenhancer’ network MLE were found to targeta defined pocket of the class II MHC molecule. Thetransient occupation of this pocket stabilizes the peptide-receptivestate in a similar way as the natural catalystHLA-DM. Several structural and computationalstudies have been carried out to describe the mechanismof this transition on the molecular level. Moreover,in vivo studies in experimental cancer models demonstratedthat the addition of MLE compound to the adjuvantgreatly improves the antigen-specific CD4+ T cellresponse. MLE compounds may therefore be usefulmolecular tools to amplify immune responses duringvaccination or therapy.Selected publications (2006 to present)Kleinewietfeld, M., Starke, M., Di Mitri, D., Borsellino, G., Battistini, L.,Rotzschke, O., and Falk, K. (2009). CD49d provides access to “untouched”human Foxp3+ Treg free of contaminating effector cells. Blood 113, 827-836.Gupta, S., Hopner, S., Rupp, B., Gunther, S., Dickhaut, K., Agarwal, N.,Cardoso, M. C., Kuhne, R., Wiesmuller, K. H., Jung, G., et al. (2008). Anchorside chains of short peptide fragments trigger ligand-exchange of class IIMHC molecules. PLoS ONE 3, e1814.Piaggio, E., Mars, L. T., Cassan, C., Cabarrocas, J., Hofstatter, M., Desbois, S.,Bergereau, E., Rotzschke, O., Falk, K., and Liblau, R. S. (2007). Multimerized Tcell epitopes protect from experimental autoimmune diabetes by inducingdominant tolerance. Proc Natl Acad Sci U S A 104, 9393-9398.Borsellino, G., Kleinewietfeld, M., Di Mitri, D., Sternjak, A., Diamantini, A.,Giometto, R., Hopner, S., Centonze, D., Bernardi, G., Dell’Acqua, M. L., et al.(2007). Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysisof extracellular ATP and immune suppression. Blood 110, 1225-1232.Hopner, S., Dickhaut, K., Hofstatter, M., Kramer, H., Ruckerl, D., Soderhall, J.A., Gupta, S., Marin-Esteban, V., Kuhne, R., Freund, C., et al. (2006). Smallorganic compounds enhance antigen loading of class II major histocompatibilitycomplex proteins by targeting the polymorphic P1 pocket. J BiolChem 281, 38535-38542.Patents (2005 to present)PCT/EP2005/010008 - „Änderung des Beladungszustands von MHCMolekülen“PCT/EP2008/008599 – “Method and kit for the rapid isolation of humanFoxp3+ Treg cells”PCT/EP2008/008660 - “Fusion protein comprising S-antigen repeatunits“Invention disclosure (07.05.2007): “Use of peptide derivates as MHCloading enhancers (MLE)”Cancer Research 135

Structure of the GroupIduna FichtnerGroup LeaderDr. Iduna FichtnerScientistsDr. Michael BeckerDr. Klaus EckertDr. Reiner ZeisigDr. Diana BehrensExperimental PharmacologyPersonalized medicine will be the future goal of treatment options in cancer therapy. To achievethis, the routine use of biomarkers for the prediction of response or inherited resistance of anindividual tumor is necessary. Patient-derived tumor grafts gain increasing interest for the definition,characterization and validation of relevant biomarkers and are especially suitable models toinvestigate the dynamic regulation of genes or proteins in a clinically related way. The group“Experimental Pharmacology” has established a variety of patient-derived models from breast, colon,lung, ovarian carcinomas and from leukemias and sarcomas. Recent research was mainly focused onnon-small cell lung carcinomas and their biomarker profiling in relation to the response to classical ortargeted therapies. Further interest of the group concentrated on the generation of nanoparticulateformulations with a potential influence on vascular metastasis or for the treatment of brainmalignancies. The continued stem cell research focused on comparisons between adult haematopoieticand embryonic stem cells. Both in vitro and in vivo methods were used to evaluate thetransdifferentiation potential of these both cell types. It could be shown that embryonic stem cellshave a higher potential for differentiation into hepatic lineage cells than adult stem cells.Potential of patient derived xenografts for theidentification of biomarkersAs recently reported, our group in cooperation with theEvangelische Lungenklinik Berlin-Buch (Dr. Merk) wasable to establish a panel of non-small cell lung cancer(NSCLC) xenografts by transplanting patient derivedsurgical specimens directly to immunodeficient miceand keeping them in low passages. These tumor graftswere characterized using the expression of epidermalgrowth factor receptor (EGFR) related markers on thegenetic (Affymetrix profiling, mutational analysis) andprotein levels. It could be shown that there was a highcongruence between the original and the xenotransplantedtumor concerning histology, immunohistochemistryand gene expression pattern. Thechemotherapeutic responsiveness of the tumor graftsto classical cytotoxic drugs (paclitaxel, gemcitabine,carboplatin, etoposide, vinorelbine) as well as towardstargeted therapeutics (cetuximab, erlotinib) resembledthe clinical situation. No correlation could be foundbetween mutations in the EGFR, p53, c-met or PI3K andresponse to therapy. However, a correlation between K-ras mutations and response to erlotinib was registered.After treatment with Cetuximab, a down-regulation ofEGFR was observed in some sensitive but never in theresistant tumor models. The lack of therapeuticresponse of the NSCLC xenografts was not related tothe expression of resistance markers like BCRP, LRP,MDR1 and MRP1 at RNA or protein level.The comparison of the genome-wide gene expressionprofiling revealed a close clustering between the originalpatient tumor and the derived xenografts with correlationcoefficients between 0.945 and 0.782 (Figure 1).193 probe sets were differentially expressed. From136 Cancer Research

Graduate StudentsJana RolffAnnika Wulf-GoldenbergMarlen KeilJane WenzelAndrea OrthmannMaria Rivera MarkelovaMaria StecklumTechnical AssistantsMonika BeckerMargit LemmLars KonkelSecretariatSylvia SchulzFigure 1. One dimensional hirarchical clustering of primary lung carcinomas and their derived xenografts (Affymetrix profiling).these, 155 probe sets mainly belonging to cell adhesion,immune response and extracellular pathways weredown regulated. These data confirm the observationthat during early xenotransplantation, human normalaccessory cells are replaced by murine tissue while thetumors themselves maintain their identity. Some ofthe NSCLC xenografts were used as intracranialgrowing models to show the potential of a novelepothilone derivative, Sagopilone, to inhibit brainmetastases in a very efficient way. Further analysesconcerning the relevance of EGFR related signalingfor response of tumors to anti-EGFR therapies areongoing.Inhibition of metastasis by nanoparticlesThe formation of metastases is not completely understoodand is the main reason for a failure in cancertherapy. We used the human MT3 breast cancer in amouse xenograft model to gain a deeper insight intothe mechanisms of metastasis. We found that thedevelopment of lung metastases is essentially dependenton the formation of fibrin clots in the vasculature asa precondition for a pulmonary arrest of tumor cells.Subsequently, intravascular micro-metastases are generatedwhich finally invade into the surrounding tissue.We could further demonstrate a prevention of fibrinclot formation in vivo by the use of dual liposomes,simultaneously encapsulating the anticancer drug,Perifosine, and the haemostatic inhibitor, Dipyridamole.These dual liposomes are potent inhibitors ofmetastasis and caused a significant reduction in thenumber of lung metastases both in an experimental(MT3) (Figure 2 a) and in a spontaneous murine 4T7breast carcinoma model. Our data suggest that thesedual liposomes may display a new principle to preventmetastasis.Cancer Research 137

Figure 22a2b2a: Dual liposomes significantly reduced the number of metastases ofhuman MT-3 breast cancer in nude mice by preventing the formationof fibrin clots with proliferating tumor cells in a lung capillary(Micrograph: blue: tumor cells, red: fibrin).2b: Mitoxantrone (MTO) loaded liposomes significantly (*) inhibitedthe intracerebral growth of MT-3 tumor (blue area in micrographs)after intravenous treatment, indicating a better transcellular transportacross the blood-brain barrier.Liposomes were also developed to inhibit metastasis inthe brain. Endothelial and epithelial barriers play animportant role in the drug exchange between bloodand tissues. Such tight cellular linings especially limitthe drug transport across the blood brain barrier.Liposomes were designed to be used as drug transportersinto the brain. They were characterized concerningtheir membrane properties, their uptake by andtheir transcytotic transport across an epithelial barrierin vitro and were tested concerning their therapeuticeffect on breast cancer metastasis in the brain. Electronparamagnetic resonance measurements revealed aclear correlation between membrane fluidity of theliposomal bilayer and transcytosis. Liposomes with thehelper lipids DOPE and Perifosine showed the highesttranscytosis across a tight monolayer resulting in a fivefoldincreased transport of a marker into the basalmedium of a transwell system in comparison to controlliposomes. Liposomes containing both the helper lipidsand Mitoxantrone as cytotoxic drug were shown tosignificantly inhibit tumor growth in the brain of nudemice by 61 % (Figure 2 b). It is expected that transcellulartransport across the blood brain barrier can furtherbe enhanced by fluid liposomes conjugated with a peptidefor an active targeting to a specific receptor at theblood brain barrier. This project is currently ongoing.Stem cell differentiation and transplantation intomouse modelsExperimental and clinical studies showed that thetransplantation of different stem cell types can contributeto the regeneration of injured tissues. The aimof our work was the evaluation and comparison of thespontaneous and directed hepatic differentiation ofadult CD34 + cord blood stem cells and human embryonicstem cells.Human embryonic SA002 stem cells showed spontaneousin vitro differentiation into mesodermal, endodermaland ectodermal cell types. Hepatocyte conditionedmedium and co-culture systems with murinehepatocytes were investigated regarding their potentialto induce stem cell differentiation. Gene expressionpattern of cultivated CD34 + cells in hepatocyte conditionedmedium resulted in an only moderate regulationof genes associated with cell cycle and cytoskeletonorganization. In contrast to the restricted transdifferentiationpotential of CD34 + cells, SA002 cells in co-culture138 Cancer Research

with murine hepatocytes showed morphologic similaritiesto hepatic progenitor cells and revealed high levelsof early endodermal and hepatic markers, measured bythe detection of AFP, SOX17, HNF4 and albumin transcripts.The in vivo properties of transplanted stem cells wereexamined regarding proliferation, engraftment and celldifferentiation after application into newborn andadult immunodeficient mice. Intrahepatic transplantationof undifferentiated embryonic stem cells into miceresulted in fast and multiple teratoma growth.Different liver injury models were established inNOD/SCID mice and characterized with biochemicalmethods. An increase of hepatic engraftment afterCD34 + cell transplantation was determined in a modelof chemically-induced liver injury.Transplanted human embryonic stem cells into immunodeficientmice differentiated into cells of the threegerm layers, including endodermal derived cells, asrevealed by real-time RT-PCR with marker expression forAFP, HNF3/4 alpha and CK19 as well as by histology.Furthermore, gene expression pattern demonstratedthat 1053 genes were up regulated more than 2 fold inthe teratomas compared to undifferentiated embryonicstem cells, including genes for neuronal developmentas well as for angiogenesis. The teratomas showed ahigh degree of similarity to those structures of embryonicstem cells cultivated and differentiated in 3-D perfusionbioreactors (cooperation with Charite Berlin).The established transplantation models are suitable forexamining the organ distribution and the regenerativepotential of stem cells.Selected PublicationsFichtner, I, Rolff, J, Soong, R, Hoffmann, J, Hammer, S, Sommer, A, Becker, M,Merk, J. (2008). Establishment of patient-derived non-small cell lungcancer xenografts as models for the identification of predictivebiomarkers. Clin. Cancer Res. 14, 6456-6468.Hoffmann, J, Fichtner, I, Lemm, M, Lienau, P, Hess-Stumpp, H, Rotgeri, A,Hofmann, B, Klar. U. (2009). Sagopilone crosses the blood-brain barrier invivo to inhibit brain tumor growth and metastases. Neuro. Oncol.11, 158-166.Rolff, J, Dorn, C, Merk, J, Fichtner, I. (2009). Response of patient-derivednon-small cell lung cancer xenografts to classical and targeted therapiesis not related to multidrug resistance markers. J. Oncol.doi:10.1155/2009/814140.Wenzel, J, Zeisig, R, Haider, W, Habedank, S, Fichtner I. (2009). Inhibition ofpulmonary metastasis in a human MT3 breast cancer xenograft modelby dual liposomes preventing intravasal fibrin clot formation. BreastCancer Res. Treat. In press.Wulf-Goldenberg, A, Eckert, K, Fichtner, I. (2008). Cytokine pretreatment ofCD34 + cord blood stem cells in vitro reduced long-term engraftment inNOD/SCID mice. Eur. J. Cell Biol. 87, 69-80.Cancer Research 139

Function and Dysfunctionof the Nervous SystemFunktion und Dysfunktion des NervensystemsCoordinator: Carmen BirchmeierSignaling Pathways and Mechanismsin the Nervous SystemCoordinator: Carmen BirchmeierImaging of the Living BrainCoordinator: Frauke ZippPathophysiological Mechanisms ofNeurological and Psychiatric DisordersCoordinator: Helmut Kettenmann

Function and Dysfunction of the Nervous SystemFunktion und Dysfunktion des NervensystemsThe nervous system is a complex, sophisticated networkthat regulates and coordinates the body’sbasic functions and activities. Disorders of the nervoussystem that manifest themselves as neurological andpsychiatric disease constitute a major challenge foraffected individuals, their families, and for society.Determining the molecular basis of normal nervous systemfunction, and discovering the changes responsiblefor inherited or acquired defects are important prioritiesin the fight against nervous system disease.Research in the Neuroscience Department of the MDCfocuses on molecular and cellular analysis of the centraland peripheral nervous system. Themes and expertisecovered by the department are broad and interdisciplinary,and comprise genetics, neurophysiology, proteomicsand system biology, biochemistry, cell biology and stemcell research. This provides many opportunities for interactionsand collaborations, and ensures an optimal environmentfor young researchers and group leaders. We arepleased that we were able to recruit three outstandingyoung scientist to the department during the past reportperiod. Björn Schroeder joined the neuroscience departmentin 2008. He trained as a postdoc in the laboratory ofLily Jan und Yuh Nung Jan at the University of CaliforniaSan Francisco, where he used expression cloning to identifythe first member of a family of calcium-activatedchloride channels. Electrophysiological experiments hadpredicted the existence of such channels, but their molecularnature was not known until Björn developed asophisticated assay that allowed their identification.James Poulet and Jan Siemens joined the department in2009. James had worked at the Ecole PolytechniqueFédérale de Lausanne in the laboratory of Carl Petersen.He uses demanding and newly developed techniquesthat allow high-resolution recording of brain activity inawake and behaving animals. Jan trained in the laboratoryof David Julius at the University of California in SanFrancisco. He identified new ion channel molecules thatallow the detection of painful and cold stimuli by sensoryneurons in the skin. He received the Sofia Kovalevskayaaward in 2008, a prestigious prize granted by theAlexander von Humboldt Foundation, that will fund hiswork at the MDC during the next five years.The SFB-TRR43 began its work in January 2008, andintensifies interactions and collaborations betweenresearchers from Berlin and Göttingen. It devotes itself tothe characterization of inflammatory mechanisms in centralnervous system disease, and is entitled ‘The Brain as aTarget of Inflammatory Processes‘. Frauke Zipp is thespokesperson of the SFB-TRR that includes 15 projects andDas Nervensystem ist ein hochkomplexes Netzwerk,das die grundlegenden Funktionen und Aktionen desKörpers koordiniert. Störungen führen zu neurologischenund psychischen Krankheiten, die für die betroffenenMenschen, ihre Familien und die ganze Gesellschaft einerhebliches Problem darstellen. Die Untersuchung der molekularenGrundlagen der normalen Nervenfunktion und dieAufklärung der Ursachen für ererbte oder erworbeneStörungen des Nervensystems sind von herausragenderBedeutung im Kampf gegen solche Krankheiten.Die Forschung im Fachbereich “Neurowissenschaft” des MDCkonzentriert sich auf die molekulare und zellbiologischeAnalyse des zentralen und des peripheren Nervensystems.Forschungsthemen und -methoden, die im Bereich behandeltwerden, sind interdisziplinär breit ausgerichtet undumfassen Genetik, Neurophysiologie, Proteomik, Systembiologie,Zellbiologie und Stammzellforschung. Dies bietet zahlreicheKooperationsmöglichkeiten und ermöglicht eine optimaleForschungsumgebung für junge Forscher und Forschungsgruppenleiter.Wir hatten das Glück, in der Berichtsperiode drei talentiertejunge Forscher in den Bereich zu integrieren. Björn Schroederkam im Jahr 2008 an das MDC. Er wurde als PostDoc imLabor von Lily Jan und Yuh Nung Jan an der Universität vonCalifornien in San Francisco ausgebildet. Mittels Expressionsklonierunggelang ihm die Identifizierung des ersten Mitgliedseiner Familie von kalziumaktivierten Chlorid-Ionenkanälen.Elektrophysiologische Messungen hatten die Existenzsolcher Kanäle vermuten lassen, aber ihre molekulare Naturwar unbekannt, bis Björn sie mit einem komplizierten Nachweisverfahrenidentifizieren konnte. James Poulet und JanSiemens kamen 2009 in den Fachbereich. James hatte zuvoran der Ecole Polytechnique Fédérale de Lausanne im Laborvon Carl Petersen gearbeitet. Er arbeitet mit neuentwickeltenaufwendigen Techniken daran, die Hirnaktivität inwachen, sich spontan verhaltenden Tieren in hoher Auflösungzu erfassen. Jan wurde in der Arbeitsgruppe von DavidJulius an der Universität von Californien in San Franciscoausgebildet. Er fand neue Ionenkanalmoleküle, die die Entdeckungvon Schmerz- und Kältereizen durch sensible Neuronenin der Haut ermöglichen. Jan wurde 2008 mit demSofya-Kovalevskaya-Preis ausgezeichnet, einem angesehenenPreis der Alexander-von-Humboldt-Stiftung, der ihm dieFinanzierung seiner Arbeit am MDC für die nächsten fünfJahre sichert.Der Sonderforschungsbereich SFB-TRR43 begann im Januar2008 mit seiner Arbeit. Unter dem Titel “Das Gehirn als Zielentzündlicher Prozesse” intensiviert er die Zusammenarbeitvon Forschern in Berlin und Göttingen bei der Charakterisie-142 Function and Dysfunction of the Nervous System

Photo: David Ausserhofer / Copyright: MDCDr. Volker Siffrin (a medical doctor in training, research group:Prof. Frauke Zipp) uses the Two-Photon-Laser-Scanning-Microscope to perform intravital imaging of the brainstem of amouse with chronic inflammation of the central nervous system.Dr. Volker Siffrin (ein Arzt in der Ausbildung, Forschungsgruppe:Prof. Frauke Zipp) verwendet das Zwei-Photon-Laser-Scanning-Mikroskop um eine intravitale Darstellung des Gehirnstammseiner Maus mit chronischer Entzündung des zentralenNervensystems zu erzeugen.is funded by the German Science Foundation with 2.5 Mio€/year. In 2008 Thomas Jentsch was awarded a collaborativeresearch grant entitled “Physiology and Pathology ofKCNQ potassium channels” worth ~1 mio € from theLeibnitz society as part of its “Pakt für Forschung”. Grantfunding started in 2009 and the grant supports a multidisciplinaryexamination of KCNQ in collaboration withGary Lewin and Dietmar Schmitz (Charité, Berlin). CarmenBirchmeier successfully established a research networkthat is funded by the Ministry of Research and Education,which devotes itself to the analysis of the stem cells ofthe adult muscle (SatNet). Further researcher participatingin the network are Nikolaus Rajewsky (MDC) SimoneSpuler and Markus Schülke-Gerstenfeld (Charité, Berlin)and Thomas Braun (MPI Bad Nauheim).During the report period, Helmut Kettenmann was electedpresident of the Federation of European NeuroscienceSocieties, and he became also a member of the AcademiaEuropaea. Also, Gary Lewin was elected as a new memberof EMBO in 2008. Erich Wanker and members of hisresearch team were awarded the Erwin Schrödinger Prizein 2008 by the Helmholtz Association of GermanResearch Centers for their work on neuroproteomics andthe generation of a first human protein-protein interactionmap. Erich Wanker’s group began to set up a newplatform named ‘Interactome’ funded by the Ministry ofResearch and Education via an extension of the NGFN.This platform will use new high throughput technologiesto analyze and validate protein interaction data.rung entzündlicher Prozesse in Krankheiten des ZentralenNervensystems. Frauke Zipp ist die Sprecherin des SFB-TRR,der 15 Projekte vereint und von der Deutschen Forschungsgemeinschaftmit 2.5 Mio Euro/Jahrfinanziert wird.Thomas Jentsch erhielt 2008 von der Leibnitz Gesellschafteine Forschungszuwendung von 1 Mio Euro für das Verbundprojekt“Physiologie und Pathologie der KCNQ Kaliumkanäle”.Die Förderung lief 2009 an, und unterstützt einemultidisziplinäre Untersuchung der KCNQ Kanäle in Zusammenarbeitmit Gary Lewin und Dietmar Schmitz (CharitéHochschulmedizin Berlin). Carmen Birchmeier etablierte2008 ein Verbundprojekt zu Stammzellen der Skelettmuskulatur,SatNet, das vom Bundesministeriums für Bildung undForschung gefördert wird; an dem Projekt partizipierenneben Nikolaus Rajewsky (MDC) auch Wissenschaftler derCharité (Simone Spuler und Markus Schülke-Gerstenfeld)und der Max-Planck-Gesellschaft (Thomas Braun, Bad Nauheim).Während der Berichtsperiode wurde Helmut Kettenmannzum Präsidenten der Föderation der Europäischen NeurowissenschaftlichenGesellschaften gewählt, und zum Mitgliedder Academia Europaea berufen. Ebenfalls 2008wurde Gary Lewin als neues EMBO Mitglied gewählt.Erich Wanker und Mitglieder seine Arbeitsgruppe erhielten2008 den Erwin Schrödinger Preis der Helmholtz-Gemeinschaftdeutscher Forschungszentren für ihre Arbeit auf demGebiet der Neuroproteomik und die Erstellung der erstenmenschlichen Protein-Protein Interaktionskarte. DieArbeitsgruppe erhielt zudem erhebliche Drittmittel desBundesministeriums für Bildung und Forschung für die Entwicklungeiner neuen „Interactom“-Plattform im Rahmendes NGFN-Projekts. Diese Plattform wird Hochdurchsatztechnologienzum Nachweis von Proteininteraktionen einsetzen.Während des Berichtszeitraums hat der Fachbereich “Neurowissenschaften”unter der Federführung von Gary Lewindie Helmholtz International Research School “MolecularNeurobiology“ etabliert. Es handelt sich dabei um eingemeinsames Programm des MDC mit der Freien Universitätund der Charité, das Doktoranden ausbilden soll, die ander Analyse neurobiologischer Prozesse arbeiten. ErfolgreicheBewerber erhalten vollfinanzierte Doktorandenstellenan einer der beitragenden Institutionen, um an einem Projektzu den molekularen Grundlagen von Funktion und Dysfunktiondes Nervensystems zu arbeiten. Der Lehrplan desProgramms beinhaltet einen zweijährigen Vorlesungszyklusüber grundlegende sowie fortgeschrittene Konzepte derNeurobiologie, einen Journal Club der Studenten, und prak-Function and Dysfunction of the Nervous System 143

The Neuroscience Department also established theHelmholtz International Research School ‘MolecularNeurobiology’. This program is a joint activity of the Max-Delbrück Centre (MDC) for Molecular Medicine, the FreeUniversity Berlin, and the Charité, and is headed by GaryLewin. The aim of the school is to provide state of the arttraining to PhD students working on the analysis of neurobiologicalprocesses. Successful PhD applicants areoffered a fully funded doctoral position either at the MDC,the Free University or the Charité, and they are expectedto pursue a research project that devotes itself to themolecular understanding of normal function and dysfunctionin the nervous system. The curriculum of theprogram includes a two years lectures series coveringbasic and advanced concepts of neurobiology, a studentjournal club, and practical experimental courses. In addition,the program offers ‘soft skills’ training, for instancecourses on communication and presentation, and fundingto allow students to attend international conferencesto present their results.Activities in the department include a biannual retreatthat is attended by students, postdocs and group leaders.In 2009, the retreat took place in a small village inBrandenburg, Hubertusstock. The beautiful and secludedenvironment there was well suited for intense but informalscientific discussion and presentation of results.Signaling pathways and mechanisms in thenervous systemSignaling systems control the establishment of geneexpression programs and cellular interactions duringdevelopment. Their analysis provides insight into themechanisms used to build the nervous system, andunravels the cause of developmental disorders thatunderlie disease. In the mature nervous system, signalingmolecules control synaptic functions or neuronal maintenance,are key molecules in neuron-glia interactions, andcontrol the transmission and processing of electricalinformation. Many groups in the NeuroscienceDepartment focus on defining the roles of signaling moleculesin the developing and adult nervous system, in thehealthy as well as in the afflicted organism.Human Bartter syndrome type IV is associated withsevere renal loss of fluid and salt as well as with congenitaldeafness and is caused by mutations in the BSNDgene. The group of Thomas Jentsch has shown previouslythat barttin, the protein encoded by BSND, is an accessory-subunit of the chloride channels ClC-Ka and –b. Bothchannels are expressed in the kidney and in the cochleartische Kurse. Überdies werden Kommunikations- und Präsentationstechnikenerlernt und Stipendien für die Teilnahmean internationalen Tagungen gewährt, auf denen die Studentenihre Ergebnisse präsentieren können.Eine weitere Fortbildungsaktivität sind die regelmäßigenKlausurtagungen, die im Jahre 2009 Studenten, PostDocsund Gruppenleiter im Brandenburger Ort Hubertusstockzusammenbrachte. Die abgelegene, landschaftlich schöneUmgebung bot einen geeigneten Rahmen für intensiveinformelle Diskussion und Vorstellung der neuesten Ergebnisse.Signalwege und Signalmechanismen imNervensystemSignalsysteme kontrollieren Genexpressionsprogramme undzelluläre Interaktionen während der embryonalen Entwicklung.Ihre Analyse gewährt Einsicht in die Mechanismen derEntstehung des Nervensystems und hilft, Entwicklungsstörungenzu verstehen, die zu neurologischen Erkrankungenführen. Im ausgereiften Nervensystem kontrollieren Signalmoleküledie synaptische Funktion und die Aufrechterhaltungder neuronalen Strukturen. Sie sind die Schlüsselmolekülefür die Interaktion von Neuronen und Gliazellen undsteuern die Übertragung und Weiterverarbeitung elektrischerSignale. Etliche Gruppen im Fachbereich konzentrierensich auf die Rolle, die Signalmoleküle im sich entwickelndenund im adulten Nervensystem spielen, sowohl im gesundenwie im pathologisch veränderten Organismus.Das klinische Bartter Syndrom des Typs IV ist gekennzeichnetdurch erhebliche Flüssigkeits- und Salzverluste über die Nierezusammen mit angeborener Taubheit, und wird durchMutationen im BSND-Gen verursacht. Die Gruppe um ThomasJentsch hatte bereits gezeigt, dass das von diesem Genkodierte Protein Barttin eine zusätzliche Beta-Untereinheitder Chloridkanäle CIC-Ka und CIC-Kb bildet. Beide Kanälesind sowohl in der Niere wie im Cochlear-Epithel der striavascularis des Innenohrs exprimiert.Die Forscher stellten nun eine konditionelle “gefloxte” barttinMutante in der Maus her, mit der das Gen gezielt nur imInnenohr deletiert wurde, um so die durch Salz- und Flüssigkeitsverlustverursachte postnatale Letalität der konventionellenbarttin Nullmutante zu vermeiden. Diese Tiere sindgenau wie Patienten mit einer BSND-Mutation angeborentaub. Die Wissenschaftler in der Arbeitgruppe von ThomasJentsch konnten zeigen, dass dies auf einen Kollaps des elektropositivenPotentials in der scala media (dem sog. endocochlearenPotential) zurückzuführen ist. Letzteres ist essentiell,um einen depolarisierenden Kalium-Einstroms durch144 Function and Dysfunction of the Nervous System

epithelium of the stria vascularis in the inner ear. Theynow generated a conditional ‘floxed’ barttin KO mouse todelete barttin exclusively in the inner ear, thereby avoidingthe massive salt and fluid loss that leads to early postnataldeath of mice carrying null-mutations in BSNDgene. Like patients with mutations of BSND, these miceare congenitally deaf. They showed that this is due to acollapse of the positive voltage in the scala media (theendocochlear potential), which is necessary to drive adepolarizing influx of potassium through mechanosensitivechannels in sensory hair cells. Outer hair cells were nolonger able to mechanically amplify sound in the innerear and eventually degenerated. The group has thus clarifiedthe pathological mechanism underlying a form ofhuman deafness and revealed a previously unknown roleof chloride channels in the generation of the endocochlearpotential (Rickheit et al., EMBO J. 27, 2907-2917).In a healthy organism, a balance is maintained betweenthe excitation and inhibition of electrical neuronalimpulses. Deregulation of this balance results in nervoussystem disorders. The group of Jochen Meier investigatespost-transcriptional processes that control the balancebetween excitation and inhibition. Glycine receptors areligand-gated ion channels that recognize the amino acidglycine, which acts as an inhibitory neurotransmitter.Jochen had previously discovered that mRNA encoding forthe glycine receptor is post-transcriptionally modified:the modified mRNA encodes a receptor that binds glycinewith a higher affinity than the receptor encoded by theunmodified RNA. The group currently characterizes thecompensatory function of these high affinity receptors inhyperexcitability disorders.The group of Ines Ibanez-Tallon has developed a novelgenetic method to manipulate neuronal activity in vivousing genetically encoded cell-surface anchored toxinsand neuropeptides. These toxins interfere with ion channelfunction and are designed to silence neurons via inhibitionof ion channels in a cell-autonomous manner, sothat only the neuron that expresses the toxin is affected.The design of these tethered toxins follows nature, asendogenous molecules exist in mammals that modulateneuronal activity in such a manner (Tekinay et al., PNAS2009). The group of Ines Ibanez-Tallon does use thesenovel tools to define the function of a particular neuronin a neuronal circuit, or to characterize the function of aclass of ion channels in a specific neuronal population.During his postdoc years, Björn Schroeder identified a prototypeof a new family of ion channel (Schroeder et al.,2008, Cell). Two members of this family, TMEM16A andTMEM16B, encode calcium activated chloride channels. Indie mechanosensitiven Kanäle der sensorischen Haarzellenzu treiben. Die äußeren Haarzellen sind so nicht mehr in derLage, den eingehenden Schall im Innenohr zu verstärken,und degenerieren schließlich. Mit diesen Arbeiten hatJentschs Gruppe einen Pathomechanismus für menschlicheTaubheit aufgeklärt und eine zuvor unbekannte Rolle vonChloridkanäle bei der Herstellung des endochochlearenPotentials aufgedeckt (Rickheit et al., EMBO J. 27, 2907-2917).Im gesunden Organismus besteht ein Gleichgewicht zwischenexzitatorischen und inhibitorischen Nervenimpulsen.Störungen dieses Gleichgewichts führen zu Erkrankungendes Nervensystems. Die Gruppe von Jochen Meier untersuchtdie post-transkriptionellen Prozesse, die das Gleichgewichtaufrecht erhalten. Glycinrezeptoren sind LigandenaktivierteIonenkanäle, die die Aminosäure Glyzin binden,welche hier als hemmender Neurotransmitter funktioniert.Jochen Meier konnte bereits zeigen, dass mRNA, die für diesenRezeptor kodiert, post-transkriptionell modifiziert wird:die modifizierte mRNA kodiert für einen Rezeptor, der einehöhere Affinität zu Glyzin aufweist. Die Arbeitsgruppeuntersucht nun die kompensatorische Funktion des hochaffinenRezeptors bei hyper–exzitatorischen Störungen.Der Arbeitsgruppe von Ines Ibanez-Tallon gelang es, neuronaleAktivität in vivo mit Hilfe genetisch kodierter, zelloberflächengebundenerToxine und Neuropeptiden zu manipulieren. DieseToxine interferieren mit Ionenkanälen und sind so gestaltet,dass sie zellautonom nur diejenigen Neuronen funktionellbeeinträchtigen, von denen sie exprimiert werden. Das Designdieser oberflächengebundenen Toxine ist dem von endogenenModulatoren neuronaler Funktion ähnlich (Tekinay et al.,PNAS 2009). Die Gruppe um Ibanez-Tallon nutzt diese neuartigenMoleküle als Werkzeuge, um die Funktion einer bestimmtenNervenzelle in einem neuronalen Schaltkreis zu bestimmen,oder um die Funktion einer Klasse von Ionenkanälen ineiner bestimmten Populationen von Neuronen aufzuklären(Auer et al., Nature Methods, 2010, in press).Während seiner Jahre als PostDoc gelang es Björn Schroeder,den Prototyp einer neuen Ionenkanal-Familie zu entdecken(Schroeder et al., 2008, Cell). Zwei bestimmte Genedieser Familie, TMEM16A und TMEM16B, kodieren für kalzium-aktivierbareChloridkanäle. Schroeder will die Funktiondieser Kanäle mit Hilfe von genetischen Experimenten in derMaus untersuchen. Besonders interessiert ihn dabei dieRolle solcher Kanäle bei Geruchs- und Geschmacksempfindungenund bei der Blutdruckregulation, sowie beim transepithelialenWasser- und Ionentransport in der Lunge. Beabsichtigtsind auch strukturelle und funktionelle Studien andiesen Kanälen mit Hilfe der sog. site-directed mutagenesisin einem transienten Expressionssystem.Function and Dysfunction of the Nervous System 145

the coming years, Björn Schröder plans to functionallycharacterize these channels using mouse genetics. In particular,he intends to determine the role of these channelsin olfaction, taste and blood pressure regulation, and isinterested in their activity during transepithelial waterand ion transport in the lung. In addition, he will performstructural and functional studies on these novel channelsusing site-directed mutagenesis and a transient expressionsystem.The ability of any living organism to sense stimuli emanatingfrom the surrounding environment is of fundamentalimportance for its well-being and survival. Thesomatosensory system allows the detection of pain andmechanical stimuli as well as changes in temperature.Sensory mechanisms not only monitor the outside worldbut also detect changes of interior parameters such asblood sugar levels, blood pressure or body temperature.Processing and integration of sensory information fromthe outside world and the interior environment allows ourbody to adjust to changes and to maintain homeostasis.The naked mole rat, also known as desert mole rat, is anunusual rodent that thrives in a harsh and hot undergroundenvironment. It is the only known poikilothermicmammal (i. e. cold blooded), it lives in colonies with aninsect-like social structure, and it is also the longest-livedrodent species known (lifetimes in excess of 25 yrs). Thisanimal has a normal acute pain response, but displays nohypersensitivity (so called hyperalgesia) to a variety ofinflammatory and chemical stimuli. Strikingly, the animalslack behavioral response to acid. The group of GaryLewin has used electrophysiology to show that primaryafferent nociceptors in naked mole-rats are insensitive toacid stimuli, consistent with the lack of acid-inducedbehavior. Acid sensitivity by sensory neurons is observedin birds, amphibians, and fish, which suggests that it hasbeen selectively lost in the naked mole-rat in the courseof its evolution. In contrast, nociceptors of the naked molerate do respond vigorously to capsaicin, a pain-inducingchemical isolated from chili peppers. Nevertheless, theactivation of capsaicin-sensitive sensory neurons innaked mole-rats does not produce pain-related behavior.The unique pain response of the naked mole-rat can provideinsights into what constitutes normal mammaliannociception. To elucidate its cause, the Lewin group is currentlyanalyzing gene variants of the naked mole rat thatencode ion channels and associated channels that arerequired for the transduction of painful stimuliJan Siemens found that three inhibitor cysteine knot (ICK)peptides from tarantula venom target a particular TRP ionchannel, TRPV1. TRPV1 functions also as the receptor forDie Registrierung von Reizen aus der Umgebung ist eineessentielle Funktion für Wohlbefinden und Überlebensfähigkeiteines jeden Organismus. Das somatosensorische Systemvermittelt dabei die Wahrnehmung von mechanischen undSchmerz-Reizen sowie die Empfindung für die Umgebungstemperatur.Sensorische Mechanismen registrieren jedochnicht nur äußere Einflüsse, sondern auch innere Parameterwie Blutzuckerniveau, Blutdruck oder die Körpertemperatur.Beide Wahrnehmungsvarianten erlauben dem Körper diegezielte Anpassung an Veränderungen und die Aufrechterhaltungder Homöostase des Organismus.Das Nacktmull, eine Rattenart, die auch als Wüstenmullbezeichnet wird, ist ein sehr ungewöhnliches Nagetier, das inunwirtlichen und heißen Böden lebt. Es ist das einzige bisherbekannte wechselwarme Säugetier, lebt in insektenähnlichensozialen Strukturen und ist die Nagerart mit der größtenLebenserwartung (mehr als 25 Jahre). Dieses Tier zeigteine normale akute Schmerzempfindlichkeit, ist jedoch nichtüberempfindlich (hyperalgesisch) gegen eine Reihe chemischerund entzündungserzeugender Reize. Auffallend ist dasFehlen einer Reaktion auf Säure. Die Forschergruppe umGary Lewin nutzte elektrophysiologische Methoden, um zuzeigen, dass die primären afferenten Nozirezeptoren imNacktmull nicht auf Säurestimuli reagieren, wie es beiVögeln, Amphibien und Fischen der Fall ist. Das legt nahe,dass der Nacktmull eine analoge Funktion in der Evolutionverloren hat. Hingegen wurde festgestellt, dass die Nozirezeptorendes Nacktmull heftig auf Capsaicin reagieren, eineschmerzauslösende Substanz, die aus Chillischoten isoliertwird. Allerdings erzeugt die Aktivierung der Capsaicin-empfindlichensensorischen Neurone nicht das sonst üblicheschmerzinduzierte Verhalten der Tiere. Diese einzigartigeSchmerzreaktion im Nacktmull vermittelt indirekt Einsichtenin die Funktionsweise der normalen Nozizeption vonSäugetieren. Um den Mechanismus weiter aufzuklären, analysiertLewin gegenwärtig Genvarianten des Nacktmulls, diefür Ionenkanäle und assoziierte Kanäle kodieren und für dieTransduktion von Schmerzreizen verantwortlich sind.Jan Siemens hat festgestellt, dass drei sogenannte inhibitorischeCystein-Knoten-Peptide (ICK) aus dem Gift der Taranteleinen bestimmten TRP-Ionenkanal, TRPV1, angreifen. DieserTRPV1 ist ein Capsaicin-Rezeptor, dessen Aktivierung einenbrennenden Schmerz erzeugt. Die ICK-Toxine wirken alsTRPV1-Agonisten, was auch erklärt, warum das TarantelgiftSchmerzen erzeugen kann. Damit liegt nun mit den ICK Toxinenein neues pharmakologisches Werkzeug vor, um dieFunktion von TRP Kanälen zu studieren. Zugleich eröffnensich neue Ansätze zur Entwicklung von Medikamenten zurTherapie von Schmerzzuständen (Siemens et al., Nature146 Function and Dysfunction of the Nervous System

capsaicin, the active component of chili peppers, whichproduces a burning and painful sensation. The ICK toxinsfunction as TRPV1 agonists, which explains how thetarantula venoms can cause pain. The ICK toxins thusconstitute new pharmacological tools to study TRP channelgating, and open new avenues to design drugs thatblock the pain pathway (Siemens et al., Nature. 444, 208-212). While we have learned a lot about biophysical propertiesof individual TRP ion channels over the last coupleof years, little is known about their functional modulationand regulation in vivo. By analogy to TRP channels in theDrosophila eye, we hypothesize that the mammalianorthologs are components of supramolecular membraneboundprotein complexes that enable the channels tofunction specifically and effectively in a context dependentmanner. Jan Siemens will use make use of genetic,biochemical and functional screening paradigms to identifycomponents of this putative modulatory complex.Sensory neurons project to the spinal cord, and innervateneurons located in the dorsal horn. Neurons in the dorsalhorn of the spinal cord integrate and relay the sensoryinformation to other parts of the nervous system. Sensoryaxonal projections into the spinal cord display a highlystereotyped pattern of T- or Y-shaped axon bifurcation atthe dorsal root entry zone. The group of Fritz Rathjenidentified molecules that control sensory axon bifurcation.These are the natriuretic peptide C (CNP), its receptor,the guanylyl cyclase Npr2, and its downstream signalingmediator, the cyclic guanosine monophosphatedependentprotein kinase I (cGKI). Bifurcation does notoccur in mice with mutations in one of these componentssuggesting that they act in a signaling cascade. This isaccompanied and by altered neuronal connectivity in thedorsal spinal cord, and by changes in the perception ofpainful stimuli (Schmidt et al., 2009, PNAS, in press).A further relay station for sensory information is providedby hindbrain neurons. Somatosensory neurons innervatethe trigeminal nucleus of the hindbrain and convey informationabout touch and pain from the face, whereas viscerosensoryneurons innervate the nucleus of the solitarytract to convey information about parameters like visceralpain or blood pressure.The group of Carmen Birchmeieridentified two transcription factors, Lbx1 and Olig3, thatare essential for the development of these two hindbrainnuclei, and that appear to play opposing roles duringtheir development. The homeobox gene Lbx1 is essentialfor generation of the trigeminal nucleus. In Lbx1 mutantmice, instead of a trigeminal nucleus, an extremely largenucleus of the solitary tract is formed. Conversely, thenucleus of the solitary tract is absent in Olig3 mutant444,208-212). Während die biophysikalischen Eigenschafteneinzelner TRP Kanäle in den letzten Jahren erforscht wurden,ist relativ wenig über ihre funktionale Modulation undRegulierung in vivo bekannt. Es wird vermutet, dass, analogzu den TRP Kanälen in den Augen von Drosophila, die Orthologein Säugetieren ebenfalls Komponenten von membrangebundenenProteinkomplexen sind, in deren Kontext dieKanäle spezifische Funktionen übernehmen. Jan Siemenswird genetische, biochemische und funktionelle Screeningverfahreneinsetzen, um weitere Bestandteile dieser vermutetenmodulatorischen Komplexe zu identifizieren.Sensorische Neuronen leiten Impulse an das Rückenmarkweiter und innervieren hier die Neuronen im Hinterhorn.Diese integrieren die Information und leiten sie weiter inandere Regionen des Nervensystems. Sensorische axonaleProjektionen ins Rückenmark zeigen ein hoch stereotypesMuster von T- oder Y-förmigen Axonaufzweigungen an derEintrittszone in die Wurzeln des Hinterhorns. Die Gruppevon Fritz Rathjen hat Moleküle gefunden, die die sensorischeAxonaufzweigung kontrollieren. Verantwortlich ist dasnatriuretischen Peptid C (CNP) mit seinem Rezeptor, derGuanylatzyklase Nrp2, und dem Signalvermittler, der Guanosinmonophosphat-abhängigenProteinkinase I (cGKI). ZurBifurkation kann es nicht kommen, wenn einer dieser Faktorendurch Mutation in der Maus inaktiviert ist, was auf ihreVerschaltung als Signalkaskade hinweist. In der Folge tretenveränderte Nervenverschaltungen im dorsalen Rückenmarkauf, und Änderungen in der Schmerzempfindung (Schmidtet al., 2009, PNAS, im Druck)Eine weitere Relaisstation für sensorische Information sinddie Neuronen des Hirnstamms. Somatosensorische Neuroneninnervieren den Trigeminuskern im Rautenhirn und vermittelndamit Information über Berührungs- und Schmerzreizeim Gesicht, während viszerosensorische Neuronen denGeschmackskern des Nervus vagus (Nucleus tractus solitarii)mit Informationen über abdominale Schmerzempfindungenund Blutdruckvariation versorgen. Die Gruppe vonCarmen Birchmeier hat zwei Transkriptionsfaktoren, Lbx1und Olig3, identifiziert, die für die embryonale Entwicklungdieser zwei Hirnstammkerne essentiell sind, und gegensätzlicheFunktionen während der Ausbildung der Region übernehmen.Das Homöobox-Gen Lbx1 ist für die Ausbildung desTrigeminuskerns erforderlich. In mutanten Mäusen ohneLbx1 bildet sich anstelle des Kerns ein extrem großer Solitartraktaus; in Olig3-mutanten Mäusen hingegen fehlt derSolitartrakt. Eine genauere Analyse der Funktion von Olig3zeigte, dass eine wichtige Funktion dieses Faktors in derUnterdrückung von Lbx1 in Hirnstammneuronen besteht(Storm et al., Development 2009, 139:295-305).Function and Dysfunction of the Nervous System 147

mice. Further analysis of the function of Olig3 demonstratedthat an important aspect of its developmentalfunction lies in the suppression of Lbx1 in hindbrain neurons(Storm et al., Development 2009 136:295-305).Recent technical advances have allowed researchers tomake high-resolution recordings of brain activity evenwhile animals are awake and behaving, which providesremarkable insights into normal brain function. Of particularinterest is the activity of neurons in the neocortex, asthey are involved in conscious movement and sensoryperception. The group of James Poulet combines electrophysiologicaland optical neural recordings to investigatethe link between neural activity, sensory perception andmotor behavior. The very first recordings from the awakehuman brain by Hans Berger (1929) revealed distinct patternsof cortical activity, or “brain states” during rest ascompared to motor activity. Using dual whole-cell recordingsfrom layer 2/3 primary somatosensory mousewhisker barrel cortex, James Poulet is investigating themechanisms and functions of changes in brain state. Hisinitial recordings have shown that the intracellular membranepotential of neurons is highly synchronized whilethe mouse is sitting still. However, when the mousemoves its whiskers to sense the environment, a desynchronizedlocal field potential and electroencephalogramare observed (Poulet and Peterson, Nature 2008). In thefuture James Poulet and his group will analyze the primarysensory and motor cortical areas controlling limbmovement. Cortical activity controlling limb movement isparticularly relevant in the emerging field of neuroprosthetics,where cortical activity from paralyzed patients isused to drive robotic limbs.Imaging of the living brain and pathophysiologicalmechanism of neurological and psychiatricdisordersDisorders of the nervous system are a major challenge ofour society. Several groups in the MDC NeuroscienceDepartment concentrate on elucidating molecular mechanismsthat lead to nervous system disease, thus aimingfor the development of rational therapies, as for instancethe design of small molecular therapeutical compounds.Multiple sclerosis is an autoimmune disease in which thepatient’s immune response attacks the central nervoussystem, leading to demyelination.Multiple sclerosis affects thus the ability of nerve cells tocommunicate with each other. The group of Frauke Zippstudies molecular mechanisms that cause multiple sclerosis,and define molecules that affect the pathophysio-Neue technische Entwicklungen haben es möglich gemacht,die Hirnaktivität wacher und verhaltensaktiver Tiere mithoher Empfindlichkeit zu registrieren., Von besonderem Interesseist hierbei die Aktivität der Neuronen des Großhirns,denn sie sind sowohl an bewussten Bewegungen wie an dersensorischen Wahrnehmung der Umgebung beteiligt. DieGruppe von James Poulet nutzt elektrophysiologische undoptische Ableitungen, um die Verbindung zwischen neuronalerAktivität, sensorischer Wahrnehmung und motorischerAntwort zu studieren. Die ersten Ableitungen eines wachenmenschlichen Gehirns durch Hans Berger (1929) enthülltenspezifische Muster kortikaler Aktivität, oder “Gehirnzustände”,abhängig von Ruhezustand oder motorischer Aktivität.Mit Hilfe paralleler Ableitungen von Zellen aus den Schichten2/3 des somatosensorischen Tasthaar-Barrel-Kortex derMaus untersucht James Poulet Mechanismus und Zweck vonÄnderungen des Gehirnstatus. Erste Ergebnisse zeigen, dassdas intrazelluläre Membranpotential stark synchronisiert ist,solange eine Maus still sitzt. Bewegt die Maus hingegen dieTasthaare, um die Umgebung zu erkunden, dann desynchronisiertdas lokale Feldpotential sowie das EEG (Poulet &Peterson, Nature 2008). Die Arbeitsgruppe will nun die Bereichedes primären sensorischen und motorischen Kortex analysieren,die die Extremitäten kontrollieren. Solche Arbeitensind besonders relevanten für das aufkommende Forschungsfeldder Neuroprosthetik, wo kortikale Impulse vongelähmten Patienten zur Steuerung von mechanischenGliedmaßen benutzt werden.Bildliche Darstellung des intakten Gehirns undpathophysiologische Mechanismen neurologischerund psychiatrischer StörungenStörungen des Nervensystems sind ein drängendes gesellschaftlichesProblem. Mehrere Gruppen des FachbereichsNeurowissenschaften am MDC konzentrieren ihre Arbeit aufdie Aufklärung molekularer Mechanismen der Entstehungvon Krankheiten des Nervensystems mit dem Fernziel, gezielteTherapien mit z.B. niedermolekularen Wirkstoffen zu entwickeln.Die Multiple Sklerose (MS) ist eine Autoimmunkrankheit, beider die Immunreaktion des Patienten das zentrale Nervensystemangreift und die Myelinscheide der Axone zerstört.Befallene Nervenzellen verlieren ihre Fähigkeit zu kommunizieren.Die Gruppe von Frauke Zipp studiert die molekularenMechanismen, die MS verursachen und sucht nach denMolekülen, die am pathophysiologischen Prozess teilnehmen.Vor einiger Zeit konnte die Gruppe zeigen, dass das Kallikrein-Kinin-Systeman der Regulation von Entzündungendes ZNS beteiligt ist, indem es die enzephalitogene T-Lym-148 Function and Dysfunction of the Nervous System

logical process. They recently showed that the kallikreinkininsystem is involved in the regulation of CNS inflammation,limiting encephalitogenic T lymphocyte infiltrationinto the CNS. By this, they identified a new moleculartarget, the kinin receptor B1, for the therapeutic treatmentof chronic inflammatory diseases such as multiplesclerosis (Schulze-Toppenhoff et al., Nature Medicine2009).Gliomas comprise the majority of cerebral tumors, andpatients diagnosed with glioma multiforme, a highlyinvasive glioma type, have a very poor prognosis. Thegroup of Helmut Kettenmann found that microglial cellsstrongly promote glioma growth and invasion, andobserved an interesting interplay between microglial andglioma cells. Glioma cells release the metalloproteaseMMP2, which degrades the extracellular matrix and promotesinvasion.This metalloprotease is, however, releasedin an inactive precursor form and requires processing foractivation. The ectoenzyme MT1-MMPT does activateMMP2, but this enzyme is not produced by glioma cells.Instead, a factor released by the glioma cells activatestoll-like receptors in the surrounding microglial cells, andupregulates microglial expression of MT1-MMP. Thus,glioma cells exploit microglial cells to promote their owninvasion. Interfering with toll-like receptors or the signalingpathways used by these receptors might reduce therapid expansion of glioma cells, and therefore microgliahave become a new target for glioma research (Markovicet al., PNAS 2009, in press).Many proteins of diverse sequence, structure and functionself-assemble into fibrillar aggregates commonlytermed amyloids. Amyloid formation is associated withprotein-misfolding disorders, and plays important roles inneurodegenerative diseases such as Alzheimer’s,Parkinson’s and Huntington’s disease. The group of ErichWanker explores if amyloid formation pathways can beredirected with the help of small molecules. They showedthat EGCG, a compound originally isolated from greentea, potently inhibits the formation of huntingtin aggregates.In collaboration with the group of Jan Bieschke,they defined the mechanisms by which EGCG affects thisprocess. The compound directly binds to the nativelyunfolded polypeptides and prevents their conversion intoamyloid oligomers and protofibrils. Thus, this substanceinterferes with an early step in the amyloid formationcascade and redirects unfolded polypeptide moleculesinto an alternative aggregation pathway before theybecome amyloidogenic (Ehrnhoefer, Bieschke and others,Nature Struct Mol Biol. 2008).phozyten-Infiltration begrenzt. Auf diese Weise konnte einneues molekulares Angriffsziel, der Kinin Rezeptor B1, für dieBehandlung chronischer inflammatorischer Prozesse wieder MS identifiziert werden (Schulze-Toppenhoff et al.,Nature Medicine 2009).Die Mehrzahl der Hirntumoren sind Gliome. Speziell dasGlioma multiforme ist eine sehr invasive Form, und davonbetroffene Patienten haben eine sehr schlechte Überlebensprognose.Die Gruppe von Helmut Kettenmann hat festgestellt,dass Mikroglia-Zellen das Wachstum von Gliomenund ihre Invasionsneigung stark befördern und dass dabeieine Wechselwirkung zwischen Mikroglia und Gliomzellenbesteht. Gliomzellen setzen die Metalloprotease MMP2 frei,die die extrazelluläre Matrix angreift und damit die Invasionins Gewebe begünstigt. Dieses Enzym wird allerdings alsinaktive Vorstufe freigesetzt und erst durch weitere Prozesseaktiviert. Das Ektoenzym MT1-MMPT aktiviert MMP2,wird in Gliomzellen jedoch nicht gebildet. Ein anderer Faktoraus den Gliomzellen hingegen aktiviert Toll-like Rezeptorenin benachbarten Mikrogliazellen, was in diesen zurExpression von MT1-MMP führt. Gliomzellen nutzen aufdiese Weise Mikroglia als Helfer für die eigene Ausbreitung.Ein therapeutischer Eingriff auf Ebene der Toll-like Rezeptorenoder ihrer Signalkaskade könnte die rasche Ausbreitungder Tumorzellen verzögern. Mikroglia könnte somit einneues Ziel der Gliomforschung werden (Markovic et al.,PNAS, 2009, im Druck).Zahlreiche Proteine verschiedenster Aminosäuresequenz,Raumstruktur und zellulärer Funktion lagern sich sich zufibrillären Aggregaten zusammen, die üblicherweise Amyloidegenannt werden. Amyloidbildung steht im Zusammenhangmit Erkrankungen, bei denen die Proteinfaltunggestört ist, und spielt bei neurodegenerativen Krankheitenwie Morbus Alzheimer, Morbus Parkinson oder MorbusHuntington eine wichtige Rolle. Die Arbeitsgruppe von ErichWanker untersucht, ob die Amyloidbildung durch Einwirkungniedermolekularer Wirkstoffmoleküle beeinflußt werdenkann. So konnte gezeigt werden, dass EGCG, eine Verbindung,die ursprünglich aus Grünem Tee isoliert worden war,die Bildung von Huntingtin-Aggregaten sehr wirksamhemmt. Zusammen mit der Gruppe von Jan Bieschke klärtedie Wanker-Gruppe den Wirkungsmechanismus dieserHemmung auf. Die Verbindung bindet direkt an noch ungefaltetePolypeptide und verhindert damit deren Umwandlungin Amyloid-Oligomere und Protofibrillen. Die Substanzgreift also in einen frühen Schritt der Amyloid-Bildungskaskadeein und leitet den Prozess in einen anderen Aggregationswegum, so dass er nicht amyloidbildend wirkt. (Ehrnhoefer,Bieschke and others, Nature Struct Mol Biol. 2008).Function and Dysfunction of the Nervous System 149

Signaling Pathways and Mechanisms in the Nervous SystemStructure of the GroupCarmen Birchmeier-KohlerGroup LeaderProf. Dr. CarmenBirchmeier-KohlerScientific ManagerDr. Michael StrehleSenior ScientistsDr. Thomas MüllerDr. Alistair GarrattScientistsDr. Dominique BröhlDr. Cyril CheretDevelopmental Biology/Signal TransductionWe analyze the functions of signaling molecules and of transcription factors indevelopment of the nervous system and muscle. For this work, we use mice as a modelorganism. The molecular genetics of mice is well developed, and homologous recombinationcombined with embryonic stem cell technology can be used to introduce deletions orinsertions into the genome. A further development of the technique, the Cre/LoxP technology,allows us now to introduce conditional mutations that are restricted to a particular celllineage. We have used these technologies to analyze genes that control myoblast fusion, andshowed that two small G proteins, Rac1 and Cdc42, are essential for the fusion process. Inaddition, we identified the function of several transcription factors in development of thenervous system. Among these is a novel factor, Insm1, that we found unexpectedly to performalso important functions in development of pancreatic beta-cells, the insulin-producingendocrine cells.The role of Insm1 in neuronal developmentRobert Storm, Jochen Welcker, Kira Balueva and ShiqiJia (in collaboration with John Jacob and JamesBriscoe, MRC, London).Insm1 (insulinoma associated antigen) encodes a Znfingerfactor that is transiently expressed in differentiatingneurons throughout the developing nervous system,as well as in endocrine cells of the pancreas andintestine. We generated mice with a targeted mutationto analyze the function of the Insm1 gene. In an initialanalysis, we identified Insm1 as a factor crucial for thedifferentiation of beta-cells in the pancreas. Impairedfunction or loss of pancreatic beta-cells causes diabetes,a prevalent humane disease throughout theworld. In the absence of Insm1, the expression programfor hormones and a plethora of genes coding for proteinsinvolved in secretion and vesicle transport wasdownregulated in beta-cells.However, Insm1 is not only required in endocrine celldevelopment, but is also a crucial component of thetranscriptional network that controls neuronal development.In Insm1 mutant mice, differentiation of sympatho-adrenalprecursors was strongly delayed, whichwas accompanied by reduced proliferation of the precursors.Whereas sympathetic neurons differentiatedlate and in reduced numbers, resulting in small sympatheticganglia, terminal differentiation of adrenal chromaffincells, the major source of noradrenaline, did notoccur. The catecholamine noradrenaline is essential forfetal heart function and survival. Due to a pronouncednoradrenaline deficiency, Insm1 homozygous mutantmice died during mid-gestation, but we could rescuethem by administration of catecholamine intermediates.Analysis of the transcriptional network governingsympatho-adrenal precursor differentiation indicatedthat Insm1 acts downstream of Mash1 (Ascl1) and Phox2b.150 Function and Dysfunction of the Nervous System

Dr. Katja Grossmann*Dr. Elena RoccaDr. Elvira Rohde (guest)Dr. Robert StormDr. Hagen WendeDr. Elena VasyutinaGraduate andUndergraduate StudentsFabian Ahrens*Kira BaluevaJustyna Cholewa-WaclawMaciej Czajkowski*Pedro Garcia Vallecillo*Shiqi JiaUlrich Koestner*Diana LenhardRobin SchneiderJochen WelckerTechniciansBettina BarbyMaria Braunschweig*Sven BuchertKarin GottschlingCarola GriffelAndrea LeschkeIvonne SchiffnerAnimal CaretakersClaudia PäselerPetra StallerowSecretariatDr. Timkehet Teffera**part of the period reportedFigure 1. Insm1 expression in the developing embryo. To analyzeInsm1 expression, we took advantage of the Insm1lacZallele in which lacZ sequences replace the Insm1-codingsequence. Insm1lacZ/+ mice. LacZ expression is detected in theentire primary sympathetic ganglion chain (arrowhead), aswell as in sensory ganglia and in the central nervous system.In a collaborative effort, we could also show that Insm1controls the differentiation of monoaminergic neuronsin the hindbrain. These neurons use either the neurotransmitterserotonin or noradrenaline, both of whichhave a wide range of complementary actions and areimplicated in the pathophysiology of many commonneurological and psychiatric disorders. In Insm1 mutantmice, serotonergic precursors located in the ventralhindbrain, began to differentiate despite the reducedexpression of the postmitotic serotonergic fatedeterminants (Pet1, Lmx1 and Gata2), but failed toproduce serotonin due to the lack of tryptophanhydroxylase 2, an enzyme essential for serotoninbiosynthesis. In brainstem noradrenergic centers ofInsm1 mutants, expression of tyrosine hydroxylase wasimpaired, resulting in aberrant noradrenergic differentiation.The bHLH factor Olig3 functions in thedetermination of neuronal fatesJustyna Cholewa-Waclaw, Robert Storm, DominiqueBröhl, Thomas Müller (in collboration with MathiasTreier, EMBL Heidelberg)The hindbrain is the part of the central nervous systemthat monitors and regulates inner organ function suchas the rate of heartbeat, blood pressure and breathing.To achieve this, hindbrain neurons receive and integrateviscerosensory information from inner organs. Thecomplex circuitry in which neurons participate is establishedduring development and depends on their spatiallyand temporally ordered appearance.One important class of genes that regulates cellulardiversity in the nervous system encodes basic helixloop-helix(bHLH) transcription factors. The Olig3 bHLHfactor is expressed in the ventricular zone of the dorsalFunction and Dysfunction of the Nervous System 151

Control Olig3 -/-ABFigure 2. Genetic lineage tracing in heterozygous and homozygous Olig3 mutant mice. Cre expression that is driven by the Olig3 locus inducesrecombination in the Rosa26R allele, allowing expression of a lacZ gene. The active, recombined lacZ gene is inherited in cells that expressed haveexpressed Olig3 (and therefore the cre recombinase) at earlier stages in development. Cells that express lacZ were identified by X-Gal staining(blue). Shown is the medulla oblongata of control (A: Olig3CreERT2/+; Rosa26R) and Olig3 mutant (B: Olig3CreERT2/-; Rosa26R) mice at E18.5.Arrowheads indicate cuneate nucleus (Cu), inferior olivary nucleus (ION), nucleus of the solitary tract (NTS) and lateral reticular nucleus (LRt).Note the absence of inferior olivary nucleus and nucleus of the solitary tract, as well as the changed distribution of neurons in the dorsal hindbrainof the homozygous Olig3 mutant mice.alar plate of the hindbrain. We found that the Olig3+progenitor domain gives rise to several neuronal subtypesin the dorsal alar plate. We used genetic lineagetracing to demonstrate that these neurons settle in thenucleus of the solitary tract and to precerebellar nuclei.The fate of class these neurons is not correctly determinedin Olig3 mutant mice, and as a consequence, thenucleus of the solitary tract does not form, and precerebellarnuclei, such as the inferior olivary nucleus, areabsent or small.The small G-proteins Rac1 and Cdc42 are essentialfor myoblast fusion in the mouseElena Vasyutina, Benedetta Martarelli, Hagen Wende(in collaboration with Cord Brakebusch, University ofCopenhagen)Skeletal muscle fibers are syncytia that arise by thefusion of myogenic cells. Mononucleated myogeniccells, the myoblasts, fuse with each other to form multinucleatedmyotubes. During development and in theadult, myoblast fusion allows generation, growth andrepair of muscle fibers. Rac1 and Cdc42 are small G-proteinsthat regulate actin dynamics. In Drosophila, RacGTPases, dRac1 and dRac2, act downstream of cell adhesionmolecules to control cytoskeletal rearrangements,and myoblast fusion.We analyzed the function of Rac1 and Cdc42 in myogenesisusing conditional mutagenesis in mice. Weshowed that in the absence of Rac1 and Cdc42,myoblast fusion is severely compromised in vivo and invitro. The deficit in fusion of Rac1 or Cdc42 mutantmyoblasts correlated with a deficit in the recruitmentof actin fibers and vinculin to myoblast contact sites.Moreover, we demonstrated that Rac1 and Cdc42 arerequired in both fusion partners. Thus, our analysisdemonstrated that the function of Rac1 is evolutionarilyconserved from insects to mammals, and that Cdc42,a molecule hitherto not implicated in myoblast fusion,is essential for the fusion of murine myoblasts.152 Function and Dysfunction of the Nervous System

Figure 3. Myoblast fusion in mice requires Rac1 and Cdc42. In themuscle of a control animal (A), single myoblasts (visualized by MyoDin red) fuse to generate multinucleated myotube. In conditional Rac1(B) or Cdc42 (C) mutant muscle, myoblasts do not fuse and myofibersremain short and thin.ControlRac1 fl/fl ;Lbx1 creCdc42 fl/fl ;Lbx1 creSelected publicationsVasyutina E, Martarelli B, Brakebusch C, Wende H, Birchmeier C (2009)The small g-proteins rac1 and cdc42 are essential for myoblast fusion inthe mouse. Proc Natl Acad Sci U S A 106: 8935-8940Storm R, Cholewa-Waclaw J, Reuter K, Brohl D, Sieber M, Treier M, Muller T,Birchmeier C (2009) The bhlh transcription factor olig3 marks the dorsalneuroepithelium of the hindbrain and is essential for the development ofbrainstem nuclei. Development 136: 295-305Wildner H, Gierl MS, Strehle M, Pla P, Birchmeier C (2008) Insm1 (ia-1)is a crucial component of the transcriptional network that controlsdifferentiation of the sympatho-adrenal lineage. Development 135:473-481Hori K, Cholewa-Waclaw J, Nakada Y, Glasgow SM, Masui T, Henke RM,Wildner H, Martarelli B, Beres TM, Epstein JA, Magnuson MA, MacdonaldRJ, Birchmeier C, Johnson JE (2008) A nonclassical bhlh rbpj transcriptionfactor complex is required for specification of gabaergic neuronsindependent of notch signaling. Genes Dev 22: 166-178Gierl MS, Karoulias N, Wende H, Strehle M, Birchmeier C (2006) The zincfingerfactor insm1 (ia-1) is essential for the development of pancreaticbeta cells and intestinal endocrine cells. Genes Dev 20: 2465-2478Function and Dysfunction of the Nervous System 153

Thomas J. JentschStructure of the GroupGroup LeaderGroup LeaderProf. Dr. Dr. Thomas J. JentschScientistsScientistsDr. Graduate MurielStudentsAuberson*Dr. Luiza Bengtsson*Dr. Pawel Fidzinski*Dr. Jens Fuhrmann*Technical AssistantsDr. Ioana NeagoeDr. Gaia NovarinoSecretariatDr. Vanessa Plans*Dr. Guillermo Spitzmaul**part Dr. Tobias of theStauberperiod reportedDr. Vitya Vardanyan*Physiology and Pathology Headof Ion TransportIon transport across cellular membranes is important for cellular homeostasis and hasintegrative functions such as transepithelial transport or neuronal signal transduction.We study these processes at various levels, from biophysical analysis of transport proteins,structure-function analysis, role in cellular functions such as cell volume regulation orendocytosis, to the role in the organism. The physiological role of ion transport proteins hasoften been gleaned from pathologies resulting from their inactivation in human diseases orin mouse models. We have discovered several human ‘channelopathies’ and have generatedand analyzed many mouse models.We focus on CLC chloride channels and transporters, KCC potassium-chloride cotransportersand KCNQ potassium channels, and are currently extending our interestto other channel classes. The mutational inactivation of these ion transport proteins ledto pathologies ranging from epilepsy, deafness, lysosomal storage disease to osteopetrosis,kidney stones and hypertension. We are particularly interested in the control of neuronalexcitability and in the role of chloride and pH in endosomes and lysosomes.Headline BlindtextHeadline BlindtextHeadline BlindtextCLC chloride channels and transportersPeoples names Peoples names Peoples names PeoplesThe CLC gene family, discovered in our laboratory innames Peoples names Peoples names1990, encodes plasma membrane chloride channelsReceptor-mediated endocytosis is the main mechanismand chloride transporters of intracellular membranes.that enables selective transport of macromolecules acrossIn the past couple of years, we identified associatedthe plasma membrane into cells. Significant progress hasβ-subunits, been made indiscovered elucidatingthat the various certainsteps vesicular of endocytosis CLCs areelectrogenic at the cellular level. Cl - /H However, + -exchangers, the physiological performedrelevance structurefunctionmany endocytic analysis, pathways and uncovered for organseveral functionnew remains patholo-elu-ofgies sive. resulting from their dysfunction.The main class of endocytic receptors is a group of proteinsAn known inner as the ear-specific LDL receptor knock-out gene family. ofNine thereceptors Cl - channel existβ-subunit in mammalian barttin organisms, explains all of which deafness sharein common humanstruc-tural motifs syndrome (Figure 1). type The prototype IV of the gene family isBartterthe LDL receptor, a protein that mediates cellular uptake ofGesa Rickheit, Hannes Maier, Anselm Zdebikcholesterol-rich lipoproteins. Since other family membersHuman Bartter syndrome type IV is associated withalso bind lipoprotein particles, contributions of these receptorsto cellular renal loss andof systemic fluid and lipidsalt, metabolism as well as arewith anticipat-con-severegenital ed. A major deafness. part ofWe ourhave workpreviously is dedicatedshown to the that functional barttin,characterization the proteinof encoded orphan receptors by the inBSND this genefamily that(soiscalled LRPs), particularly focusing on their diverse roles inlipid homeostasis.mutated in that disease, is an accessory β-subunit oftheSortilinschloridearechannelsa secondClC-Kagroupandof endocytic-Kb. Bothreceptorschannelsthatarerecently attracted our attention because of structural andexpressed in the kidney and in the epithelium of thefunctional similarities with LRPs. The founding member ofstria vascularis of the cochlea.We now generated a conditional‘floxed’ barttin KO mouse to delete barttinthis gene family is SorLA, a receptor that shares structuralelements found in the LDL receptor and that also bindsspecificallylipoproteinsin(Figurethe inner1). In addition,ear, therebySorLAavoidingincludesthea domainmassiveidentified salt and in the fluidvacuolar loss that protein leads sorting to early10postnatalproteindeath. (Vps10p), Like a sorting patients, protein thesein mice Yeastare that congenitally directs enzymes deaf.We fromshowed the Golgi that to the thisvacuole. due to Initially a collapse considered of theapositivemosaicvoltage receptorin of the LDL scala receptor media gene (the family, endocochlear subsequent potential) identificationisofnecessary four otherto mammalian drive a depolarizing proteins withinflux Vps10p-ofwhichpotassium domain (Sortilin, through SorCS-1, mechanosensitive -2, and -3) suggested channels the existence in sensoryhair cells. Outer hair cells were no longer able tomechanically Sortilin amplify sound in the inner ear and eventuallydegenerated. We have thus clarified the pathologicalmechanism underlying a form of human deafnessand have revealed a previously unknown role ofchloride channels in the generation of the endocochlearpotential.154 Function and Dysfunction of the Nervous System

Dr. Rubén Vicente García*Dr. Stefanie WeinertDr. Anselm Zdebik*Graduate Students/Postdocs after graduationPhilipp Lange*Carsten PfefferLena WartoschGraduate StudentsEun-yeong Bergsdorf*Gwendolyn Billig*Matthias Heidenreich*Sabrina Jabs*Maren Knoke*Lilia Leisle*Kristin Natho*Patricia PrestonGesa Rickheit*Patricia SejaTechnical AssistantsAnyess von Bock*Alexander FastInga Freyert*Nicole KrönkeIna LauterbachRainer LebenJanet LieboldRuth ParejaPatrick Seidler*Stefanie Wernick*Silke ZillmannStudentsVerena Perneczky*Cathleen Rohleder*Florian WagnerResearch CoordinatorDr. Dietmar Zimmer*Animal CarePetra Göritz*SecretariatPia Philippi**part of the period reportedFigure 1. Model for potassium recycling in theinner ear. The fluid space of the scala media isin direct contact with the apical membranesof inner hair cells (shown in green) and outerhair cells (red). Its unusually high K + concentrationand positive voltage (+100 mV), both generatedby the epithelium of the stria vascularis,are needed to drive a depolarizing K + currentthrough mechanosensitive channels intothose sensory hair cells. Potassium then leaveshair cells at their basal pole and is transportedback to the stria.ClC-7 is needed for normal protein degradation inrenal proximal tubulesLena Wartosch, Jens Fuhrmann, Tobias StauberClC-7 is a lysosomal Cl - /H + exchanger broadly distributedacross tissues. We have previously shown that itsdisruption leads to osteopetrosis and lysosomal storagedisease in mice and men. We now generated a conditionalClC-7 KO that allowed us, on the one hand, tofollow the CNS pathology much further and to showthat it is owed to a cell-intrinsic effect on neurons. Toinvestigate the lysosomal storage disease that isobserved in the kidney, we selectively inactivated ClC-7in proximal tubules. Combined endocytosis/proteindegradation experiments in vivo showed for the firsttime that the lack of ClC-7 significantly slows proteindegradation. The enlargement of lysosomal-like compartmentsin KO proximal tubules, however, is not dueto excessive protein accumulation because it could notbe prevented by the simultaneous KO of ClC-5 that iscrucial for endocytotic uptake.Role of the vesicular chloride transporterClC-3 in neuroendocrine cellsTanja Maritzen, Damien Keating, Ioana Neagoe,Anselm ZdebikClC-3 is a Cl - /H + -exchanger that is expressed on endosomesand synaptic vesicles. We have previously shownthat its KO leads to a severe neurodegeneration whichresults in a complete absence of the hippocampus aftera few months. However, ClC-3 is broadly expressed inmany tissues. Newly generated antibodies allowed us forthe first time to investigate its tissue expression byimmunocytochemistry. We found that ClC-3 is highlyexpressed in several neuroendocrine tissues, includingadrenal glands and pancreatic islets, where it isexpressed in all secretory cell types. ClC-3 was detected inendosomes in synaptic-like microvesicles, but not in largedense core vesicles that are responsible e.g. for insulin oradrenalin secretion in β-cells or chromaffin cells, respectively.Nonetheless, stimulated secretion of either hormonewas reduced in ClC-3 KO cells, suggesting an indirecteffect of ClC-3 on large dense core vesicle exocytosis.Function and Dysfunction of the Nervous System 155

Figure 2. Cellular model for ionic transport across the stria vascularis. In this multilayered epithelium, potassium is secreted across the apicalmembrane of marginal cells through KCNQ1/KCNE1 K + -channels. Potassium is taken up basolaterally through the combined transport activity ofthe Na,K-ATPase and the NaK2Cl cotransporter NKCC1. The latter transporter needs Cl - channels for recyling. Our work has shown that this task iscarried out by ClC-Ka/barttin and ClC-Kb/barttin Cl - channels. If the β-subunit is missing as in patients with Bartter syndrome type IV or in ourKO mouse model, potassium secretion is impaired and the K + -concentration in the extracellular space between marginal and intermediate cellsincreases. This leads to a dramatic drop of the endocochlear potential, which is largely generated by a K + -diffusion potential through Kir4.1K + -channels in the apical membrane in intermediate cells. This drop in potential leads to deafness.Figure 3. Selective degeneration of outer hair cells in barttin KO mice. The drop in endocochlear potential owed to a loss of barttin leadsto a functional impairment of outer hair cells and later also to their degeneration, as shown by immunocytochemistry (upper row; innerhair cells stained in red, outer hair cells in green) and hematoxin-eosin staining (below, outer hair cells indicated by arrows).156 Function and Dysfunction of the Nervous System

Determinants of anion-proton coupling inmammalian endosomal CLCsAnselm Zdebik, Eun-Yeong Bergsdorf, Michael Pusch,Giovanni ZifarelliSimilar to the bacterial EcClC-1 protein, and in contrastto their previous classification as Cl - -channels, ClC-4and ClC-5 are antiporters that exchange chloride forprotons. By mutagenesis and biophysical analysis, weshowed that a glutamate at the cytoplasmic face of thetransporter is important for proton coupling. It apparentlyserves as a proton acceptor. Cl - /H + -transport persistswhen this residue is mutated to other titratableamino-acids, but transport is altogether stopped whenit is mutated e.g. to alanine. Transport is restored whena centrally located ‘gating glutamate’ is neutralized,which eliminates Cl/proton coupling at a centralexchange site and therefore eliminates the need of protonsfor Cl transport.Residues important for nitrate transport in plantand mammalian CLC proteinsEun-Yeong Bergsdorf, Anselm ZdebikAtClC-a from the plant Arabidopsis has been shown byBarbier-Brygoo and colleagues to function as a NO3 - /H +exchanger with which plants accumulate the nutrientnitrate into their vacuoles. This requires tightly coupledcountertransport. However, when nitrate is transportedby mammalian endosomal ClC-4 and ClC-5, it is largelyuncoupled from protons. Sequence comparison identifieda serine in a highly conserved signature sequenceof ClC-4/5 that is exchanged by a proline in AtClC-a.Mutating the serine of ClC-5 to proline led to tightlycoupled nitrate/proton exchange, whereas the planttransporter lost its preference for nitrate when its prolinewas mutated to serine. The ClC-0 Cl channel fromelectric fish also gained nitrate selectivity when thisproline was introduced. We have thus identified aresidue crucial for anion selectivity and have also beenable for the first time to functionally express a plantCLC in animal cells.KCl and NaK2Cl cotransportersCarsten Pfeffer, Guillermo Spitzmaul, Patricia Seja,Valentin Stein, Hannes MaierWe have previously knocked-out all KCl-cotransporterisoforms (KCC1-4) in mice which led to specific andhighly interesting phenotypes. We are continuing ourstudies on KCCs with conditional KOs. Our major focusis on KCCs expressed in neurons, where KCC2 in particularlowers the cytoplasmic chloride concentration.Such a low concentration is necessary for the inhibitoryaction of GABA and glycine, which act on ligand-gatedchloride channels. We are currently investigating variousmouse lines in which KCC2 has been inactivated inspecific sets of neurons.We have also studied the transporter that is the majorplayer in elevating cytoplasmic chloride in neuronsbefore the expression of KCC2 kicks in, namely theNaK2Cl cotransporter NKCC1. We found that NKCC1 isan important, though not the only, transporter elevatingintraneuronal chloride. The excitability and spontaneousnetwork activity of hippocampal slices werereduced in the first 10 days after birth. This correlatedwith a delay in synapse maturation. However, in contrastto speculations by others, no morphologicalchanges were observed.KCNQ potassium channelsMatthias Heidenreich, Guillermo Spitzmaul, VityaVerdanyan, Pawel FidzinskiThere are five different isoforms of KCNQ (Kv7) potassiumchannels, KCNQ1- KCNQ5. KCNQ2-KCNQ5 mediate‘M-currents’ that regulate neuronal excitability. We hadpreviously shown that KCNQ2 and KCNQ3 underlie aform of human epilepsy and that dominant KCNQ4mutations are a cause of human deafness and havepublished a few years ago a mouse model for KCNQ4deafness. We are now investigating possible vestibularphenotypes in these mice and are generating othermouse models.Selected PublicationsBlanz J., Schweizer M., Auberson M., Maier H., Muenscher A., Hübner C.A.,Jentsch T.J. (2007). Leukoencephalopathy upon disruption of the chloridechannel ClC-2. J. Neurosci. 27, 6581-6589.Maritzen T., Keating D.J., Neagoe I., Zdebik A.A., Jentsch T.J. (2008). Role ofthe vesicular chloride transporter ClC-3 in neuroendocrine tissue. J.Neurosci. 28, 10587-10598.Rickheit G., Maier H., Strenzke N., Andreescu C.E., De Zeeuw C.I., ZdebikA.A., Jentsch T.J. (2008). Endocochlear potential depends on chloridechannels: mechanism underlying deafness in Bartter syndrome IV. EMBOJ. 27, 2907-2917.Pfeffer C.K., Stein V., Keating D.J., Maier H., Rinke I., Rudhard Y., HentschkeM., Rune G., Jentsch T.J., Hübner C.A. (2009). NKCC1-Dependent GABAergicExcitation Drives Synaptic Network Maturation During EarlyHippocampal Development. J. Neurosci. 29, 3419-3430.Bergsdorf, E.-Y., Zdebik A.A., Jentsch T.J. (2009). Residues important fornitrate/proton coupling in plant and mammalian CLC transporters. J. Biol.Chem. 284, 11184-11193.Function and Dysfunction of the Nervous System 157

Structure of the GroupGroup LeaderFritz G. RathjenScientistsRené JüttnerChristopher PatzkeHannes SchmidtLuminita Stoenica*Fritz G. RathjenNeuronal ConnectivityThe functioning of the nervous system is critically dependent on the correct wiring ofneurons which is primarily established during embryonic and early postnataldevelopment. To generate precise circuits neuron extends an axon and dendrites which areguided to their targets by growth cones. This highly motile structure at the tip of axons ordendrites is steered by molecular guidance cues located in the local environment. In thetarget area neurons then establish synapses that are the fabric of communication betweenneurons. Our research group focuses currently on the following molecular aspects on theformation of neuronal connectivity: branching of axons and dendrites and formation andmaturation of synapses.Axonal and dendritic branching are key steps inregulating neuronal connectivityOne important prerequisite to generate the complexcircuits of the mature nervous system is the arborizationof axons and dendrites. This key process enables anindividual neuron to innervate multiple targets wherebyinformation from neurons in various locations canbe integrated. Branching is therefore a critical step inthe formation of complex neuronal circuits. Despite itsimportance the extracellular signals and the intracellularsignaling machinery that regulate branching haveremained poorly understood. To investigate theseprocesses we concentrated on sensory axons projectinginto the spinal cord and on cortical neurons.A cGMP signaling cascade is essential for branchingof sensory axonsSensory axons enter the spinal cord at the dorsal rootentry zone where they branch into a rostral and a caudalarm. These two arms remain confined to lateralregions of the cord and grow over several segments inboth directions. Collaterals are then generated by buddingfrom these stem axons which extend into the graymatter of the cord. Sensory axons therefore reveal atleast two branching modes: splitting of the growthcone when arriving at the cord followed by budding ofcollaterals (interstitial branching) from the stem axons.By genetic ablation and by single axon tracing werevealed that a cGMP-dependent signaling cascade isimportant for sensory axon branching at the dorsal rootentry zone. In the absence of either the natriuretic peptideC (CNP), the receptor guanylyl cyclase Npr2 or thecGMP-dependent kinase I (cGKI) axons are unable toform a T-shaped branch when entering the spinal cord(Figure 1 and Figure 2). Instead axons turn in rostral orcaudal directions. The other branching mode – the formationof collaterals – is not regulated by this signalingcascade.Our current efforts concentrate on the identification offurther upstream and downstream components toestablish a complete picture of this signaling cascadeas well as on other axon systems that regulate branchingby this signaling cascade.158 Function and Dysfunction of the Nervous System

PhD studentsJadwiga CholewaRogerio CraveiroVincent Ramillon*Agne StonkuteGohar Ter-Avetisyan*Philip Tröster*Master of Science studentsMie Kristensen*Katharina Seifferth*Steven Sievers*Daniela Spohr*Technical assistantsHannelore DrechslerMadlen DriesnerMechthild HenningSecretariatBirgit Cloos (part time)*part of the period reportedFigure 1. A cGMP signaling cascade important for axonalbifurcation.Our starting point was the cGKI (encircled) which we showed to beimportant for branching. During the past funding period we usedgenetic ablation techniques to characterize the receptor guanylylcyclase Npr2 and its ligand CNP (natriuretic peptide C) and showedthat these components are essential for bifurcation. Furthermore,several phosphorylation targets have been identified by biochemicalapproaches; however, it is currently unknown whether they are alsoimportant for bifurcation.Figure 2. Impairment of bifurcation in the absence of cGMP signaling.A shows a single bifurcating sensory axon in a normal mouse embryo while B reveals an axon in the absence of CNP signaling which turnsbut does form T-shaped branches.The transmembrane protein CALEB is implicatedin synapse maturationBy a detailed biochemical and histological characterizationwe revealed that CALEB is primarily localized in thesomato-dendritic compartment of a neuron and partiallyco-localizes with synapses (Figure 3). It is stronglyexpressed in early postnatal stages of the mouse whenthe majority of synapses and dendritic trees areformed.Dendrites are the input regions of a neuron. In manycases they arborize into a tree like structure by anextensive and complex branching program. Earlystages of dendrite development might be regulated byintrinsic programs specific for a neuron type whichmight then be modulated by different external signalsdetected by specific receptor proteins. During thedevelopment of dendritic trees CALEB appears to berequired for specific aspects of synapse maturation. Forexample, CALEB-deficient synapses displayed higherFunction and Dysfunction of the Nervous System 159

paired-pulse facilitation, less depression during prolongedrepetitive activation and a lower rate of spontaneouspostsynaptic currents that are based on a lowerrelease probability of neurotransmitters at early butinterestingly not at mature postnatal stages.CALEB is a transmembrane protein composed of an N-terminal segment that contains chondroitinsulfatechains followed by an acidic stretch, and an EGF-likedomain, a transmembrane and a cytoplasmic segment.The EGF-like domain of CALEB is related to the EGFdomains of the neuregulins, TGFα or EGF itself andtherefore CALEB might be considered to be a memberof the EGF-family of differentiation factors. CALEBappears to be generated as a precursor protein thatbecomes converted in a truncated transmembraneform with an exposed EGF domain. In slices and in culturesthis conversion occurs at the cell surface which isfacilitated by membrane depolarization and calciuminflux through voltage gated calcium channels.The understanding of the cell biological function ofCALEB in the development of neuronal circuits is ofgreat interest since genetic linkage studies identifiedCALEB as a putative susceptibility gene for schizophreniain humans which is considered as a disorder ofdevelopment.Figure 3. Localization of CALEB on dendrites.CALEB (green) is primarily found on structures that are positive forMAP2 – a marker for dendrites – and appears to be absent fromaxons which are stained by anti-neurofilament (red).Neural cell adhesion proteins and their functionin the formation of neural networksCell adhesion proteins of the Ig superfamily, the cadherinsor the neurexins are thought be essential for theformation of neuronal circuits. Currently we are characterizingthe Ig superfamily member CAR (coxsackieadenovirusreceptor) that is strongly expressed atdevelopmental stages and then becomes down regulatedin the mature brain. CAR is a transmembrane proteincomposed of two Ig-like domains and an intracellularsegment that becomes alternatively spliced. Ourelectrophysiological recordings and calcium imagingmeasurements using wildtype and knockout neuronsreveal that CAR is not simply an adhesion protein. Incontrast, manipulation of CAR by using a specific ligandindicates that CAR affects intracellular calcium signalingand thereby influences the development of neuronalnetworks. Binding as well as structural studiesdemonstrate homophilic and heterophilic interactionsof CAR for which specific extracellular domains areimportant.160 Function and Dysfunction of the Nervous System

Selected PublicationsSchmidt, H., Stonkute, A., Jüttner, R., Koesling, D., Friebe, A., F.G. and Rathjen,F.G. (2009) C-type natriuretic peptide (CNP) is a bifurcation factor forsensory neurons, PNAS, 106, 16847-16854.Lisewski, U., Shi, Y., Wrackmeyer, U., Fischer, R., Chen, C., Schirdewan, A.,Jüttner, R., Rathjen, F., Poller, W., Radke, M.H. Gotthardt, M. (2008). The tightjunction protein CAR regulates cardiac conduction and cell-cellcommunication, J. Exp. Med, 205, 2369-2379.Schmidt, H., Stonkute, A., Jüttner, R., Schäffer, S., Budgereit, J., Feil, R.,Hofmann, F., and Rathjen, F.G. (2007). The receptor guanylyl cyclase Npr2 isessential for sensory axon bifurcation within the spinal cord. J. Cell Biol.,179, 331-340.Jüttner, R., Moré, M.I., Das, D., Babich, A., Meier, J., Henning, M., Erdmann, B.,Müller, E.C., Otto, A., Grantyn, R., and Rathjen, F.G. (2005). Impaired synapsefunction during postnatal development in the absence of CALEB, an EGFlikeprotein processed by neuronal activity. Neuron, 46, 233-245.Function and Dysfunction of the Nervous System 161

Gary R. LewinStructure of the GroupGroup LeaderProf. Dr. Gary R. LewinScientistsDr. Jing Hu*Dr. Alexey KozlenkovDr. Stefan LechnerDr Nevena Milenkovic*Dr Simone Pifferi*Dr Kate PooleDr. Ewan St. John SmithMolecular Physiology of SomaticSensationSomatic sensation includes all those sensations that we consciously feel after stimulation ofthe body, e.g. touch, warmth, cooling, or even limb movement. We experience thesesensations as a direct result of the activation of sensory neurons that are located in the dorsalroot ganglia (DRG). In our group we are interested in the molecular mechanisms that allowthese neurons to transduce these varied stimuli. Sensory neurons can, for example, detectchanges in temperature of the skin in non-noxious (not painful) as well as the noxious range(painful heat, or cold). They can also detect gentle movement of the skin as well as intensemechanical stimulation of the skin that is normally harmful. The nature of the transductionmolecules involved together with the developmental events that lead to specification of theappropriate sensory neuron sub-types are actively investigated the lab.Molecular Basis of MechanotransductionYinth Andrea Bernal-Sierra, Liudmilla Lapatsina, StefanLechner, Alexey KozlenkovMechanotransduction is the process whereby receptorproteins present in the endings of sensory neurons areable to detect mechanical stimulation of the tissuethey innervate. We have used information from geneticexperiments with the nematode worm C.elegans toidentify possible vertebrate candidate proteins thatmight detect mechanical stimuli. Genetic screens fortouch insensitive worms have turned up around 15genes whose function is necessary to confer touch sensitivity.These genes were named mec for mechanicallyinsensitive and we have focused on identifying a rolemammalian orthologs of these genes in touch sensation.The mec genes in C.elegans have been proposed towork together in a mechanotransduction complex. Anessential component of this complex is the membraneprotein MEC-2 that forms a hairpin in the membraneand might regulate the activity of the mechanotransducingchannel. We have cloned new vertebrateshomologues of mec genes and have created mousemutant alleles to characterize the in vivo function ofthese genes. MEC-2 is a member of a large family ofproteins that contain a stomatin-like domain. A memberof this family called SLP3 (stomatin like protein-3)was cloned by our group, and we subsequently generateda mouse model with a null mutation of the SLP3locus. In SLP3 mutant mice many mechanoreceptors (ortouch receptors) in the skin do not work in the absenceof the SLP3 protein. In order to analyze touch sensationin mice we also developed a novel behavioral assay fortouch driven behavior in rodents. This assay is based onthe ability of mice to detect and react to gratings, whichare fine enough to have a textured quality.We were verypleased to find that SLP3 mutant mice have severedeficits in their ability to detect such textured surfaces.Current work in the lab focuses on the role of relatedmembers of the stomatin-domain family in mechanotransduction,structure function studies and the identificationof further essential interaction partners forSLP3.162 Function and Dysfunction of the Nervous System

Graduate StudentsYinth Andrea Bernal-Sierra*Li-Yang ChiangHenning FrenzelRegina Hartl*Liudmilla LapatsinaSören MarkworthDamir Omerbasic*Rui WangTechnical AssistantsKathleen Barda *Liana Kosizki*Anke ScheerHeike ThränhardtAnja Wegner*SecretariatManuela Brandenburg*part of the period reportedA single sensory neuron growing on a check patterned substrate. Top right (A) shows the stripes of laminin printedonto a glass surface. Note that different proteins can be printed in the vertical (red) or horizontal axis (green). (B )Phase contrast picture of a sensory neuron growing on the patterned substrate. Note that the neuron only growsneurites along the stripes to form a window like pattern of growth. In this picture the neuron was recorded with apatch pipette (RE) and the neurites can be mechanically stimulated with a nanomotor (MS). (C) The neurons wasfilled with a yellow dye via the recording pipette to show that neurites belong to the indicated cell. (D) Colour combineshowing the pattern together with the yellow neurons and its neurites.Neuronal nanodetection and micro-patterning,engineering sensory neurons for functionLi-Yang Chiang, Jing Hu, and Kate PooleThe mechanosensitive ion channels that are expressedby sensory can be measured using high-resolution electrophysiologytechniques. We have recently shown thatsuch ion channels in the membranes of cultured DRGneurons can be activated by stimuli in the nanometerrange. We have also gathered considerable evidencethat the mechanosensitive channels are actuallyopened via a protein tether that attaches to laminincontainingextracellular matrices. In order to study theinfluence of different extracellular matrices on mechanotransductionand to quantify the tiny forces that arerequired to open mechanosensitive channels we havestarted to a use variety of new micro-fabrication techniques.For example, we have used micro-patterning ofmatrix molecules in order force neurons in culture toadopt morphologies that better match the in vivo situation.We can also use such patterning to test the localinfluence of specific matrix molecules on transductionability or axon branching behavior. We have shown thatFunction and Dysfunction of the Nervous System 163

Model summarizing the three waves of mechanosensitivity acquisition in different sensory neuron subtypes. Developmental stage is depictedfrom top to bottom for mechanoreceptors (blue) and nociceptors (red). Cartoons (right) depict the innervation of the limb with the knowndistribution of the neurotrophins NT-3 and NGF. Figure reproduced from Lechner et al. 2009 EMBO J.sensory neurons can be made to grow to produce highlystructured patterns in vivo (see Figure 1). Anotherapplication of micro-engineering is to make neuronsgrow on three dimensional surfaces that allow us togauge the forces needed to open mechanosensitive ionchannels in single cells.Touch, hearing and the development ofmechanosensationHenning Frenzel, Regina Hartl, Stefan Lechner, NevenaMilenkovic, Simone PifferiHereditary deafness is a relatively common phenomenonand a large number of genes have been identifiedthat when mutated lead to deafness in mouse andman. We are working with several deaf mutant mice toexamine whether genes required for normal mechanotransductionin the inner ear may also be required fornormal cutaneous sensation. Our data indicate thatmembers of the unconventional myosin protein familyhave a common function in sensory neurons and in haircells, mechanotransducing cells of the inner ear. In bothcell types these proteins may function to regulate theadaptation of the mechanotransduction channels. Weare currently working on further hearing genes thatmay also affect cutaneous mechanosensation. Thesame genes as we study in the mouse are also mutatedin humans and it is possible that the perception ofcutaneous touch stimuli is altered in such patients. Weare measuring pyschometric functions in normals andhearing impaired people in order to describe quantitativelydifferences in the perception of touch.We are alsocarrying out a large twin study, to examine the heritabilityof touch acuity in humans. Initial results suggestthat genetic factors are very important in determininghow good our sense of touch is.We are also pursuingthe hypothesis that some of the genetic factorsinfluencing touch may also directly affect the secondmechanosensory sense, hearing.We have been interested in the development ofmechanosensation for many years and it was remarkablehow little was known in this area. We have recentlyshown, in a very detailed study, that sensory neuronsacquire their competence to detect mechanical stimulivery early in embryonic development. Interestingly, very164 Function and Dysfunction of the Nervous System

distinct developmental mechanisms are used to inducesuch competence in neurons that underlie touch sensationas opposed to nociception (Painful stimuli). Forexample, we have shown that NGF plays a critical role inthe acquisition of transduction competence by nociceptors.Tuning pain sensitivityStefan Lechner, Nevena Milenkovic, Rui WangNociception is describes our ability to respond to potentiallyor actually damaging stimuli. An important aspectof the biology of nociception is that after injury peopleand animals become much more sensitive to sensorystimulation than before injury. This phenomenon issometimes called sensitization and it is often desirableto block this process after inflammation to prevent painbecoming pathologically severe. We are interested inthe cellular and molecular basis of sensitization. Werecently discovered that some endogenous chemicals,such as ATP and UTP that are released from damagedcells during inflammation can potently increase themagnitude of the mechanosensitive current in sensoryneurons. This would have the effect of making nociceptorsinnervating inflamed tissue more sensitive tomechanical stimuli, and such a phenomenon mayunderlie the tenderness that follows inflammation.Identification of the mechanotransducer as a target ofinflammation indicates that, this as yet unknown ionchannel, may be an excellent molecular target to blockin order to treat pain after inflammation.Sensitization also happens to heat stimuli, a familiarexample is the increased sensitivity to even moderatetemperature that we experience following UV inducedsunburn.We recently identified the ligand of the tryosinekinase receptor c-Kit as a very potent factor inincreasing the sensitivity of nociceptors to noxiousheat. There are already clinically available potent blockersof c-Kit e.g. Gleevec, that are used to treat a varietyof cancers. We are present carrying out a patient basedstudy to determine if Gleevec and related compoundshave significant analgesic effects when used in man.This study is being carried out within the ECRC at theMDC and at the Charité university hospital.The naked mole rat is an unusual subterranean rodentin many respects. It is the only known poikilothermicmammal (ie. cold blooded), it lives in colonies with aninsect-like social structure, and it is also the longestlivedrodent species known (lifetimes in excess of 25yrs). Interestingly, although this animal has normalacute pain responses it displays no hypersensitivity (socalled hyperalgesia) to a variety of inflammatory andchemical stimuli. What is particularly striking in thenaked mole rat is that the animals completely lack aneuronal or behavioral response to acid. We suspectthat at the heart of this specialized adaptation lies indistinct gene variants encoding ion channels and associatedchannels that are required for the transductionof painful stimuli.We are at present cloning and characterizinggenes coding ion channels from the nakedmole rat to address this issue. We have cloned andstarted to characterize the naked mole rat capsaicinreceptor, an ion channel called TRPV1 as well as the tyrosinekinase receptor trkA the activation of which by NGFcan potently potentiate TRPV1. We are presentlyembarking on a large scale molecular characterizationof the naked mole rat transcriptome using so-calleddeep sequencing technologies (In collaboration withWei Chen, BIMSB).Selected PublicationsLechner SG, Frenzel H, Wang R & Lewin GR. (2009). Developmental wavesof mechanosensitivity acquisition in sensory neuron subtypes duringembryonic development. Embo J 28, 1479-1491.Lechner SG & Lewin GR. (2009). Peripheral sensitisation of nociceptors viaG-protein-dependent potentiation of mechanotransduction currents. JPhysiol 587, 3493-3503.Milenkovic N, Frahm C, Gassmann M, Griffel C, Erdmann B, Birchmeier C,Lewin GR & Garratt AN. (2007). Nociceptive tuning by stem cell factor/c-Kit signaling. Neuron 56, 893-906.Park TJ, Lu Y, Juttner R, Smith ES, Hu J, Brand A, Wetzel C, Milenkovic N,Erdmann B, Heppenstall PA, Laurito CE, Wilson SP & Lewin GR. (2008).Selective inflammatory pain insensitivity in the African naked mole-rat(Heterocephalus glaber). PLoS Biol 6, e13.Wetzel C, Hu J, Riethmacher D, Benckendorff A, Harder L, Eilers A,Moshourab R, Kozlenkov A, Labuz D, Caspani O, Erdmann B, Machelska H,Heppenstall PA & Lewin GR. (2007). A stomatin-domain protein essentialfor touch sensation in the mouse. Nature 445, 206-209.The Naked Mole Rat a pain free mammal?Damir Omerbasic, Ewan St. John SmithFunction and Dysfunction of the Nervous System 165

Structure of the GroupInes Ibañez-TallonGroup LeaderDr. Ines Ibañez-TallonScientistsDr. Silke FrahmDr. Julio Santos-TorresGraduate StudentsAnnika StürzebecherMartin LaquaSebastian AuerMarta SlimakLeiron FerrareseTechnical AssistantsBranka KampfrathDaniela KurzhalsSecretariatDr. Timkehet TefferaMolecular Neurobiology ofCell-surface Channels and ReceptorsIon channels and neurotransmitter receptors control cellular communication betweennerve cells influencing neuronal excitability and synaptic transmission. Our group isinterested in dissecting the contribution of individual channels and receptors on neuronalfunction in the mammalian nervous system. We use a combination of molecular, behavioral,electrophysiological, and genetic tools for the functional analysis of ion channels in thedevelopment of the nervous system and in disease, using mice as a model organism.Genetic control of neural circuitsContemporary genetic methods allow cell-selective targetingwithin complex neural circuits. We have recentlydeveloped a novel genetic method to manipulate ioniccurrents in vivo using genetically encoded cell-surfaceanchored toxins and neuropeptides, based on the discoveryof endogenous prototoxins. The ongoing projectsin our group are aimed at silencing or manipulatingspecific ion channels in defined neuronal circuits.The questions we are addressing are: how a particularclass of ion channels in one cell population contributesto the function of a given neuronal circuit, and whethersilencing one cell population has an impact in only thatcircuit and/or also affects the next circuit. Anotherquestion of interest is whether these functions can berestored upon reversibly inhibiting the expression ofthe cell-surface toxin or peptide. To approach thesequestions, we are focusing on specific neuronal circuitsin which manipulation of certain ion channels couldhelp dissecting the cascade of events that leads tochronic pain, hearing impairment, and nicotine mediatedeffects. To target these circuits, we are using geneticapproaches such as BAC and knock-in transgenesis andlentiviral vectors to achieve cell-specific and stableexpression in specific neuronal populations in vivo.Endogenous protoxin modulators of nicotinicacetylcholine receptorsThe cholinergic modulators lynx1 and lynx2 are aunique class of cell-surface regulatory molecules. Thesemolecules are members of the Ly6 superfamily thatalso includes the three-finger fold snake venom toxins,α- and κ-bungarotoxin. Both lynx1 and lynx2 form stableassociations with nicotinic acetylcholine receptors(nAChRs) and alter their function in vivo. Lynx1-like moleculesare well conserved across species, both in structureand function, suggesting the importance of cellsurfacemodulators of nicotinic receptors in nature.Cell-surface tethered toxin and peptidemodulatorsWe have developed a novel strategy, termed “tetheredtoxins and peptides” to characterize neuronal circuitsusing the evolutionary derived selectivity of venompeptide toxins and endogenous peptide ligands, suchas lynx1 prototoxins. Peptide toxins from predatory animalsthat have been routinely employed for neuroscienceresearch do not normally exist as cell-surfaceanchored molecules. Using the scaffold of the lynx1-likegene family, i.e. secretory signal and consensussequences for GPI processing and recognition, it is possibleto produce a series of tethered toxins (t-toxins)that are highly effective modifiers of neuronal activity.166 Function and Dysfunction of the Nervous System

Applications of the tethered toxin and neuropeptide strategy. Endogenous peptide ligands, natural toxins, and synthetic peptides that are modifiedversions of ligands or toxins, can be integrated into recombinant membrane-attached fusion constructs and applied in vitro in transfected ortransduced cells in cell-culture, or in vivo in transgenic or virus-transduced animals. The t-peptide retains the specificity of the toxin/peptide ligandin a region or cell specific expression allowing for controlled manipulation of distinct subtypes of ion channels and receptors in a given neuronalcircuit without affecting other channels/receptors in the cell.Approximately 40 different chimeric t-toxins derivedfrom the venom of several predatory animals havebeen cloned and their activity characterized on voltageand ligand-gated ion channels. The t-peptide strategyhas also been successfully extended to other bioactivepeptides, such as ligand peptides for constitutive activationof GPCRs, illustrating the general applicability ofthis approach for cell-surface modulation of receptors(Figure).Tethered toxins and peptides are being used for verydiverse applications pertaining to experimental animalphysiology. Because of their mode of action at the cellsurface,membrane-anchored peptide molecules actonly on ion channels and receptors present in themembrane of the cell that is expressing the t-toxin or t-peptide, and not on identical receptors present onneighboring cells that do not express the tethered construct.We are applying the t-toxin strategy combinedwith transgenesis and viral-mediated geneticapproaches for investigations regarding the physiologyof neuronal circuits in the mouse nervous system.Selected PublicationsAuer S, Stürzebecher A.S., Jüttner R., Santos-Torres J., Hanack C., FrahmS., Liehl B., Ibañez-Tallon I. Silencing neurotransmission with membranetetheredtoxins. Nature Methods in pressHruska M, Keefe J, Wert D, Tekinay AB, Hulce JJ, Ibañez-Tallon I, Nishi RProstate stem cell antigen is an endogenous lynx1-like prototoxin thatantagonizes alpha7-containing nicotinic receptors and preventsprogrammed cell death of parasympathetic neurons. J Neurosci. 2009Nov 25;29(47):14847-54.Holford M, Auer S, Laqua M, Ibañez-Tallon I. Manipulating neuronalcircuits with endogenous and recombinant cell-surface tetheredmodulators. Front Mol Neurosci. 2009;2:21. Epub 2009 Oct 30.Tekinay AB, Nong Y, Miwa JM, Lieberam I, Ibañez-Tallon I, Greengard P,Heintz N. (2009) A role for LYNX2 in anxiety-related behavior. Proc NatlAcad Sci. 106: 4477-82Miwa J.M., King S. L., Stevens T. R., Caldarone B. J. , Ibañez-Tallon, I,Fitzsimonds R. M., Pavlides C., Picciotto M.R., Heintz N. (2006). Balancingneuronal activity and survival via the cholinergic modulator lynx1.Neuron 51: 587-600Function and Dysfunction of the Nervous System 167

Jochen C. MeierStructure of the GroupGroup LeaderJun.-Prof. Dr. Jochen C. MeierScientistsDr. Sabrina EichlerGraduate StudentsBenjamin FörsteraAline WinkelmannTechnical AssistantsCarola BernertJosephine GroschRNA Editing and HyperexcitabilityDisordersIn a healthy organism, a balance is maintained between the excitation and inhibition ofelectrical impulses generated by neurons in the brain. Deregulation of this balance results innervous system disorders. A core aspect of our work concerns the study of the brain at themolecular level, by investigating post-transcriptional enzymatic processes known in research as"RNA editing" and "splicing". We search for disease-associated alterations in post-transcriptionalprocesses in the nervous system. Within this context, we are more closely scrutinizing glycineand GABA(A) receptors and gephyrin – key components of the molecular machine responsiblefor inhibition of electrical impulses in the brain.Synaptic and tonic inhibition – Glycine receptordynamics from the point of view of gephyrinNeurotransmitter receptors are highly mobile entitieswithin the neuronal plasma membrane. Enrichment ofpostsynaptic domains with neurotransmitter receptorstherefore reflects a dynamic equilibrium between lessmobile, synaptic and highly mobile, non-synaptic receptors.The diffusion rate is slowed down by reversibleglycine receptor binding to the postsynaptic scaffoldingprotein gephyrin. These receptors contribute to synapticinhibition of action potential generation whereashighly mobile receptors, which escaped postsynapticanchoring, are involved in tonic inhibition of neuron firing.In the past, we could identify several novel splicevariants of gephyrin, which were uncovered to adoptspecific functions in the hippocampus. There, certaingephyrin splice variants were identified negative regulatorsof postsynaptic receptor stabilization at inhibitorysynapses, providing us with novel experimentalstrategies for treatment of hyper-excitability disorders,such as temporal lobe epilepsy.Deciphering the molecular basis of Molybdenumcofactor biosynthesisGephyrin has enzymatic activity. It is a multidomainprotein that emerged from fusion of two bacterial proteins,MogA and MoeA. These Escherichia Coli proteinscontribute to the biosynthesis of molybdenum cofactor(Moco), which is an essential component of cellularredox reactions. Mammalian gephyrins are still able tosynthesize Moco because the enzymatic activity of theE. Coli homologous domains is preserved. In mammals,the most important Molybdenum enzyme is sulfite oxidase,which catalyzes the last step in the degradation ofsulfur-containing amino acids and sulfatides. HumanMoco deficiency is a hereditary metabolic disorder characterizedby severe neurodegeneration resulting inearly childhood death. We have identified a numbergephyrin splice variants deficient in Moco synthesis.Therefore, another aspect of our work concerns themolecular and functional dissection of alternativelyspliced gephyrins, using truncated and mutant expressionconstructs in a variety of cell types.168 Function and Dysfunction of the Nervous System

A B CD E FRNA editing emerges as a compensatory albeitpathophysiological mechanism in hyperexcitabilitydisordersTonic inhibition of neuron firing plays a pivotal role inbrain information transfer because it provides a globalcontrol of neuronal excitability. A large body of evidencehas implicated impaired hippocampal GABAergic inhibitionin enhanced susceptibility of neurons to becomehyperexcitable and to generate epileptiform discharges.Glycine receptors have recently been involvedin hippocampal tonic inhibition, and we could isolatemRNAs encoding gain-of-function glycine receptorswith substantially increased apparent affinities forglycine (Figure A-C) and taurine, rendering them perfectlyadjusted to mediating tonic inhibition. We couldestablish that high affinity glycine receptors arise frompost-transcriptional C-to-U RNA editing (Figure D), asdemonstrated by the absence of encoding genomicsequences (Figure E). Furthermore, the C-to-U RNA editinginhibitor zebularine was found to be effective ontonic glycinergic currents elicited in hippocampal neurons(Figure F). Having these tools at hand, functionalanalysis is carried out at a cellular level of investigation,using high affinity receptor expression in primary hippocampalneurons, and at a systemic level, using highaffinity receptor screening of resected hippocampifrom mesial temporal lobe epilepsy (TLE) patients. Sofar, our data point to a compensatory and homeostatic,but pathophysiological, role of high affinity glycinereceptor activation in the course of TLE. Therefore, weare interested in developing this novel functionalglycine receptor property into novel pharmacologicalapproaches to the treatment of hyperexcitability disorders.Selected PublicationsLegendre, P, Förstera, B, Jüttner, R, Meier, JC. (2009). Glycine receptorscaught between genome and proteome – Functional implications ofRNA editing and splicing. Front. Mol. Neurosci. 2:23.Eichler, SA, Förstera, B, Smolinsky, B, Jüttner, R, Lehmann, T-N, Fähling, M,Schwarz, G, Legendre P, Meier, JC. (2009). Splice-specific roles of glycinereceptor α3 in the hippocampus. Eur. J. Neurosci. 30, 1077-1091.Eichler, SA, Kirischuk, S, Jüttner, R, Schäfermeier, PK, Legendre, P, Lehmann,T-N, Gloveli, T, Grantyn, R, Meier, JC. (2008). Glycinergic tonic inhibition ofhippocampal neurons with depolarizing GABAergic transmission elicitshistopathological signs of temporal lobe epilepsy. J. Cell. Mol. Med.12,2848-2866.Smolinsky, B, Eichler, SA, Buchmeier, S, Meier, JC*, Schwarz, G*. (2008).Splice-specific functions of gephyrin in molybdenum cofactorbiosynthesis. J. Biol. Chem. 283,17370-17379. * Equal contribution.Meier, JC, Henneberger, C, Melnick, I, Racca, C, Harvey, RJ, Heinemann, U,Schmieden V, Grantyn, R. (2005). RNA editing produces P185L amino acidsubstitution in glycine receptor α3 resulting in high agonist potency.Nature Neurosci. 8,736-744.Function and Dysfunction of the Nervous System 169

Björn Christian SchroederStructure of the GroupGroup LeaderDr. Björn Christian SchroederGraduate StudentsAlena MaulTechnical AssistantsAriane GieseManuela ReichSecretariatSylvia OlbrichStart of the group: January 2009Signaling and Transport ProcessesThe work of our group is aimed at understanding signal processes and epithelial transporton the molecular level. Currently we focus on the functional characterization of therecently identified TMEM16 family of ion channels with 10 members. Some of them arecalcium activated chloride channels (CaCCs), known to be involved in various types of sensorytransduction like vision, olfaction and pain perception, muscle contraction and blood pressureregulation as well as secretion of hormones and airway fluids. Mutations in one TMEM16 genecan cause Gnathodiaphyseal Dysplasia, a rare bone disease, while other family members areheavily unregulated in some cancer. It has also been suggested to use openers of TMEM16channels to bypass the defect cystic fibrosis chloride channel in lung.IntroductionCaCCs have first been described in salamander photoreceptorsin 1982 and in many other cells in the followingyears. They activate in response to elevated levels of calciumions in cells. The effect of the chloride conductancedepends on the intracellular chloride concentration.Its opening can cause depolarization, faster repolarization,the prevention of action potentials or chloridesecretion. Their function has been studied extensivelyin some cell types. E.g. in olfactory receptor neuronsthey act as an amplifier to ensure neuronal depolarization.The role in some smooth muscle cells is toproduce depolarization that opens voltage dependentcalcium channels to produce contraction, and in manyepithelia, e.g. salivary and lacrimal glands, chloridesecretion through CaCCs drives fluid secretion.However the molecular identity of these channelsremained elusive over the last decades. In 2008, as apostdoctoral researcher at the University of California –San Francisco, my research lead to the discovery thatmembers of the TMEM16 family of membrane proteinsencode CaCCs – making TMEM16s the latest addition tothe small group of ion channel families.So far two members of the TMEM16 family, TMEM16Aand TMEM16B, have been shown to encode CaCCs. Theirfunction and regulation can now be studied on themolecular level. In addition we are interested in the roleof the other members of the TMEM16 family.Structure / function and cell physiology ofTMEM16 proteinsTo perform structure and function studies on theTMEM16 family using site-directed mutagenesis a transientexpression system is required. A very convenientone is the oocyte system in combination with two electrodevoltage clamp. It allows screening of many geneticallymodified ion channel constructs in short time. Wenow establish oocytes of the salamander Ambystomamexicanum as alternative to the traditionally usedXenopus oocytes. The later ones can not be used withCaCCs as they, in contrast to salamander oocytes,express large amounts of endogenous CaCCs. We havegenerated several TMEM16 mutants, which will be analyzedwith this system as well as in transfected HEK-293cells using patch clamp techniques.Some of the constructs we generated are fusion proteinswith the fluorescent reporter’s eGFP and cherry.We use them to search for changes in the sub cellularlocalization of TMEM16 proteins after various stimuli orafter coexpression with potential interaction partners.170 Function and Dysfunction of the Nervous System

CaCC in olfactory receptor neurones Binding of odorants to specific G-protein coupled receptors activates an adenylate cyclase (AC) through theG-protein β-subunit Golf. The produced cAMP binds to and opens cyclic nucleotide gated channels and calcium enters the cell. The initial depolarizationcaused by the opening of this unselective cation channels is than amplified by the opening of CaCCs. It has been estimated that the chloridecurrent can be up to 30 times larger than the cation current.We generated antibodies against TMEM16A and othermembers of the family and could show that TMEM16Aand F show highly polarized expression in epithelialcells and some neurons. This is consistent with calciumactivated chloride currents in native cells which displayin an asymmetric fashion.TMEM16 in vivo studiesTo understand the in vivo function of TMEM16 proteinsand their relationship to CaCCs we, in collaborationwith the group of Christian Hübner, University of Jena,have started to generate TMEM16 knock out mice. To dothis we have used floxed constructs. This will give usthe option of a tissue specific knock out by crossing thefloxed lines with appropriate Cre lines. Currently, it isonly possible to speculate about the experiments necessaryfor the analysis as they will depend mainly onthe observed phenotypes. For the analysis we plan totest physiological functions known or hypothesized todepend on CaCCs including olfaction, taste and bloodpressure regulation. In addition we are interested in therole of TMEM16A in transepithelial water- and iontransport of the lung. Its expression in airway epitheliamakes it a good candidate to be the channel that couldbypass the defective CFTR chloride channel in patientssuffering from cystic fibrosis.We hope that our research will not only help to understandthe physiological role of CaCCs, but also lead tonew treatments of diseases like hypertension, asthmaor cystic fibrosis.Selected PublicationSchroeder, BC, Cheng, T, Jan, YN, Jan, LY. (2008). Expression cloning ofTMEM16A as a calcium-activated chloride channel subunit. Cell. 134,1019-1029.Function and Dysfunction of the Nervous System 171

Structure of the GroupJan Siemens(Sofia Kovalevskaya Prize)Group LeaderDr. Jan Siemens**Graduate StudentsChristina Hanack*Kun Song*Technical AssistantsJana Rossius*SecretariatManuela Brandenburg**part of the period reported** start of the group: May 2009Sensory NeurobiologyThe ability of any living organism to probe and sense stimuli emanating from thesurrounding environment is of fundamental importance for its well-being and survival.While a lot of progress has been made in elucidating sensory systems such as vision andolfaction, our understanding of the somatosensory system, giving rise to the detection of(painful) mechanical stimuli as well as changes in temperature, is lacking behind.Sensory mechanisms not only monitor the outside world but also detect changes of interiorparameters such as blood sugar levels, osmolarity, body temperature and many others.Processing and integration of sensory information from the outside world and the interiorenvironment allows our body to maintain homeostasis.Our research focuses on understanding sensory mechanisms at the molecular level.Previous WorkAs a postdoctoral fellow in the lab of David Julius at theUniversity of California San Francisco, I focused on thermo-sensoryion channels of the pain pathway. My projectshave centered on describing new pharmacologicaltools to study and manipulate these ion channels andon genetic methods to determine their physiologicalroles in vivo.Bites and stings from venomous creatures can producepain and inflammation but little is known of howvenom ingredients activate the pain pathway. In thesomatosensory system, sensors for painful (and thermal)stimuli include the TRP channel family membersTRPV1, TRPM8, and TRPA1. I found that three inhibitorcystein knot (ICK) peptides from tarantula venom targetTRPV1, the capsaicin receptor. In contrast to the predominantrole of other ICK toxins as channel inhibitors,these newly discovered toxins -termed vanillotoxinsfunctionas TRPV1 agonists, which explains how venomscan cause pain. Moreover, these toxins constitute newpharmacological tools to study TRP channel gating andgenerate drugs that block the pain pathway.TRPM8 is activated by menthol, a product of the mintplant used in chewing gum and toothpaste to evoke acooling sensation. Low temperatures activate TRPM8 invitro, further suggesting that TRPM8 is a cold receptor.To test this hypothesis in vivo, we generated TRPM8deficient mice and examined their response to coldstimuli. We found that these mice were not able to discriminatebetween cold and warm temperatures inbehavior assays and displayed dramatically reducedresponses to cold stimuli at the cellular level. Thesefindings validate the hypothesis that TRP channels participatein the sensation of thermal stimuli in theperipheral nervous system.TRPA1 is a detector of environmental irritants such asacrolein, an airway irritant present in tear gas, vehicleexhaust and smoke. We found that 4-Hydroxynonenal(HNE), a molecule produced in response to tissue injury,is a potent activator of TRPA1, demonstrating that thision channel is also an important sensor for endogenousinflammatory factors.In summary, my postdoctoral studies have advancedour understanding of TRP channel function in inflammationand peripheral temperature sensation.172 Function and Dysfunction of the Nervous System

Current ResearchI remain fascinated by mechanistic aspects of sensoryneuroscience and my research team will continue tofocus on understanding sensory mechanisms at themolecular level. We are investigating 3 areas in thisfield:Identification of sensory TRP channel modulators.By analogy to TRP channels in the Drosophila eye, wehypothesize that the mammalian orthologs are componentsof supramolecular membrane-bound proteincomplexes that enable the channels to function specificallyand effectively in a context dependent manner.We are using genetic, biochemical and functionalscreening paradigms to identify components of thisputative modulatory complex.Identification of molecules involved in developmentaland functional aspects of mechanosensation.The ability of cells to detect and transduce mechanicalstimuli is a fundamental biological process that underliestouch, pain and control of muscle tension. In vertebrates,a subpopulation of somatosensory neurons isspecialized to detect these different mechanical stimulibut how they accomplish this task is at present notunderstood. Similarly, the developmental program thatallows these neurons to differentiate into a diversity ofcell types with very specific mechanosensitive traits islargely unknown.We are employing a multidisciplinary approach gearedtowards the identification and characterization of moleculesthat are necessary for the development andfunction of mechanosensory neurons. Using the mouseas a model system, we have conducted a genetic screenthat will allow us to isolate molecules such as transcriptionfactors and signaling molecules involved inthe differentiation of mechanosensory neurons.Subsequently, we will test their potential to drive differentiationof neuronal precursors into specific mechanosensoryneurons using stem cell biology.Molecular mechanisms underlying temperaturedetectionand core body temperature regulation.Temperature detection and regulation is of vital importanceto any homeothermic organism. In order to maintaintemperature homeostasis it is necessary for theautonomic nervous system to monitor small fluctuationsin core body temperature and initiate counterThe picture depicts Embryonic Stem (ES-) cells that have been differentiatedinto neuronal precursors in vitro. The cells have been stainedwith antibodies for the neurofilament nestin (red), the cell proliferationmarker Ki-67 (green). The cell nuclei are labeled by Dapi (blue).Our goal is to differentiate these precursors into sensory neuron lineages,thereby generating largely homogenous cell populations thatcan be used for biochemical experimentation.measures to prevent temperature fluctuations beyonda tightly controlled set point. Key brain centers concernedwith temperature control are the preoptic areaand anterior hypothalamus (PO/AH). These hypothalamicregions harbor neurons that not only detectchanges in core body temperature, but are also believedto receive and integrate input from ascendingsomatosensory pathways carrying information fromperipheral temperature sensors.The molecular machinery underlying central temperaturedetection by hypothalamic neurons is currentlyunknown. We will characterize the thermal responseproperties generated by hypothalamic PO/AH neurons,with the goal of elucidating mechanisms underlyingtheir temperature sensitivity.Selected PublicationsSiemens, J, Zhou, S, Piskorowski, R, Nikai, T, Lumpkin, EA, Basbaum, AI, King,D, Julius, D. (2006). Spider Toxins activate the Capsaicin Receptor toproduce Inflammatory Pain. Nature. 444, 208-212.Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum AI, Stucky CL,Jordt SE, Julius D. (2007). The Menthol Receptor TRPM8 is the PrincipalDetector of Environmental Cold. Nature. 448, 204-208.Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B,Imamachi N, Andrè E, Patacchini R, Cottrell GS, Gatti R, Basbaum AI,Bunnett NW, Julius D, Geppetti P. (2007). 4-Hydroxynonenal, anEndogenous Aldehyde, causes Pain and Neurogenic Inflammationthrough Activation of the Irritant Receptor, TRPA1. Proc Natl Acad Sci. 104,13519-24.Function and Dysfunction of the Nervous System 173

Structure of the GroupGroup LeaderDr. James Poulet*PostdocsJean-Sebastian Jouhanneau*Nevena Milenkovic*Birgit Voigt*James Poulet*start of the group : July 2009Neural Circuits and BehaviorRecent technical advances are allowing researchers to make high resolution recordings andmanipulations of brain activity even while animals are awake and behaving. These data areproviding remarkable insights into normal brain function. Of particular interest is the activity ofneurons in neocortex, as they are involved in conscious movement and sensory perception. Ourlab combines electrophysiological and optical neural recordings with genetically targetedmanipulations of neural activity in mice during trained behavior to investigate the link betweenneural activity, sensory perception and motor behavior.Brain states during behaviorThe very first recordings from the awake human brainby Hans Berger (1929) revealed distinct patterns of corticalactivity during different behavioral states.Different “brain states” were thought to reflect changesin the synchrony of cortical activity and be fundamentalto sensory perception, sensorimotor coordinationand learning. However, it was unknown how corticalsynchrony is reflected in the intracellular membranepotential (V m ) dynamics of behaving animals. With CarlPetersen (EPFL, Switzerland) we showed, using dualwhole-cell recordings from layer 2/3 primarysomatosensory mouse whisker barrel cortex, that theV m of nearby neurons is highly correlated while themouse is sitting still. However, when the mouse movesits whiskers, to sense the environment, an internallygenerated state change reduces the V m correlation(Figure 1).We went on to show that the rate of action potentialfiring was surprisingly low in layer 2/3 pyramidal neurons.Single action potentials were driven by a large,brief and specific excitatory input that was not presentin the V m of neighbouring cells. Action potential initiationoccurs with a higher signal-to-noise ratio duringwhisker movement as compared to quiet periods. Thechange in brain state therefore may increase the overallinformation coding capacity of the brain.We have gone on to investigate the neural mechanismsunderlying the change in brain state. So far we haveshown that the state change is not the result of sensoryfeedback as it persists even after cutting the primarysensory neurons that innervate the whisker pad (Figure2). We are presently investigating the role of the thalamusin regulating cortical state changes.Sensorimotor integration in the mouse forepawcortical regionOur lab will focus on the primary sensory and motorcortical areas associated with mouse forepaw. Neuronalactivity in cortical regions controlling limb movement isparticularly relevant in the emerging field of neuroprosthetics,where cortical activity from paralyzed patientsis used to drive robotic limbs.We will train mice to maketargeted reaching movements while recording andmanipulating cortical neural activity.Role of acetylcholine in cortical processing andbehaviorThe neocortex is densely innervated by axon fibers containingacetylcholine (ACh). Neurons containing AChhave received much attention, perhaps mainly becauseof their degradation in Alzheimer’s disease but also dueto their putative role in cognitive processing such as174 Function and Dysfunction of the Nervous System

Figure 1. (a) Anatomical reconstruction of two layer 2/3 pyramidal neurons in mouse barrel cortex. (b)Simultaneous dual whole-cell recording of cells shown in A while the mouse is sitting still (quiet) and duringwhisking movements (whisk). (c) Cross correlation of V m in these two neurons shows high correlation duringquiet periods and a reduced correlation during whisker movements. (from Poulet and Petersen, 2008).Figure 2. (a) Single image from high speed(500Hz) whisker filming showing measurementof whisker angle (b) Schematic ofwhisker sensory pathway, scissors indicateposition of bilateral ION cut. (c) Membranepotential (V m ) dynamics undergo a changein state during whisking (red) as comparedto quiet periods (black), irrespective ofwhether the IONs were intact (black) or (d)cut (gray).memory, attention, plasticity and modulation of sensoryresponsiveness. Despite the widespread and importantroles of ACh, the underlying in vivo circuit andsynaptic mechanisms of its action in the central nervoussystem have remained elusive. In this project, wewill selectively manipulate the activity of ACh neuronsusing novel genetically-targeted techniques to revealthe precise role of ACh in cortical processing and ultimatelybehavior.Selected PublicationsPoulet JFA, Petersen CC (2008) Internal brain state regulates membranepotential synchrony in barrel cortex of behaving mice. Nature. 454:881-5.Borgdorff AJ, Poulet JFA, Petersen CC. (2007) Facilitating sensoryresponses in developing mouse somatosensory barrel cortex. JNeurophysiol. 97:2992-3003.Poulet JFA and Hedwig B (2006) The cellular basis of a corollarydischarge. Science 311, 518-22.Hedwig B. and Poulet JFA (2004) Complex auditory behaviour emergesfrom simple reactive steering. Nature 430, 781-785.Poulet JFA and Hedwig B (2002) A corollary discharge mechanismmaintains auditory sensitivity during sound production. Nature 418,872-876.Function and Dysfunction of the Nervous System 175

Imaging of the Living BrainStructure of the GroupFrauke ZippGroup LeaderProf. Dr. Frauke ZippScientistsPD Dr. Seija LehnardtOA Dr. Friedemann PaulDr. Carmen Infante-DuarteDr. Volker SiffrinDr. Jason MillwardDr. Ulf Schulze-TopphoffDr. Raluca NiesnerMolecular NeurologyThe group works on immune regulation, neural cell damage and neuronal protectionmechanisms in the context of inflammatory pathologies of the nervous system such asmultiple sclerosis. Methodology ranges from murine and human lymphocyte and brain sliceculture to models of neuroinflammation, in vivo multi-photon-microscopy and magneticresonance imaging. The overall goal is to translate resulting knowledge into proof-of-conceptclinical trials. The work is mainly performed within two collaborative research centers: SFB-TRR43 and SFB 650.The role of the immune system in inflammatoryneurodegeneration and repairNeuronal damage has only recently been shown toinfluence the pathology in inflammatory diseases ofthe central nervous system such as meningitis and multiplesclerosis (MS). We have meanwhile experimentaland clinical data for a marked loss of lower motor neuronsin MS patients. We regularly find dying spinalmotor neurons surrounded by CD3+ (CD4+ as well asCD8+) T cells expressing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in postmortemtissue from MS patients. Based on these findingsand on experimental data in experimental autoimmuneencephalomyelitis (EAE), our study indicates thatdamage to lower motor neurons and TRAIL-mediatedinflammatory neurodegeneration in the spinal cordcontribute to MS pathology (Vogt et al. 2009).Performing two-photon laser scanning microscopy inthe brainstem of living mice suffering from EAE, we furthermorevisualize T cells migrating into the centralnervous system (CNS) and directly attacking neuronsand their processes. Both in mouse and man, we havevery recently in collaboration with M. Bader shown thata system known to play a role in cardio-vascular medicine,namely the kallikrein-kinin-system, is of relevancefor the transmigration of proinflammatory T cell subsetsinto the CNS (Schulze-Topphoff et al, in press).Bradykinin receptor 1 inhibits infiltration, especially of Thelper 17 cells (TH17) which are proinflammatory andencephalitogenic. Prior to their effector function, the Tcells exert distinct behavior at the blood brain barrierwith CD4+ T cell compartmentalization in theparenchyma along CNS vessels. This process is dependenton the chemokine CXCR4, while key adhesion moleculesseem to have no major function in this process(Siffrin et al. 2009). This is in contrast to their essentialrole in the transmigration step, in which integrin-mediatedadhesion is necessary to overcome endothelialbarriers.Within the perivascular compartment, the lymphocytescan interact with each other and/or otherimmune cells as a prerequisite for the sequential stepsinvolved in the process of inflammation and immuneregulation. Collectively, these findings support the ideathat compartmentalization of activated CD4+ T lymphocytesin the target organ serves a similar purpose asin lymph nodes, i.e. increasing the chance of T cell receptorand co-receptor engagement with the whole spectrumof possible outcomes between tolerance andimmunity.176 Function and Dysfunction of the Nervous System

Dr. Jan-Leo RinnenthalHelena RadbruchRobert GlummChristina LillGraduate StudentsNicole HentschelMagdalena PaterkaIvo PanovJosephine HerzTina LeuenbergerTechnical AssistantsNathalie AsselbornThordis HohnsteinBibiane SeegerCornelia GrähningRobert GüntherSecretariatMarion GojowySFB assistant coordinatorChristine KutschbachWith regard to potential repair properties of theinflamed CNS, we have studied the cell-fate decision ofneural progenitor cells (NPC) being affected by theredox state with oxidizing conditions favouring differentiationin astrocytes, whereas reducing conditionsfavour neuron formation. To analyse the molecularmechanism underlying these effects, we investigated agene called Hairy/enhancer of split 1 (Hes1), a transcriptionalrepressor of Mash1, which, in turn, is responsiblefor the activation of a neuron-specific transcriptionprogram.We found that under oxidizing conditions, thehistone deacetylase Sirt1 and Hes1 form a complex thatbinds to and deacetylates histones at the Mash1 promoter.These events cause downregulation of Mash1expression and block neuronal differentation. In areducing environment, the Hes1–Sirt1 complex is notobserved. Instead, Hes1 recruits transcription activatorssuch as CREB binding protein to the Mash1 promoter,and this drives NPC towards a neuronal fate(Prozorovski et al. 2008). The influence of the redoxstate on NPC cell-fate decisions was eliminated byremoval of Sirt1 activity either by RNAi or through theuse of Sirt1 inhibitors. Thus, through its action at theMash1 promoter, Sirt1 seems to act as a cell-fate decisionswitch in NPCs in vitro, with increased Sirt1 activitycausing increased differentiation of NPCs into astrocytesat the expense of neurons. Using healthy miceand EAE, in vivo investigations followed. Injectingyoung mice with an oxidizing agent increased thenumber of newly differentiated Sirt1-positive astrocytesin the brain. Performing in utero electroporationof GFP-marked RNAi constructs against Sirt1 to depleteSirt1 in collaboration with Robert Nitsch, an increasedproportion of Mash1-positive cells was found in postnatalanimals treated with an oxidizing agent. EAEinduces an oxidizing environment and reactive astroglyosis.Whereas Sirt1 expression was low in unaffectedbrain regions, inflamed areas with an abundance ofinvading leukocytes contained an increased number ofcells positive for both GFAP and Sirt1.Selected PublicationsWuerfel J.,M. Haertle, H.Waiczies, E. Tysiak, I. Bechmann, K.D.Wernecke, F.Zipp§, F. Paul§. Perivascular spaces – MRI marker of inflammatory activityin the brain? Brain 2008, 131: 2332-2340Prozorovski T., U. Schulze-Topphoff, R. Glumm, J. Baumgart, F. Schröter, O.Niemann, E. Siegert, I. Bendix, O. Brüstle, R. Nitsch, F. Zipp§, O. Aktas§(2008) Sirt1 critically contributes to the redox-dependent fate of neuralprogenitors. Nature Cell Biology, 10: 385-394Siffrin V, Brandt AU, Radbruch H, Herz J, Boldakowa N, Leuenberger T, WerrJ, Hahner A, Schulze-Topphoff U, Nitsch R, Zipp F. Differential immune celldynamics in the CNS cause CD4+ T cell compartmentalization. Brain2009, 132: 1247-1258Schulze-Topphoff U., A. Prat, T. Prozorovski, V. Siffrin, M. Paterka, J. Herz, I.Bendix, I. Ifergan, I. Schadock, M.A. Mori, J. Van Horssen, F. Schröter, M.H.Han, M. Bader, L. Steinman, O. Aktas, F. Zipp. Activation of kinin receptor B1limits encephalitogenic T lymphocyte recruitment to the central nervoussystem. Nature Medicine 2009, 15: 788-793Vogt J, Paul F, Aktas O, Müller-Wielsch K, Dörr J, Dörr S, Bharathi BS,Glumm R, Schmitz C, Steinbusch H, Raine CS, Tsokos M, Nitsch R§, Zipp F§.Lower motor neuron loss in multiple sclerosis. Annals of Neurology2009, in press§equally contributingFunction and Dysfunction of the Nervous System 177

Pathophysiological Mechanisms of Neurological and Psychiatric DisordersStructure of the GroupHelmut KettenmannGroup LeaderProf. Dr. Helmut KettenmannScientistsDr. Anika Bick-SanderDr. Rainer GlassDr. Katrin FaerberDr. Vitali MatiashDr. Christiane Nolte (part time)Dr. Daniel Reyes-HaroCellular NeurosciencesOur goal is to understand the role of glial cells in physiology and pathology. We focus onquestions as to how neuronal activity is sensed by astrocytes, how astrocytes communicateamong each other, and how they feedback on neurons. A second focus addresses the role ofconnexin proteins in oligodendrocytes function, the cells which are the myelin forming cells inthe brain. Thirdly, we study the expression of transmitter receptors in microglial cells and howactivation of these receptors influences microglial function. This is of particular interest withinthe context of pathology and we are currently studying this question in stroke and gliomas. Afourth line of research addresses the question as to how glioma cells interact with the intrinsicbrain cells, specifically microglia and stem cells. We are aiming to understand this interaction ona molecular level, in particular with the hope of identifying tools which impair glioma invasion.The central nervous system contains two major cellpopulations, neurons and glial cells. The neurons areregarded as the elements mediating the electricalactivity in the brain. As a consequence, neuroscienceresearch of the past has focused on this cell type. Thefunctional role of glial cells is not as obvious: while theywere first described as cells providing only structuralsupport to neurons, a series of more recent studies onglial cell function has attracted the attention of theneuroscience community. It has become evident thatglial cells are essential for the proper functioning of thebrain. The different types of glial cells fulfil distincttasks. Oligodendrocytes are the myelin-forming cells ofthe central nervous system and ensure a rapid signalconduction in the white matter. The role of astrocytes isless well defined; they provide guiding structures duringdevelopment and represent important elements forcontrolling the composition of the extracellular spacemediating signals between the brain endothelium andthe neuronal membrane. They form intimate contactwith synapses and neuronal activity results in astrocyteresponses. Microglial cells are immuno-competent cellsin the brain and their functional role is best defined asthe first responsive elements during pathologic events.The present research program is focused on four topics:(1) the role of astrocytes in information processing (2)the impact of connexion expression for oligodendrocytesfunction (3) the response of microglial cells tobrain injury and (4) the interaction of gliomas withmicroglia and stem cells.Mechanisms of neuron-astrocyte interactionsThis project aims to understand signaling mechanismsbetween astrocytes and neurons. We recently havefocused on two preparations, the barrel cortex and themedial nucleus of the trapezoid body. The Calyx of Heldis a giant glutamatergic terminal on the principal neuronsin this nucleus. It has been used as a modelsynapse to study mechanisms of transmitter releaseand synaptic plasticity since both, pre- and postsynapticelements can be simultaneously recorded using physiologicaltechniques. We have studied the morphologicalarrangements and the properties of the astrocyteswhich are in close contact with the Calyx. We use brainslices containing the medial nucleus of the trapezoidbody and have established to simultaneously record178 Function and Dysfunction of the Nervous System

Graduate StudentsLarisa BulavinaBruno BenedettiGiselle CheungSridhar ChirasaniMinChi KuJitender KumarJochen MüllerMarta MaglioneJulia ParnisStefanie SeifertKristin StockGrietje TessmannKatyayni VinnakotaTechnical AssistantsBrigitte Gerlach (part time)Irene HauptKarin Heufelder (part time)Regina PiskeNadine Scharek (part time)Michaela Seeger-ZografakisFigure 1. Stereological counting ofmicroglial cells in the dentate gyrus ofB6/129svj mouse. Microglia are stained withanti-Iba1 antibodies and visualized withDAB (Olympus, 100x). Nuclei are counterstainedwith toluidine blue.from neurons and astrocytes. We obtained evidencethat two types of astrocytes perceive the Calyx activity.One type of astrocyte is characterized by a complexmembrane current pattern and these cells receivesynaptic input mediated by glutamate. The other typeof astrocyte characterized by a passive membrane currentpattern exhibits currents which are due to glutamateuptake. Ultrastructural inspection revealed thatboth types of astrocytes are in direct contact with both,the pre- and postsynaptic membrane. Moreover, wecould identify glial postsynaptic structures on the cellwith complex current pattern. One goal of this study isto determine how astrocytes integrate synaptic inputfrom defined synapses (funded by a Schwerpunktprogrammof the DFG).The sensory input of the whiskers in rodents is representedin the somatosensory cortex. Each whisker projectsinto a defined cortical area, the barrel field. Theseareas are morphologically delineated and can be recognizedin acute brain slices without additional staining.The barrel cortex is a well established model for plasticitysince removal of whiskers results in changes of thebarrel fields. After stimulation in the cortical layer 4, theinput to the barrel field, we can record responses inastrocytes and in neurons by using Ca 2+ imaging andpatch-clamp recording. While the neuronal activityspreads beyond barrel borders, the astrocyte activity isrestricted to the barrel field. We are now particularlyinterested in the question how astrocyte activity feedsback on neuronal activity. In an ongoing study we try toperturb astrocyte function while simultaneouslyrecording neuronal activity.How does connexin expression affect oligodendrocytefunction?In a collaboration with Prof. Klaus Willecke’s group inBonn we are studying the expression of connexins, themolecular substrate for gap junctions in oligodendrocytes.Gap junctions are communication channelsbetween cells which allow the exchange of moleculesbetween cells, but also serve for intercellular signaling.They can also connect different compartments within agiven cell. By using different combinations of mouselines with connexion deletions, we determine which ofthe connexins are essential to form gap junctionsamong oligodendrocytes and among astrocytes andoligodendrocytes. This also has an important clinicalimpact. Mutations in connexins expressed by oligodendrocytescan lead to defects in myelin formation, animportant function of oligodendrocytes. We are currentlystudying a mouse mutant which mimics apatient mutation which leads to leukodystrophy. WeFunction and Dysfunction of the Nervous System 179

Figure 2. Iba1 positive microglia cells over-expressMT1-MMP when associated with experimentalgliomas. Mouse brains injected with GL261 gliomacells expressing EGFP (green) were studied for themicroglia marker Iba1 (blue) and for the metalloproteaseMT1-MMP (magenta). (A) In the control (gliomanon-injected) hemisphere, the level of MT1-MMPexpression is low. (B) The density of microglial cells ismuch higher in the vicinity of gliomas than in thenormal brain; moreover, MT1-MMP is over-expressedin microglia associated with gliomas; labelling forMT1-MMP is especially intense in microglia makingclose contact with glioma cells, whereas GL261glioma cells express only very low levels of MT1-MMP;a magnified 3D reconstructed micrograph of thetumor area is shown in the insert.are, therefore, interested in the question how couplingdetermines myelin formation and maintenance(Funded by Deutsche Forschungsgemeinschaft).What are the physiological features of microglialcells in brain tissue?Microglial cells are the pathologic sensors and representthe immune cells of the central nervous system.During any kind of disease or any pathological eventsuch as after trauma, stroke or in multiple sclerosis, theresting microglial cell transforms into an activated formcharacterized by an ameboid morphology. Activatedmicroglia can proliferate, migrate to the site of injury,phagocytose, and release a variety of factors likecytokines, chemokines, nitric oxide and growth factors.They also express a variety of receptors for chemokinesand cytokines as expected from a macrophage-like cell.We have addressed the question whether microgliawould also express receptors to sense neuronal activity.We have recently developed an in situ model whichallows us to study the physiological responses of restingand activated microglia. This enables us to characterizethe functional receptors and the physiologicalphenotype of microglia in situ. Using this approach, wecould identify microglial receptors for GABA, the majorinhibitory transmitter of the CNS. Activation of theGABA B receptors suppressed indicators of microglialactivation such as the release of IL-6. A similar reductionin proinflammatory mediators was found withactivation of purinergic receptors and of adrenergicreceptors. GABA also triggers an indirect effect onmicroglia since GABA receptor activation in other glialcells and neurons trigger an elevation in extracellularpotassium which is sensed by microglial cells.This leadsto an enhanced release of the cytokine MIP-1α.Microglia expresses a variety of purinergic receptorsand the expression pattern undergoes changes duringdevelopment and in pathology. We have found an interestinginterplay between purinergic and adenosinereceptors to control microglial migration. In the extracellularspace, ATP is rapidly degraded to ADP, AMP andadenosine. In the brain, two prominent ectonucleotidases,cd39 (NTPDase1) degrading ATP to AMP and cd73(5`-nucleodidase) degrading AMP into adenosine, areexclusively expressed by microglial cells and even haveserved as microglial-specific markers. We found thatATP fails to migration in microglia deficient for cd39.However, the effects of ATP on migration in cd39 deficientmicroglia can be restored by co-stimulation withadenosine or by addition of a soluble ectonucleotidase.We also tested the impact of cd39 deletion in a modelof ischemia, in an entorhinal cortex lesion and in thefacial nucleus after facial nerve lesion. The accumulationof microglia at the pathological sites was markedlydecreased in cd39 deficient animals. We concludethat the co-stimulation of purinergic and adenosinereceptors is a requirement for microglial migration andthat the expression of cd39 controls the ATP/adenosinebalance (funded by DFG).Do microglial cells influence glioma cells?Gliomas comprise the majority of cerebral tumors andpatients have a poor prognosis since there is essentiallyno concept for successful treatment. Gliomas includeastrocytomas, oligodendrogliomas, and the mostmalignant (and untreatable) brain tumor, the glioblastomamultiforme. We have found that microglial cellsstrongly promote glioma growth and invasion. There isan interesting interplay between microglial and gliomacells. Glioma cells release the metalloprotease MMP2180 Function and Dysfunction of the Nervous System

Figure 3. Oligodendrocytic coupling in the corpus callosum of WTmice. Confocal image of the biocytin/streptavidin-Cy3 (red) labellednetwork immunostained for the oligodendroglial markers NG2 (blue)and CNPase (green). A single oligodendrocyte was injected with thegap junction permeable tracer biocytin by whole-cell patch clamp.Biocytin staining revealed a network consisting of 39 cells. The majorityof coupled cells was identified as CNPase expressing oligodendrocytes,only 5% as NG2 positive while 13% was negative for both markers.Within the CNPase positive population 42% of the coupled cellswere weakly expressing NG2.which is important for degradation of extracellularmatrix and promotes invasion. This metalloprotease is,however, released in an inactive, larger form and itneeds to be cleaved to acquire its activity. This cleavageis accomplished by the ectoenzyme MT1-MMP. A factorreleased by the glioma cells activates toll-like receptorsin the surrounding microglial cells and triggers theexpression of the MT1-MMP. Thus, glioma cells exploitmicroglial cells to promote their invasion. Glioma cellsobviously need microglia since they can not produceMT1-MMP themselves: A forced expression MT1-MMP inglioma cells leads to their death. Thus interfering withTLR receptors or their intracellular pathways mightreduce the rapid expansion of glioma cells andmicroglia have become a new target for gliomaresearch.This research is funded by a binational BMBF grant withBozena Kaminska, Warsaw (DLR 01GZ0701).Do stem cells influence glioma cells?We have previously observed that gliomas attract neuralprecursor cells from the subventricular zone. Thesecells migrate over large distances and enwrap thetumor yet they do not originate from the tumor properas was previously suspected. This intrinsic anti-tumorigenicresponse is strongly related to age in an animalmodel and occurs only during youth when neural precursorcells are more active. Consequently, in older animalsthis interaction does not occur. The precursor cellsinhibit tumor growth and addition of exogenous precursorsprolongs the survival rate in older animals. Theproliferative response of NPCs to glioblastomasdepended on the expression of D-type cyclins. In youngmice, NPCs express the cyclins D1 and D2, but theexpression of cyclin D1 is lost during aging, and in adultNPCs only cyclin D2 remains. In young and adult cyclinD2-deficient mice we observed a reduced supply ofNPCs to glioblastomas and the generation of largertumors compared with wild-type mice. We concludethat cyclin D1 and D2 are nonredundant for the antitumorresponse of subventricular NPCs. Loss of a single D-type cyclin results in a smaller pool of proliferatingNPCs, lower number of NPCs migrating to the tumor,and reduced antitumor activity. We are currently characterizingfactors by which stem cells suppress tumorgrowth. Interestingly, there are two distinct mechanisms,one targeting the glioma cell mass inducing celldeath. The second mechanism is targeted towardsglioma stem cells, a small population of cells within thetumor mass identified by the antigen CD 133. Thesecells are highly aggressive and are therefore novel targetsfor therapeutic approaches.This research is funded by a binational BMBF grant withBozena Kaminska, Warsaw (DLR 01GZ0701).Selected publicationsMarkovic, DS., Vinnakota, K, Chirasani, S, Synowitz, M, Raguet, H, Stock, K,Sliwa, M,, Lehmann, S, Kälin, R, van Rooijen, N, Holmbeck, K, Heppner, FL,Kiwit, J, Matyash,V, Lehnardt, S, Kaminska, B, Glass, R, and Kettenmann H.(2009) Glioma induce and exploit microglial MT1-MMP expression fortumor expansion, PNAS, in pressMüller, J, Reyes-Haro, D, Pivneva, T, Nolte, C, Schaette, R, Lübke, J, andKettenmann H. (2009) The principal neurons of the medial nucleus ofthe trapezoid body and NG2+ glial cells receive coordinated excitatorysynaptic input. J Gen. Physiol., in pressCheung, G, Kann, O, Kohsaka, S, Faerber, K, and Kettenmann H. (2009)GABAergic activities enhance macrophage inflammatory protein 1 alpharelease from microglia (brain macrophages) in postnatal mouse brain. JPhysiol. 587,753-768.Hanisch, UK and Kettenmann, H. (2007) Microglia – active sensor andversatile effector cells in the normal and pathologic brain, Nat. Neurosci.10,1387 – 1394.Pocock, JM and Kettenmann H. (2007) Neurotransmitter receptors onmicroglia, Trends Neurosci. 30, 527-535.Function and Dysfunction of the Nervous System 181

Erich E. WankerStructure of the GroupGroup LeaderProf. Dr. Erich WankerScientistsDr. Vinayagam ArunachalamDr. Tachu BabilaRaphaele FoulleDr. Ralf Friedrich*Christian HänigDr. Maciej Lalowski*Dr. Katja Neugebauer*Dr. Albrecht Otto*Dr. Christine Petersen*Dr. Spyros Petrakis*Proteomics and Molecular Mechanismsof Neurodegenerative DisordersProteins are central to almost all cellular functions, such as metabolism, regulation of cellgrowth and communication between cells. They are synthesized from amino acid buildingblocks and must fold into unique three-dimensional structures in order to become functionallyactive. Misfolded proteins are a normal feature of this process. Under physiological conditionsmisfolded proteins are efficiently degraded. When cells are challenged with environmentalstress or genetic mutations, however, protein misfolding increases dramatically and changesvarious cascades of cellular function, which leads to the manifestation of pathologicalphenotypes. More than 35 systemic and neurological diseases are caused by the formation ofabnormally folded protein species, among them the late-onset neurodegenerative disordersAlzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD). To date, onlysymptomatic treatments with limited effectiveness are available for these illnesses.The main objective of our work is to elucidate themolecular mechanisms of protein misfolding diseases.Several lines of experimental evidence indicate thatabnormal protein assembly in vitro and in vivo is a multistepprocess, involving the formation of on- and offpathwayaggregation intermediates such as sphericaloligomers or worm-like protofibrils. However, the structure,size and morphology of these potentially highlytoxic structures have not been fully clarified. Moreover,it remains unclear how misfolded protein species interactwith their cellular environment and cause cellulardysfunction and toxicity. Evidence was presentedrecently that abnormal protein aggregates alter thefunction of complex cellular processes such as membranesignaling or protein degradation mediated by theubiquitin proteasome system (UPS). However, theimpact of these changes on cell function remains largelyunknown. Also, it remains unclear why certain typesof cells accumulate misfolded proteins while others donot.Chemical compounds that are able to modulate proteinmisfolding pathways in vitro and in vivo are highly valuabletools to analyze the complex protein assemblyprocess. They are valuable starting points for therapydevelopment. Besides small molecules we are alsohighly interested in finding proteins or peptides thatdirectly influence the amyloid formation cascade. Theyare identified using high throughput protein interactiontechnologies such as an automated yeast twohybrid(Y2H) system. Recently, a network of dysregulatedprotein-protein interactions (PPIs) for HD was createdusing bioinformatic strategies. This study allowedthe identification of the neuron-specific protein CRMP1,a protein that dramatically influences polyglutamine(polyQ)-mediated huntingtin aggregation in cell-freeand in vivo disease model systems.Finally, we aim to develop innovative technologies anddatabases for systematic proteomics research. An interactomedatabase termed UniHI was established at theMDC that contains more than 250,000 human interactionsand allows scientists to link individual proteins toing cascades and disease processes.182 Function and Dysfunction of the Nervous System

Dr. Pablo Porras-MillanDr. Ellen Ramminger*Dr. Tamás RaskóDr. Sean-Patrick Riechers*Dr. Michael Schmidt*Dr. Martin StrödickeDr. Bernhard Suter*Dr. Thomas WiglendaAffiliated ScientistsDr. Matthias Futschik* (HU Berlin)Graduate and UndergraduateStudentsAnup ArumughanYacine Bounab*Gautam Chaurasia* (HU Berlin)Anja Fritzsche*Antje Haug*Manuela JacobNancy Keil*Matthias KönnShuang Li*Annekathrin MöllerMonique Rothe*Jenny Russ*Maliha Shah*Anne WagnerKatja WelschHeike Wobst*Systematic investigation of predicteddysregulated huntingtin interactionpartners in a HD Drosophila model. (A)Dysregulated huntingtin PPI networkpredicted by bioinformatic analysis ofPPI and gene expression data fromhuman tissues and clinical case-controlstudies. (B) Expression of the proteinsCRMP1, CRMP, PACSIN1, Profilin2 andChic reduce mutant huntingtin-mediatedphotoreceptor degeneration in aHD model, whereas expression of theprotein PRPF40a has the oppositeeffect.Identification and characterization of small moleculesthat modulate protein misfolding pathwaysIn previous studies, we have identified the natural compound(-)-epigallocatechin gallate (EGCG) as a modulatorof polyQ-mediated huntingtin aggregation(Ehrnhoefer et al., 2006). We continued these studieswith the disease proteins causative of PD and AD. α-synuclein and amyloid-β amyloidogenesis was investigatedusing biochemical, biophysical and cell biologicalmethods. We found that EGCG efficiently prevents fibrilformation of both polypeptides in cell-free model systems,supporting the previous results with the huntingtinprotein and hinting at a generic mechanism.Further studies subsequently revealed that EGCG isable to redirect the amyloid fibril formation pathway. Itbinds to α-synuclein and amyloid-β monomers andstimulates the assembly of off-pathway, highly stableoligomers, which are non-toxic for mammalian cells.These investigations suggest that EGCG functions as achemical chaperone that recognizes natively unfoldedpolypeptides and influences their ability to spontaneouslymisfold and self-assemble into amyloidogenicprotein aggregates (Ehrnhoefer and Bieschke, 2008).Most recently, we studied whether EGCG disassemblespreformed amyloid fibrils. Our data indicate that EGCGhas the ability to convert preformed, mature α-synucleinand amyloid-β fibrils into smaller, amorphous proteinaggregates that are non-toxic for mammaliancells. These findings suggest that EGCG is a potentremodelling agent of mature amyloid fibrils and supportour hypothesis that the compound has chemicalchaperone function.Several lines of experimental evidence indicate thatsoluble, pre-fibrillar assemblies of the amyloid-βpolypeptide rather than mature, end-stage amyloid fibrilscause neuronal dysfunction and memory impairmentin Alzheimer’s disease. This suggests that accelerationof fibrillogenesis might reduce the levels of toxicaggregation intermediates. To address this question,we searched for chemical compounds that promotespontaneous amyloid-β formation. Using a filter assay,the natural dye orcein and related substances wereidentified which directly bind to small transient amyloidoligomers and stimulate their assembly intomature amyloid fibrils. These structures have alteredsurface properties and are non-toxic for mammaliancells. Our results suggest that conversion of smallFunction and Dysfunction of the Nervous System 183

Scientific-Technical StaffAnja Fritzsche*Ronny Kalis*Sandra NeuendorfStephanie PlaßmannKirstin RauAnke ThiemeFranziska Schiele*Martina ZenknerTechnical AssistantsNouhad Benlasfer*Anna Happe-Kramer*Daniela KleckersSusanne Köppen*Susanne RautenbergAlexandra RedelDana Rotte*Kati Scharf*Nancy SchugardtJan TimmCarsta Werner*Projekt Management(NGFN2 SMP Protein)Dr. Patrick Umbach*Project Management(NGFN-Plus/NeuroNet)Dr. Paul Schultze-Motel*aggregation intermediates into large amyloid structuresmight be a powerful therapeutic approach totreat protein misfolding diseases.In order to investigate whether the small moleculesthat modulate amyloid formation pathways in vitro andin vivo are useful for therapy development, a drug discoveryprogram for AD and HD was started in 2007 inthe framework of the GO-Bio initiative of the GermanFederal Ministry for Education and Research. The aim ofthis program is to promote the establishment of innovativestart-up companies out of the academic sector. Inthe first funding phase of the Go-Bio project, we havediscovered a number of novel drug candidates for bothAD and HD and developed them up to in vivo proof ofconcept in transgenic mouse models of the two diseases.At the moment, derivatisation and further in vivotesting are in progress in the framework of lead optimisation.Identification of proteins that modulate polyQmediatedhuntingtin aggregationHD is an inherited neurodegenerative disorder that iscaused by an expansion of a polyQ tract in the proteinhuntingtin, which leads to a characteristic accumulationof insoluble Htt aggregates in affected neuronsand eventually to cellular dysfunction and toxicity.However, the molecular pathways underlying brainspecific,polyQ-induced neurodegeneration in HD arestill unknown. Recently, a large number of interactionpartners were identified that associate with the N-terminaldomain of huntingtin, which harbours the aggregation-pronepolyQ tract. We hypothesized that perturbationof functional huntingtin protein complexes inneurons induces protein misfolding and neurotoxicity.To identify tissue-specific, dysregulated huntingtin proteininteractions, a bioinformatic approach was developed.By filtering publically available protein-proteininteraction (PPI) data with information from geneexpression studies of brain and non-brain tissues aswell as clinical case-control studies, a brain-specifichuntingtin PPI network was created, linking 14 potentiallydysregulated proteins directly or indirectly to thedisease protein. Analysis of published data confirmedthe predictive value of this network modelling strategy.Moreover, systematic investigations with in vitro andDrosophila model systems of HD demonstrated thatthe potentially dysregulated huntingtin interactionpartners influence polyQ-mediated protein misfoldingand neurodegeneration. The neuron-specific proteinCRMP1 e.g. is recruited to inclusion bodies with aggregatedhuntingtin protein in brains of HD transgenicmice and efficiently inhibits polyQ-mediated huntingtinexon 1 aggregation in cell free assays. Our resultsoffer a new strategy for identifying perturbed, tissuespecifichuman PPIs and modulators of protein misfoldingand aggregation (Bounab et al., 2009, submitted).Development of interactome databases andnovel quality standards for systematic proteininteraction studiesHuman protein interaction maps have become importanttools of biomedical research for the elucidation ofmolecular mechanisms and the identification of newmodulators of disease processes. We developed a comprehensiveinteractome database termed UnifiedHuman Interactome (UniHI). It provides researcherswith a comprehensive integrated platform to query andaccess human PPI data. Since its first release, UniHI hasconsiderably increased in size. The latest update ofUniHI includes over 250,000 interactions between~23,000 unique proteins collected from 14 majorsources. However, this wealth of data also poses newchallenges for researchers due to the size and complexityof interaction networks retrieved from the database.We therefore developed several new tools to query, analyzeand visualize human PPI networks. Most importantly,UniHI now allows the construction of tissue-specificinteraction networks and focused searches ofcanonical pathways. This will enable researchers to targettheir analysis and to prioritize candidate proteinsfor follow-up studies. UniHI 4 can be accessed athttp://www.mdc-berlin.de/unihi (Chaurasisa et al.,2008).184 Function and Dysfunction of the Nervous System

Project Management (GO-Bio)Dr. Annett BöddrichSigrid Schnögl*part of the period reportedAdministrative AssistantsErika PischSilke Spading*Several attempts have been made to systematicallymap PPI networks. However, it remains difficult toassess the quality and coverage of existing data sets. Incollaboration with the research group of Prof. MarcVidal from Harvard Medical School, Boston, we havedeveloped an approach to identify the most suitablequality parameters of currently available human interactomemaps. We found that high-throughput yeasttwo-hybrid (HT-Y2H) screening yields more accurateinteraction data for human proteins than non-systematicstudies of small numbers of individual interactions.This suggests that HT-Y2H screening is a powerfulmethod to map a significant portion of the humaninteractome. We estimated that the human interactomecontains ~130,000 binary interactions, most ofwhich remain to be mapped by systematic screenings.High quality data and accurate estimates of interactionnumbers are crucial to establish the magnitude of thetask of comprehensively mapping the human interactome(Venkatesan et al., 2009).Constructing directed protein interaction networksfor activated EGF/Erk signalingEpidermal growth factor (EGF) signaling through extracellular-signalregulated kinases (Erks) is a complexprocess involving a series of protein-protein interactions(PPIs) that propagate information from the plasmamembrane to transcription factors. To obtain aglobal view of EGF/Erk signaling and to predict potentialmodulators, we created a network connecting 1126proteins via 2626 PPIs using automated yeast twohybrid(Y2H) interaction mating. From this interactionmap, a network of activated signaling was generatedusing a naïve Bayesian classifier, in which informationon shortest PPI paths from membrane receptors totranscription factors was exploited to predictinput/output relationships between interacting proteins.Analysis of the resulting network model revealedregulatory motifs typical for information processingsystems. Moreover, it allowed predictions of potentialmodulators of EGF/Erk signaling, which were validatedin mammalian cell-based assays. This generic experimentaland computational approach provides a frameworkfor elucidating causal connections between proteinsand facilitates the identification of factors modulatingthe flow of information in signaling networks(Vinayagam et al., 2009, submitted).Selected PublicationsEhrnhoefer, DE, Bieschke, J, Boeddrich, A, Herbst, M, Masino, L, Lurz, R,Engemann, S, Pastore, A, Wanker, EE. (2008) Redirecting aggregationpathways: small molecule-mediated conversion of amyloidogenicpolypeptides into unstructured, off-pathway oligomers. Nat Struct MolBiol. 15(6), 558-66.Zolghadr, K, Mortusewicz, O, Rothbauer, U, Kleinhans, R, Goehler, H,Wanker, EE, Cardoso, MC and Leonhardt, H. (2008) A fluorescent twohybrid(F2H) assay for direct visualization of protein interactions in livingcells. Mol Cell Proteomics 7(11), 2279-87.Chaurasia, G, Malhotra, S, Russ, J, Schnögl, S, Hänig, C, Wanker, E andFutschik, M. (2009) UniHI 4: New tools for query, analysis andvisualization of the human protein-protein interactome. Nucleic AcidsRes. 37(Database issue), D657-60.Venkatesan, K, Rual, .J-F Vazquez, A, Stelzl, U, Lemmens, I, Hirozane-Kishikawa, T, Hao, T, Zenkner, M, Xin, X, Goh, K-I, Yildirim, MA, Simonis, N,Heinzmann, K, Gebreab, F, Sahalie, JM, Cevik, S, Simon, C, de Smet, A-S,Dann, E, Smolyar, A, Vinayagam, A, Yu, H, Szeto, D, Borick. H, Dricot, A,Klitgord, N, Murray, RR, Lin, C, Lalowski, M, Timm, J, Rau, K, Boone, C, Braun,P, Cusick, ME, Roth, FP, Hill, DE, Tavernier, J, Wanker, EE, Barabási, A-L andVidal, M. (2009) An empirical framework for binary interactome mapping.Nat Methods. 6(1), 83-90.Palidwor, GA, Shcherbinin, S, Huska, MR, Rasko, T, Stelzl, U, Arumughan, A,Foulle, R, Porras, P, Sanchez-Pulido, L, Wanker, EE, and Andrade-Navarro,MA. (2009) Detection of alpha-rod repeats using a neural network andapplication to huntingtin. PLoS Comput Biol. 5(3):e1000304.Function and Dysfunction of the Nervous System 185

Structure of the GroupJan Bieschke(Delbrück Fellow)Group LeaderDr. Jan BieschkeScientistsDr. Ralf FriedrichGraduate StudentsKatja WelschMaliha Shah*Heike Wobst*(Master student)Technical AssistantsGerlinde GrelleSusanne Kostka*part of the periodreportedAging-related Protein Misfolding andDetoxification MechanismsThe misfolding of endogenous protein or peptide fragments to form cytotoxic depositsmade from fibrillar protein aggregates characterizes amyloid diseases. The misfoldedpolypeptides, which are believed to be the root cause of the pathologies, are specific for eachdisease, such as amyloid β (Aβ) in Alzheimer’s disease (AD), α-synuclein in Parkinson’s disease(PD) or fragments of the huntingtin protein in Huntigton’s disease (HD). The specific natureand mechanism of the cytotoxicity are yet unknown. However, a wealth of evidence points tosmaller oligomeric aggregates rather than large fibrils bein the crucial species.Both AD and PD mostly occur sporadically without knowngenetic components. They have the patient’s age as thesingle most important risk factor. Yet, little is known howaging influences them. Our lead hypothesis states thattoxic protein aggregates result from an imbalance in thedynamic equilibrium between aggregation and clearanceof misfolded proteins rather than from a slow stochasticprocess, as has previously been assumed.The mechanistic details of protein misfolding, the autocatalyticreplication of misfolded protein aggregates,and possible detoxifying mechanisms are the majorpoints of our research (Fig. A). We found that misfoldedAβ aggregates are detoxified by two opposing activitiesunder the control of two interconnected aging-relatedpathways, the Insulin/IGF-receptor pathway on onehand and the stress response / heat shock reponsepathways controlled by HSF-1 on the other hand (Fig B).The primary detoxification pathway appears to involvedisassembly of misfolded aggregates, a secondary pathwaythe induced aggregation of small Aβ aggregatesinto larger, less-toxic structures.Endogenous Modifiers of Amyloid FormationWe aim to identify key components of the cellulardetoxification machinery and study the influence ofendogenous proteins on aggregate assembly and disassemblyusing several screening based methods:a. Cellular aggregation assays and siRNAinterferenceKatja WelschAggregation-prone fragments of huntingtin areexpressed in mammalian cells (N2A, PC12). Target genesfrom a library of proteins that are related to proteinfolding or to the aging pathways are simultaneouslydownregulated by siRNA during aggregation or afterthe completion of aggregation to assess their influenceon amyloid formation and removal by the cells.b. Genome-wide functional screening foramyloid modulatorsRalf Friedrich, Maliha ShahProteins are recombinantly expressed from a genomichuman protein library (ca. 14.000 constructs) and addedto in vitro aggregation assays and disaggregationassays (Fig C, D) using the Aβ peptides and theα-synuclein protein. Aggregation and disaggregationkinetics are used to identify proteins that influenceaggregation or that have a disaggregating activity and186 Function and Dysfunction of the Nervous System

on-pathway or off-pathway to amyloid formation(Fig. A). We aim to understand the relationshipbetween aggregation state and cytotoxicity of thesespecies and test their capacity to inducue homologousand heterologous amyloid formation.To that end we are, on one hand, focussing ondetailed mechanistic studies using biophysical andbiochemical assays, fluorescence techniques andatomic force microscopy. On the other hand we arecorrelating these results with aggregate uptake andtoxicity assays in mammalian cell culture, to whichaggregate sub-populations are added.Mechanisms of Anti-Amyloid Drug ActionJ. Bieschke, G. Grelle; collaboration withE. WankerThe flavonoid (-)-epigallocatechin-gallate (EGCG)from green tea and related substances were found tobe potent inhibitors of amyloid formation for a varietyof polypeptides such as Aβ, α-synuclein and huntingtin.We found that EGCG prevents amyloid formationby a unique mechanism. It stimulates the productionof off-pathway oligomers early in the aggregationprocess and thus diverts the misfoldingprocess away form the toxic species (Fig. 1A). Thesesubstances can thus be used to probe the mechanismof amyloid formation and toxicity.(A) Amyloid formation cascade. Unfolded or natively folded proteinpartially misfolds and associates in a multi-step process viaoligomer and protofibril formation to form amyloid fibrils. EGCGredirects unfolded monomer into benign stable aggregates. (B)Schematic representation of detoxifying machinery controlled bythe insulin signaling pathway. (C,D) Aggregation and disaggregationassays can be used to identify aggregation inhibitors and promotersin vitro and from mammalian cells.Selected PublicationsEhrnhoefer DE*, Bieschke J*, Boeddrich A, Herbst M, Masino L, Lurz R,Engemann S, Pastore A, Wanker EE. (2008). EGCG redirectsamyloidogenic polypeptides into unstructured, off-pathway oligomers.Nature Struct Mol Biol. 15, 558-66.Bieschke J, Zhang Q, Bosco DA, Lerner RA, Powers ET, Wentworth P Jr,Kelly JW. (2006). Small molecule oxidation products trigger diseaseassociatedprotein misfolding. Acc Chem Res. 39, 611-9.Cohen E*, Bieschke J*, Perciavalle RM, Kelly JW, Dillin A. (2006).Opposing activities protect against age-onset proteotoxicity. Science313, 1604-10.hit proteins are being validated in mammalian cell cultureexperiments.Amyloid Aggregate-Toxicity Relationship in Aβand tau-ProteinHeike WobstUsing model substances, such as EGCG and lipid oxidationproducts, as well as specific oligomer formationprotocols, we create fibrillar and oligomeric species ofAβ peptides and tau-protein fragments that are eitherBieschke J, Weber P, Sarafoff N, Beekes M, Giese A, Kretzschmar H.(2004) Autocatalytic self-propagation of misfolded prion protein. ProcNatl Acad Sci U S A. 101, 12207-11.Bieschke J & Cohen E et al. A kinetic assessment of the C.elegansamyloid disaggregation activity enables uncoupling of disassemblyand proteolysis. Protein Sci. 2009 [Epub ahead of print]Function and Dysfunction of the Nervous System 187

Berlin Institute of Medical SystemsBiology (BIMSB)Coordinator: Nikolaus Rajewsky

Berlin Institute for Medical SystemsBiology, BIMSBThe MDC has launched a major expansion of its currentscientific activities in the area of systems biology: TheBerlin Institute for Medical Systems Biology.The aim ofthis initiative is to consolidate current scientific trends suchas increasing interdisciplinarity, and the availability of hugeamounts of data from new types of experiments and highthroughputgenomic platforms. This information needs tobe integrated into a systematic and quantitative understandingof all scales of life. The BIMSB is creating a researchenvironment for excellent scientific groups with interdisciplinaryand systems biology approaches at the MDC.The Berlin Institute for Medical Systems Biology will closelycollaborate with research institutions and universities inBerlin, and a new research building is planned to be constructedin Berlin-Mitte, most likely at the ‘Campus Nord’ ofthe Humboldt University. Promising talks have already beenheld between the MDC, HU-Berlin and the Berlin Senate.The Federal Ministry of Education and Research (BMBF) hasallocated 7.5 million euros for the period of 2008-2010 andthe Senate of Berlin is complementing this with up to 4.4million euros. During this period young research groups andan international exchange program will start their researchand activities in Berlin-Buch. Major investments in high-endtechnologies will enable the BIMSB to set up state-of-theartscientific technology platforms for genomics, proteomics,metabolomics and bioinformatics. The funding forthe BIMSB was announced on May 5th 2008 by Germany’sEducation and Research Minister Annette Schavan as part ofthe BMBF initiative “Advanced Research and Innovation inthe New States” (Spitzenforschung und Innovation in denneuen Ländern). During the initial phase, the first juniorgroups have been recruited and an international exchangeprogram has been started with the Center for FunctionalGenomics of New York University (NYU), USA. This has beenaccompanied by major investments in technology platformsin the areas of genomics, proteomics, metabolomics andbioinformatics, which are used by MDC and external groups.The scientific missionBasic and biomedical research has provided a wealth ofinformation about the role of individual genes in various diseases.Despite these discoveries and the progress made, ithas been extremely challenging using this knowledge tounderstand disease and design and improve treatments. It isbecoming increasingly clear that disease processes involvecomplex interactions between hundreds of genes, proteinsand metabolites. Especially the most prevalent and devastatingdiseases such as cancer, cardiovascular disease, dia-Mit dem zunächst auf Projektbasis operierenden BerlinerInstitut für Medizinische Systembiologie(BIMSB) und dem geplanten Ausbau als integralerBestandteil des MDC hat das MDC eine bedeutende Entwicklungin der biomedizinischen Forschung, das Gebiet der Systembiologie,aufgegriffen. Das Ziel ist die Konsolidierung dergegenwärtig wissenschaftlichen Trends zu verstärkter Interdisziplinarität,der rasanten Genierung von sehr großenDatenmengen durch die immer stärkere Ausnutzung undWeiterentwicklung modernster Hochdurchsatz-Technologienund anderer Technologien, wie z. B. modernster Imaging-Verfahren.Daraus ergibt sich die Notwendigkeit der Integrationdieser Vielzahl an Informationen, um ein systematisches, qualitativesund quantitatives Verständnis der Beziehungeninnerhalb und zwischen den verschiedenen Ebenen biologischerOrganisation, von der Funktion einzelner Gene bis hin zukomplexen Wechselwirkungen in Zellen, Organen und Organismen,zu erreichen. Im BIMSB werden exzellente wissenschaftlicheForschungsgruppen mit interdisziplinärer und systembiologischerAusrichtung zusammengeführt. Durch dieenge Vernetzung innerhalb des MDC mit den bestehendenMDC-Gruppen können vorhandene Stärken mit neuesten Entwicklungenoptimal verbunden und ausgenutzt werden.Bereits jetzt schon unterstreichen erste Ergebnisse erfolgreicherZusammenarbeit, die auch in diesem Bericht zu findensind, die Berechtigung und Richtigkeit des eingeschlagenenWegs. Darüber hinaus wird die enge Kooperation des MDC mitden anderen Forschungseinrichtungen in Berlin und insbesondereden Berliner Universitäten über das BIMSB vertieft undstark ausgebaut. Daher hat sich das MDC entschlossen, dasneue Forschungsgebäude zentral in der Mitte Berlins, voraussichtlichauf dem Campus-Nord der Humboldt-Universität zuBerlin, zu errichten. Erste vielversprechende Gespräche zwischenMDC, HU-Berlin und dem Berliner Senat wurden bereits geführt.Für die Startphase des BIMSB hat das Bundesministerium fürBildung und Forschung (BMBF) für den Zeitraum von 2008 bis2010 7,5 Millionen ¤ bereitgestellt. Um 4,4 Millionen ¤ hat derSenat von Berlin diese Mittel ergänzt. Die Bereitstellung derMittel für das BIMSB als Teil der BMBF-Initiative „Spitzenforschungund Innovation in den neuen Ländern“ wurde am 5.Mai 2008 durch die Ministerin für Bildung und Forschung, Dr.Annette Schavan, verkündet. Während dieser Startphase wurdendie ersten Nachwuchsforschungsgruppen rekrutiert undein internationales Austauschprogramm mit dem Zentrumfür Funktionelle Genomik der New York University (NYU), USA,gestartet. Durch große Investitionen in Technologieplattformenim Bereich Genomik, Proteomik, Metabolomik und Bioinformatikwurde eine ausgezeichnete infrastrukturelle Basisgeschaffen, die von den Gruppen des MDC und externen Gruppengenutzt wird.190 Berlin Institute for Medical Systems Biology

etes, metabolic diseases and neurodegenerative disordersare multifactorial. Such complex systems can in most casesonly be understood by quantitative models that predict theinteractions and functions of numerous components. Neverbefore has it been possible to collect complete data from aminimal amount of biological data about the condition of acell, organ, or organism at the levels of the genome, proteome,and metabolome. This information can only beunderstood with the help of mathematical models. ThusSystems Biology is by nature highly interdisciplinary andcombines molecular biology, biochemistry, mathematics,physics and engineering.The importance of Systems Biology in biomedical research isparticularly apparent in the field of post-transcriptionalgene regulation and RNA biology (Special issue, Cell 2009).Additionally many new classes of non-coding and smallRNAs (such as microRNAs, piRNAs, and many others) havebeen recently discovered and have been shown to act in theregulatory networks in all multicellular organisms.Furthermore, the human genome encodes hundreds of proteinswith RNA binding domains. These proteins function inregulating mRNA localization, mRNA turnover, protein synthesisand other crucial steps that control gene expression.Small RNAs and RNA binding proteins have been shown tobe in many ways relevant players in regulation of phenotypesin health and disease and have therefore a hugepotential for medical applications. In short, the grand challengeand overall scientific mission for the BIMSB is to decipherthe ‘post-transcriptional regulatory code’ and to directlyintegrate it with other major cellular regulatory mechanisms,in particular transcriptional regulatory circuits, signaltransduction pathways, protein-protein interactions networks,and post-translational modifications.Advanced technologies like next generation sequencing andmass-spectrometry, combined with imaging approachesand biochemical methods, will allow genome-wide quantitativeanalysis of regulatory mechanisms and resolution ofRNA function on many different levels.BIMSB research groups, scientific platforms andthe international PhD exchange programYoung scientists leading independent research groups andscientific platforms in genomics, proteomics and quantitativebiology have been appointed at the BIMSB. A few examplesof the ongoing work reveal how their research can complementand expand on the MDC’s overall mission of “molecularmedicine.”Das wissenschaftliche ProgrammIn der biomedizinischen Grundlagenforschung konnten in denvergangenen Jahrzehnten eine Fülle an Informationen überdie Rolle einzelner Gene bei Erkrankungen gewonnen werden.Trotz dieser Entdeckungen und dem damit einhergehendenErkenntnisfortschritt ist es immer noch ein großes Problem,dieses Wissen für das Verständnis von komplexen Erkrankungenund damit für die Entwicklung neuer und die Verbesserungbestehender Therapien zu nutzen. Es hat sich klargezeigt, dass Krankheitsprozesse aus der Wechselwirkung vonHunderten von Genen, Proteinen und Stoffwechselproduktenentstehen. Besonders die häufigsten und bedrohlichstenErkrankungen wie Krebs, kardiovaskuläre Erkrankungen, Diabetes,metabolische und neurodegenerative Störungen sindmultifaktoriell bedingt. Solche komplexen Systeme können inden meisten Fällen nur mit Hilfe mathematischer Modelle verstandenwerden, die die Interaktionen und Funktionen derzahlreichen Komponenten integrieren und damit eine Vorhersageder Auswirkungen auf das Gesamtsystem ermöglichen.Systembiologie ist hoch interdisziplinär und umfasst nebenMolekularbiologie und Biochemie vor allem Mathematik, Physikund Ingenieurwissenschaften. Nie zuvor war es möglich,den gesamten Datensatz zum „Zustand“ einer Zelle/Organ/Organismus zu einem bestimmten Zeitpunkt mit einem Minimuman biologischem Material auf Genom-, Transkriptom-,Protein- und Metabolomebene zu erhalten. Die daraus resultierendengroßen Datenmengen sind ohne mathematischesVerständnis nicht zu bewältigen. Letztendlich schafft diesesHerangehen jedoch völlig neue Möglichkeiten für eine systematischeAnalyse verschiedener Zustände und damit einneues Verständnis der Mechanismen komplexer Systeme.Die Bedeutung einer systembiologischen Herangehensweiselässt sich besonders anhand der auf diese Weise gewonnenenErkenntnisse auf dem Gebiet der post-transkriptionalen Genregulationund RNS-Biologie (Spezialausgabe von Cell, 2009)illustrieren. Darüber hinaus wurden in letzter Zeit neue Klassennicht-kodierender RNS (microRNS, piRNS usw.) entdeckt,die die regulatorischen Netzwerke aller multizellulären Organismenbeeinflussen. Es konnte nachgewiesen werden, dasskleine RNS-Moleküle und RNS-bindende Proteine wichtigeRegulationsfaktoren für die Ausbildung verschiedener Phänotypenin normalen und kranken Organismen sind. Dasmenschliche Genom kodiert für Hunderte von Proteinen mitRNS-Bindungsdomänen. Diese Proteine regulieren die Syntheseund die Lokalisierung der entsprechenden Boten-RNS, dieProteinsynthese selbst und andere wichtige Schritte der Genexpression.Diese Befunde eröffnen völlig neue Möglichkeitenfür die klinische Anwendung. Kurz gesagt, die wissenschaftlicheMission des BIMSB ist es, den „post-translationalen Code“zu entziffern und ihn mit anderen zellulären Regulationsebenen,insbesondere der transkriptionalen regulatorischen Kreisläufe,den Signalketten und Signaltransduktionswegen, Pro-Berlin Institute for Medical Systems Biology 191

Markus Landthaler is an outstanding scientist in the field ofpost-transcriptional regulation and RNA binding proteins.His research aims for a systems level understanding of posttranscriptionalregulation and a transcriptome-wide highresolutionmap of RNA-protein interaction. MarkusLandthaler’s detailed research profile appears on page 62.The Genomics Platform is lead by Wei Chen, an expert innext-generation sequencing technologies and their applicationto systems wide approaches in genomic research.Christoph Dieterich heads the Bioinformatics Platform andwill perform and support the computational side ofgenome-wide analysis and bioinformatics. The third platform,headed by Stefan Kempa, will enable to MDC andBIMSB research groups to analyse the proteome andmetabolome by mass-spectromentry in a high-throughputformat. The integration of the technologies of all scientificplatforms will support systems-wide understanding of complexregulatory mechanisms.Another focus of the BIMSB is the international and interdisciplinarytraining of young scientists, especially PhD students.A first step has been to develop a meaningfulexchange program jointly operated by the MDC and NewYork University (NYU) in the United States. Other internationalpartnerships and scientific exchange programs arebeing negotiated, e.g. with Kyoto Medical School, Japan, andfurther systems biology institutes in the USA. Within theMDC-NYU PhD exchange program students will carry outcollaborative projects between the Center for FunctionalGenomics in New York and the BIMSB in Berlin and mayspend up to 50% of their time in either location. They are comentoredby NYU and MDC faculty and can participate inprojects and classes at both institutions. In 2009 this collaborationhas already led to important publications and manyothers are in the pipeline. Additionally, the BIMSB is veryactive in coordinating scientific workshops for communicationand collaborative efforts with local, national and internationalpartners.PartnersBy definition, systems biology must be open to the integrationof a wide range of expertise. The BIMSB’s strategic concepthas been developed in collaboration with virtually alllocal universities and scientific institutions in Berlin as wellas other partners in Germany. Several projects funded by theBMBF, Helmholtz Association, the DFG and other sources areongoing between BIMSB investigators and collaboratorsthroughout Berlin.tein-Protein-Interaktions-Netzwerken und posttranslationalenModifikationen zu verzahnen.Modernste Sequenzierungs- und Massenspektrometrietechnologienin Verbindung mit modernen bildgebenden Verfahrenund biochemischen Methoden werden eine genomweitequalitative und quantitative Analyse der Regulationsmechanismen,insbesondere der Aufklärung der RNS-Funktion aufden verschiedenen Hierarchieebenen, ermöglichen.BIMSB Forschergruppen, wissenschaftlichePlattformen und das Internationale PhD-Austausch-ProgrammInnerhalb der erst kurzen Laufzeit konnten bereits herausragendejunge Wissenschaftler als Leiter unabhängiger Forschungsgruppenund wissenschaftlicher Plattformen imBereich Genomik, Proteomik und quantitative Biologie rekrutiertwerden. Die hier im Bericht aufgeführten Beispiele der laufendenForschung zeigen, wie ihre Arbeiten die Gesamtmissiondes MDC in der „Molekularen Medizin“ ergänzt und erweitert.Markus Landthaler ist ein hervorragender Wissenschaftler aufdem Feld der post-transkriptionalen Regulation und der RNSbindendenProteine. Er arbeitet an einem systemischen Verständnisder post-transkriptionalen Regulation und an einertranskriptom-weiten hochauflösenden Karte von RNS-Protein-Interaktionen.Sein Forschungsprofil wird im vorderen Teildes Berichtes detailliert dargestellt (Seite 62).Die Genomik-Plattform wird von Wei Chen geleitet, einemExperten für die neue Generation von Sequenziertechnologienund ihre Anwendung in der systemweiten Genomforschung.Christoph Dieterich leitet die Bioinformatik-Plattform und wirddie algorithmisch-bionformatische Seite der genomweiten Analysenabdecken. Die dritte Plattform, geleitet von Stefan Kempa,wird den MDC- und BIMSB-Gruppen ermöglichen, das Proteomund das Metabolom mit Hilfe von Massenspektrometrie imHochdurchsatzformat zu analysieren. Die Integration diesermethodischen Plattformen wird ein systemweites Verständniskomplexer regulatorischer Mechanismen unterstützen.Ein weiterer Schwerpunkt des BIMSB ist eine internationalausgerichtete interdisziplinäre Ausbildung junger Wissenschaftler,speziell von Doktoranden. Ein erster Schritt ist dieEtablierung des Austauschprogramms, das gemeinsam vomMDC und von der New York University (NYU) in den USA organisiertwird und in 2009 gestartet wurde. Weitere internationalePartnerschaften sind in Vorbereitung, z.B. mit der KyotoMedical School in Japan und Systembiologie-Instituten in denUSA. Im MDC-NYU-Programm werden gemeinsame Projektezwischen dem Zentrum für Funktionelle Genomik in New Yorkund dem BIMSB in Berlin durchgeführt, so dass die Teilnehmerbis zu 50% der Zeit in der jeweils anderen Institution verbringenkönnen. Dies wird von den beiden Fakultäten gemeinsambegleitet und beinhaltet die Teilnahme an Seminaren in beidenInstitutionen. Diese Kooperation hat in 2009 bereits einePublikation in ‚Nature Methods’ ergeben. Insgesamt wird das192 Berlin Institute for Medical Systems Biology

A number of MDC investigators have substantially contributedto the scientific concept and are involved throughcollaborative projects; these include Jana Wolf, MatthiasSelbach, Norbert Hübner, Erich Wanker, Miguel Andrade-Navarro, Martin Falke, and investigators from the LeibnizInstitute for Molecular Pharmacology (FMP).The BIMSB investigators have additionally establishedindustry collaborations for joint projects and technologydevelopment (i.e. with Applied Biosystems, Qiagen andRoche).International collaborations are initiated or established withthe NIH-supported centers for systems biology, the NewYork University and the Rockefeller University. Additionally,the European Molecular Biology Laboratory and EU-fundednetworks and ESF policy agendas have greeted, supportedand integrated the BIMSB in discussions on future systemsand RNA biology efforts on a national and European level.Future perspectivesThe BIMSB will become an institutional branch of the MDCin Berlin-Mitte, a location that will more closely interconnectthe MDC with experimental and theoretical institutions inBerlin including universities, the Charité University hospital,research institutions and the DFG Research Center Matheonand other non-university research institutes. This interdisciplinaryinterface between universities and research institutionswill strengthen the scientific profile of Berlin.The structure and size of the new building in Berlin-Mittewill offer research space for up to 25 research groups plus 5technology platforms, ample space for visiting scientists andextensive workshop and conference facilities.In 2009, the performance of the existing BIMSB activities aswell as the overall scientific concept of the BIMSB werereviewed by an international panel of experts in systemsbiology, and RNA biology. The panel strongly supported theMDC in proceeding on this endeavour and expanding andestablishing the BIMSB, with this scientific mission, the proposedsize, funding, and the location in Berlin-Mitte.BIMSB durch die Organisation wissenschaftlicher Workshopsund anderer Veranstaltungen den Erfahrungsaustausch unddie Zusammenarbeit mit regionalen, nationalen und internationalenPartnern fördern.Systembiologie steht für die Integration eines breiten Wissensaus unterschiedlichsten Fachgebieten. Das strategische Konzeptdes BIMSB wurde in enger Abstimmung mit nahezu allenuniversitären und außeruniversitären Forschungseinrichtungenim Land Berlin und anderen Partnern in Deutschland erarbeitet.Die Basis für die weitere enge Zusammenarbeit bildenbereits jetzt schon mehrere vom BMBF, der DFG und aus anderenQuellen finanzierte Kooperationsprojekte.Viele MDC-Wissenschaftler haben entscheidend zu diesenneuen Entwicklungen und zum wissenschaftlichen Konzeptbeigetragen und sind in verschiedene gemeinsame wissenschaftlicheProjekte eingebunden, unter ihnen Jana Wolf, MatthiasSelbach, Norbert Hübner, Erich Wanker, Miguel Andrade-Navarro, Martin Falcke und weitere Kollegen aus dem Leibniz-Institut für Molekulare Pharmakologie (FMP). Darüber hinaushaben die Wissenschaftler des BIMSB auch gemeinsameKooperationsprojekte mit der Industrie zur Technologie-Entwicklung(z.B. mit Applied Biosystems, Qiagen, Roche) gestartet.Internationale Kooperation in größerem Umfang wurdenmit den NIH-geförderten Zentren für Systembiologie an derNew York University und der Rockefeller University initiiert.Das European Molecular Biology Laboratory (EMBL), EUfinanzierteForschungsnetzwerke auf dem Gebiet Systembiologieund verschiedene ESF-Gremien haben das MDC zumgeplanten substantiellen Ausbau der Systembiologie im Rahmendes BIMSB beglückwünscht.ZukunftsperspektivenDas BIMSB als Teil des MDC wird durch seinen Standort in Berlin-Mittedas MDC insgesamt noch enger mit den experimentellenund theoretischen Institutionen der Universitäten, derCharité-Kliniken, dem DFG-Forschungszentrum Matheon undweiteren außeruniversitären Forschungsinstituten verbinden.Es wird als interdisziplinäre Schnittstelle zwischen Universitätenund außeruniversitären Institutionen das wissenschaftlicheProfil Berlins stärken. Das neue Gebäude in Berlin-Mittewird Platz für bis zu 25 Forschergruppen und 5 Technologieplattformenbieten, ergänzt durch Seminar- und Konferenzräume.Sowohl die bisherige Leistung als auch das wissenschaftlicheZukunftskonzept wurden im Frühjahr 2009 einer umfassendenBegutachtung durch ein internationales Expertenteamunterzogen. Die Gutachterkommission setzte sich aus weltweitanerkannten Wissenschaftlern auf dem Gebiet der RNS-Biologieund der Systembiologie zusammen. Die Gutachterkommissionhat diese bisherigen Aktivitäten und das wissenschaftlicheKonzept des MDC zur Errichtung des BIMSB begeistert begrüßtund nachhaltig unterstützt. Sowohl Größe und Umfang desgeplanten Instituts als auch der vorgesehene Standort in Berlin-Mittewurden als angemessen und notwendig erachtet.Berlin Institute for Medical Systems Biology 193

Wei ChenStructure of the GroupGroup LeaderDr. Wei ChenScientistsDr. Yuhui HuDr. Andreas DoeringGraduate StudentsNa LiHui KangWei SunYongbo WangRaghu Bhushan (till June 2009)Novel sequencing technology,miRNA regulation and humanmolecular geneticsThe recent introduction of massive parallel sequencing technology has revolutionizedgenomic research. These so-called next generation sequencing platforms, such asRoche/454, Illumina/solexa and ABI/Solid system can sequence DNA orders of magnitudefaster and at much lower cost than conventional Sanger method. With their incrediblesequencing capacity, my lab has been focused on developing and implementing variousgenomic assays based on this new generation of sequencers. Apart from offering servicesto the institute as a core facility provider, we are now applying the assays in studyingtranscriptional and posttranscriptional regulation of miRNA genes, as well as identifyinggenetic factors underlying human diseases.Transcriptional and posttranscriptional regulationof miRNA genesYuhui Hu, Andreas Doering, Na Li, Hui Kang, Wei SunMiRNAs are small non-coding RNAs that control theexpression of target genes at the posttranscriptionallevel. Recently, more and more miRNAs have been implicatedin a variety of biological processes.Whereas muchattention has been focus on finding the target genesregulated by miRNAs, little is known about the systemwhich regulates miRNA expression. One major focus ofmy lab is to study transcription and posttranscriptionalregulation of miRNA genes. In the study of transcriptionalregulation, we are involved in genome wide discoveryof miRNA promoters in pre-B cells using ChIPseq,mRNA-seq and small RNA sequencing methods.It has been demonstrated that the Drosha or Dicer processingof individual miRNA can be regulated. Though,it is yet not known that how generalized the phenomenaare.We are therefore interested in studying the posttranscriptionalregulation of miRNAs, especially regulationof Dicer processing at the genomic level bygenome-wide profiling of miRNA precursor (premiRNA)and mature miRNA from the same sample andcomparing their relative abundance across differentsamples. Currently, we are developing a novel assay toefficiently profile pre-miRNA based on new sequencingtechnology.Characterisation of breakpoints in disease-associatedbalanced chromosome rearrangementsYuhui Hu, Andreas DoeringBalanced chromosome rearrangements (BCRs) cancause genetic diseases by disrupting or inactivatingspecific genes, and the characterization of breakpointsin disease-associated BCRs has been instrumental inthe molecular elucidation of a wide variety of geneticdisorders. However, mapping chromosome breakpointsusing traditional methods is rather laborious and timeconsuming.In addition, the resolution is often insufficientto unequivocally identify the disrupted gene. To194 Berlin Institute of Medical Systems Biology

Technical AssistantsClaudia LangnickMirjam FeldkampRegine SchwartzStart of group: January 2009overcome these limitations, we have developed novelmethods based on new sequencing technology to characterizethe breakpoints in an efficient manner. We arenow implementing our method in large-scale breakpointmapping and gene finding.De novo transcriptome sequencing using 454pyrosequencingYongbo WangNew sequencing technologies are not only robust toolsfor the investigation of transcriptome in model organisms,but also manifest great potential in studying nonmodelorganisms. To facilitate comprehensive transcriptomecharacterization, we are developing methodsfor transcriptome sequencing using 454 pyrosequencing.With long read length and high accuracy, it is particularlysuitable for de novo sequence assembly. To befit for 454 sequencing, our methods consist of steps toremove poly A+ tails and cDNA library normalization.The sequencing data would provide a comprehensivereference resource for further functional studies.Selected publicationsFu X, Fu N, Guo S, Yan Z, Xu Y, Hu H, Menzel C, Chen W*, Li Y, Zeng R,Khaitovich P*. (2009). Estimating accuracy of RNA-Seq and microarrayswith proteomics. BMC Genomics. 10, 161-169*shared corresponding authorEnder C, Krek A, Friedländer MR, Beitzinger M, Weinmann L, Chen W,Pfeffer S, Rajewsky N, Meister G. (2008). A Human snoRNA withMicroRNA-Like Functions. Mol Cell. 32(4), 519-528Kuss AW, Chen W. (2008) MicorRNAs in brain function and disease. CurrNeurol Neurosci Rep. 3, 190-197.Friedländer MR, Chen W, Adamidi C, Maaskola J, Einspanier R, Knespel S,Rajewsky N. (2008). Discovering microRNAs from deep sequencing datausing miRDeep Nat Biotechnol. 26(4), 407-415.Chen W, Kalscheu V, Tzschach A, Menzel C, Ullmann R, Schulz M, ErdoganF, Li N, Kijas Z, Arkesteijn G, Pajares IL, Goetz-Sothmann M, Heinrich U,Rost I, Dufke A, Grasshoff U, Glaeser BG, Vingron M, Ropers HH.(2008)Mapping translocation breakpoints by next-generation sequencing.Genome Res. 18: 1143-1149Berlin Institute of Medical Systems Biology 195

Stefan KempaStructure of the GroupGroup LeaderStefan KempaScientistsGuido MastrobuoniGraduate StudentMatthias PietzkeUndergraduate StudentChristin HessTechnical AssistantJulia DiesbachSecretariatSabrina DeterStart of group: April 2009Integrative metabolomics andproteomicsWithin the past decades biochemical data of single processes, metabolic and signalingpathways has been collected. Advances in technology led to improvements of sensitivityand resolution of bioanalytical techniques. These achievements build the basis of so-called‘genome wide’ analyses.High throughput techniques are the tool for such largescale “-omics” studies allowing the obtainment of acomplete picture of a determinate cell state, concerningits metabolites, transcripts and proteins. For exampletwo-dimensional gas chromatography coupled to timeof-flightmass spectrometry (GCxGC-TOF-MS) is a promisingtechnique to overcome limits of complexmetabolome analysis using one dimensional GC-TOF-MS. However, single level study of a living organism(transcripts, proteins or metabolites) cannot give acomplete understanding of the mechanism regulatingbiological functions. The integration of transcriptomics,proteomics and metabolomics data in the newlyemerging field of System Biology, combined with existingknowledge, allows connecting biological processeswhich were treated as independent so far. In this contextthe aim of our group is to apply metabolomics andproteomics techniques for absolute quantification andanalysis of turnover rates of proteins and metabolitesusing stable isotopes; in addition, the further developmentof workflows for data analysis and integrativestrategies will be in the focus of our interest.Functional analysis of cancer metabolismMatthias Pietzke, Julia Diesbach, Markus Landthalerand Jana WolfBeside the enormous diversity of cancers, all tumor cellsseem to share the same metabolic disorder. Already in1924 the famous Nobel Prize biochemist Otto Warburgdescribed an anaerobic type of metabolism performedby cancer tissues. This phenomenon, called Warburgeffect, is still under investigation and the benefit forcancer cells from this metabolic behavior is stillobscure; but it might stem from the onset of cancerogenesis.To unravel the regulatory mechanisms inducingthis metabolic disorder we perform integrativetranscriptomic, proteomic and metabolomic studies.These analyses will allow understanding the molecularprocesses leading to the observed metabolic alterationsof cancer cells and could help to improve anti cancertherapies.Planaria as model organismGuido Mastrobuoni, Julia Diesbach, SebastianMackowiak and Catherine Adamini (Rajewsky Group)The planarian Schmidtea mediterranea is a well-establishedmodel organism at BIMSB. These freshwater flatwormscan regenerate all lost body tissue after amputationdue to a population of pluripotent somatic stemcells called neoblasts that constitute up to 30 percentof the total organism cells.In parallel to other studies carried at BIMSB, aimed tounderstand the role of small RNAs in the planariandevelopment and regeneration of their germ cells, ourgroup is involved in collaborative projects for the functionalannotation of Planaria genome using proteomicdata (figure 1).Once a full genome has been assembled, the main challengelies in its annotation, i.e., in identifying the protein-codinggenes and other functional units that areencoded in the genome. Whole-genome annotation196 Berlin Institute of Medical Systems Biology

has become a standardized data flow and initial sets ofall encoded proteins can be generated in a computerassistedand automated way.The integration of metabolomic and proteomics technologiesinto the annotation process gives an experimentalvalidation of in silico gene models as well as toimproved accuracy of existing gene models.BIMSB systems databaseGuido Mastrobuoni, Matthias Pietzke and ChristophDieterichModern “-omics” technologies like transcriptomics, proteomicsand metabolomics are established at BIMSB.Those techniques generate large amounts of data andcover a wide range of biological processes. To analyzeand store such large amounts of data from these technologieswe are establishing the BIMSB systems database.The database will consist of several modules fordata input and subsequent data analysis and comparison,thus it will connect the analytical platforms withcomputational biologists (see figure 2). It will help tostructure and synchronize large datasets from multipleanalytical platforms and will build up the basis for integrativeanalyses.Figure 1. Functional categories of identified planarian proteins. Totalprotein extract from whole worm was enzymatically digested andthe resulting peptide mixture analyzed by LC-MS/MS on an Orbitrapmass spectrometer. The first analysis allowed the matching ofaround 35% of MS/MS peptide fragmentation spectra to proteinsequences from a database of predicted gene products.Selected PulicationsMay P, Christian JO, Kempa S and Walther D (2009) ChlamyCyc: anintegrative systems biology database and web-portal forChlamydomonas reinhardtii. BMC Genomics 4;10:209Kempa S, Hummel J, Schwemmer T, Pietzke M, Strehmel N, Wienkoop S,Kopka J and Weckwerth W (2009) An automated GCxGC-TOF-MS protocolfor batch-wise extraction and alignment of mass isotopomer matrixesfrom differential 13C-labelling experiments: a case study forphotoautotroph-mixotroph grown Chlamydomonas reinhardtii cells. JBasic Microbiol. 49(1):82-91Kempa S, May P, Wienkoop S, Usadel B, Christian N, Rupprecht J, Weiss J,Recuenco Munoz L, Ebenhöh O, Weckwerth W and Walther D (2008)Metabolomics and Proteomics Assisted Genome Annotation andAnalysis of the Draft Metabolic Network of Chlamydomonas reinhardtii.Genetics 179: 1–10Kempa S, Krasensky J, Dal Santo S, Kopka J and Jonak C (2008) A centralrole of abscisic acid in stress regulated carbohydrate metabolism. PLoSONE 3(12):e3935Kempa S, Rozhon W, Šamaj J, Erban A, Baluška F, Becker T, Haselmayer J,Schleiff E, Kopka J, Hirt H and Jonak C (2007) A plastid localized glycogensynthase kinase 3 modulates stress tolerance and carbohydratemetabolism. Plant J. 49(6):1076-90Figure 2. Scheme of BIMSB systems database. The database consists ofseveral modules from data input, analysis, storage and subsequentanalyses. It will be a tool to analyze large datasets from different analyticalplatforms and will allow interspecies comparison on a quantitativelevel. The aim of the database is to connect the analytical platformswith the computational biologists at the BIMSB.Berlin Institute of Medical Systems Biology 197

Structure of the GroupChristoph DieterichGroup LeaderDr. Christoph DieterichProgrammersMatthias DodtDominic TölleGraduate StudentsSebastian FröhlerZisong ChangSystem administratorAndreas KuntzagkSecretariatSabrina DeterStart of group:April 2009Bioinformatics inOuantitative BiologyBioinformatics is a highly dynamic discipline, which operates at the interface of life sciences,computer science and formal sciences. Currently, emerging technologies in nucleic acidssequencing, mass spectrometry and imaging revolutionize biology. For the first time, a holisticquantification of biological systems at the level of genomes, transcripts, proteins andmetabolites is in reach. Bioinformatics supports this technology-driven transition of biologyinto a truly quantitative science.We use existing and develop novel methods for high-throughput data acquisition, processing,model building and inference. Computational studies are backed up by experimental workwithin our group and within active collaborations.Computational approaches to study eukaryoticgene regulationThe expression of a gene is directly regulated by interactionsof proteins and nucleic acids or by RNA-RNAinteractions. We work on statistical methods for regulatorymotif (e.g. binding site) and module (e.g. promoterelements) prediction. Lately, we explore the use ofsupervised sequence segmentation approaches to pinpointenhancer regions (HM-SVMs). The evolution ofbinding sites is another important aspect, we payattention to. Long-ranging interactions between differentgenomic loci are still enigmatic, yet are of tremendousimportance to gene regulation. We support thisimportant endeavor by an experimental design algorithmfor the chromatin conformation capture (3C)technique.Pristionchus – a model system in ecologyPristionchus pacificus has been established as a satellitesystem to Caenorhabditis elegans over the last years. P.pacificus is an omnivorous nematode and lives in anecromenic association with scarab beetles, which isthought to constitute an evolutionary intermediatebetween free living nematodes and true parasites. Tocomplement our biological knowledge, we sequencedthe P. pacificus genome and found it to be enriched forgene families involved in stress response and metabolismof xenobiotics. Additionally, there are severalinstances of lateral gene transfer. For example, cellulasegene predictions were confirmed by experiment andshown to be integrated into the host biology by activityassays. Another set of predicted protein-coding genesdid not show any similarity to the known protein universeand is currently being confirmed by transcriptomesequencing and mass spectrometry approaches.Genome evolution – content, order and variationPhylogenetic profiling of gene content may guide us instudying species adaptation to certain environments.Similarly, gene order is constrained by several partlyunknown factors. We invented algorithms for identifyinggene order conservation without assigning orthologya priori. We are able to look deep into the history ofgenome structure evolution since these approaches donot rely on a given whole-genome alignment. We arelikewise interested in reconstructing the order of theseevents (in collaboration with Jian Ma, UCSC). Sequencevariation (SNPs and Indels) is another layer of genome198 Berlin Institute of Medical Systems Biology

Phylogeny of cellulase genes from P. pacificus and plant parasitic nematodes. The phylogeny of cellulases indicates that plant parasitic nematodes(green) and P. pacificus (red) have acquired cellulases independently by horizontal gene transfer. The circular phylogram and branch length estimates(expected number of amino acid substitutions) are based on a multiple alignment of the conserved region (70 amino acids) around the C-terminalactive site. Only nematode cellulases with >10% sequence divergence are shown. Additionally, best non-nematode protein matches to P.pacificus cellulases are shown.evolution. We detect sequence variation from highthroughputsequencing approaches and predict potentialeffects on the phenotype with expressive graphicalmodels.Selected PublicationsDieterich, C. & Sommer, R. J. (2009), ‘How to become a parasite – lessonsfrom the genomes of nematodes.’, Trends Genet 25(5), 203-209.Roeseler, W.; Tian, H.; Witte, H.; Yang, S.-P.; Wilson, R. K. & Sommer, R. J.(2008), ‘The Pristionchus pacificus genome provides a unique perspectiveon nematode lifestyle and parasitism.’, Nat Genet 40(10), 1193-1198.Rödelsperger, C. & Dieterich, C. (2008), ‘Syntenator: Multiple gene orderalignments with a gene-specific scoring function.’, Algorithms MolBiol 3, 14.Dieterich, C. & Sommer, R. J. (2008), ‘A Caenorhabditis motif compendiumfor studying transcriptional gene regulation.’, BMC Genomics 9, 30.Dieterich, C.; Clifton, S. W.; Schuster, L. N.; Chinwalla, A.; Delehaunty, K.;Dinkelacker, I.; Fulton, L.; Fulton, R.; Godfrey, J.; Minx, P.; Mitreva, M.;Berlin Institute of Medical Systems Biology 199

The Experimental andClinical Research Center (ECRC)Director: Friedrich Luft

Experimental and Clinical Research Center (ECRC)Experimental and Clinical Research Center (ECRC)The ECRC´s mission is to accelerate the translationof discoveries from fundamental researchinto new procedures for diagnoses, prevention,and therapies for the most common diseases thatthreaten our society. (The focus of the ECRC´sresearch is on cardiovascular and metabolic diseases,cancer, and neurological disease – corresponding tothe research programs of the MDC). The ECRC is ajoint institutional infrastructure dedicated to supportingcollaborative research projects and patientorientedresearch and was launched in 2007 by theMDC and the Charité.The ECRC creates multiple links between basicresearchers and clinicians, thus fostering the bidirectionaltransfer of knowledge between the two“worlds”, which is considered a key factor for successful“translational” research.The ECRC provides scientists from the Charité andMDC extensive opportunities for carrying out translationalprojects:· Clinicians or basic researchers can apply for independentproject groups to conduct a translationalproject within the ECRC. Funding is providedfor three years (extension possible). The fundingdecision is based on a competitive evaluationinvolving external experts.· Clinicians and researchers of the MDC can jointlysubmit proposals for collaborative projects whichcan be funded through the ECRC after a competitiveevaluation (KKP program). The projects areconducted at the ECRC and/or the MDC.· Within the ECRC, the MDC and Charité jointlysponsor a training program for clinicians (KAP, seebelow) in basic molecular biology, in which clinicianstake a break from their duties and join oneof the MDC’s laboratories. This promotes longtermcollaborations between the home and host.· Clinicians direct out-patient clinics and carry outclinical studies within the ECRC;· Clinicians and scientists have access to clinicalresearch center (outpatients clinics, beds for clinicaltrials, see below) and technology platforms,(e.g. ultrahigh-field MR instruments, GMP facility,biobanking).Das ECRC hat zum Ziel, die Übertragung vonErkenntnissen der Grundlagenforschung inneue Verfahren zur Vorbeugung, Diagnose undTherapie von Krankheiten unserer Gesellschaft zubeschleunigen. Der Schwerpunkt der Forschung imECRC liegt auf Herz-Kreislauf- und Stoffwechselkrankheiten,Krebs- und neurologischen Erkrankungen undentspricht den Forschungsprogrammen des MDC.Seit seiner Gründung im Jahr 2007 bildet das ECRC einegemeinsame institutionelle Infrastruktur von Charitéund MDC zur Unterstützung von Kooperationsprojektenund patientenbezogener Forschung. Es zielt daraufab, vielfältige Verbindungen zwischen Grundlagenforschernund Klinikern herzustellen, um auf diese Weiseden Wissensaustausch zwischen den beiden „Welten“zu fördern.Das ECRC eröffnet Wissenschaftlern von Charité undMDC weitreichende Möglichkeiten zur Durchführungtranslationaler Projekte:· Kliniker oder Grundlagenforscher können sich umdie Förderung einer unabhängigen Projektgruppebewerben, um ein Translationsprojekt im ECRCdurchzuführen. Die Förderung wird auf der Basiseiner kompetitiven Begutachtung für drei Jahremit der Möglichkeit zur Verlängerung vergeben.· Kliniker und Grundlagenforscher können in einemebenfalls kompetitiven Verfahren gemeinsam dieFinanzierung kooperativer Projekte beantragen(Klinisches Kooperationsprogramm, KKP).Entsprechende Projekte werden in Räumen desECRC und/ oder des MDC durchgeführt.· MDC und Charité finanzieren im Rahmen des ECRCein gemeinsames Ausbildungsprogramm(Klinisches Ausbildungsprogramm, KAP) für Klinikerin der molekularen Grundlagenforschung. So könnenKliniker für einige Zeit ein Projekt in MDC-Laboratorien bearbeiten. Dadurch wird die Basisfür eine spätere längerfristige Zusammenarbeitgeschaffen.· Kliniker betreiben Hochschulambulanzen am ECRCund führen klinische Studien durch.· Klinische und experimentelle Forscher habenZugang zu einem Klinischen Forschungszentrum202 The Experimental and Clinical Research Center

Photo: David Ausserhofer / Copyright: MDCProf. Dr. Walter Rosenthal (MDC Scientific Director), Dr: Annette Schavan(German Minister of Education and Research), State Secretary Dr. Hans-Gerhard Husung (Berlin Senate for Education, Science, and Research), and Prof.Dr. Friedrich Luft (ECRC Director) (from left to right) at the dedication of the 7Tesla ultra-high field magnetic resonance imaging (MRI) scanner and spectroscopefacility on January 20, 2009 in a new research building of the MDC.Prof. Dr. Walter Rosenthal (Wissenschaftlicher MDC-Vorstand),Bundesforschungsministerin Dr. Annette Schavan, Staatssekretär Dr. Hans-Gerhard Husung (Berliner Senatsverwaltung für Bildung, Wissenschaft undForschung) und Prof. Dr. Friedrich Luft (ECRC-Direktor) (v. l.) bei der Einweihungdes 7-Tesla-Magnetresonanztomographen am 20. Januar 2009 in einem neuenForschungsgebäude des Max-Delbrück-Centrums für Molekulare Medizin (MDC)Berlin-Buch.Scientific projectsTypically, ECRC projects have arisen in two ways. Somebegan as studies of molecular, cellular, or animalmodel systems related to disease, often at a very basicmechanistic or structural level. The second type ofproject began as work in which physicians were servingas caregivers for patients, who served as the startingpoint to probe mechanisms underlying particularhealth problems. A good example of the latter is thediscovery by the group of Ludwig Thierfelder (MD) ofa connection between mutations in the plakophilingene and a condition called arrhythmogenic rightventricular cardiomyopathy. This gene was identifiedin the basic research lab of Walter Birchmeier (PhD) atthe MDC, during a study of proteins involved in WNTsignaling. Working together, the labs identified amutation that greatly increases the likelihood of suddencardiac arrest in affected people. This was developedinto a diagnostic tool to identify family membersat risk, who could be saved by the implantationof a pacemaker. These life-saving insights would nothave been obtained without the constellation of a clinicianinterested in underlying disease mechanisms,(Hochschulambulanzen, Probandenstation mitBetten für klinische Studien, siehe unten) und zutechnolgischer Infrastruktur (bspw. Ultrahochfeld-Magnetresonanztomographie, GMP-Labor,Biobank)Wissenschaftliche ProjekteTypischerweise sind ECRC-Projekte auf zweierleiWegen entstanden: Ein Teil der Projekte hat mit oftmalssehr elementaren, mechanistischen oder strukturellenUntersuchungen auf molekularer bzw. zellbiologischerEbene oder an Modellorganismen begonnen.Ein anderer Teil der Projekte resultiert aus der Betreuungvon Patienten. Für den zweiten Weg ist die Entdeckungeiner Verbindung zwischen Mutationen im Plakophilin-Genund der arrythmogenen rechtsventrikulärenKardiomyopathie durch die Gruppe von LudwigThierfelder (Kliniker) ein gutes Beispiel. Das Gen wurdein der Forschergruppe von Walter Birchmeier (Zellbiologe)am MDC im Zusammenhang mit Arbeiten anProteinen des sog. Wnt-Signalweges der embryonalenEntwicklung entdeckt. Im Rahmen einer Zusammenarbeitwurde eine Mutation bei Patienten arrythmogenenrechtsventrikulären Kardiomyopathie gefunden,die das Risiko eines plötzlichen Herzstillstandes starkerhöhte. Auf der Basis dieser Entdeckung wurde eindiagnostischer Test entwickelt, um innerhalb betroffenerFamilien Träger der Mutation zu identifizieren.Diese Personen können jetzt durch die Implantationeines Herzschrittmachers geschützt werden. Dieselebensrettenden Entdeckungen wären nicht gemachtworden, wenn es nicht das Zusammenspiel zwischeneinem an den Krankheitsmechanismen interessiertenKliniker mit seinen Patienten und einem Zellbiologengegeben hätte. Das Beispiel zeigt in eindrucksvollerWeise, wie das Konzept des ECRC zu einer Überwindungder traditionellen Trennung zwischen Grundlagenforschungund klinischer Medizin beitragen kann.Alle klinischen Abteilungen der Charité und Forschungsgruppendes MDC haben die Möglichkeit, anECRC-Projekten teilzunehmen. Allerdings erhalten sieForschungsflächen im ECRC erst nach erfolgreicherBeantragung und Evaluierung durch Fachgutachter.Mehrere klinische Abteilungen forschen unter demDach des ECRC: Hämatologie und Onkologie (BerndThe Experimental and Clinical Research Center 203

his patients, and a basic research group. The casedescribed is a prime example how the ECRC can overcometoday´s principal problems deriving from theclassical separation of basic science and clinical applications.All clinical departments at the Charité and basicresearchers of the MDC are eligible to participate inECRC activities, but laboratory and clinical space isavailable only after proposals have been submitted topeer review and approved. Several major clinicalgroups are carrying out their work under the ECRCframework: the Department of Hematology-oncology(Bernd Dörken), the Department of Oncological surgeryand the Charité Comprehensive Cancer Center(Peter Schlag), the Department of Neurology (FraukeZipp, Simone Spuler), the Department of Cardiology(Rainer Dietz, Ludwig Thierfelder) and the Departmentof Nephrology-hypertension (Friedrich C. Luft).The ECRC also serves as the base for DFG-sponsoredClinical Research Groups in skeletal muscle diseases(directed by Simone Spuler) and malignant lymphoma(supported up to 2009; directed by ClemensSchmitt).Some examples of clinical work currently underwaywithin the ECRC are projects involving new types oftherapies. For example, the clinicians JörgWestermann and Antonio Pezzutto at the Charité,along with the laboratories of MDC/Charitéresearchers Bernd Dörken and Thomas Blankenstein,are carrying out phase 1 and phase 2 trials of immunetherapies involving chronic myeloid leukemia andrenal carcinomas.Main components of the ECRCThe main physical components of the ECRC are a clinicalresearch center (CRC) for studies involvingpatients and healthy humans, and a high-field magneticresonance facility for the examination ofhumans and model animals. A third building to housethe experimental research center (ERC), whose focusis disease-oriented experimental research, is planned.The CRC, which is located on the Campus Buch in theCharité research building (the former Robert-Rössle-Clinic). Two outpatient clinics have already beenestablished there: one for neuroimmunological dis-Dörken), Onkologische Chirurgie und Charité ComprehensiveCancer Center (Peter Schlag), Neurologie (FraukeZipp, Simone Spuler), Kardiologie (Rainer Dietz, LudwigThierfelder), Nephrologie und Bluthochdruck(Friedrich C. Luft). Das ECRC ist auch Basis der DFGgefördertenklinischen Forschergruppen auf demGebiet von Erkrankungen der Skelettmuskulatur (Leiterin:Simone Spuler) und des malignen Lymphoms(gefördert bis 2009; Leiter: Clemens Schmitt).Einige laufende Projekte betreffen die Entwicklungneuer Therapiekonzepte. Zum Beispiel führen die KlinikerJörg Westermann und Antonio Pezzuto zusammenmit MDC-Forschungsgruppen von Bernd Dörken undThomas Blankenstein klinische Studien (Phase I und II)zur Immuntherapie chronischer myeloischer Leukämieund Nierenkarzinomen durch.Komponenten des ECRCDas ECRC verfügt über ein Gebäude für klinischen Forschungeinschließlich Studien mit Patienten und Probanden(CRC) und ein Gebäude für Ultrahochfeld-Magnetresonanz-Bildgebungfür Untersuchungen amMenschen und an Modellorganismen. Ein drittesGebäude für die krankheitsorientierte, experimentelleForschung (ERC) ist geplant.Das CRC ist im Charité-Forschungsgebäude (ehemalsRobert-Rössle-Klinik) auf dem Campus Buch untergebracht.Im CRC werden mehrere Hochschulambulanzenbetrieben, eine für Neuroimmunologische Erkrankungen(mit Schwerpunkt Multiple Sklerose) und dieandere für Muskelkrankheiten. Letztere wurde im Rahmender DFG-geförderten Forschergruppe eingerichtetund behandelt im Jahr etwa 1200 Patienten mit seltenenMuskelerkrankungen.Weitere Hochschulambulanzenwerden 2010 eingerichtet werden. Ergänzend könnendie Forscher des ECRC an allen Standorten der CharitéForschungsprojekte betreiben, die eine stationäreAufnahme von Patienten erfordern.Das CRC verfügt über Untersuchungsräume und spezielleUntersuchungsmethoden wie Mikroneurographie,Mikrodialyse, eine Kammer für Stoffwechseluntersuchungen,eine Hypoxie-Kammer sowie Vorrichtungenfür invasives hämodynamisches Monitoring.Die zur Verfügung stehenden bildgebenden Verfahren204 The Experimental and Clinical Research Center

eases (with a special focus on multiple sclerosis) andanother for muscle disease. The latter was establishedin the context of a DFG-financed clinicalresearch group and deals with ca. 1200 patients withrare muscle diseases per year. Further outpatient clinicsof the Charité will be established in 2010. In addition,ECRC researchers may conduct inpatient studieson all other Charité campuses.The CRC has examining rooms, specialized procedurefacilities such as microneurography, microdialyis, ametabolic chamber, normobaric hypoxia facilities,and tools for invasive hemodynamic monitoring.Available imaging technologies include ultrasound,echocardiography, Doppler tools, and access to highfieldMRI. Personnel include study nurses, dietitians,biostatisticians, and ancillaries. The unit also has anerstwhile bone marrow transplant unit with appropriatesafeguards. A “good manufacturing practice”(GMP) laboratory has been established to provide cellulartherapies. At present, preparations are underwayto launch a Phase I study for tumor immune therapyusing dendritic cells, financed by the HelmholtzAssociation.CRC physicians are trained in internal medicine, neurology,clinical pharmacology, and oftentimes a combinationof these specialties. Credit towards certificationin internal medicine, neurology, and clinical pharmacologycan be granted to clinical trainees participatingin the ECRC.The CRC encompasses not only clinical researchgroup projects but will include the HelmholtzEpidemiological Cohort study, for which 40,000human subjects will be recruited from the Berlinregion, from a total of 200,000 who are being recruitedthroughout Germany. Additionally, the CRC hasthe capacity to develop several outpatient clinics. Twooutpatient clinics have already been establishedthere: one for neuroimmunological diseases (with aspecial focus on multiple sclerosis) and another formuscle disease. The latter was established in the contextof a DFG-financed clinical research group anddeals with ca. 1200 patients with rare muscle diseasesper year. Further outpatient clinics of the Charité willbe established in 2010. In addition, ECRC researchersmay conduct inpatient studies on all other Charitécampuses.umfassen Ultraschall, Echokardiographie, Doppler-Sonographie, und Hochfeld-Magnetresonanztomographie.Zum Personal gehören u. a. Studienschwestern,Diätassistenten und Biostatistiker. Ein GMP(„good manufacturing practice“) -Labor für die Zelltherapiewurde eingerichtet. Gegenwärtig wird einePhase I-Studie zur Immuntherapie mit dendritischenZellen vorbereitet, die von der Helmholtz-Gemeinschaftfinanziert wird.Die ärztlichen Mitarbeiter des CRC erhalten eine Ausbildungin Innerer Medizin, Neurologie oder KlinischerPharmakologie, meist als Kombination dieser Fächer,die sie sich auf ihre Facharztausbildung anrechnen lassenkönnen.Im CRC werden nicht nur die Projekte klinischer Forschungsgruppendurchgeführt sondern es wird auchdie epidemiologische Kohortenstudie der Helmholtz-Gemeinschaft beherbergen, für die aus der BerlinerRegion 40000 Probanden rekrutiert werden sollen. Insgesamtsollen deutschlandweit 200000 Probanden indie Kohorte eingeschlossen werden.Die Ultrahochfeld-Magnetresonanz-Anlage des MDCmit einem 7 Tesla-Ganzkörper-Magnetresonanztomograph(MRT) für die Anwendung am Menschen undeinem experimentellen 9,4 Tesla-MRT ist im Januar2009 eröffnet worden. Bis Ende 2009 wurde dasGebäude um ein zweites Stockwerk für die Unterbringungeines klinischen 3 Tesla-MRT erweitert. Damitstehen nunmehr insgesamt 913 m Forschungsflächefür die MR-Bildgebung zur Verfügung. Der Physiker,Thoralf Niendorf, ein führender Experte auf demGebiet der MR-Technologie mit Erfahrung im akademischenund industriellen Bereich wurde als Leiter derUltrahochfeld-MR-Anlage berufen.In unmittelbarer Nachbarschaft der Ultrahochdfeld-MR-Anlage wird das MDC im Frühjahr 2010 mit demNeubau für das ERC beginnen. Nach Fertigstellungwerden im ERC weitere ca. 2600 m 2 hochwertige undflexibel nutzbare Labor- und Büroflächen für die translationaleForschung zur Verfügung stehen.AusbildungszieleEin wichtiges Motiv für die Gründung des ECRC wardie Notwendigkeit, eine neue Generation von For-The Experimental and Clinical Research Center 205

The ultrahigh field MR facility was opened in January2009. By the end of 2009, the facility has beenenlarged by a second floor to house a new 3 Tesla clinicalscanner in addition to a 9.4 Tesla animal scannerand a 7 Tesla whole body scanner, resulting in a totalof 913 m of research space for MR technologies.Thoralf Niendorf was appointed director of the facilityin 2009. A physicist by training, Thoralf Niendorf isa leading expert in MR imaging with a research experiencein academia and industry.To house the ERC, the MDC will start construction ona new building in close proximity to the MR facility inspring 2010. The ERC building will comprise ca. 2600m 2 of highly flexible laboratory and office space fortranslational research projects.Educational missionAn important motivation for the project is the needfor a new breed of researcher: a “physician-scientist”equally at home in the clinic and the basic researchlab. Translational research involves all levels of biologicalorganization, from the structures and functionsof single molecules to the overall health of an organismover the long term, Physicians and basicresearchers have complementary perspectives andexpertise that need to be combined – ideally in a singleperson – to design meaningful basic experimentsthat will shed light on disease processes in patients.The centerpiece of the educational activities is atraining program for clinicians in basic molecularbiology, jointly sponsored by the MDC and Charité, inwhich clinicians take “time out” from their clinicalduties and join a laboratory run by an MDC basic scientist.Entry into the program (Kliniker Ausbildungsprogramm,or KAP, in German) is gained through acompetitive application procedure. Proposals arepeer-reviewed with intermittent follow-up.Successful applications commonly lead to long-termcollaborations between a clinical group and the MDCbasic science laboratory hosting the applicant.schern auszubilden, die gleichermaßen in der Klinik wieim Forschungslabor zu Hause sind. Die translationaleForschung spannt den Bogen von der Struktur undFunktion einzelner Moleküle bis zur ganzheitlichenFunktion des Organismus. Ärzte und experimentelleBiologen haben einander ergänzende Denkweisen undErfahrungen, die vereinigt werden müssen – idealerweisein einer Person – wenn Experimente so konzipiertsein sollen, dass sie zu neuen Einsichten über Krankheitsprozessein Patienten führen können.Eine Schlüsselkomponente des ECRC ist daher ein Trainingsprogramm,um den „Physician Scientist“ mit dementsprechenden Expertise-Profil auszubilden. Ein zentralesElement ist hierbei das Ausbildungsprogramm fürKliniker in der Molekularbiologie, das von Charité undMDC gemeinsam gestaltet wird. Hier können Klinikereine „Auszeit“ von ihren klinischen Pflichten nehmenund in einem Labor arbeiten, das von einem MDC-Forschergeleitet wird. Der Zugang zum Kliniken Ausbildungsprogramm(KAP) ist über ein kompetitives Bewerbungsverfahrengeregelt. Der Projektfortschritt wirddurch eine Zwischenevaluation überprüft. Gleichzeitigbildet der Gastaufenthalt oftmals die Ausgangsbasisfür eine längerfristige Zusammenarbeit zwischen demKAP-Stipendiaten und der gastgebenden Abteilung.PerspektivenDas ECRC wird wichtige Infrastrukturkomponenten fürweitere Projekte bereitstellen, die sich gegenwärtig imPlanungsstadium befinden, bspw. ein Deutsches Zentrumfür Kardiovaskuläre Forschung (DZHK) und einDeutsches Institut für Kardiovaskuläre Forschung(DIHK, wie in der Einleitung zum Research Reporterwähnt). Wenn das DIHK wie vorgesehen auf demCampus Buch angesiedelt wird, könnte es von derunmittelbaren Nachbarschaft des ECRC mit seinentechnologischen Plattformen und seiner Forschungsinfrastrukturprofitieren.PerspectivesThe ECRC will provide vital infrastructure for someother projects that are currently being planned, suchas the establishment of a German Center forCardiovascular Research (Deutsches Zentrum für206 The Experimental and Clinical Research Center

Kardiovaskuläre Forschung, DZHK) and a GermanInstitute for Cardiovascular Research (DeutschesInstitut für Kardiovaskuläre Forschung, DIHK) whichhave been proposed by the MDC and Charité. The proposalforesees the construction of the DIHK on theBerlin-Buch campus, which would put it in the immediatevicinity of the ECRC and its technological platformsand infrastructure.Structure of the ECRCDirectorFriedrich C. LuftChief AdministratorRegina JüngerProgram ManagerCornelia MaurerSteering committeeWalter Rosenthal (MDC Director)Annette Grüters-Kieslich (Charité Dean)Cornelia Lanz (MDC Administrative Director)Gerrit Fleige (Administrative Head of the CharitéFaculty)ECRC Council (chairman)Thomas SommerSelected publications of ECRC participantsMathas, S, Kreher, S, Meaburn, KJ, Jöhrens, K, Lamprecht, B, Assaf, C,Sterry, W, Kadin, ME, Daibata, M, Joos, S, Hummel, M, Stein, H, Janz, M,Anagnostopoulos, I, Schrock, E, Misteli, T, Dörken, B. (2009) Gene deregulationand spatial genome reorganization near breakpoints prior toformation of translocations in anaplastic large cell lymphoma. ProcNatl Acad Sci U S A. 106, 5831-6.Stein, U, Walther, W, Arlt, F, Schwabe, H, Smith, J, Fichtner, I, Birchmeier,W, Schlag, PM. (2009) MACC1, a newly identified key regulator of HGF-MET signaling, predicts colon cancer metastasis. Nat Med. 15, 59-67.Prozorovski, T, Schulze-Topphoff, U, Glumm, R, Baumgart, J, Schröter, F,Ninnemann, O, Siegert, E, Bendix, I, Brüstle, O, Nitsch, R, Zipp, F, Aktas, O.(2008) Sirt1 contributes critically to the redox-dependent fate of neuralprogenitors. Nat Cell Biol. 10, 385-94.Hauck, L, Harms, C, An, J, Rohne, J, Gertz, K, Dietz, R, Endres, M, vonHarsdorf, R. (2008) Protein kinase CK2 links extracellular growth factorsignaling with the control of p27(Kip1) stability in the heart. Nat Med.14, 315-24.Organisationsstruktur des ECRCDirektorFriedrich C. LuftKaufmännische LeiterinRegina JüngerProgramm-ManagerinCornelia MaurerLenkungsausschussWalter Rosenthal (Wissenschaftlicher Vorstand, MDC)Annette Grüters-Kieslich (Dekanin, Charité)Cornelia Lanz (Administrativer Vorstand, MDC)Gerrit Fleige (Kaufmännischer Leiter der Fakultät,Charité)ECRC-Rat (Vorsitzender)Thomas SommerAusgewählte Publikationen von Forschern des ECRCMathas, S, Kreher, S, Meaburn, KJ, Jöhrens, K, Lamprecht, B, Assaf, C,Sterry, W, Kadin, ME, Daibata, M, Joos, S, Hummel, M, Stein, H, Janz, M,Anagnostopoulos, I, Schrock, E, Misteli, T, Dörken, B. (2009) Gene deregulationand spatial genome reorganization near breakpoints prior to formationof translocations in anaplastic large cell lymphoma. Proc NatlAcad Sci U S A. 106, 5831-6.Stein, U, Walther, W, Arlt, F, Schwabe, H, Smith, J, Fichtner, I, Birchmeier, W,Schlag, PM. (2009) MACC1, a newly identified key regulator of HGF-METsignaling, predicts colon cancer metastasis. Nat Med. 15, 59-67.Prozorovski, T, Schulze-Topphoff, U, Glumm, R, Baumgart, J, Schröter, F,Ninnemann, O, Siegert, E, Bendix, I, Brüstle, O, Nitsch, R, Zipp, F, Aktas, O.(2008) Sirt1 contributes critically to the redox-dependent fate of neuralprogenitors. Nat Cell Biol. 10, 385-94.Hauck, L, Harms, C, An, J, Rohne, J, Gertz, K, Dietz, R, Endres, M, vonHarsdorf, R. (2008) Protein kinase CK2 links extracellular growth factorsignaling with the control of p27(Kip1) stability in the heart. Nat Med.14, 315-24.Machnik, A, Neuhofer, W, Jantsch, J, Dahlmann, A, Tammela, T, Machura,K, Park, JK, Beck, FX, Müller, DN, Derer, W, Goss, J, Ziomber, A, Dietsch, P,Wagner, H, van Rooijen, N, Kurtz, A, Hilgers, KF, Alitalo, K, Eckardt, KU,Luft, FC, Kerjaschki, D, Titze, J. (2009) Macrophages regulate salt-dependentvolume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism. Nat Med. 15, 545-52.Machnik, A, Neuhofer, W, Jantsch, J, Dahlmann, A, Tammela, T, Machura,K, Park, JK, Beck, FX, Müller, DN, Derer, W, Goss, J, Ziomber, A, Dietsch, P,Wagner, H, van Rooijen, N, Kurtz, A, Hilgers, KF, Alitalo, K, Eckardt, KU,Luft, FC, Kerjaschki, D, Titze, J. (2009) Macrophages regulate saltdependentvolume and blood pressure by a vascular endothelialgrowth factor-C-dependent buffering mechanism. Nat Med. 15, 545-52.The Experimental and Clinical Research Center 207

Structure of the GroupSimone SpulerGroup LeaderProf. Dr. Simone SpulerScientistsPD Dr. Ute ZachariasPD Dr. Hans KnoblauchDr. Verena SchöwelDr. Stephanie AdamsDr. Miriam CarlMuscle Research Unit and ClinicalResearch Group (CRG) 192Simone Spuler directs CRG 192 for skeletal muscle diseases (speaker, Friedrich C. Luft) at theCharité. CRG 192 is home to groups at all Charité campuses; however, much of the researchactivities, including her own research team is located at the ECRC. Within the ECRC, she directsan outpatient clinical for >1000 patients with genetic and acquired skeletal muscle diseases.CRG 192 is in the process of expanding by founding a graduate school (Graduiertenkolleg) fordoctoral and postdoctoral students in conjunction with the University of Paris. Simone Spulerhas focused her attention on dysferlin. The dysferlinopathies encompass a large variety ofneuromuscular diseases characterized by the absence of dysferlin in skeletal muscle and anautosomal recessive mode of inheritance. Three main phenotypes are known, Miyoshimyopathy, limb girdle muscular dystrophy type 2B, and distal myopathy with anterior tibialonset.Dysferlin is a large protein located at the sarcolemmaand is involved in membrane repair after microinjury, aphysiological consequence of normal muscle activity.Patients with mutations in the dysferlin gene experienceprogressive muscle weakness in early adulthoodleading to loss of ambulation within 10-15 years. Soonafter the dysferlin gene was discovered in 1998, inflammatorychanges were observed to be an intrinsic part ofthe disease spectrum in dysferlinopathies. The Spulerlaboratory was the first to demonstrate involvement ofthe immune system. They observed that the complementinhibitory factor CD 55 is selectively downregulatedon dysferlin-deficient skeletal muscle leading toincreased vulnerability to complement attacks.Modulation of the complement cascade may open atherapeutic strategy.The team was also the first to demonstrate that dysferlinmutations lead to misfolding and aggregation ofdysferlin and muscle amyloidosis. In many aspects, dysferlinopathiesresemble other protein misfolding diseasessuch as Alzheimer’s disease and Parkinson’s disease.Dr. Verena Schöwel is working on a strategy to rescuemissense-mutated dysferlin from degradation andrelocating the molecule to the plasma membrane. Dr.Ute Zacharias is investigating the effect of the potentmodulator of skeletal muscle growth, myostatin, ondysferlin-deficient human myotubes. These efforts areleading to novel treatments to relocate the availableprotein. An effort is underway to apply this new knowledgeto in vivo models.Muscular dystrophies are closely related to generalizeddisturbances in metabolism. Years ago, muscular dystrophieswere considered to be endocrinological diseases.This aspect was later neglected due to the enormousprogress in visualization techniques, genetictools, and molecular biology. The structural abnormalitiesof skeletal muscle disorders were described andcategorized. Furthermore, many new genes were identifiedthat cause myopathies once mutated. However, the208 The Experimental and Clinical Research Center

Graduate StudentsJoanna SchneiderSina GloyManager of sponsored programsSusanne WisslerTechnical AssistantsStephanie MeyerAnne SchulzeAntje DrägerHundreds of myoblasts can be generated from a single human musclefiber. Mouse anti-human desmin ab (green) and Hoechst stain.mechanism of progressive muscle weakness andreplacement of muscle by connective tissue is still notwell understood. Today, we have increasing evidencethat important aspects of muscular dystrophies mayindeed be endocrinological. In an interdisciplinaryapproach involving pharmacologists, biochemists,endocrinologists and neurologists we investigate glucoseand lipid metabolism in vivo and in vitro in molecularlydefined groups of patients. Some muscular dystrophies,Dunnigan’s for example, feature partial lipodystrophy,early onset of type 2 diabetes mellitus, andnon-alcoholic steatosis. The Spuler group is workingtogether with other ECRC investigators belonging toCRG 192 to elucidate the faulty metabolism of thesedisorders. Joanna Schneider is working on FRET analysisusing glucose sensitive vectors.After muscle injury, muscle satellite cells have the fullpotential to proliferate, differentiate, and conductrepair. However, in tissue culture these cells rapidly losethese properties. Because satellite cells would be agood target for gene therapy, it is important to investigatetheir regenerative potential in vitro. Much informationis available from mouse models. Human satellitecells have not been characterized in any meaningfuldetail. Dr. Stephanie Adams and Sina Gloy isolate andcharacterize human satellite cells. They are focusing inparticular on Notch and Wnt signaling pathways. Thepotential of this work is particularly relevant for theECRC with the advent of the GMP stem cell laboratory,a core facility for all disciplines at the ECRC and MDC.When the time comes, the movement to translate newfindings will be rapid.Selected publicationsSpuler S, Kalbhenn T, Zabojszcza J, van Landeghem FK, Ludtke A, Wenzel K,Koehnlein M, Schuelke M, Lüdemann L, Schmidt HH. Muscle and nervepathology in Dunnigan familial partial lipodystrophy. Neurology. 2007Feb 27;68(9):677-83.Spuler S, Carl M, Zabojszcza J, Straub V, Bushby K, Moore SA, Bähring S,Wenzel K, Vinkemeier U, Rocken C. Dysferlin-deficient muscular dystrophyfeatures amyloidosis. Ann Neurol. 2008 Mar;63(3):323-8.Schmidt S, Vieweger A, Obst M, Mueller S, Gross V, Gutberlet M,Steinbrink J, Taubert S, Misselwitz B, Luedemann L, Spuler S. Dysferlindeficientmuscular dystrophy: gadofluorine M suitability at MR imagingin a mouse model. Radiology. 2009 Jan;250(1):87-94.Knoblauch H, Geier C, Adams S, Budde B, Rudolph A, Schulz-Menger J,Spuler A, Ben Yaou R, Nürnberg P, Voit T, Bonne G, Spuler S. Emery-Dreifuss syndrome in a novel FHL1 mutation. Ann. Neurol. 2009 (in press)Gueneau L, Bertrand AT, Jais JP, Salih MA, Stojkovic T, Wehnert M,Hoeltzenbein M, Spuler S, Knoblauch H, Saitoh S, Verschueren A,Tranchant C, Beuvin M, Lacene E, Romero NB, Heath S, Zelenika D, Voit T,Eymard, Ben Yaou R, Bonne G. Mutations of FHL1 gene cause Emery-Dreifuss muscular dystrophy. Am J Hum Genet 2009 (in press)The Experimental and Clinical Research Center 209

Ralph KettritzStructure of the GroupGroup LeaderProf. Ralph KettritzGraduate students and clinical fellowsDr. Adrian SchreiberDr. Mira ChoiDr. Julia BontschoDr. Uwe JerkeDr. Astrid BergmannClaudia EulenbergNeutrophil biology in healthand diseaseThis group focuses on the neutrophilic granulocyte. Neutrophils form an essential part of theinnate immune system. Neutrophils circulate in the blood stream and migrate into tissuesduring inflammation and also in some cancers. This process is initiated by signals, such asInterleukin-8 (IL-8), Interferon-gamma (IFN-γ), and complement fraction C5a. The fundamentalfinding that some systemic vasculitidis are caused by autoantibodies directed at neutrophilproteinase 3 and myeloperoxidase, sparked our interest in neutrophils. These antineutrophilcytoplasmic antibodies (PR3 and MPO ANCA) cause a common syndrome featuring upper andlower airway disease as well as rapidly progressive “pauci-immune” glomerulonephritis (ANCAvasculitis). In addition, our group is interested in all aspects of neutrophil function.A mouse model of ANCA vasculitisAdrian Schreiber, a DFG-sponsored research fellow, participatedin the development of a mouse model duringhis fellowship at the University of North Carolina withCharles Jennette. They immunized MPO gene-deficientmice with MPO inducing MPO ANCA. Then, these micewere irradiated and underwent bone-marrow transplantwith MPO+/+ marrow. The animals now had MPOANCA and MPO in their neutrophils and developed aclassic MPO ANCA vasculitis, including glomerulonephritis.Werecently made the novel observation that the C5areceptor mediates neutrophil activation in this model.Supernatants from ANCA-stimulated neutrophils activatedthe complement cascade in normal serum in vitro,producing C5a. This conditioned serum primed neutrophilsfor ANCA-induced activation and C5aR blockadeabrogated this effect. In our mouse model, bone marrowtransplants were done from C5a-/- and C5a+/+ mice.Theresults were convincing and showed that the complementsystem is pivotal to MPO ANCA vasculitis raisingimportant therapeutic possibilities.The CD177 glycoprotein NB1 and PR3 cell-surfacepresentationThe serine protease proteinase 3 (PR3) is a mainautoantigen in ANCA vasculitis. PR3 surface presentationon neutrophilis, the main effector cells, is pathogenicallyimportant. PR3 is presented by the NB1 (CD177)glycoprotein, but how the presentation develops duringneutrophil differentiation is not known. The Kettritzteam is intensively studying the relationship betweenNB1 and PR3. An N-terminally unprocessed PR3 (proPR3)is produced early during neutrophil development andpromotes myeloid cell differentiation. The group recentlytested whether or not it depended on NB1 duringneutrophil differentiation and if PR3 and proPR3 couldboth be presented by NB1. NB1-mediated PR3 presentationdepended on PR3 N-terminal processing implicatingthe PR3-N-terminus as NB1-binding site. Kettritzand associates are currently pursuing this promisingarea of investigation further.The adage is “starve a fever, feed a cold.”Physicians spend much of their time fighting fever;even William Osler thought that the issue was important.We asked whether or not fever regulates neutrophilbehavior in a series of studies. Mira Choi was primarilyresponsible for this work. The group tested thehypothesis that exposure of mice to fever-like temperaturesabrogates neutrophil recruitment and NF-kappaBactivation in a mouse model of skin inflammation. Mice210 The Experimental and Clinical Research Center

Associated scientists and clinical scientistsProf. Friedrich C. LuftManager of sponsored programsSusanne WisslerTechnical AssistantsSusanne RolleSylvia Krügerthat were exposed to 40° C for 1 hour showed stronglyreduced GM-CSF- and IL-8-induced neutrophilic skininflammation. In vitro heat exposure abrogated migrationtoward both cytokines by down-regulating PI3-K/Akt. Furthermore, neutrophils on fibronectin showedabrogated NF-κB activation in response to GM-CSF andIL-8 after heat. Less NF-κB activation was seen in theinflammatory lesions of mice exposed to fever-like temperaturesas demonstrated by in situ hybridization forIκBα mRNA. These new findings suggest that heat mayhave anti-inflammatory effects in neutrophil-dependentinflammation. Thus, we believe we should let feversbe!ANCA from a patient with active vasculitis shows a cytoplasmicimmunofluorescence staining pattern in permeabilized neutrophils(left). Necrotizing crescentic glomerulonephritis in a mouse model ofANCA vasculitis (right).Is platelet dust more important than stardust?Birgit Salanova and the group tested the notion thatplatelets could “spill over” functional glycoproteinIIb/IIIa (GPIIb/IIIa) receptors onto neutrophils viaplatelet-derived microparticles (PMPs). The groupobserved that acquired GPIIb/IIIa receptors co-localizedwith beta2-integrins and cooperated in NF-κB activation.They showed that Src and Syk non-receptor tyrosinekinases, as well as the actin cytoskeleton, controlNF-kappaB activation. When granulocyte macrophagecolony-stimulating factor-treated neutrophils wereincubated on fibronectin, strong NF-κB activation wasobserved, but only after loading with PMPs. Currentlyavailable GPIIb/IIIa inhibitors were effective. The dataimplicate GPIIb/IIIa receptors as new therapeutic targetsin neutrophil-induced inflammation.BK channels and neutrophil functionThe group (in collaboration with Maik Gollasch (ECRC)and William Nauseef (University of Iowa) was involvedin an “intensive” controversy with a group at UniversityCollege London, regarding the importance of BK channelsin neutrophil “burst” reactions. To test this notion,Kirill Essin and the group directly assessed the role of BKchannels in neutrophil function, including the NADPHoxidase. Neutrophils lacking BK channels (BK-/-) hadnormal NADPH oxidase activity in response to receptorindependentand phagocytic challenges. Furthermore,NADPH oxidase activity of neutrophils and macrophageswas normal after treatment with BK channelinhibitors. The group concluded that the BK channel isnot required for NADPH oxidase activity in neutrophils.The contentious argument has been dropped.Selected PublicationsSchreiber, A, Xiao, H, Jennette, JC, Schneider, W, Luft, FC, Kettritz, R. (2009)C5a receptor mediates neutrophil activation and ANCA-induced glomerulonephritis.J Am Soc Nephrol. 20, 289-298.Choi, M, Salanova, B, Rolle, S, Wellner, M, Schneider, W, Luft, FC, Kettritz, R.(2008) Short-term heat exposure inhibits inflammation by abrogatingrecruitment of and nuclear factor-{kappa}B activation in neutrophilsexposed to chemotactic cytokines. Am J Pathol. 172, 367-777.von Vietinghoff, S, Choi, M, Rolle, S, Luft, FC, Kettritz, R. (2007) Febrile temperaturescontrol antineutrophil cytoplasmic autoantibody-induced neutrophilactivation via inhibition of phosphatidylinositol 3-kinase/Akt.Arthritis Rheum. 56, 3149-3158.Salanova, B, Choi, M, Rolle, S, Wellner, M, Luft, FC, Kettritz, R. (2007) Beta2-integrins and acquired glycoprotein IIb/IIIa (GPIIb/IIIa) receptors cooperatein NF-kappaB activation of human neutrophils. J Biol Chem. 282,27960-27969.Essin, K, Gollasch, M, Rolle, S, Weissgerber, P, Sausbier, M, Bohn, E,Autenrieth, IB, Ruth P, Luft, FC, Nauseef,WM, Kettritz, R.(2009) BK channelsin innate immune functions of neutrophils and macrophages. Blood 113,1326-1331.The Experimental and Clinical Research Center 211

Structure of the GroupJeanette Schulz-MengerGroup LeaderProf. Dr. Jeanette Schulz-MengerScientistsDr. Steffen BohlDr. Philipp BoyeDipl.-ing Matthias DieringerDr. Florian v. Knobelsdorff-BrenkenhoffCardiovascular MagneticResonanceThe CMR group at the Franz-Volhard-Klinik has focused its research on the in vivoassessment of functional and structural myocardial abnormalities related toinflammatory diseases and coronary heart disease. We perform clinical research using a1.5 clinical MRI-scanner with dedicated cardiac software. We developed new approaches forthe differentiation of tissue changes in myocardial diseases. The application as researchtools and the translation into a clinical setting are the main interests of the group. Thesuccessful work led to the introduction of the University Professorship in Cardiology forNoninvasive Imaging focused on Cardiovascular magnetic resonance (CMR).In 2009 the Ultra-High-Field Facility was opened giving us the possibility to extend ourresearch field by developing translational research tools applying advanced imagingmodalities.Myocardial InjuryThe diagnosis of myocardial inflammation in differentdiseases and the assessment of myocardial tissuechanges during follow-up is a challenging task in cardiovascularresearch and clinical cardiology. Clinicalpresentation of patients with myocarditis or myocardialinvolvement in systemic disorders often mimicsother disorders and may vary from flu-like symptomsor subclinical disease to acute heart failure and suddencardiac death. Cardiovascular magnetic resonance(CMR) has the capability to differentiate between thevarious forms of myocardial injuries (e.g. edema,hyperemia and fibrosis). In the late 90s we developedan approach for the noninvasive detection of acutemyocarditis by CMR. In 2008/09 we published thenoninvasive detection of myocardial edema and thecapability of a multi-sequential approach for followup.(1)Myocardial inflammation also has a high prognosticimpact in different systemic diseases. Howeverearly assessment is difficult. We used CMR technologyin patients with systemic lupus erythematosus andChurg Strauss syndrome and were able to detectmyocardial involvement in those patients with preservedleft-ventricular function. (2) It is well known,that that development of fibrosis is of potential prognosticimpact in hypertrophy. Using contrastenhanced CMR we were able do differentiate the patternin various non-ischemic disease (3) and visualizefibrotic tissue in hypertrophy caused by different diseases(4) Furthermore we tried to get insight the gender-relatedremodeling process in hypertrophic cardiomyopathy.(5)Coronary Artery Disease and ArteriosclerosisThe detection of coronary artery stenosis is a growingfield in CMR. Quantitative analysis of perfusion is acrucial step for evaluation of significant ischemia anddevelopment of new therapeutic strategies. In 2009we could show that the lipid-apheresis has an impacton myocardial perfusion in young patients with elevatedLPa. In 2009 we published, that the quantitativedual bolus perfusion is of higher accuracy having thepotential to reduce the sample size. The field is ourstep into Ultra-High-Field research. In collaboration212 The Experimental and Clinical Research Center

Dr. André RudolphDr. med. Dipl.-Phys. Wolfgang UtzDr. Ralf WassmuthDr. Anja ZagrosekGraduate StudentsSascha AischeSana El-MammoudHoang GiangMaria KraussHannes KutzaMartin PofahlMarcel ProthmannJuius TraberTechnical AssistantsDenise KleindienstKerstin KretschelEvelyn PolzinStudy nurse/Project assistanceAnnette Köhlerwith the University of Oxford we published our firstpaper on CMR-driven infarct-detection in mices. (6)We are working on establishing the method at theMDC, Technical improvement will take place at all levelsof research in a strong collaboration with thegroup of the Ultra-Highfield-FacilitySelected publicationsZagrosek A, Abdel-Aty H, Boyé P, Wassmuth, R. Messroghli, DR. Utz, W.Rudolph, A. Bohl, S. Dietz, R. Schulz-Menger, J. Cardiac MagneticResonance Monitors Reversible and Irreversible Myocardial Injury inMyocarditis. J Am Coll Cardiol CVI 2009;2(2):131-138Bohl S, Wassmuth R, Abdel-Aty H, Rudolph, A. Messroghli, D. Dietz, R.Schulz-Menger, J.Delayed enhancement cardiac magnetic resonanceimaging reveals typical patterns of myocardial injury in patients with variousforms of non-ischemic heart disease. Int J Cardiovasc Imaging. Aug2008;24(6):597-607.Rudolph A, Abdel-Aty H, Bohl S, Boye, P. Zagrosek, A. Dietz, R. Schulz-Menger, J. Noninvasive detection of fibrosis applying contrast-enhancedcardiac magnetic resonance in different forms of left ventricular hypertrophyrelation to remodeling. J Am Coll Cardiol. Jan 20 2009;53(3):284-291.Schulz-Menger J, Abdel-Aty H, Rudolph A, Elgeti, T. Messroghli, D. Utz, W.Boye, P. Bohl, S. Busjahn, A. Hamm, B. Dietz, R. Gender-specific differencesin left ventricular remodelling and fibrosis in hypertrophic cardiomyopathy:Insights from cardiovascular magnetic resonance. Eur J Heart Fail.Sep 2008;10(9):850-854.Bohl S, Lygate CA, Barnes H, Medway, D. J. Stork, L. A. Schulz-Menger, J.Neubauer, S. Schneider, J. E.Advanced Methods for Quantification ofInfarct Size in Mice Using Three-Dimensional High-Field Late GadoliniumEnhancement MRI. Am J Physiol Heart Circ Physiol. Feb 13 2009.The Experimental and Clinical Research Center 213

Maik GollaschStructure of the GroupGroup LeaderProf. Maik GollaschGraduate students andclinical fellowsJohanna SchleifenbaumCarolin KöhnMirija WechslerMarwan MannaaDr. Alexander WutzlerAssociated scientistsDr. Kirill EssinDr. Galyna DubrovskaTechnical AssistantsYoland-Marie AnistanDiana HeroldManager of sponsoredprogramsSusanne WisslerSmooth Muscle Cell Electrophysiology,Ion Channel, and Transporter FunctionIon channels and electrophysiologyThis group focuses on ion channels, primarily in vascularsmooth muscle cells (VSMC), to clarify mechanismscontributing to hypertension and cardiovascular disease.Calcium-activated potassium (BK) channels havereceived special attention. The group collaborated withRalph Kettritz to study the notion that BK channels areinvolved in neutrophil activation. A knockout mousemodel showed convincingly that BK channels are notinvolved in this process. The group investigated the roleof transient receptor potential (TRP) ion channels inagonist-independent G(q 11) protein-coupled receptoractivation. Their work showed that G(q 11)-coupledreceptors could function as membrane stretch receptorsin VSMC. A close collaboration with Wolf-HagenSchunck at the MDC has turned the group’s focustowards eicosanoids. The group found that p450eicosanoids such as epoxyeicosatrienoic acids arevasodilatory, largely through their ability to activateendothelial NO synthase and NO release. Furthermore,in collaboration with Huang Yu in Hong Kong, theyfound that endothelium-derived contracting factors aredependent on cyclooxygenase-2. Another area ofresearch is directed towards identifying the role of theperivascular fat as a modulator of arterial tone, withspecific emphasis on the resistance vasculature.BK channels and neutrophil functionThe group (in collaboration with Ralph Kettritz (ECRC)and William Nauseef (University of Iowa) was involvedin an “intensive” controversy with a group at UniversityCollege London, regarding the importance of BK channelsin the mediation of neutrophil “burst” reactions. Totest this notion, Kirill Essin and the group directlyassessed the role of BK channels in neutrophil function,including the NADPH oxidase. Neutrophils lacking BKchannels (BK-/-) had normal intracellular and extracellularNADPH oxidase activity in response to both receptor-independentand phagocytic challenges. Furthermore,NADPH oxidase activity of neutrophils andmacrophages was normal after treatment with BKchannel inhibitors. Although BK channel inhibitors suppressedendotoxin-mediated tumor necrosis factoralphasecretion by bone marrow-derived macrophages,cells from BK-/- and wild-type mice responded identicallyand exhibited the same ERK, PI3K/Akt, and NF-κBactivation. The group concluded that the BK channel isnot required for NADPH oxidase activity in neutrophils.The contentious argument has been dropped.Epoxyeicosatrienoic acids (EETs)EETs serve as endothelial-derived hyperpolarizing factors(EDHF), but may also affect vascular function byother mechanisms. The Gollasch team identified anovel interaction between EETs and endothelial NOrelease using soluble epoxide hydrolase (sEH) -/- and+/+ mice. EDHF responses to acetylcholine in pressurizedisolated mesenteric arteries were neither affectedby the sEH inhibitor, N-adamantyl-N’-dodecylurea(ADU), nor by sEH gene deletion. However, the EDHFresponses were abolished by catalase and byapamin/charybdotoxin (ChTx), but not by iberiotoxin,nor by the cytochrome P450 inhibitor PPOH. All fourEETs (order of potency: 8,9-EET >14,15-EET approximately5,6-EET >11,12-EET) and all 4 dihydroxy derivatives(14,15-DHET approximately 8,9-DHET approximately11,12-DHET >5,6-DHET) produced dose-dependentvasodilation. Endothelial removal or L-NAME blocked8,9-EET and 14,15-DHET-dependent dilations. Theeffects of apamin/ChTx were minimal. 8,9-EET and14,15-DHET induced NO production in endothelial cells.ADU (100 µg/ml in drinking water) lowered blood pres-214 The Experimental and Clinical Research Center

Epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoicacids (DHETs) stimulate the productionof nitric oxide (NO) in endothelial cellsto induce vasorelaxation. Red blood cells are arich source of EETs and may utilize this mechanismto produce vasorelaxation in the microcirculation(from the cover page of ArteriosclerThromb Vasc Biol. 2009 Jan;29(1)).sure in angiotensin II-infused hypertension, but not inL-NAME-induced hypertension. Blood pressure andEDHF responses were similar in L-NAME-treated sEH+/+ and -/- mice. The data indicate that the EDHFresponse in mice is caused by hydrogen peroxide, butnot by P450 eicosanoids. Moreover, P450 eicosanoidsare vasodilatory, largely through their ability to activateendothelial NO synthase (eNOS) and NO release.Endothelial-derived contracting factors (EDCFs)Hypertension and vascular dysfunction result in theincreased release of EDCFs, whose identity is poorlydefined. The Gollasch team tested the hypothesis thatendothelial cyclooxygenase (COX)-2 can generateEDCFs and identified the possible EDCF candidate. Theyshowed that endothelium-dependent contractionswere triggered by acetylcholine (ACh) after inhibitionof nitric oxide production and they were abolished byCOX-2 but not COX-1 inhibitors or by thromboxaneprostanoidreceptor antagonists. The cation channelblocker, 2-amino-ethoxydiphenyl borate eliminatedendothelium-dependent contractions and ACh-stimulatedrises in endothelial cell [Ca(2+)](i). RT-PCR andWestern blotting showed COX-2 expression mainly inthe endothelium. Enzyme immunoassay and high-performanceliquid chromatography-coupled mass spectrometryshowed release of prostaglandin(PG)F(2alpha) and prostacyclin (PGI(2)) increased byACh; only PGF(2alpha) caused contraction at relevantconcentrations. COX-2 expression, ACh-stimulated contractions,and vascular sensitivity to PGF(2alpha) wereaugmented in aortae from aged hamsters. Humanrenal arteries also showed thromboxane-prostanoidreceptor-mediated ACh- or PGF(2alpha)-induced contractionsand COX-2-dependent release of PGF(2alpha).The results support a critical role of COX-2 in endothelium-dependentcontractions in this species with anincreased importance during aging and, possibly, asimilar relevance in humans.Selected publicationsEssin, K, Gollasch, M, Rolle, S, Weissgerber, P, Sausbier, M, Bohn, E,Autenrieth, IB, Ruth, P, Luft, FC, Nauseef, WM, Kettritz, R. (2009) BKchannels in innate immune functions of neutrophils and macrophages.Blood. 113, 1326-1331.Hercule, HC, Schunck, WH, Gross, V, Seringer, J, Leung, FP, Weldon, SM, daCosta Goncalves, ACh, Huang, Y, Luft, FC, Gollasch, M. (2009) Interactionbetween P450 eicosanoids and nitric oxide in the control of arterial tonein mice. Arterioscler Thromb Vasc Biol. 29, 54-60.Wong, SL, Leung, FP, Lau, CW, Au, CL, Yung, LM, Yao, X, Chen, ZY, Vanhoutte,PM, Gollasch, M, Huang, Y. (2009) Cyclooxygenase-2-derived prostaglandinF2alpha mediates endothelium-dependent contractions in theaortae of hamsters with increased impact during aging. Circ Res. 104,228-35.Hegner, B, Lange, M, Kusch, A, Essin, K, Sezer, O, Schulze-Lohoff, E, Luft, FC,Gollasch, M, Dragun, D. (2009) mTOR regulates vascular smooth musclecell differentiation from human bone marrow-derived mesenchymalprogenitors. Arterioscler Thromb Vasc Biol. 29, 232-238.Mederos, Y, Schnitzler, M, Storch, U, Meibers, S, Nurwakagari, P, Breit, A,Essin, K, Gollasch, M, Gudermann, T. (2008) Gq-coupled receptors asmechanosensors mediating myogenic vasoconstriction. EMBO J. 27,3092-3103.The Experimental and Clinical Research Center 215

Technology PlatformsComputational Biology and Data MiningMathematical Cell PhysiologyMass SpectrometryPreparative FlowcytometryElectron MicroscopyTransgenics

Miguel AndradeStructure of the GroupGroup LeaderDr. Miguel AndradeScientistsDr. Enrique MuroDr. Jean-Fred FontaineDr. Adriano BarbosaDr. Nancy MahGraduate StudentMartin SchaeferTechnical AssistantMatthew HuskaSecretariatSylvia OlbrichTimkehet Teffera**part of the period reportedComputational Biology and Data MiningOur group focuses on the development and application of computational methods that areused to research the molecular and genetic components of human disease. Often, we workwith data from high throughput gene expression, proteomics, and protein-protein interactionexperiments performed at a genomic level. Our primary research tools, besides hardware andsoftware, are the increasing collection of public repositories of biological information such asmolecular sequence and structure databases, literature databases like MEDLINE, and otherresources related to human disease such as the OMIM database. The results of our work aredistributed as software or online web services.Prediction of transcript 3’UTR endsMethodologies for the prediction of gene transcriptsfrom genomic and expression data are still under development,and the increase in the amount of transcriptdata in public databases offers a chance to improvesuch methods. We observed that the databases ofexpressed sequence tags (ESTs) contain abundant evidenceof alternative 3’UTR ends that are currentlyabsent from the public database records for manygenes, or invalidate many transcript ends included indatabases like RefSeq or Ensembl. We proposed andexperimentally verified a method to predict transcriptends using EST data and analysis of poly-adenylationsignals (Muro et al., 2008). The results of the analysis ofthe complete human and murine genomes are availableas data tables and through the TranscriptomeSailor web tool [http://www.ogic.ca/ts/], which allowsexamination of particular genomic regions for predictionsand evidence.Analysis of gene expression instem cell differentiationGene expression repositories generated for a particularobjective within a single laboratory overcome some ofthe problems that pervade such data such as platformheterogeneity and poor sample quality. The Stem CellGenomics Project (http://www.ottawagenomecenter.ca/projects/stemcellgenomics) used this approach toproduce a resource of gene expression experimentsthat follow stem cell differentiation. In this context, weproduced a database of stem cell microarray data[StemBase; http://www.stembase.ca/] describing geneexpression (from cDNA microarrays and experiments ofserial analysis of gene expression) in more than 200samples of stem cells and their derivatives in mouseand human (Sandie et al., 2009). We illustrated how touse these data to study stem cell biology at three levels:cells, genetic networks, and particular genes. The databaseprovides a variety of querying mechanisms for differenttasks including the detection of gene markers[http://www.ogic.ca/projects/markerserver/] and theuse of the UCSC Genome Browser to display the data ingenomic context.Identification of protein repeatsSince 1995, when we found the first homology of theHuntington’s disease protein (huntingtin) to other proteinsequence to be a repeat of around 40 amino acids(HEAT repeat; Andrade and Bork, 1995), few advanceshave been made in the characterization of theserepeats in huntingtin or in other proteins. In the case ofhuntingtin, this enormously hampers the elucidation ofits normal function and of the mechanisms that triggerHuntington’s chorea. To approach this problem, wedeveloped a neural network [ARD; http://www.ogic.ca/218 Technology Platforms

projects/ard/] to detect repeats like HEAT, Armadillo,and PBS, that form similar structures composed ofalpha-helices (which we termed alpha-rods) (Palidworet al., 2009). This method allowed us to detect novelinstances of this structure, for example in human proteinsSTAG1-3, SERAC1, and PSMD1-2 & 5. Application ofthe method to human huntingtin and comparison toorthologs allowed us to delimit three alpha-rods inhuntingtin (See Figure 1) whose intra-molecular interactionswe characterized experimentally using yeasttwo hybrid and co-immunoprecipitation of proteinfragments encoding the domains.Protein domain analysisSome of our work focuses on the search for particularprotein functions using protein sequence comparisonand other bioinformatics tools. A recent example is oursearch for proteins regulating mitochondrial morphologywhere we did a computational screen for proteinsequences predicted as mitochondrial and containingRING domains (characteristic of ubiquitinatingenzymes). We experimentally characterized one ofthem, which we named MAPL (mitochondrial anchoredprotein ligase), which is located in the outer membraneof the mitochondria. Observation of MAPL-YFP led tothe first observation of mitochondrially derived vesicles,which fuse with peroxisomes. The protein containsa domain that we named BAM (Besides a Membrane)(Andrade-Navarro et al., 2009), which presents a complicatedtaxonomic distribution, being scattered inarchaea and bacteria, and present in most eukarya (butnot in fungi). We deduce that this domain has beengenerated along the eukaryotic lineage and has beenhorizontally transferred multiple times to and betweenprokaryotic lineages. We hypothesize that it must havea function that confers prokaryotes with a selectiveadvantage without being crucial.Data and text miningDatabases of molecular sequences are a very usefulsource of functional information for genes and proteinsbut nevertheless they are often incomplete or not up todate. This motivates us and many others working in thefield of computational biology to develop tools that“mine” resources that contain textual descriptions ofresearch results, chiefly, the millions of abstracts referringto the biomedical literature deposited in the MED-LINE database. In this respect, we recently developed ascoring system to rank all abstracts in MEDLINE accordingto a training set that can consist of ten of thousandsof abstracts (Fontaine et al., 2009); this is usefulUsing a computational method on a sequence alignment of huntingtinwith its orthologs (output in the background) we predicted thatthe human huntingtin contains three elongated domains (alpha-rods,yellow cylinders), which are involved in intra-molecular (left) and intermolecular(right) interactions, liberally represented in this cartoon.when a researcher has a small dataset of references ofinterest and wants to find “more of the same”.Algorithms such as neural networks or support vectormachines are too computationally intensive for thistask so we opted for a simpler solution that could bedefined as text indexing. We implemented andevaluated this method using a linear naïve Bayesianclassifier in a web server [MedlineRanker; http://cbdm.mdc-berlin.de/~medlineranker/]. This tool usesas input a set of MEDLINE abstracts and optionally abackground to compare to, and outputs discriminatewords and scored abstracts.Selected PublicationsAndrade-Navarro, MA, Sanchez-Pulido, L , McBride, HM. (2009).Mitochondrial vesicles: an ancient process providing new links toperoxisomes. Current Opinion in Cell Biology. 21:560-567.Jean-Fred Fontaine, Adriano Barbosa-Silva, Martin Schaefer, Matthew R.Huska, Enrique M. Muro, and Miguel A. Andrade-Navarro (2009).MedlineRanker: flexible ranking of biomedical literature. Nucl. Acids Res.2009 37: W141-W146.Palidwor, GA, Shcherbinin, S, Huska, MR, Rasko, T, Stelzl, U, Arumughan, A,Foulle, R, Porras, P, Sanchez-Pulido, L, Wanker, EE, Andrade-Navarro, MA.(2009). Detection of alpha-rod repeats using a neural network andapplication to huntingtin. PLoS Comp. Biol. 5, e1000304.Sandie, R, Palidwor, GA, Huska, MR, Porter, CJ, Krzyzanowski, PM, Muro, EM,Perez-Iratxeta, C, Andrade-Navarro, MA. (2009). Recent developments inStemBase: a tool to study gene expression in human and murine stemcells. BMC Research Notes. 2, 39.Muro, E.M., R. Herrington, S. Janmohamed, C. Frelin, M.A. Andrade-Navarro*, and N.N. Iscove*. (2008). Targeting probes to gene 3'-ends byautomated EST cluster analysis. Proc. Natl. Acad. Sci. 150:20286-20290.*Co-senior authors.Technology Platforms 219

Martin FalckeStructure of the GroupGroup LeaderPD Dr. Martin FalckeScientistsDr. Mihaela EnculescuStart of group: March 2009Graduate StudentsKevin ThurleyR. Bianca SprincenatuThomas SchendelJuliane ZimmermannDiploma StudentMichael FaberMathematical Cell PhysiologyThe core facility group „Mathematical Cell Physiology“ develops mathematical models ofcellular processes. Current projects comprise second messenger signaling systems (cAMP,Ca 2+ ), membrane potential dynamics, pH-regulation, cardiac myocyte cell models, actin dynamicsand cell motility. The group’s service to experimental groups is to develop quantitativeformulations of their hypotheses in form of mathematical models. We provide that service on avariety of levels from assistance in usage of modeling tools to complete model formulation andtesting. The group works currently with the labs of T. Jentsch’s, I. Morano’s and H. Kettenmann’s.We also conduct independent research. Projects on IP3-induced Ca 2+ -signaling, cAMP-signalingand actin dynamics were the focus in 2008/2009. All studies reported here were closecollaborations with experimental groups.In many cell types, the inositol trisphosphate receptorIP 3 R is one of the important components controllingintracellular calcium dynamics, and an understandingof this receptor is necessary for an understanding of thecontrol of gene expression, secretion, muscle contractionand many other processes controlled by calcium.IP 3 -induced Ca 2+ -signaling comprises the structural levelschannel molecule IP 3 R, channel cluster and clusterarray on cell level. We investigated behavior on all 3 levels.Based on single-channel data from the type-1 inositoltrisphosphate receptor, we showed that the mostcomplex time-dependent model that can be unambiguouslydetermined from steady-state data is onewith three closed states and one open state (1). Becausethe transitions between these states are complex functionsof calcium concentration, each model state mustcorrespond to a group of physical states. We found thatthe main effect of [Ca 2+ ] is to modulate the probabilitythat the receptor is in a state that is able to open, ratherthan to modulate the transition rate to the open state.Another study to which we provided the simulationsdeals with the cluster level (2). It showed that low concentrationsof IP 3 cause IP 3 Rs to aggregate rapidly andreversibly into small clusters of about four closely associatedIP 3 Rs. At resting cytosolic [Ca 2+ ], clustered IP 3 Rsopen independently, but with lower open probability,shorter open time, and less IP 3 sensitivity than loneIP 3 Rs. Increasing cytosolic [Ca 2+ ] reverses the inhibitioncaused by clustering, IP 3 R gating becomes coupled, andthe duration of multiple openings is prolonged.Clustering both exposes IP 3 Rs to local Ca 2+ rises andincreases the effects of Ca 2+ . Dynamic regulation of clusteringby IP 3 retunes IP 3 R sensitivity to IP 3 and Ca 2+ , facilitatinghierarchical recruitment of the elementaryevents that underlie all IP 3 -evoked Ca 2+ signals.Two studies deal with the cellular level. Our group hadpredicted in 2003/2004 on theoretical grounds, thatCa 2+ spiking exhibits a random sequence of interspikeintervals instead of a regular oscillation and that theaverage length of interspike intervals and their standarddeviation will sensitively depend on the cytosolicCa 2+ buffering capacity. That was verified experimentallyfor spontaneous spiking in astrocytes, microglia andPLA cells and also for stimulated spiking in HEK cells (5).The effect of Ca 2+ buffers on individual spikes was investigatedin a pure simulation study in collaboration withI.Parker’s experimental group (4). It provided new explanationsfor previously measured time courses of fluorescencesignals upon photo-uncaging of IP 3 . There is alow cluster density regime and a high cluster density220 Technology Platforms

Simulation of line-scan images of Ca 2+ release upon a step increase ofIP3 in Xenopus oocytes. Fluorescence increases with the cytosolic free[Ca 2+ ]. The color indicates low fluorescence with blue and high fluorescencewith red. The control simulation uses 40 µM fluorescent dye asthe only exogenous Ca 2+ buffer and the other panels additionally theindicated amount of slow EGTA or fast BAPTA. We see the typicalshortening of the fluorescence signal by competition between EGTAand the dye and the prolongation of release by BAPTA.regime. In the high density regime, buffers with slowbinding rates like EGTA and a K D in the order of magnitudeof cytosolic Ca 2+ resting levels shape the timecourse of fluorescence signals by buffer competition butdo not shape release at IP 3 R clusters. Buffers with fastbinding rates shape release by delaying Ca 2+ -dependentinhibition of IP 3 Rs. That causes larger spikes of the numberof open channels and prolongs release. The low densityregime comprises the effect on spike sequencesdescribed above and termination of repetitive spiking bydecoupling of clusters by large [EGTA] or [BAPTA].Our modeling of actin based cell motility aims at suggestingmechanisms which explain the velocitydynamics observed with bacterial propulsion, the morphodynamictypes of protrusions observed at the leadingedge of a variety of motile cells and to link the typesto the state of signaling pathways. The similarity of themolecular components involved in both groups ofprocesses suggests it to be possible to describe bothbacterial propulsion and the dynamics of protrusion bymodels which are distinguished essentially only byparameter values but consist otherwise of very similarequations. We were able to derive such a model from ahypothesis on the relevant processes and to demonstratethe existence of the observed dynamic regimeslike steady and oscillatory movement. The research ofthe group will now establish the link between cell signalingand morphodynamic types.We are also developing a model of cardiac myocytes. Itmodels individual L-type and ryanodine receptor channelswith stochastic state transitions and will still beable to simulate cellular dynamics because of the useof multiscale techniques. The goal of this modelingeffort is to simulate excitation contraction couplingwith realistic intracellular Ca 2+ and membrane potentialdynamics. The model will serve to investigate conditionsand dynamics of Ca 2+ alternans and membranepotential alternans in first studies. It will then beadapted and enhanced according to the needs ofexperimental research at the MDC.Selected PublicationsGin, E, Falcke, M, Wagner II, LE, Yule, DI, Sneyd, J. (2009). A Kinetic Modelof the Inositol Trisphosphate Receptor Based on Single-Channel Data.Biophys.J. 96, 4053-4062.Taufiq-Ur-Rahman, Skupin, A, Falcke, M, Taylor, CW. (2009). Clustering ofInsP3 receptors by InsP3 retunes their regulation by InsP3 and Ca 2+ .Nature 458, 655-659.Skupin, A, Kettenmann, H, Winkler, U, Wartenberg, M, Sauer, H, Tovey, S,Taylor, CW, Falcke, M. (2008). How does intracellular Ca 2+ oscillate: bychance or by the clock?. Biophys. J. 94, 2404-2411.Zeller, S, Rüdiger, S, Engel, H, Sneyd, J, Warnecke, G, Parker, I, Falcke, M.Modeling of the modulation by buffers of Ca2+ release through clustersof IP3 receptors. Biophys. J., in pressEnculescu, M, Gholami, A, Falcke, M. (2008). Dynamic regimes andbifurcations in a model of actin based motility. Physical Review E 78,031915Technology Platforms 221

Structure of the GroupGroup LeaderDr. Gunnar DittmarGraduate StudentGünther KahlertTechnical AssistantsMijo StanicStart of group: January 2008Gunnar DittmarMass SpectrometryCellular signalingCells interact with their environment and react to differentstimuli and changes in the environment. Thesereactions can either be based on transcriptional activityor chemical modifications of existing proteins, so calledpost-translational modifications. In order to gaininsight into the regulation of these protein-interactionnetworks it is necessary to identify proteins, their modificationsand to measure the protein expression levelswithin a cell. This requires the identification of severalhundreds or thousands proteins and their quantificationin a complex mixture. In addition the informationhas to be collected in a time efficient way. All theserequirements are matched by modern mass spectrometryand result in the methods rise to be the defaultmethod for large-scale protein identification in life sciences.The core facility mass spectrometry offers a wide rangeof mass spectrometry methods for the identificationand quantification of proteins and peptides. Besides theidentification of proteins in gel slices, the mass spectrometrycore facility uses a number of proteomic techniquesin different collaborations with groups at theMDC. We are working closely with these groups to optimizethe methods for the different projects.Targeted proteomicsMonitoring the regulation of proteins under differentconditions can provide deeper insight in the regulatorynetwork, which underlies a signal transduction cascade.For many cascades the major players in thesepathways are already identified. Incorporating thisknowledge into a targeted strategy allows focusing tomonitor changes in the concentration of these players,avoiding the sequencing of unrelated, not regulatedproteins in the cell. A method that allows the selectionof a limited number of proteins is multiple reactionmonitoring (MRM). The technique has the advantage ofa high sensitivity combined with short run times on theliquid chromatography systems. This opens the possibilityof measuring large quantities of different samplesin a short time period and quantifying all componentsof the cascade.Several collaborations of the core facility within theMDC are now based on multiple reaction monitoringexperiments. The core facility has access to two differentmass-spectrometers capable for this type of experiments(Q-Trap 5500 and Q-Trap 4000).Non-targeted proteomic approachesMetabolic labeling of cells offers great advantages forthe quantification of proteins. The cells of interest aretherefore cultured in media, which contain isotopiclabeled (non radioactive) amino acids. The additionalmass of the amino acids can later be detected by massspectrometry. Comparison of two different samples ispossible by analyzing the sample on a high accuracymass spectrometer (LTQ-Orbitrap). The results of thesemeasurements are relative ratios for each proteindetected in the two different samples. This techniquesis currently used by the core facility for the quantificationof immuno-precipitations and large scale proteinidentifications.For samples, which cannot be cultured in media containingheavy amino acids, another technique is available.This technique relies on a highly reproducible liquidchromatography, since two different chromatographicruns have to match. Measurements of nonlabeledsamples are carried out on a Q-TOF premier as aMS E experiment.Identification of post-translational modificationsBesides the translational regulation of proteins, anotherlayer of regulation exists, which is mediated by post-222 Technology Platforms

Development of an MRM method for quantification of peptides.Starting from the initial MS/MS spectrum of the peptide of interestthe MRM-transitions are constructed. Elution profile of different peptidesdeducted from MRM measurements, which lead to the quantificationof the peptide of interest.translational modifications. For the understanding ofthe molecular mechanisms, which regulate these proteins,it is necessary to gain insight into the differentpost-translational modifications of proteins. The corefacility actively pursues the development of new methodsfor the identification of modification sites by ubiquitin-likeproteins. For the identification of phosphorylationsites the core facility now provides specializedmethods, e.g. phospho-ion scan with polarity switchingfor improved sensitivity.Selected PulicationsDittmar, G. and Selbach, M. Novel insights into proteomic technologiesand their clinical perspective. Genome Med (2009) vol. 1 (5) pp. 53.Guenther, U.-P., Handoko, L., Varon, R., Stephani, U., Chang-Yong, T.,Mendell, J., Lützkendorf, S., Hübner, C., von Au, K., Jablonka, S., Dittmar, G.,Heinemann, U., Schütz, A. and Schülke, M. Clinical variability in distalspinal muscular atrophy type 1 (DSMA1): determination of steady-stateIGHMBP2 protein levels in five patients with infantile and juveniledisease. J Mol Med (2009) vol. 87 (1) pp. 31-41.Schwanhäusser, B., Gossen, M., Dittmar, G. and Selbach, M. Globalanalysis of cellular protein translation by pulsed SILAC. Proteomics(2009) vol. 9 (1) pp. 205-9.Technology Platforms 223

Structure of the GroupGroup LeaderDr. Anje SporbertStart of group: March 2008Anje SporbertConfocal and 2-Photon MicroscopyIn March 2008, a microscopy core facility was established at the MDC focusing on confocal and2-Photon fluorescence microscopy. Starting with two confocal laser scanning microscopes inone room the facility provides now an instrumentation of seven different microscopes eachoptimised for specific applications in three rooms: four confocal laser scanning microscopes withdifferent configurations (one equipped with an 2-Photon laser), one wide-field fluorescencemicroscope with two lasers for TIRF-Illumination, one multifocal-2-Photon laser scanningmicroscope and one wide-field fluorescence microscope with UV-laser for laser-assisted microdissectionand catapulting. All microscopes are centrally located in rooms equipped with asterile bench, a CO 2 -cell incubator, a small lab bench and gas supplies for the microscope stageincubators offering an ideal environment also for live specimen experiments.The aim of the Microscopy Core Facility (MCF) is to provide researchers at the MDC a choice ofhigh-end fluorescence microscopes and the necessary support to enable them to performadvanced imaging experiments with different specimens ranging from fixed cells and tissuesections to live cells and organisms.Potential applicationsIn confocal microscopy lasers are scanned line-wiseover the sample to excite the fluorophores in the specimen.The emitted fluorescence is detected with a digitaldevice via a pinhole in front of it. This pinholerestricts the detected fluorescence to light originatingonly from the focal plane.The benefits of this techniqueare (i) a better separation of spectrally overlapping fluorophoresas often used in multi-colour fluorescencelabellings in many immuno-fluorescence techniquesand (ii) a higher axial resolution by the optical z- sectioningeffect which is especially prominent in thickerspecimen.The confocal microscopes in the MCF are equipped withdifferent sets of lasers and objectives to offer optimalconditions for multi-colour fluorescence imaging ofstructures ranging from the subcellular level to themorphology of small organisms (see Figure A/B).Stage incubators on three of the four confocal microscopesallow for live specimen imaging by controllingthe temperature and CO 2 -environment. This is a prerequisitefor short and long time-lapse experiments toobserve, i.e. the development of zebrafish/mouseembryos (Figure B), the migration of cells, the movementof organelles (Figure C) or the different stages ofthe cell cycle.The possibility to direct the laser beam to a definedposition and illuminate only this area in the sampletogether with the development of fluorescent proteinswith new properties opens up a range of exciting experimentaltools for live specimen at the confocal microscopes:a) Photobleaching a specified area in the specimenand observing how fast the fluorescence recoversinto that area (“FRAP”) gives indications on the224 Technology Platforms

mobility or turnover of the underlying biologicalstructures.b) Photoactivation or photoswitching of fluorescentproteins (such as paGFP, Dendra, EosFP) in a definedarea of the specimen allows for the highlighting of asubset of cells or a fraction of the protein of interestand to follow its migration or movement over time(see Figure C).In 2-Photon microscopy a pulsed IR-laser is used toexcite the fluorophore at about half the normal wavelength(2-Photon effect). This IR light can penetratedeeper into biological samples and is less harmful for it.Therefore, 2-Photon microscopy is especially beneficialfor imaging of thicker specimen as mouse embryos,zebrafish embryos, brain sections, tissue preparations.The fixed stage setup at the multifocal-2-Photon microscopeallows even for the intravital imaging of, i.e. traffickingcells of the lymphatic or vascular system or themigration of cells into certain tissues.TIRF-(Total Internal Reflection Fluorescence) Microscopyis an optical technique for the selective imaging of cellmembranes or structures in its vicinity in live cells sinceonly fluorophores close to the coverslip (penetrationdepth of 100-700nm) are excited. TIRF cannot imagedeep into a specimen. Therefore, it is an ideal techniqueto observe dynamic processes associated with the cellmembrane or the cell surface, i.e. the release and transportof vesicles, cell adhesion molecules and membraneproteins, endocytic and exocytic processes, receptor-ligandinteractions.The Laser microdissection microscope uses the energyof a pulsed UV-laser to cut out selected cells or tissueareas and lifting them up (catapulting) to capture it ina collection device. Parts of chromosomes, individuallive or fixed cells, parts of tissue sections or small organismscan be isolated with this technique and processedfurther, i.e. by recultivating isolated cells/ cell clusters,extracting DNA or RNA. The MCF also offers guidance tofind the optimal conditions for the intended experiments(i.e., selection of optimal fluorophores, correctspecimen preparation) and help with the followingimage processing and image analysis.From subcellular structures to the morphology of small organisms,from the localisation of proteins to the mobility and function ofproteins.A: F-actin staining displaying the structure of M-band and Z-disk infixed rat cardiomyocytes.B: cross section of stained zebrafish embryo trunk (left, courtesy ofC. Otten/S. Seyfried) and whole mount mouse embryo (right,courtesy of F. Spagnoli).C: live CHO cell expressing APP-paGFP after perinuclearphotoactivation showing APP vesicle trafficking.Selected Publications*Görisch SM, *Sporbert A, Stear JH, Grunewald I, Nowak D, Warbrick E,Leonhardt H, Cardoso MC (2008). Uncoupling the replicationmachinery: replication fork progression in the absence of processiveDNA synthesis. Cell Cycle.7,1983-90. * equal contributionSchmidt V, Sporbert A, Rohe M, Reimer T, Rehm A, Andersen OM,Willnow TE. (2007) SorLA/LR11 regulates processing of amyloidprecursor protein via interaction with adaptors GGA and PACS-1. J BiolChem., 282, 32956-64.Sporbert A, Domaing P, Leonhardt H, Cardoso MC (2005). PCNA acts as astationary loading platform for transiently interacting Okazakifragment maturation proteins. Nucleic Acids Res., 33, 3521-8.Sporbert A, Gahl A, Ankerhold R, Leonhardt H, Cardoso MC (2002). DNApolymerase clamp shows little turnover at established replication sitesbut sequential de novo assembly at adjacent origin clusters. Mol Cell,10, 1355-65.Technology Platforms 225

Structure of the GroupGroup LeaderHans-Peter RahnStudent AssistantJens HartwigSecretariatDaniela KeynerStart of group: January 2009Hans-Peter RahnPreparative FlowcytometryFluorescence-activated cell sorting (FACS) is a specialized type of flowcytometry. It wasdesigned in 1971 by Len Herzenberg to provide a fast physical separation method forfractionating a heterogeneous mixtures of cells into distinct subsets, based upon the lightscattering and fluorescent characteristics of different cell types.The FACS Core Facility assists researchers at the MDC with two state-of-the-art, digital highspeed“FACSAria” sorters and one analog high-speed “FACSVantage SE” sorter from BD. Thefacility has been used by more than 30 scientific groups at the MDC. Sorted cells are oftenused for microarray- or sequencing-based gene expression analysis, quantitative real-timePCR, DNA sequencing, live cell imaging or adoptive transfer experiments in animals.Recent Projects:Sophisticated FACS applications are used in theresearch group of Frank Rosenbauer, with the theme“Cancer, Stem Cells, and Transcription Factors”. Theinterest of this group is to integrate hematopoitic stemcell characteristics with underlying genetic and epigeneticregulatory mechanisms. To identify specific differentiationstates of hematopoetic and leukemic cells, thegroup developed new staining methods using up tonine colors. Routinely, seven color sorts of mouse bonemarrow cells are used to analyse hematopoetic cells invivo and in vitro.The “Molecular Tumor Genetics and Immunogenetics”group of Martin Lipp investigates the function of T- andB- cells during the development of acute and chronicinflammation. The group focuses on distinct memoryand effector T cell populations which are important forthe development of antibody-based adaptive immuneresponses. In this connection central memory, effector/memoryund follicular B helper T cells are enrichedby FACS using specific patterns of surface markers onthese cells.The “Hematoloy, Oncology and Tumorimmunology”group of Bernd Dörken “Hematoloy, Oncology andTumorimmunology” uses FACS enrichment of tumorand primary murine hematopoetic cells in order toanalyse the function of ectopically expressed genes incomparison to the endogenous expression levels inlymphoma cells.Together with the research group of Manfred Gossen,FACS was used to create long term stable transgeniccell clones, without the influence of additional anitbioticresistance genes.To study early embryogenesis employing high-throughputgenomics as well as biochemistry assays, the“Systems Biology” lab of Nikolaus Rajewsky devised anew method to collect large amounts of preciselystaged C. elegans embryos by FACS. This method willmake a contribution towards a more complete understandingof gene regulatory networks during early C.elegans development.226 Technology Platforms

Left: Inside view of a FACSAria2 (flow cell with sort blockand deflection plates),Right: Schematic view of theseparation principleSelected publicationsBroske, A.M., Vockentanz, L., Kharazi, S., Huska, M.R., Mancini, E., Scheller,M., Kuhl, C., Enns, A., Prinz, M., Jaenisch, R., et al. (2009). DNA methylationprotects hematopoietic stem cell multipotency from myeloerythroidrestriction. Nat Genet 41, 1207-1215.Rasheed, A.U., Rahn, H.P., Sallusto, F., Lipp, M., and Muller, G. (2006).Follicular B helper T cell activity is confined to CXCR5(hi)ICOS(hi) CD4 Tcells and is independent of CD57 expression. Eur J Immunol 36, 1892-1903.Mathas, S., Kreher, S., Meaburn, K.J., Johrens, K., Lamprecht, B., Assaf, C.,Sterry, W., Kadin, M.E., Daibata, M., Joos, S., et al. (2009). Gene deregulationand spatial genome reorganization near breakpoints prior to formation oftranslocations in anaplastic large cell lymphoma. Proc Natl Acad Sci U S A106, 5831-5836.Kaufman, W.L., Kocman, I., Agrawal, V., Rahn, H.P., Besser, D., and Gossen, M.(2008). Homogeneity and persistence of transgene expression byomitting antibiotic selection in cell line isolation. Nucleic Acids Res 36,e111.Stoeckius, M., Maaskola, J., Colombo, T., Rahn, H.P., Friedlander, M.R., Li, N.,Chen, W., Piano, F., and Rajewsky, N. (2009). Large-scale sorting of C.elegans embryos reveals the dynamics of small RNA expression. NatMethods 6, 745-751.Technology Platforms 227

Structure of the GroupGroup LeaderDr. Bettina ErdmannBettina ErdmannTechnical AssistantsMarianne VannauerMargit Vogel (part-time)Electron MicroscopyMethods of ultrastructural research are increasingly being used again to characterizegenetically modified specimens. During the last years, the electron microscopy (EM)facility has established and extended a panel of approaches, protocols and the technical basisfor morphological studies for a wide range of research projects. Based on two transmissionelectron microscopes (Zeiss 910, FEI Morgagni), both equipped with high resolution CCDcameras,we offer conventional plastic embedding for phenotyping, immunogold labeling withcryo methods as well as negative staining. In this way, collaborations with more than 25research groups in the house were performed, using the major model systems like cultured cells,mouse, yeast, zebrafish and biopsie probes from clinical projects. Only some typical examplesfor EM service are mentioned here:Phenotyping/ultrastructural characterizationBy using Lumox dishes for cell growth, we were able toretain the spatial arrangement of cultured cells duringEM processing. With this approach one can easily identifydifferentiation of stem cells even at the light miscroscopicallevel (Fig. a, collaboration with K. Eckert). Athigher magnifications we could detect cellular componentslike myofibrils and desmosomes in multilayeredcell cultures of cardiomyocytes (Fig. b, collaborationswith B. Gerull and C. Özcelik). Furthermore, we found awidened endoplasmic reticulum and signs of stress inglioma cell cultures (collaboration with H.Kettenmann). Fibroblast cell lines with different oncogenesrevealed remarkable changes in the number ofautophagosomes (collaboration with C. Schmitt).A range of research projects to `Function and Dysfunctionof the Nervous System` analyze model organismswith defects in nerve fibers and myelin structure.Together with C. Birchmeier, G. Lewin and A. Garratt auseful approach was developed to determine the numberof axons in peripheral nerves and the degree ofmyelination (g-ratio). A lot of phenotypes of mice andmole rats were described in this way (Fig. d), and thereforewe recently try to establish a new software modulefor the automatical detection of myelin figures.Other types of phenotyping at the ultrastructural levelreveal details that cannot be resolved by fluorescencelabeling and confocal microscopy. We identified andcounted the number of satellite cells in the developingmuscle in different mutant mice (Fig. e, collaborationwith C. Birchmeier). In contrast to fibroblasts orendothelial cells in this tissue, these satellite cells arelocated under the basal membrane close to the musclecells.Immunocytochemical labelingThe available expertise in the use of cryosectioningtechniques according to Tokuyasu was extended andincreasingly applied for immunolabeling of tissues.Although it often remains difficult to find an acceptablecompromise between fixation, structure preservationand the retaining of antigenicity, in some casesthese methods yield excellent results even in complextissue like brain. So we could detect a reduced numberof amyloid precursor protein (APP) molecules in Golgifields in the hippocampus of Sorla-deficient mice (Fig. c,collaboration with T. Willnow). Using Lamp1 as marker,we quantified with the same method lysosomalchanges in kidneys of chloride channel knock out mice(collaboration with T. Jentsch). In peroxisomes of the228 Technology Platforms

Examples fort the use of EM methods for phenotyping and immunolabelinga. cell culture of human embryonic stem cells, line SA 002, embeddingin Epon as for EM, semithin section (1µm), light microscopy,bar = 50µma spontaneous differentiation of cells towards high prismatic,epithelial-like structures becomes visible (arrowhead)d. saphenous nerve of a mole rat, embedding in Epon, ultrathinsection, bar = 2µmthe number of myelinated and unmyelinated (arrowheads) axonscan be counted, and g-ratios can be calculated from myelin andaxon perimeters (coulored lines)b.cell culture of neonatal rat cardiomyocytes, embedding in Epon,ultrathin section, bar = 1µmcell components like growing myofibrils and cell-cell-contacts(arrowhead) are detectablee. limb muscle of a mouse embryo, embedding in Epon, ultrathinsection, bar = 500 nmidentification of satellite cells (sc) among muscle cells (mc) and othercell types is done finally by electron microscopy, as depicted by thebasal membrane covering both cell types (arrowheads)c. hippocampus of an adult mouse, ultrathin cryosection accordingto Tokuyasu, bar = 250 nmimmunolabeling of amyloid precursor protein (APP) in Golgi fields,12 nm colloidal goldheart, we firstly localized the soluble epoxide hydroxylase(sEH), an enzyme with putative importance to preventheart failure (collaboration with W.-H. Schunck).Selected publicationsWetzel, C, Hu, J, Riethmacher, D, Benckendorff, A, Harder, L, Eilers, A,Moshourab, R, Kozlenkov, A, Labuz, D, Caspani, O, Erdmann, B, Machelska,H, Heppenstall, PA, Lewin, GR. (2007). A stomatin-domain proteinessential for touch sensation in the mouse. Nature 445, 206-209.Vasyutina, E, Lenhard, DC, Wende, H, Erdmann, B, Epstein, JA, Birchmeier,C. (2007). RBP-J (Rbpsuh) is essential to maintain muscle progenitor cellsand to generate satellite cells. PNAS 104, 4443-4448Rohe, M, Carlo, A-S, Breyhan, H, Sporbert, A, Militz, D, Schmidt, V, Wozny, C,Harmeier, A, Erdmann, B, Bales, KR, Wolf, S, Kempermann, G, Paul, SM,Schmitz, D, Bayer, TA, Willnow, TE, Andersen, OM. (2008) Sortilin-relatedreceptor with A-type repeats (SORLA) affects the amyloid precursorprotein-dependent stimulation of ERK signaling and adult neurogenesis.J Biol. Chem. 283, 14826-14834König, O, Rüttiger, L, Müller, M, Zimmermann, U, Erdmann, B, Kalbacher, H,Gross, M, Knipper, M. (2008) Estrogen and the inner ear: Megalinknockout mice suffer progressive hearing loss. FASEB J. 22, 410-417Gramlich, M, Michely, B, Krohne, C, Heuser, A, Erdmann, B, Klaassen, S,Hudson, B, Magarin, M, Kirchner, F, Todiras, M, Granzier, H, Labeit, S,Thierfelder, L, Gerull, B. (2009, 47, 352-358) Stress-induced dilatedcardiomyopathy in a knock-in mouse model mimicking human titinbaseddisease. J Mol Cell Cardiol 47, 352-358Technology Platforms 229

Structure of the GroupGroup LeaderDr. Boris JerchowSecretariatChristine KrauseBoris JerchowTechnical AssistantsKatja BeckerTianwu QiaoAndrea LeschkeTransgenicsThe Transgenic Core Facility (TCF) offers expertise in all kinds of mouse Assisted ReproductiveTechnologies (ART). Starting at the planning phase, we support scientists from the MDCand their external collaborators with the design and layout of their projects. One focus of ourwork is the generation of genetically modified mouse lines. This is accomplished either bygene targeting in embryonic stem (ES) cells and subsequent generation of chimeric mice fromrecombinant ES cell clones or by microinjection of plasmid or BAC type transgenes into thepronuclei of fertilized oocytes. During the last year, we had the chance to develop a relatedtechnique together with Lajos Mates from the group of Zsuzsanna Izsvak: After coinjection of atransgene flanked by specific inverted repeats together with RNA encoding an optimizedversion of the Sleeping Beauty Transposase, SB100, into oocytes, an enzyme-mediatedintegration of the transgene takes place (refer to selected publication). Compared toconventional microinjection this technique leads to the integration of single transgenesinstead of the integration of an uncontrollable number of copies as a concatamer and yields ahigher rate of transgenic founders. The size of the transgene is a limitation of this technique,though, and only plasmid type sized transgenes can be integrated into the genome.A second focus of our work is the conservation of preciousmouse lines and the rederivation of conservedlines. This service has been continuously expanded duringthe last years both in quantity and in terms of thedifferent protocols that have been optimized in the lab.We can now offer the long term cryopreservation ofpre-implantation embryos as well as sperm in liquidnitrogen. Due to the growing number of mouse modelsworldwide, we see an increase in the number of organizations,who offer to send frozen material instead oflive mice. We therefore see an increased demand forrevitalization of lines conserved by third parties followinga variety of protocols. In this context we were successfulto establish a new protocol developed by theJackson Laboratories that make even recalcitrant lineslike C57BL/6 amenable for in vitro fertilization. In addition,we have a laser device at our disposal that can beused to penetrate the outer zona pellucida of the oocyteto facilitate sperm entry and thereby achieve fertilizationeven in cases, where this does not spontaneouslyhappen.In addition to the above, we have just taken on the rederivationof hygienically compromised lines by embryotransfer. Rederivation by hysterectomy is therefore discontinued.There are individual projects that do not fit into any ofthe above that can be supported by the TCF and we willhelp whenever it comes to the production, isolation,manipulation, culture or retransfer of pre-implantationembryos. Moreover, we can give advice on cloning andtargeting strategies, BAC preparation, ES cell culture, EScell strain background, coat color genetics, ES cell derivation,and more.Besides ongoing projects for our clients, the currentcenter of our work is the optimization of ES cell deriva-230 Technology Platforms

Oviducts lined up in pre-implantation embryo culture medium before zygote collection.tion and culture conditions and the work with C57BL/6derived ES cell lines, namely the generation of coatcolor chimeras with these lines. Furthermore, we aim atimplicating additional steps in the quality control ofmaterials received before employing them in the generationof genetically modified subjects. By thesemeans we hope to achieve a refinement that will notonly improve our service but also reduce cost and at thesame time reduce the number of animals we need tocomplete a project.Selected publicationMátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, GrzelaDP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C,Pryputniewicz D, Miskey C, Fletcher B, Vandendriessche T, Ivics Z, Izsvák Z(2009). Molecular evolution of a novel hyperactive Sleeping Beautytransposase enables robust stable gene transfer in vertebrates. NatureGenetics 41, 753-761.Record 2008:· 18 lines conserved· 32 lines from recombinant ES cells· 12 transgenic lines· 1 recombinant ES cell line· 1 conserved line (third party) rederivedTechnology Platforms 231

AcademicsAkademische Aktivitäten

Academic Appointments 2008-2009Berufungen 2008-2009Appointments at the MDC/Joint AppointmentsThe MDC has established an official cooperation agreementwith the Humboldt University Berlin (HUB) and theFree University of Berlin (HUB) which permits jointappointments. Many of the scientists appointed to theMDC are interested in a joint appointment with one ofthe universities of Berlin. Through this academic link,they wish to participate actively in teaching as well asensure access to the Berlin universities for their Mastersand PhD students. The MDC and the Berlin universities,likewise, open up the possibility to their employees to dodoctoral studies and to qualify as lecturers and professorsin the corresponding Faculties.Since 2002, when the 5 th Amending Act of the FrameworkLaw Governing Universities (Hochschulrahmengesetz)went into effect, the MDC has been able to appoint juniorprofessors jointly with the Berlin Universities. Inrespect to the collaboration between the MDC and theHUB in 1994, a Supplementary Agreement was concludedin 2002 which allows for the appointment of juniorprofessors similar to the guidelines for joint appointmentsto conventional professorships. Likewise, the MDCsigned a Supplementary Agreement with the FUB inDecember 2002.In June 2003, the Charité – Universitätsmedizin Berlinbecame the common medical faculty of both the FUBand HUB and, since then, most of the appointments atthe MDC have been done in cooperation with the Charité,reflecting the close connection between the twoestablishments. Currently, the following joint MDC-Charité positions exist at the MDC: ten W3/C4 Professors,three W2/C3-Professors, and two Junior Professors.In addition, together with the FUB, the MDC has appointedtwo W3/C4-Professors and, with the HUB two W3/C4-Professors. Furthermore, 12 appointed scientists (W3/C4und W2/C3) from the Charité and one scientist (W3) fromthe HUB are simultaneously group leaders at the MDC.2008MDC Research Group Leader Dr. M. Cristina Cardoso hasaccepted an appointment to become a W3 professor ofmolecular cell biology in the biology department of theTechnical University Darmstadt. There the Lisbon-borncell biologist will intensify her research on the regulation,Berufungen an das MDC/GemeinsameBerufungenDas MDC hat mit der Humboldt-Universität zu Berlin(HUB) und der Freien Universität Berlin (FUB) Kooperationsverträgeabgeschlossen, die gemeinsame Berufungenerlauben. Viele der an das MDC berufenen Wissenschaftlerinnenund Wissenschaftler sind an einer gemeinsamenBerufung mit einer der Berliner Universitäten interessiert.Sie möchten durch diese akademische Anbindung aktiv ander Lehre teilnehmen, den Zugang für ihre Diplomanden/innenbzw. Doktoranden/innen zu den Berliner Universitätensichern und ihren Mitarbeiterinnen und Mitarbeiterndie Möglichkeit eröffnen, sich an den entsprechendenFakultäten zu promovieren und zu habilitieren.Seit 2002 kann das MDC, auf Grundlage des 5. Änderungsgesetzeszum Hochschulrahmengesetz, gemeinsam mitBerliner Universitäten Junior Professoren berufen. Auf derGrundlage der Vereinbarung über die Zusammenarbeitzwischen dem MDC und der HUB zu Berlin aus dem Jahre1994 wurde 2002 eine Ergänzungsvereinbarung abgeschlossen,die die Ernennung von Junior-Professoren analogder Leitsätze für gemeinsame Berufungen auf konventionelleProfessuren ermöglicht. Das MDC hat im Dezember2002 in gleicher Weise mit der FUB eine Ergänzungsvereinbarungbeschlossen.Seit der Gründung der Charité – UniversitätsmedizinBerlin als gemeinsame Medizinische Fakultät derFUB und der HUB im Juni 2003 erfolgen aufgrund derengen Kooperation von MDC und Charité die meistenBerufungen als gemeinsame Berufungen mit der Charité.Zur Zeit arbeiten am MDC insgesamt zehn gemeinsam mitder Charité berufene W3/C4-Professoren, drei W2/C3-Professorenund zwei Juniorprofessoren, zwei gemeinsam mitder FUB berufene W3/C4-Professoren und zwei gemeinsammit der HUB berufene W3/C4-Professoren und ein W2-Professor. Darüber hinaus sind 12 berufene Wissenschaftler(W3/C4 und W2/C3) der Charité und ein berufenerWissenschaftler (W3) der HUB gleichzeitig Forschungsgruppenleiteram MDC.2008MDC-Forschungsgruppenleiterin Dr. M. Cristina Cardosohat einen Ruf auf eine W3-Professur für Molekulare Zellbiologieim Fachbereich Biologie der Technischen UniversitätDarmstadt angenommen. Dort wird die aus Lissabonstammende Zellbiologin ihre Forschung über die Regulati-234 Academics

eplication and reprogramming of the epigenome, whichis of special significance for cancer research. In 1995 Dr.Cardoso came from Harvard Medical School (Boston,USA) to Berlin-Buch as research group leader at the FranzVolhard Clinic of the Charité; in 1997 she was appointedresearch group leader at the MDC.The biophysicist Dr. Jing Hu (MDC) has accepted the positionof junior group leader at the Werner Reichardt Centrefor Integrative Neuroscience in Tübingen. There shecontinues to work on the molecular and cellular mechanismsof mechanosensation and pain. Dr. Hu came to theMDC in 2001 as Alexander von Humboldt Research Fellowin the research group of Professor Gary Lewin, after completingher PhD at the Institute of Biophysics of the ChineseAcademy of Sciences in Beijing. 2008 she received aMax Delbrück Scholarship (formerly named HelmholtzScholarship) of the MDC for her achievements in the fieldof pain research.The director of the Franz Volhard Center for CardiovascularDiseases and Nephrology of the Charité, CampusBerlin, Professor Jens Jordan, was appointed directorof the Department of Clinical Pharmacology at the HannoverMedical School in April 2008. Professor Jordan wasa Helmholtz fellow at MDC before he accepted the newposition at the Volhard Clinic in 2002. Since that year heis also extraordinary member of the Drug Commission ofGerman Physicians.Professor Helmut Kettenmann (MDC) is the new presidentof the Federation of the European NeurosciencesSocieties. He took over the position at the annual meetingof FENS on July 16, 2008 in Geneva (Switzerland) fromProfessor Richard Morris (University of Edinburgh, UK),who had held the position since 2006. The FENS wasfounded in Berlin in 1998. It represents 28 neurosciencesocieties in Europe and has about 16,000 members.Since July 1, 2008, Dr. Olaf Rötzschke (MDC) has beenheading an independent research group at the “SingaporeImmunology Network (SIgN)”. The recently openedinstitute is the youngest spin-off of the international“Biopolis” research campus in Singapore, which focuseson basic research and different aspects of translationalresearch in the life sciences.Biochemist Dr. Björn Christian Schroeder from the Universityof California, San Francisco, USA, has beenappointed as a junior group leader at the MDC. InNovember 2008, he started his research group in Berlin-on, Replikation und Reprogrammierung des Epigenoms, dievon besonderer Bedeutung für die Krebsforschung ist, weiterführen. Dr. Cardoso kam 1995 von der Harvard MedicalSchool (Boston, USA) als Gruppenleiterin an die Franz-Volhard-Klinikder Charité, 1997 hatte sie eine Forschungsgruppeam MDC erhalten.Die Biophysikerin Dr. Jing Hu (MDC) hat den Ruf auf eineJuniorgruppenleiterstelle am Werner Reichardt Centre forIntergrative Neuroscience (CIN) in Tübingen angenommen.Sie erforscht dort die molekularen und zellulären Mechanismenvon Berührung und Schmerz. Dr. Hu war nach ihrerPromotion in Biophysik an der Chinesischen Akademie derWissenschaften in Peking 2001 als Alexander von HumboldtResearch Fellow an das MDC in die Forschungsgruppevon Prof. Gary Lewin gekommen. 2008 hatte sie einMax-Delbrück-Stipendium (vormals Helmholtz-Stipendium)des MDC erhalten.Der Leiter des Franz-Volhard-Zentrums für Herzkreislauf-Erkrankungen, Nephrologie der Charité, Campus Berlin-Buch, Prof. Jens Jordan, ist im April 2008 als Direktor derAbteilung für Klinische Pharmakologie an die MedizinischeHochschule Hannover berufen worden. Er war am MDCHelmholtz-Fellow, bevor er 2002 die Aufgabe in der Volhard-Klinikübernahm. Seit 2002 ist er zudem außerordentlichesMitglied der Arzneimittelkommission der DeutschenÄrzteschaft.Prof. Helmut Kettenmann (MDC) ist neuer Präsident derFöderation der Europäischen NeurowissenschaftlichenGesellschaften (FENS). Er übernahm das Amt auf der Jahrestagungder FENS am 16. Juli 2008 in Genf (Schweiz) vonProf. Richard Morris (Universität Edinburgh, Großbritannien),der die Position seit 2006 innehatte. Die FENS wurde1998 in Berlin gegründet. Sie vertritt 28 neurowissenschaftlicheFachgesellschaften Europas und hat rund16.000 Mitglieder.Dr. Olaf Rötzschke (MDC) leitet seit 1. Juli 2008 eine unabhängigeForschergruppe am „Singapore Immunology Network(SIgN)“. Das 2006 eröffnete Institut ist die jüngsteAusgründung des internationalen „Biopolis“ Forschungscampusin Singapur, der sich mit Grundlagenforschungund verschiedenen Aspekten der „translationalen“ Forschungauf dem Gebiet der Lebenswissenschaft befasst.Der Biochemiker Dr. Björn Christian Schroeder von der Universityof California, San Francisco, USA, ist als Nachwuchsgruppenleiteran das MDC berufen worden und hat imNovember 2008 seine Arbeit in Berlin-Buch aufgenom-Academics 235

Buch which is part of the Excellence Initiative “Neuro-Cure”. In the USA, Dr. Schroeder discovered and cloned anew family of ion channels. By studying these channels,scientists hope to get new findings about physiologicalprocesses in different organs.The administrative director of the MDC, Dr. StefanSchwartze, has been named new chancellor of the Universityof Münster. He received his certificate of appointmentin Münster on January 3, 2008 after the senate ofthe university, in an extraordinary session on December19, 2007, nominated the 41-year-old jurist for appointmentby the North Rhine-Westphalian Science Minister.He assumed his new position on February 1, 2008 for aneight-year term of office.Cell biologist Professor h. c. Thomas Sommer (MDC) hasaccepted the appointment to become full professor (W3)for cellular biochemistry of the Faculty of Mathematicsand Natural Sciences at the Humboldt University (HU)Berlin. Since 1999, Professor Sommer has been a researchgroup leader at the MDC. His area of expertise is proteindegradation. Since 2004, he has served as Vice ScientificDirector of the MDC.Diabetes researcher Dr. Francesca Spagnoli of RockefellerUniversity in New York, USA, has come as Helmholtz JuniorGroup Leaders to the MDC and the Charité. Her majorfield of interest is stem cell research, and she is currentlyinvestigating the embryonic development of beta cells inthe Langerhans’ islets of the pancreas.Diabetes researcher Dr. Matthew Poy of the Swiss FederalInstitute of Technology (ETH) Zurich, has come asHelmholtz Junior Group Leaders to the MDC and theCharité. He investigates the processes that lead to theoutbreak of diabetes. He was able to show, that microR-NAs play a crucial role in the regulation of metabolicprocesses.As of August 1, 2008 Cornelia Lanz is the new AdministrativeDirector of the MDC. She comes to her new positionfrom Lübeck University of Applied Sciences, where shewas chancellor for the last four years until July 31, 2008.At the MDC she succeeds Dr. Stefan Schwartze, who wasappointed chancellor of the University of Münster.men. Er ist mit seiner MDC-Forschungsgruppe in die BerlinerExzellenzinitiative NeuroCure eingebunden. In den USAentdeckte und klonierte er eine neue Familie von Ionenkanälen.Von der Analyse dieser Gruppe von Kanälen erhofftsich die Forschung neue Erkenntnisse über physiologischeProzesse in vielen Organen.Der administrative Vorstand des MDC, Dr. StefanSchwartze, ist am 3. Janur 2008 zum Kanzler der WestfälischenWilhelms-Universität Münster ernannt worden. DerSenat der Universität hatte den 41-jährigen Juristen am 19.Dezember 2007 in einer außerordentlichen Sitzung demnordrhein-westfälischen Wissenschaftsminister zur Ernennungvorgeschlagen. Er trat sein neues Amt zum 1. Februar2008 an. Seine Amtszeit beträgt acht Jahre. Dr. Schwartzewar am 1. August 2003 vom Bundesministerium für Bildungund Forschung (BMBF) als Verwaltungschef an dasMDC gekommen.Der Zellbiologe Prof. h. c. Thomas Sommer (MDC) hat denRuf auf die W3-Professur für das Fachgebiet „Zelluläre Biochemie“der Mathematisch-NaturwissenschaftlichenFakultät I der Humboldt-Universität zu Berlin angenommen.Prof. Sommer ist seit 1999 Forschungsgruppenleiteram MDC. Sein Spezialgebiet ist der Proteinabbau. Seit 2004ist er außerdem stellvertretender wissenschaftlicher MDC-Vorstand.Die Diabetesforscherin Dr. Francesca Spagnoli von derRockefeller Universität in New York, USA, ist als Leiterineiner Helmholtz-Nachwuchsforschergruppe an das MDCund die Charité gekommen. Dr. Spagnoli arbeitet auf demGebiet der Stammzellforschung und erforscht die Beta-Zellender Bauchspeicheldrüse.Der Diabetesforscher Dr. Matthew Poy ist von der EidgenössischenTechnischen Hochschule (ETH) Zürich, Schweiz,als Helmholtz-Nachwuchsforschergruppenleiter an dasMDC und die Charité gekommen. Dr. Matthew Poy untersuchtdie Prozesse, die zur Entstehung von Diabetes führen.Er konnte zeigen, dass kleine microRNAs, kurze Ribonukleinsäurestränge,eine entscheidende Rolle bei der Regulationvon Stoffwechselprozessen spielen.Die frühere Kanzlerin der Fachhochschule Lübeck, CorneliaLanz, ist seit 1. August 2008 neuer administrativer Vorstanddes MDC. Frau Lanz, die die vergangenen vier Jahre bis zum31. Juli 2008 in Lübeck arbeitete, tritt die Nachfolge von Dr.Stefan Schwartze an, der zum Kanzler der UniversitätMünster ernannt worden war.236 Academics

2009Physician Professor Walter Rosenthal has been appointedas Scientific Director of the MDC. The former director ofthe Leibniz-Institut für Molekulare Pharmakologie (FMP)succeeds cancer researcher Professor Walter Birchmeierwho has served as the MDC Scientific Director for thepast five years and who now focuses again to his MDCresearch group.At their annual meeting in March, 2009 the members ofthe German Society for Chrystallography (DGK) electedProfessor Udo Heinemann to be Chairman of the Board.The term of office is three years. Professor Heinemannsucceeds Professor Wolfgang Neumann from HumboldtUniversity Berlin. The DGK has more than 1000 members.In January, Dr. Wei Chen, head of the research groupApplied Bioinformatics at the Max Planck Institute (MPI)of Molecular Genetics, Berlin, has been appointed toMDC´s Berlin Institute for Medical Systems Biology(BIMSB). Dr. Chen heads the technology platform“Genomics”, which uses state-of-the-art parallelsequencing technologies. They are considerably fasterand more cost-effective than conventional sequencingequipment. With the new generation of sequencers, Dr.Chen has established a number of different methods toinvestigate the function of genesDr. Markus Landthaler from Rockefeller University in NewYork, USA, has been appointed to the Berlin Institute forMedical Systems Biology (BIMBS). He began his work asjunior research group leader at the MDC at the beginningof March. His research area is concerned with microRNAsand RNA binding proteins, which together are instrumentalin regulating protein synthesis in the cells. Thisregulation takes place in complex networks, the understandingof which can explain the mechanisms of diseasepathogenesis and development. For this purpose, Dr.Landthaler has established special RNA-protein crosslinkingexperiments which he combines with the newtechnologies in the BIMSB, deep sequencing and massspectrometry, to elucidate a system-wide under-standingof RNA-based and protein-based post-transcriptionalregulation mechanisms.Dr. Christoph Dieterich of the Max Planck Institute (MPI)of Developmental Biology, Tübingen, came to the BerlinInstitute for Medical Systems Biology (BIMSB) of theMDC in April 2009. He heads the technology platform2009Der Pharmakologe Prof. Walter Rosenthal und bisherigeDirektor des Leibniz-Instituts für Molekulare Pharmakologie(FMP) ist seit Januar 2009 neuer wissenschaftlicherVorstand des MDC. Er ist Nachfolger von KrebsforscherProf. Walter Birchmeier, der dieses Amt fünf Jahre innehatte und sich wieder verstärkt seiner Forschungsgruppeam MDC widmet.Auf ihrer Jahrestagung haben die Mitglieder der DeutschenGesellschaft für Kristallographie (DGK) am 10. März2009 Prof. Udo Heinemann zum neuen Vorstandsvorsitzendengewählt. Die Amtsperiode beträgt drei Jahre. Prof.Heinemann löst Prof. Wolfgang Neumann von der Humboldt-UniversitätBerlin ab. Die DGK hat mehr als 1 000Mitglieder.Dr. Wei Chen, Leiter der Forschungsgruppe AngewandteBioinformatik am Max-Planck-Institut (MPI) für MolekulareGenetik, Berlin, ist im Januar 2009 an das Berlin Institutefor Medical Systems Biology (BIMSB) des MDC gekommen.Dort leitet er die wissenschaftliche Technologieplattform„Genomics“, die modernste, parallel arbeitendeSequenziertechnologien einsetzt, die erheblich schnellerund kostengünstiger sind als herkömmliche Sequenzierapparate.Mit den neuen Maschinen hat Dr. Chen im BIMSBeine Reihe verschiedener Ansätze für die Erforschung derFunktion von Genen eingerichtet.Im März 2009 hat der Molekularbiologe Dr. Markus Landthalervon der Rockefeller Universität in New York, USA,seine Arbeit als Nachwuchsgruppenleiter am Berlin Institutefor Medical Systems Biology (BIMSB) des MDC aufgenommen.Arbeitsgebiet von Dr. Landthaler sind kleineRibonukleinsäuren (microRNAs) sowie RNA-bindende Proteine,die zusammen maßgeblich die Proteinsynthese inZellen regulieren. Diese Regulation spielt sich in komplexenNetzwerken ab, deren Verständnis auch die Mechanismender Entwicklung und Krankheitsentstehung erklären. Erhat hierzu spezielle RNA-Protein-Kreuzvernetzungsexperimenteetabliert, die er mit den neuen Technologien imBIMSB, der Tiefensequenzierung und der Massenspektrometrie,kombiniert. Damit will er ein systemweites Verständnisvon RNA- und Protein-basierten posttranskriptionalenRegulationsmechanismen erarbeiten.Dr. Christoph Dieterich vom Max-Planck-Institut (MPI) fürEntwicklungsbiologie, Tübingen, ist im April 2009 an dasBerlin Institute for Medical Systems Biology (BIMSB) desAcademics 237

“Bioinformatics in Quantitative Biology“. He and his colleaguesdevelop computer methods and strategies toevaluate data from gene analyses, mass spectrometryand imaging procedures. The objective is to achieve abetter understanding of biological systems.Dr. Stefan Kempa from the MPI of Molecular Plant Physiology,Potsdam, came to the Berlin Institute for MedicalSystems Biology (BIMSB) of the MDC in April 2009. Heheads the technology platform “Integrative Proteomicsand Metabolomics”. It uses the latest technologies ofmass spectrometry to investigate metabolic processesand the proteome in cell cultures, organs, and wholeorganisms.The physicist Professor Thoralf Niendorf has beenappointed to the chair for Experimental Ultra High FieldMagnetic Resonance Imaging (MRI) at the Experimentaland Clinical Research Center (ECRC) of MDC and Charité.The specialist for imaging tech-niques from RWTHAachen University became head of the MRI facility ofECRC on Campus Berlin-Buch on August 10, 2009. Theresearch facility, which has one of the world’s strongestmagnetic resonance tomographs, a 7 Tesla whole-bodyMRI scanner, was dedicated in January 2009 by FederalEducation and Research minister Annette Schavan.The neuroscientist Dr. James Poulet has been appointedto the Berlin excellence cluster and as of July 1, 2009began work there in Berlin-Mitte. Dr. Poulet, whoseresearch is being funded by the MDC, which along withother institutions is participating in NeuroCure, is studyinghow neuronal regulatory circuits control behavior.Until the summer of 2009 he was a postdoc at École PolytechniqueFédérale de Lausanne, Switzerland, where hesucceeded in recording intracellular activity between twonerve cells in the cerebral cortex of a conscious animal forthe first time.Formerly at the Humboldt University of Berlin, Dr. JanaWolf joined the MDC as a junior group leader as part ofthe Helmholtz Association’s initiative “Systems Biology”.Dr. Wolf’s group will receive additional funding throughthe BMBF’s program “Research Units in Systems Biology(FORSYS) ”. Her work focuses on mathematical modelingof cellular processes.MDC gekommen und baut dort die Technologieplattform‚Bioinformatik und Quantitative Biologie’auf. Er und seineMitarbeiter entwickeln Computermethoden und Analysestrategien,um die Daten von Genanalysen, Massenspektrometrieund bildgebenden Verfahren auswerten zu können.Ziel ist ein besseres Verständnis biologischer Systeme.Dr. Stefan Kempa vom MPI für Molekulare Pflanzenphysiologie,Potsdam, ist im April 2009 an das Berlin Institute forMedical Systems Biology (BIMSB) des MDC gekommen undbaut dort die Technologieplattform „Integrative Proteomicsand Metabolomics“ auf. Dort setzt er die neuestenTechnologien der Massenspektrometrie ein, um Stoffwechselvorgängeund das Proteom in Zellkulturen, Organen undganzen Organismen zu untersuchen.Der Physiker Prof. Thoralf Niendorf ist auf einen Lehrstuhlfür Experimentelle Ultrahochfeld-Magnetresonanz-Tomographie(MRT) an das Experimental and Clinical ResearchCenter (ECRC) des MDC und der Charité berufen worden.Der Spezialist für bildgebende Verfahren von der Rheinisch-Westfälischen Technischen Hochschule (RWTH) Aachenhat im August 2009 die Leitung der MR-Anlage des ECRCauf dem Campus Berlin-Buch übernommen. Die Forschungsanlagemit einem der weltweit stärksten Magnetresonanz-Tomographen,einem 7-Tesla Ganzkörper-MRT,war im Januar 2009 von BundesforschungsministerinAnnette Schavan eingeweiht worden.Der Neurowissenschaftler Dr. James Poulet ist an das BerlinerExzellenzcluster „NeuroCure“ berufen worden und hatdort in Berlin-Mitte Anfang Juli 2009 seine Arbeit aufgenommen.Dr. Poulet, der über das MDC finanziert wird,erforscht wie neuronale Regelkreise Verhalten kontrollieren.Er war bis Sommer 2009 Postdoktorand an der ÉcolePolytechnique Fédérale de Lausanne, Schweiz, wo es ihmals erstem gelang, die intrazellluläre Aktivität zweier Nervenzellender Großhirnrinde im wachen Tier aufzuzeichnen.Im Rahmen der Initiative Systembiologie der Helmholtz-Gemeinschaft wechselte Dr. Jana Wolf, Humboldt-Universitätzu Berlin, als Nachwuchsgruppenleiterin an das MDC.Die Nachwuchsgruppe wird zusätzlich über die BMBF Ausschreibung„Forschungseinheiten der Systembiologie-FOR-SYS“ finanziert. Ihre Arbeit konzentriert sich auf die mathematischeModellierung zellulärer Prozesse.238 Academics

AwardsPreise20082009Martin BergmannWilhelm P. Winterstein-Preis, Deutsche Herzstiftung e. V.Gudrun ErzgräberBundesverdienstkreuz, Bundesrepublik DeutschlandMartin Janz und Stephan MathasCurt-Meyer-Gedächtnispreis, BerlinerKrebsgesellschaft e. V.Friedrich LuftFranz-Volhard-Medaille, Deutsche Gesellschaft fürNephrologieRobert Tigerstedt Award, International Society ofHypertensionLehrbär, Reformstudiengang Medizin der Charite –Universitätsmedizin BerlinNikolaus Rajewsky1. Forschungspreis, Deutsche Gesellschaft fürGentherapie e. V.Jan-Erik SiemensSofja Kovalevskaja-Preis, Alexander vonHumboldt-StiftungNorbert HübnerWissenschaftspreis für „MedizinischeGrundlagenforschung“, GlaxoSmithKline StiftungFriedrich LuftRoss McIntyre Award, University of NebraskaRaluca NiesnerRahel-Hirsch-Stipendium, Charité –Universitätsmedizin BerlinKlaus RajewskyMax-Delbrück-MedailleJens ReichCarl-Friedrich-von-Weizsäcker-Preis, NationaleAkademie der Wissenschaften Leopoldina undStifterverband für die Deutsche WissenschaftFrancesca SpagnoliGrant: European Research Council (ERC)Thomas WillnowEhrendoktorwürde, Universität Aarhus, DänemarkErich Wanker, Ulrich Stelzl, Christian Hänig,Gautam Chaurasia, Matthias FutschikErwin-Schrödinger-Preis, Hermann-von-Helmholtz-Gemeinschaft Deutscher Forschungszentren e. V.(Photo: E. Fesseler/Copyright: Helmholtz-Gemeinschaft Deutscher Forschungszentren)Recipients of the Erwin Schrödinger Prize 2008Prof. Erich Wanker (MDC), Dr. Ulrich Stelzl (MPI forMolecular Genetics), Prof. Jürgen Mlynek (Presidentof the Helmholtz Association), Dipl.-Ing. ChristianHänig (MDC), Gautam Chaurasia, M.Sc., and Dr.Matthias Futschik (both HUB)(from left to right)Preisträger des Erwin Schrödinger-Preis 2008Prof. Erich Wanker (MDC), Dr. Ulrich Stelzl (MPI fürmolekulare Genetik), Prof. Jürgen Mlynek (Präsidentder Helmholtz-Gemeinschaft), Dipl.-Ing. ChristianHänig (MDC), M.Sc. Gautam Chaurasia und Dr.Matthias Futschik (beide HUB)(v.l.)Academics 239

Delbrück FellowsDelbrück-StipendiumDelbrück Fellowships at the MDC are intendedto allow promising young scientists to carryout their own independent research. DelbrückFellows have demonstrated that they are capable ofconducting high quality research.Fellows are associated with MDC host research groupsand, therefore, receive lab space, infrastructure, and aresearch budget. The host research group guaranteesthe Fellow’s independence in terms of research topic.In addition to MDC support, Fellows are expected toapply for external funding sources. Fellowships aretypically granted for between three and 5 years.Eligible are post-doctoral scientists with a strong recommendationfrom an MDC group leader. Applicationsare received by the MDC Scientific Director andreviewed by a selection committee. During the reportperiod, the MDC supported 6 Delbrück Fellows.Delbrück-Stipendien sind am MDC eingerichtet,um die frühe Unabhängigkeit junger, erfolgsversprechenderWissenschaftler zu ermöglichen. Siesind für Wissenschaftler vorgesehen, die bereits nachgewiesenhaben, dass sie hervorragende eigenständige,wissenschaftliche Arbeit leisten.Die Delbrück-Stipendiaten sollen an bestehendeArbeitsgruppen des MDC Berlin-Buch angegliedert werden,in diesem Rahmen wird ihnen Raum und Infrastruktursowie ein Sachmittelbudget zur Verfügunggestellt. Die gastgebende Forschungsgruppe garantiertStipendiaten/Stipendiatin thematische Unabhängigkeit,die selbstständige Einwerbung von Drittmittelnwird erwartet. Das Stipendium wird in der Regel für eineLaufzeit von drei bis fünf Jahren gewährt.Bewerben können sich Postdoktoranden auf Empfehlungeines Forschungsgruppenleiters aus dem MDC Berlin-Buchbeim MDC-Vorstand. Es folgt dann eine Begutachtungin den MDC Gremien. Im Berichtszeitraum hatdas MDC insgesamt 6 Stipendiaten gefördert.240 Academics

International PhD ProgramInternationales PhD-ProgrammThe MDC considers the training of new generationsof researchers as a basic prerequisite forsustainable development and international scientificsuccess. In 2003, an International PhD Programin “Molecular Cell Biology” was successfully establishedby the MDC and the Humboldt University (HU).Since that time, the MDC’s offerings to graduate studentshave significantly expanded through the establishmentof several new programs and rising numbersof applicants.In 2007 the MDC and its partners – the HU, the FreieUniversity (FU), and the FMP – were awarded a 6-yeargrant (3,6 Mio ¤) to establish the Helmholtz GraduateSchool in Molecular Cell Biology (HGS-MCB). ThisGraduate School offers a unified interdisciplinary platformfor structured PhD training at the MDC and theconsolidation of various graduate activities, such astraining, supervision, measures to assist internationalstudents, quality control, and the creation of an alumninetwork. Training in research skills is further complementedby a structured Soft Skills DevelopmentProgramme, ensuring that PhD students receive awell-rounded education that is essential for successin the modern scientific world. An annual review ofthe progress of all PhD students is carried out by PhDProject Committees that consist of 3 - 4 researchgroup leaders from different research areas. Qualitycontrol and recording of all training activities will becarried out using the system of credits, analogous tothe European Credit Transfer System, and students’achievements are summarized in the MDC PhDCertificate.In addition, close collaboration between the MDC andthe FU led to the establishment of the HelmholtzResearch School in Molecular Neurobiology(‘MolNeuro’) in 2007, funded with 1.8 Mio ¤ over 6years. The training curriculum focuses on basic andadvanced concepts of Molecular Neurobiology combinedwith a Soft Skill Development Programme thatis organized by the Helmholtz Association for doctoralstudents from the MDC and other HelmholtzResearch Schools. In addition the students organizean annual student conference called the Berlin BrainDays.Die Ausbildung junger Nachwuchswissenschaftlerist für die zukünftige Entwicklung des MDCund seinen internationalen wissenschaftlichenErfolg unverzichtbar. Aus diesem Grund richteten 2003das MDC und die Humboldt-Universität zu Berlin (HU)das Internationale PhD-Programm “Molekulare Zellbiologie”ein, das sich in den folgenden Jahren erfolgreichetablieren konnte.Seitdem hat sich das Angebot für die Doktorandendurch den Aufbau neuer Doktorandenprogrammeerweitert: 2007 erhielten das MDC zusammen mit seinenPartnern-HU, Freie Universität Berlin (FU) sowie dasLeibnitz Institut für Molekulare Pharmakologie (FMP)die Zusage die Helmholtz Graduate School „MolecularCell Biology“ zu gründen. Sie wird aus dem Impuls- undVernetzungsfonds des Präsidenten der Helmholtz-Gemeinschaft mit insgesamt 3,6 Mio Euro über 6 Jahregefördert und ist die Grundlage für eine interdisziplinäre,strukturierte Ausbildung aller Doktoranden amMDC. Das Ausbildungs-Curriculum beinhaltet nebendem gesamten Spektrum der molekularen Zellbiologieauch die Entwicklung zusätzlicher Schlüsselqualifikationen,sogenannter „Soft Skills“. Es stellt sicher, dass dieDoktoranden eine vielseitige und umfassende Ausbildungerhalten, die für den Erfolg in der modernen Wissenschaftsweltentscheidend ist.Die jährliche Leistungsbewertung aller Doktorandenwird von PhD-Projektausschüssen durchgeführt, die sichaus drei bis vier Forschungsgruppenleitern aus verschiedenenForschungsbereichen zusammensetzen. Die Qualitätskontrolleund die Dokumentation aller Ausbildungsmaßnahmenwerden entsprechend dem europäischenLeistungspunktsystem ECTS (European CreditTransfer System) durchgeführt. Nach Abschluss ihrerPromotion erhalten die Absolventen ein MDC-Promotionszertifikatmit ihren Leistungen und können in dasAlumni-Netzwerk aufgenommen werden.Die enge Zusammenarbeit zwischen MDC und FUführte 2007 zur Gründung der „Helmholtz ResearchSchool Molecular Neurobiology (HRS „MolNeuro“), diemit 1,8 Millionen Euro für sechs Jahre gefördert wird.Das Programm bietet eine fachspezifische Ausbildungim Bereich der molekularen Neurobiologie, die durchzentrale Helmholtz-Soft Skill Kurse, die mit DoktorandenAcademics 241

Yinth Andrea Bernal-Sierra, a PhD student in the Research School MolecularNeurobiology (Research group: Prof. Gary Lewin), examines cultured neuronswith a fluorescence microscope.Yinth Andrea Bernal-Sierra, eine Doktorantin der Research School MolecularNeurobiology (Forschungsgruppe: Prof. Gary Lewin), untersucht kultivierteNeuronen mit einem Fluoreszenzmikroskop.The Helmholtz Research School in TranslationalCardiovascular and Metabolic Medicine (TransCard)has been funded for 2009-2015. It fosters translationalresearch with targeted education of graduate students,scholarships, travel grants, and IT-infrastructure.PhD students in the Cardiovascular andMetabolic Disease Program are affiliated with bothbasic science and clinical research groups and activelydevelop the program by organizing seminars, retreats,and conferences. The Program offers lectures, summerschools, and e-learning and encourages interactionsnot only between students and faculty in Berlin, butalso with our international partner universities.In 2009 the Berlin Institute Medical Systems Biologyand the Center for Genomics and Systems Biology ofthe New York University started their joint PhD-Exchange- Programme. The students are working oncollaborative projects with a scientific focus on posttranscriptionalgene regulation in the elucidation ofPhoto: David Ausserhofer / Copyright: MDCanderer „Helmholtz Research Schools“ veranstaltet werden,ergänzt wird. Jährlich organisieren die Doktorandenmit anderen Doktorandenprogrammen aus demNeurobiologie Bereich in Berlin die Berlin Brain Days.Im Januar 2009 hat ein weiteres Helmholtz-Kolleg, die“Helmholtz Research School in Translational Cardiovascularand Metabolic Medicine” (TransCard), seine Arbeitaufgenommen. Es wird für die nächsten 6 Jahre mit insgesamt1,8 Millionen Euro durch den Impuls und Vernetzungsfondsder Helmholtz-Gemeinschaft gefördert. DasProgramm bietet eine fachspezifische Ausbildung durchVorlesungen, Summer Schools und E-Learning im Herz-Kreislauf-Bereich sowie der Metabolischen Medizin, diedurch zentrale Helmholtz-Soft Skill Kurse ergänzt werden.Ebenfalls 2009 startete das gemeinsame Doktorandenprogrammdes Berlin Institute Medical Systems Biology(BIMSB) und dem Zentrum für Genomics und Systembiologieder New York Universität. Doktoranden diesesProgramms bearbeiten Kooperationsprojekte zwischenWissenschaftlern des MDCs sowie der Universität NewYork. Alle Doktoranden arbeiten sowohl in Berlin alsauch in New York.242 Academics

iological systems. All the participating students splittheir time between Berlin and New York.Currently, approximately one quarter of all MDC PhDstudents are selected through a vigorous internationalrecruitment process. Around 180 PhD students areworking towards their PhD degree at the MDC and 70PhD students visit the MDC per year to work on collaborativeprojects. An annual scientific symposium (PhDRetreat) is organized by the PhD students of the MDCand FMP to promote further networking between thestudents.During the 2009 PhD student selection process, 779applications from 69 countries were reviewed. Ofthose applicants, 23 PhD students were accepted intothe different Programs. The MDC also has a FellowshipCommittee that awards scholarships to outstandingcandidates who develop and present a research project.The degree of Dr. rer. nat. (German equivalent of a PhDdegree) is either awarded by the partner universitiesor may be obtained through the student home university.Eligible are graduate students of the life sciencesholding a degree comparable to the German Diplomaor a MSc with a research thesis is typically required forapplicants.Am MDC arbeiten ständig rund 180 Doktorandinnenund Doktoranden. Etwa weitere 70 im Jahr besuchendas MDC, um an Partnerprojekten mitzuarbeiten. DieDoktoranden des MDC und des FMP organisieren jährlichein wissenschaftliches Symposium (Doktoranden-Klausurtagung) in Brandenburg sowie einen Wettbewerbfür den besten Vortrag, das beste Poster auf demCampus.Zurzeit wird etwa ein Viertel aller MDC-Doktoranden ineinem strengen internationalen Bewerbungsverfahrenausgewählt. Während des Auswahlverfahrens 2009bewarben sich 779 Kandidaten aus 69 Ländern. Aus derGruppe der Bewerber wurden insgesamt 23 Doktorandinnenund Doktoranden in die unterschiedlichen Programmeaufgenommen. Zusätzlich vergibt der Stipendienausschussam MDC in jedem Jahr Stipendien, fürdie sich Doktoranden und Post-Doktoranden mitherausragenden Forschungsprojekten bewerben können.Zur Bewerbung berechtigt sind Absolventen derLebenswissenschaften mit einem deutschen Diplomoder ein Masterabschluss (Master of Science - MSc) einschließlicheiner MSc-Forschungsarbeit. Den Doktorandenwird der Titel Dr. rer. nat. (die deutsche Entsprechungzum PhD) von einer der Partneruniversitäten verliehen.Ebenso können die Studierenden an einer ihrerHeimatuniversität promovieren.Academics 243

Congresses and Scientific MeetingsKongresse und Wissenschaftliche Tagungen200811 January Max Delbrück Lecture , Prof. Michael Botchan, University of California, Berkeley, USAOrganizer: Prof. M. Gossen, MDC18 January Neujahrsempfang des Campus Berlin-BuchOrganizer: MDC/BBB GmbH08 February Max Delbrück Lecture, Prof. Ruth Lehmann, Developmental Genetics Program, Skirball Instituteand Howard Hughes Medical Institute New York, University School of Medicine, New York, USAOrganizer: Prof. N. Rajewsky, MDC16 February HNO-Symposium des Helios KlinikumsOrganizer: Helios–Klinikum Berlin-Buch26-29 March International Conference “Invasion and Metatasis“Organizer: Prof. W. Birchmeier, MDC24-25 April LaborbaukonferenzOrganizer: R. Streckwall, MDC14-17 May International Conference „Development and Function of Somatosensation and Pain”Organizer: Prof. G. Lewin, MDC26-28 May International Symposium “Adoptive T Cell Therapy”Organizer: Prof. W. Uckert, MDC07 June 13. Bucher Nephrologisches SymposiumOrganizer: Prof. F. Luft, MDC/FVK12-14 June International Berlin Summer Meeting “Computational & Experimental Molecular Biology”Organizer: Prof. N. Rajewsky, MDC14 June Lange Nacht der WissenschaftenOrganizer: Campus Berlin-Buch17 June Max Delbrück Lecture, Prof. K. Chien, MGH Cardiovascular Research Center,Massachusetts General Hospital, Richard B. Simches Research Center, Boston, USAOrganizer: Dr. D. Besser, MDC19 - 21 June International Conference “Transposition and Animal Biotechnology”Organizer: Dr. Zs. Izsvak, MDC27 June Festveranstaltung zu Ehren der Verabschiedung von Dr. Gudrun Erzgräber, Geschäftsführerinder BBB GmbH und Verleihung des BundesverdienstkreuzesOrganizer: Campus Berlin-Buch07 -11 July Basis Gene Mapping /Linkage Analysis CourseOrganizer: Forschungsgruppe Bioinformatik, MDC mit Dr. M. Nothnagel, Uni Kiel11 July Max Delbrück Lecture, Dr. Helen H. Hobbs HHMI, UT Southwestern Medical Center Dallas, USAOrganizer: Dr. Daniel Besser, MDC08-10 October 15 th Annual Meeting of the German Society of Gene Therapy, DG-GT e.V. & 6 th AnnualWorkshop “Viral Vectors and Gene Therapy” of the Gesellschaft für Virologie, GfVOrganizer: Prof. W. Uckert, MDC mit Charité und HU Berlin29 October Symposium: Junior Group Leader Position Systems Biology am MDCOrganizer: Prof. N. Rajewsky, MDC29-30 October NMR-Symposium: “Challenging Biological Systems Systems by NMR Spectroscopy“Organizer: Prof. H. Oschkinat, FMP07 November Max Delbrück Lecture, Prof. Dr. Jonathan Weissman Howard Hughes MedicalInstitute San Francisco, USAOrganizer: Prof. Th. Sommer, MDC244 Academics

07-08 November 7. Bucher HämatologieforumOrganizer: Beckman Coulter GmbH + Prof. W.-D. Ludwig, RRK12-14 November International Symposium on Chemical BiochemistryOrganizer: Prof. Dr. H. Oschkinat, FMP01-02 December “Capital Market Day 2008“ Fa. Fresenius SE,Organizer: Bad Homburg04-05 December “Brain Tumor Meeting“Organizer: Helmut Kettenmann, MDC mit Helios Klinikum Berlin und Charité Campus,Virchow-Klinikum08 December Information workshop “EMBO: promoting excellence in the molecular life sciences in Europe”Speaker: Dr Jan Taplick, EMBO Deputy Executive Director, EMBO Fellowship ProgrammeManager and Information workshop ‘Career Pathways’Organizer: PhD Program at the MDC, Dr. O. Seumenicht and Dr. J. Droese10-12 December Berlin Brain Days 2008 veranstaltet von sieben, in Berlin angesiedelten,neurowissenschaftlichen Promovierendenprogrammen200920 January Neujahrsempfang des Campus Berlin-BuchOrganizer: MDC/BBB GmbH11 November -15 March Ausstellung mit Zeichnungen von Berliner Schülern anlässlich des 200. Geburtstages von DarwinOrganizer: Gläsernes Labor der BBB GmbH21 April - 30 June Ausstellung “Frauen in Naturwissenschaft und Technik”MDC FrauenbeauftragteOrganizer: Dr. H. Haase23-24 April Laborbaukonferenz R. StreckwallOrganizer: MDC12 June Wissenschaftshistorisches Kolloquium anläßlich des 80. Geburtstages vonProf. Dr. em. Heinz Bielka und des 70. Geburtstages von Prof. Dr. em. Jens ReichOrganizer: MDC13 June Lange Nacht der WissenschaftenOrganizer: Campus Berlin-Buch19 June Max Delbrück Lecture, Dr. Neal Copeland, Institute of Molecular and Cell Biology, SingaporeOrganizers: Carmen Birchmeier and Walter Birchmeier20 June 14. Bucher Nephrologisches SymposiumOrganizer: Prof. F. Luft, FVK03 July NEW MDC SCIENTISTS present their work Max Delbrück Center for Molecular Medicine (MDC),Organizer: Berlin Institute for Medical Systems Biology (BIMSB)10 July Max Delbrück Lecture , Dr. Stephen Cohen, Temasek Life Sciences Laboratory, SingaporeOrganizer: Prof. Th. Sommer, MDC22-26 September International EMBO Conference Series “Ubiquitin and Ubiquitin-like Modifiersin Health and Disease”Organizer: Prof. T. Sommer, MDC08-10 October International Berlin Summer Meeting “Computational & Experimental Molecular Biology”Organizer: Prof. N. Rajewsky, MDC09-13 November Basis Gene Mapping /Linkage Analysis Course Forschungsgruppe BioinformatikOrganizer: MDC mit Dr. M. Nothnagel, Uni Kiel2009 December Berlin Brain Days 2009 veranstaltet von sieben, in Berlin angesiedelten,neurowissenschaftlichen Promovierendenprogrammen.Academics 245

SeminarsSeminare2008Speaker Institute TitleThomas Bayer Department of Psychiatry, University Neurodegeneration in Alzheimer’s disease:Medical Center, Göttingen, Germanynovel findings from mouse modelsThomas Biederer Yale University, New Haven , USA SynCAM adhesion molecules: Workinghand-in-hand to organize synapsesGerard Borst Erasmus University Rotterdam, Development of the calyx of Held synapseRotterdam, NetherlandsGrzegorz Bulaj University of Utah, USA Discovery and Development of Venom-Derived Sodium Channel Blockers asTherapeutics for PainFederico Calegari CRTD, Center for Regenerative Therapies, Role of cell cycle length in mammalienDresden, GermanyneurogenesisDavid Callen The University of Adelaide Australia, Adelaide Chromosome16 Breast Cancer TumorAustraliaSuppressorsWei Chen Max Planck Institute for Molecular Medicine, Application of Illumina/Solexa sequencingBerlin, Germanytechnology in genetic researchCyrille J. Cohen Bar-Ilan University, Ramat Gan, Israel Engineering the T-lymphocyte immuneresponse against cancerJürgen Cox Max Planck Institut für Biochemie, Mass spectrometry-based proteomics withMartinsried, Germanyhigh peptide identification rates,individualized p.p.b.-range mass accuraciesand proteome-wide quantificationKarin de Visser Dept. Molecular Biology, The Netherlands The inflammatory tumor microenvironmentCancer Institute, Amsterdam, Netherlands and its impact on cancer developmentIvan Dikic Goethe University Frankfurt/Main, Frankfurt, Targeting Ubiquitin networksGermanyValentin Djonov Department of Medicine Gross Anatomy and Novel insights into intussusceptiveVascular Biology, University of Fribourg,angiogenesisSwitzerlandAndreas Ettinger MPI für Molekulare Zellbiologie und Genetik, Trash or Treasure? – the Midbody inDresden, GermanyDifferentiation and CancerReinhard Fässler Max Planck Institute of Biochemistry MartinsriedGermanyGenetic analysis ofintegrin signaling in miceJonathan Flint Wellcome Trust Centre for Human The genetic architecture of complex traits inGenetics, Oxford, United Kingdomthe mouseJean-Marc Fritschy University of Zurich, Zurich, Switzerland GABAA receptors and homeostatic synapticplasticityArmin Giese LMU Munich, Munich, Germany Prions and Parkinson: From single particleanalysis of protein aggregation to newtherapeutics246 Academics

Speaker Institute TitleMagdalena Götz GSF – National Research Center for Neurogenesis from glial cells: new views onEnvironment and Health,reactive gliosis and repairNeuherberg, GermanyFrancois Guillemot Nat. Institut for Medical Research, London, Transcriptional control of neurogenesis andUnited Kingdomneuronal migration in the mouse brainJana Hartmann TU Munich, Munich, Germany mGluR-dependent signaling in cerebellarPurkinje cellsRonald Hay University of Dundee, England How SUMO targets proteins for proteasomaldegradationMatthias W. Hentze EMBL, Germany Understanding metabolic regulatory networks:iron metabolism and its diseasesEric Honoré Institut de Pharmacologie, Moléculaire et Sensing pressure with K2P channelsCellulaire, Physiopathologie CardiovasculaireCNRS, Valbonne, FranceSebastian Jessberger Institute of Cell Biology, ETH Zurich, Fate plasticity of adult neural stem cells andZurich, Switzerlandmolecular mechanisms of neuronal maturationSven-Eric Jordt Yale University School of Medicine, TRP channels in thermosensation andNew Haven, USArespiratory reflex controlOktay Kirak Whitehead Institut for Biomedical Research, When T Cells Meet EggsCambridge, United KingdomOleg Krishtal Bogomoletz Institute Kiev, Ukraine Enigma of P2X3 receptors: how to reconcile theproperties with putative function?Thomas Kuner Institut für Anatomie und Zellbiologie Synaptic Inhibition Accelerates OdorUni Heidelberg, GermanyDiscrimination in MiceMarkus Landthaler, The Rockefeller University, Decoding posttranscriptional regulatoryNew York, USAnetworksPeter Laslo Leeds Institute for Molecular Medicine Gene regulatory networks that regulate(University of Leeds), Leeds, United Kingdom macrophage developmentCatherine Lubetzki Hôpital de la Salpêtrière Paris, France Success or failure of myelin repairMarcy MacDonald Massachusetts General Hospital, USA Genetics-directed approach to Huntington’sdisease therapeuticsHarvey MacMahon MRC Laboratory of Molecular Biology, Sculpting Cell Membranes: UnderstandingCambridge, United Kingdompathways of endocytosis and exocytosisMarco Mank MPI, Martinsried, Germany Improved Genetically-Encoded CalciumIndicators Based on Troponin CPeter McNaughton University of Cambridge, United Kingdom Why pain gets worse – role of the pacemakercurrent Ih in nociceptionEdwin Monuki University of California, Irvine, USA The Morphogen and Selector Gene Network inthe Dorsal TelencephalonAcademics 247

Speaker Institute TitleKiyoshi Mori Kyoto University Graduate School of Medicine, Biology of a siderophore-binding protein –ADepartment of Medicine and Clinical Science, common molecular mechanism underlyingKyoto, Japanmesenchymal-epithelial transition, kidneyprotection, innate immunity and anemiaRichard I. Morimoto Northwestern University, USA Regulating Proteostasis in Aging andNeurodegenerative DiseaseTobias Moser University of Goettingen, Molecular physiology of the hair cellGoettingen, Germanyribbon synapsePierluigi Nicotera University of Leicester, Leicester, Subcellular programmes forUnited KingdomneurodegenerationStephen Nutt WEHI Institute of Medcial Research, The transcriptional regulation ofAustralialymphocyte differentiationRaz Palty Department of Physiology, Zlotowski Center NCLX is an essential component offor Neuroscience, Beer-Sheva, Israelmitochondrial Na+/Ca2+ exchangeLeena Peltonen National Public Health Institute Helsinki, Genes behind rare and common neurologicalFinlanddiseases: Lessons from a genetic isolateAlexandre Prat Université de Montréal, Montreal, Canada Leukocyte adhesion to and trafficking acrossbrain ECsBernd Pulverer United Kingdom Scientific PublishingMaike Sander University of California, Temporal Control of Progenitor CellCalifornia, United StatesPluripotency in the PancreasMartin Schmelz Universität Heidelberg, Modulation of axonal excitability andHeidelberg, Germanypain – single fiber recordings inhuman and pigRalf Schneggen- Swiss Institute of Technology, Switzerland Calcium regulation of transmitter release at aburger larger CNS synapseGunnar Schütz Bayer Schering Pharma AG, Germany Molecular MRI: Magnetic Resonance AnimalImaging in Basic ResearchBarbara Seliger Marthin Luther Universität Halle, Many ways lead to MHC class I abnormalitiesHalle ,GermanyStephan Sigrist University of Wuerzburg, Germany Shedding light on synapse assembly andplasticityPeter Sonderegger University of Zurich, Zurich, Switzerland Local proteolysis at CNS synapses – a key forcognitive functions ?Theresia Stradal Signaling and Motility Group Helmholtz Regulation of different actin basedCentre for Infection Research (HZI),protrusions at the cell peripheryBraunschweig, GermanyUrsula Strobl German Research Center for Environmental The Epstein-Barr viral proteins LMP1 andHealth, Helmholtz Zentrum München,EBNA2 and their cellular homologues CD40Neuherberg, Germanyand Notch in B cell proliferation anddifferentiation248 Academics

Speaker Institute TitleAlbrecht Stroh Technical University/ Neurosciences, Modulation of embryonic stem cellMunich, Germanydifferentiation with automated temporallyprecise optogenetic stimulationHaruo Sugi University of Tokyo, Tokyo, Japan Electron microscopic demonstration ofcross-bridge recovery stroke in living musclethick filaments using the gas environmentalchamberHolger Taschen- Max Planck Institute for Biophysical Chemistry, Developmental plasticity at the calyx of Heldberger Goettingen, Germany synapseAndrei Thomas- Prof. of Pathology at the University of microRNAs and oncogenic signaling:Tikhonenko Pennsylvania, USA searching for miRaculous cancer curesMatthias Treier EMBL, Heidelberg, Germany A Sall protein complex mediates pluripotencyBahar Tunctan Mersin University Turkey, Turkey Interactions between cytochrome P450 4A,cycloxygenase and nitric oxide synthaseduring endotoxemia: therapeutic implicationsfor inflammatory diseasesDavid Van Vactor Harvard Medical School, Boston, USA Tyrosine Kinase and Phosphatase Regulationof Synapse Development in DrosophilaDavid Van Vactor Harvard Medical School, Boston, USA Tyrosine Kinase and Phosphatase Regulation ofSynapse Development in DrosophilaDevadasan Velmurugan University of Madras, Center of Adv. Studies Contributions to Macromolecular Phasing,in Crystallography and Biophysics, IndiaStructure Analysis and ModellingRichard Warth Universität Regensburg, Germany Task K+ Channels: adrenal gland developmentand functionDeborah J. Watson University of Pennsylvania, Department Normal and pathotropic migrationof Neurosurgery, Philadelphia, USAof SVZ neural progenitors in rodent, canine andfeline modelsAllan Weissman NCI Frederick, Maryland, USA RING Finger Ubiquitin Ligases in ER-AssociatedDegradation and as Targets in CancerMarius Wernig Whitehead Institute for Biomedical Research, Stem Cells and Epigenetic ReprogrammingCambridge, United KingdomKlaus Willecke University of Bonn, Bonn, Germany Expression and function of connexins – studieswith targeted mouse mutantsJochen Wittbrodt EMBL, Heidelberg, Germany Seeing is believing: Novel insights intovertebrate eye developmentJana Wolf Institute of Biology, Humboldt Mathematical modelling of signal transductionUniversity Berlin, Germanyfor the analysis of oncogenic mutationsAcademics 249

2009Speaker Institute TitleDavid G. Attwell University College London, London, Germany Neurotransmitter signaling tooligodendrocyte lineage cells: its role in CNSdevelopment and neurological disorderJoao Barata Cancer Biology Unit, Instituto de Medicina PTEN inactivation in primary T cell acuteMolecular, Lisbon, Portugalleukemia: More than the canonsHeinrich Betz MPI für Hirnforschung, Frankfurt, Germany How to make inhibitory synapsesKnut Biber University Medical Center Groningen, IL-6-type cytokines and adenosine receptorsGroningen, Netherlandsin neuroprotectionThomas Brand Theodor-Boveri-Institut für Biowissenschaften Molecular signals that control induction andBiozentrum, Universität Würzburg, Germany left- right asymmetry of proepicardiumformation in the chick embryoMichael Brecht Bernstein Center, Berlin, Germany Cellular mechanisms underlying hippocampalactivity in freely moving rodentsJoel Buxbaum The Scripps Research Institute, La Jolla, CA, USA Can amyloid precursors chaperone eachother? Transthyretin and neurodegenerativediseasesMichael Dodt TU Dresden / MPI for Cell Biology and Genetics Generation and evaluation of impulse-mediaDresden, Germanyfor the computer aided visual therapyDavid L. Feldman Novartis Institutes for Biomedical Research Inc. Renin inhibitor treatment – new insightsE. Hanover, NJ, USAVincent Fleury Laboratoire Matière et Systèmes Complexes Hydrodynamic effects in animalParis, Francemorphogenesis: does evolution follow astreamline?Gilles Fortin Institut de Neurobiologie Alfred Fessard, Central control of breathing: views from theGif sur Yvette, FranceembryoYasujuki Fujita MRC, University of London, London, Charaterization of the interface betweenUnited Kingdomnormal and transformed epithelial cellsTony Green Head, University of Cambridge Department Molecular pathogenesis of theof Haematology, Cambridge Institute for myeloproliferative disordersMedical Research, Cambridge, United KingdomRobert Grosse Universität Heidelberg, Germany Formin-controlled mechanisms of cellmorphology and invasionManuel Guzman Complutense University, Madrid, Spain Mechanisms of cannabinoid-induced cancercell deathUwe-Karsten Hanisch Georg-August-Universität, Göttingen, Microglial responses to endogenous TLRGermanyagonistsEric B. Haura The H. Lee Moffitt Cancer Center and Mechanisms and biomarkers of tyrosineResearch Institute, Tampa, Florida, USAkinase inhibitors identified through phosphoproteomicsAndreas Herrlich Whitehead Institute for Biomedical Research, A shRNA-based approach to identify novelCambridge, USAkinases and phosphatases that regulate EGFligand cleavage250 Academics

Speaker Institute TitleSergey Kasparov Department of Physiology and Pharmacology, Use of cell-specific viral gene expression inUniversity of Bristol, United KingdomneuroscienceFrank Kirchhoff MPI für Experimentelle Medizin, Göttingen, Interactions of neurons and glia in the centralGermanynervous system – transgenic mice andtwo-photon imagingAndré Kléber Institute of Physiology, University of Bern, Role of Cx43 in ventricular myocardiumSwitzerlandKaiqin Lao Applied Biosystems, Foster City, USA mRNA Seq: Whole-transcriptome analysis of asingle cellUwe Maskos Institut Pasteur, Paris, France Dissecting the nicotinic control over thedopamine system in vivoDies Meijer Erasmus University, Rotterdam, Netherlands Claw Paw meets the Adams family: A novelsignaling axis in peripheral nervedevelopmentAlex Meissner Harvard Stem Cell Institute, Cambridge, USA Epigenomics and PluripotencySlavoljub Milosevic Institut of Molecular Immunology, München, Identification of antigens, epitopes and T-cellGermanyreceptors for vaccine development andadoptive immunotherapyGerhard Moldenhauer Deutsches Krebsforschungszentrum Development of a L1-CAM (CD171)-specificHeidelberg, Germanyanti-cancer antibodyPeter Mombaerts Max-Planck Institute of Biophysics Frankfurt Germany Olfaction TargetedEmma Morris University College London Hospital NHS Trust, TCR Gene Transfer: Novel Immunotherapy forLondon, Great BritainLeukaemiaManabu Murakami Akita University School of Medicine, Japan Modified sympathetic nerve system activitywith over-expression of the voltagedependentcalcium channel beta3 subunitUwe Ohler Institute for Genome Sciences and Policy, High-throughput data generation andDuke University, Durham, NC, USAcomputational models for eukaryoticpromotersShgeo Okabe Department of Cellular Neurobiology, Visualization of synapse formationUniversity of Tokyo, Tokyo, Japanand remodeling in excitatoryMichael Olivier Medical College of Wisconsin, USA Why do we get fat? Integrated genomic andproteomic analysis of human obesityTobias Pischon German Institute of Human Nutrition (DIfE), Obesity, Adipokines and Cardiovascular RiskPotsdam-Rehbrücke, GermanyAlexey Ponomarenko Abt. für Klinische Neurobiologie, Generation of rhythms in the hippocampus:Neurologische Universitätsklinik, Heidelberg, insights from genetic modifications of theGermanynetwork clocksRichard Ransohoff Neuroinflammation Research Center, Early cortical demyelination gives a view ofCleveland Clinic, Cleveland, USAMS pathogenesis from the outside-inAcademics 251

Speaker Institute TitleJuri Rappsilber Wellcome Trust Centre for Cell Biology, The protein composition of chromatinEdinburgh, ScotlandIngmar Riedel-Kruse California Institute of Technology, Synchrony among sperms, neurons, and genesPasadena, USARafael Roesler Federal University of Rio Grande do Sul, The gastrin – releasing peptide receptor as aPorto Alegre, Braziltherapeutic target in cancer and neurologicaldisordersDavid Rubinsztein Cambridge Institute for Medical Research, Autophagy and NeurodegenerationCambridge, United KingdomPierre Savatier INSERM U846 Stem Cell and Brain Research Dissecting the mechanisms of STAT3 andInstitute, Bron, FranceNanog-dependent self-renewal in mouseembryonic stem cellsPeter Scheiffele University of Basel, Basel, Switzerland Molecular Mechanisms of Neural CircuitAssemblyGünther Schütz DKFZ, Heidelberg, Germany The nuclear receptor Tailless is crucial foradult neurogenesis and brain tumorformationNicholas D. Socci Memorial Sloan Kettering Cancer Center, Deep sequencing of small RNA profiles inNew York, USAcancerUwe Strähle University of Heidelberg and Institute Adult neurogenesis in the zebrafishof Toxicology and GeneticsForschungszentrum Karlsruhe, GermanyShunichi Takeda Center for Frontier Medicine, Kyoto University, Reverse genetic study of DNA damageJapanresponse by using the chicken DT40 cell line,and its application to chemical genetics andSILAC-based proteomic analysisDominic Tölle EBI / Cambridge University, Cambridge, Meredys, a particle-based stochasticUnited Kingdomsimulation softwareKlaus Unsicker University of Heidelberg, Germany Role of GDF-15, a novel member of the TGF-βs,in motoneuron maintenance and peripheralmyelinationPierre Van der Ludwig Institute for Cancer research, Is it possible to correct the anergy of humanBruggen Brussel, Switzerland tumor-infiltrating lymphocytes?Cinzia Volontè Institute of Neurobiology and Molecular Once upon a time ...... P2 receptors, P1Medicine , Rome, Italyreceptors, ectonucleotidases and nucleoside/nucleotide transporters got all together ina compartment at the plasma membrane .......and the purinergic signaling beganManfred S. Weiss EMBL Outstation, DESY, Hamburg, Germany Structural analysis of enzyme complexes inbiosynthetic pathways and Use of long X-raywavelengths in macromolecular crystallographyHerbert Zimmermann University of Frankfurt, Frankfurt, Germany Purinergic signaling in adult neurogenesis252 Academics

OverviewÜberblick

The Helmholtz AssociationDie Helmholtz-GemeinschaftThe Helmholtz AssociationThe Helmholtz Association (HGF) is the largest scientificorganization in Germany with 28,000 employeesin 16 research centers and an annual budget of around2.1 billion Euros. The research centers of the HelmholtzAssociation are divided into six research sectors: Energy,Earth and Environment, Health, Key Technologies,Structure of Matter, and Transport and Space.Eight centers have a focus on the field of Healthresearch in six research programs: cancer, cardiovascularand metabolic diseases, functions and dysfunctionsof the nervous system, infections and immunity,environmentally-linked health problems and the systemicanalysis of multifactorial diseases. The most significantresearch activities are clustered at fiveHelmholtz Health centers: the Deutschen Krebsforschungszentrum(DKFZ) in Heidelberg, theHelmholtz Zentrum München für Gesundheit undUmwelt (HMGU), the Helmholtz-Zentrum für Infektionsforschung(HZI) in Braunschweig, and at the MDCas well as at the future Deutschen Zentrum für NeurodegenerativeErkrankungen (DZNE) in Bonn. Otherimportant health-related work is underway at the followingHelmholtz centers: the ForschungszentrumJülich (FZJ), Helmholtz-Zentrum für Schwerionenforschung(GSI) in Darmstadt, the ForschungszentrumGeesthacht (GKSS) and the Helmholtz-Zentrum fürUmweltforschung (UFZ) in Leipzig. The focus of thesecenters lies in carrying out excellent, dynamic basicresearch and translating what is learned to the clinic.The MDC participates in three thematic programs, andis the coordinator of the program in Cardiovascularand Metabolic Diseases.Program-Oriented FundingSince the establishment of the Helmholtz e.V. in thefall of 2001, research within the Helmholtz Associationhas been strategically restructured. The six researchfields (see above) encompass different research programsthat are conducted by one or more of the individualcenters.Central to the research reform is the Program-OrientedFunding (POF) mechanism. Funding now goes toscientific programs (on a competitive basis) ratherthan to the centers. Every program is evaluated by aninternational panel of experts. This evaluation is theDie Helmholtz-GemeinschaftDie Helmholtz-Gemeinschaft ist mit ihren rund 28 000 Mitarbeiterinnenund Mitarbeitern in 16 Forschungszentrensowie einem Jahresbudget von rund 2,8 Milliarden Euro diegrößte Wissenschaftsorganisation Deutschlands.Die Forschungszentren der Helmholtz-Gemeinschaft arbeitenin sechs Forschungsbereichen: Energie, Erde undUmwelt, Gesundheit, Schlüsseltechnologien, Struktur derMaterie sowie Verkehr und Weltraum.Im Forschungsbereich Gesundheit kooperieren 8 Helmholtz-Zentren in 6 Forschungsprogrammen: Krebsforschung, Herz-Kreislauf- und Stoffwechselerkrankungen, Funktion und Dysfunktiondes Nervensystems, Infektion und Immunität,Umweltbedingte Störungen der Gesundheit sowie SystemischeAnalyse von multifaktoriellen Erkrankungen. Die wichtigstenForschungsaktivitäten sind an 5 Helmholtz-Gesundheitszentrenangesiedelt: am Deutschen Krebsforschungszentrum(DKFZ) in Heidelberg, am Helmholtz ZentrumMünchen für Gesundheit und Umwelt (HMGU), am Helmholtz-Zentrumfür Infektionsforschung (HZI) in Braunschweigund am MDC sowie in Zukunft auch am DeutschenZentrum für Neurodegenerative Erkrankungen (DZNE) inBonn. Darüber hinaus leisten die Helmholtz-Zentren: ForschungszentrumJülich (FZJ), Helmholtz-Zentrum fürSchwerionenforschung (GSI) in Darmstadt, ForschungszentrumGeesthacht (GKSS) und Helmholtz-Zentrum fürUmweltforschung (UFZ) in Leipzig, wichtige Beiträge. DerFokus liegt auf einer starken, exzellenten und dynamischenGrundlagenforschung und der Übertragung der gewonnenenErkenntnisse in die Klinik. Das MDC ist an drei Programmenbeteiligt. Das Programm‚ Herz-Kreislauf- und Stoffwechselerkrankungen’wird federführend vom MDC getragen.Die Programmorientierte FörderungIm Herbst 2001 wurde die gesamte Forschung der Helmholtz-Gemeinschaftneu strukturiert. Die Helmholtz-Gemeinschaft hat ihre Forschungsaktivitäten in den sechsoben genannten großen Bereichen gebündelt und innerhalbdieser Forschungsbereiche thematische Programme definiert,die von einem oder mehreren Zentren getragen werden.Ressourcen werden nicht mehr einzelnen Institutionen, sondernzentrenübergreifenden Forschungsprogrammen, diesich untereinander im Wettbewerb befinden, zur Verfügunggestellt. Eine strategische Begutachtung bildet die Basis fürdie Finanzierung der Forschungsprogramme. Diese Aufgabeübernehmen renommierte Experten aus aller Welt. Ihre254 Overview

asis for funding decisions made by the federal andstate governments and is conducted every five years.One important result of the last evaluation was theestablishment of two strategic initiatives that wererecommended by the panel. They concern TranslationalResearch, which aims to speed up the process bywhich the results of basic research have an impact onclinical applications, and the launch of a large populationstudy known as the “Helmholtz Cohort”, whichwill take place over the next two decades, with theaim of improving prevention and early detection ofmajor diseases.The first funding period ran from 2003 to 2008. Thesecond program period will likewise run five years,beginning in 2009 or 2010 (depending on the time ofthe evaluation). Evaluations for the second periodbegan in spring 2008 (Transport and Space, Health,Earth and Environment) and in spring 2009 (Energy,Structure of Matter, and Key Technologies).Evaluation of the centersThe MDC was extremely successful in all of its programsin the second POF evaluation, receiving thehighest marks from the evaluators.The results impressivelyconfirm the results of the evaluation of the centersfrom October 2006.In addition to program evaluations, evaluations of theHelmholtz centers at the research group level are conducted.The last review of the MDC research groupswas conducted in October 2006 and confirmed thepositive development of the institute. Sixteen (16)international and well-renowned experts spent twodays reviewing the research groups at the MDC. Theresults of the evaluation report were overwhelminglypositive, the reviewers clearly impressed by the MDC’sachievements. Eighty-three percent (83%) of the 40research groups received top-ratings: either “Excellent”(48%) or “Outstanding” (35%), respectively. Inaddition, the reviewers ranked the MDC among on ofthe top non-university research centers in the area oflife sciences in Germany. The MDC is unique as it hoststhree research programs (cardiovascular diseases, cancer,and neurosciences), allowing MDC scientists totranscend traditional boundaries and study themolecular causes of diseases in an interdisciplinaryway.Gutachten bilden die Grundlage für die Entscheidung, inwelcher Höhe und in welcher Aufteilung Bund und Länderdie einzelnen Programme fördern. Ein weiteres Ergebnis desletzten Begutachtungsprozesses sind zwei programmübergreifendestrategische Initiativen, die auf Empfehlung derGutachter gefördert werden. Dies sind die TranslationaleForschung, um Ergebnisse aus der Grundlagenforschungschneller für die klinische Anwendung zu nutzen, sowie derAufbau einer großen Populationsstudie „Helmholtz-Kohorte“über die kommenden zwei Jahrzehnte, um Vorbeugungund Frühdiagnostik bei den großen Volkskrankheiten zu verbessern.Die erste Förderperiode lief von 2003 bis 2008. Die zweiteProgrammperiode wird ebenfalls fünf Jahre betragen,beginnend ab 2009 bzw. 2010, abhängig vom Zeitpunkt derjeweiligen Begutachtung. Die Begutachtungen für die zweiteFörderperiode erfolgten im Frühjahr 2008 (Verkehr undWeltraum, Gesundheit, Erde und Umwelt) und im Frühjahr2009 (Energie, Struktur der Materie, Schlüsseltechnologien).Begutachtung der ZentrenDas MDC hat in allen drei Programmbegutachtungen imRahmen der zweiten Förderperiode äußerst erfolgreichabgeschnitten und Höchstnoten von den Gutachtern erhalten.Die Ergebnisse dieser Begutachtungen bestätigten eindrucksvolldas Ergebnis der Zentrumsbegutachtung vomOktober 2006.Zwischen den Begutachtungen im Rahmen der programmorientiertenFörderung finden die Begutachtungen der einzelnenZentren auf der Ebene der Forschungsgruppen statt.Die Forschungsgruppen des MDC wurden zuletzt im Oktober2006 begutachtet. Dabei konnte die positive Entwicklungdes MDC eindrucksvoll bestätigt werden. 16 Expertenaus dem In- und Ausland haben an zwei Tagen die Arbeitenaller Forschungsgruppen des MDC evaluiert und sich vonden wissenschaftlichen Leistungen des MDC beeindrucktgezeigt. Von den insgesamt 40 begutachteten Forschungsgruppendes MDC wurden insgesamt 48 %, also knapp dieHälfte, als „excellent“, und 35% der Gruppen als „outstanding“(“herausragend”) eingestuft. Gleichzeitig hoben dieGutachter hervor, dass das MDC als eines der größten außeruniversitärenForschungszentren auf dem Gebiet derLebenswissenschaften in Deutschland auch zu den erfolgreichstenzählt. Die besondere Ausrichtung des MDC mit seinenForschungsschwerpunkten Herz-Kreislaufforschung,Krebsforschung und Neurowissenschaften macht es nachAuffassung der Gutachter möglich, die molekularen Ursachenkrankheitsübergreifend zu verstehen.Overview 255

The Berlin-Buch CampusDer Campus Berlin-BuchBiotechnology Park with Innovation andFounders’ CenterThe basic and clinical research that takes place onCampus Berlin-Buch and the potential of the neighboringhospitals provide a stimulating environmentfor the development of the BiotechPark with itsInnovation and Founders’ Center (IGZ). The Biotech-Park along with the IGZ have developed into one of thelargest such centers in Germany.To date, more than 60million euros have been invested in the expansion andmodernization of the campus infrastructure, theshared facilities, and the biotechnology park. This sumincludes GA funds from the program “Joint task –improvement of the regional economic structure” andERDF funds (European Regional Development Funds).Overall, since the mid-nineties, the investment volumeof the companies located on campus has amounted toapproximately 180 million euros.Around 50 small and middle-sized companies, 38 ofwhich are in the biomedical sector, are currentlyengaged in research and production in the Biotech-Park. Approximately 26,000 square meters of rentalcommercial space are provided for the young firms,which altogether have about 750 employees. Currentoccupancy is at 86%. During the reporting period, twonew companies moved to the BiotechPark. In addition,a number of existing companies expanded, such asBavarian Nordic, Glycotope, Celares and Invitek. Theincrease in the number of firms choosing to locate inthe BiotechPark is an indication of the attractivenessof Campus Berlin-Buch as a preferred location.In its role as operation and development company forthe Campus Berlin-Buch, BBB Management GmbHcontinued and expanded its networking activities onall levels in 2006. As part of BBB’s endeavor to supportcampus companies and strengthen the image of thelocation, Campus Berlin-Buch was showcased atnumerous national and international events and fairs.Event programs were organized and carried out for 26visitor groups comprising representatives from science,business, politics and the media as well as forindividual visitors from 11 countries. To promote sustainedcampus development, BBB worked intensivelyto heighten the profile of Berlin-Buch as a HealthRegion. BBB Management GmbH Campus Berlin-BuchBiotechnologiepark mit Innovations- undGründerzentrumGrundlagenforschung und klinische Forschung auf demCampus sowie benachbarte Kliniken bilden ein stimulierendesUmfeld für die Entwicklung des BiotechParksmit seinem Innovations- und Gründerzentrum (IGZ).Der BiotechPark mit IGZ hat sich zu einem der GrößteninDeutschland entwickelt. In den Ausbau und dieModernisierung derCampusinfrastruktur, der Gemeinschaftseinrichtungendes Campus undin den Biotechnologieparksind bislang mehr als 60 Millionen Euroinvestiertworden. Die Fördermittel kommen aus derGemeinschaftsaufgabe zur Verbesserung der regionalenWirtschaftsstruktur (GA) und ausdem EuropäischenFonds für Regionale Entwicklung (EFRE). Die angesiedeltenUnternehmen selbst haben seit Mitte der 90´erJahre rund 180 Millionen Euro investiert.Im BiotechPark forschen und produzieren auf einer Flächevon rund 26.000 Quadratmetern gegenwärtig 50kleine und mittelständige Unternehmen. 38 davon sindim Bereich der Biomedizin tätig. Die Firmen beschäftigenetwa 750 Mitarbeiter. Zwei Unternehmen siedeltensich neu an. Die aktuelle Auslastung im BiotechParkliegt bei etwa 86%. Die gewachsene Attraktivität desCampus zieht insbesondere externe Ansiedlungen an.Zugleich expandierte eine Reihe von ansässigen Firmendes Campus, wie Bavarian Nordic, Glycotope, Celaresund Invitek.Als Betreiber- und Entwicklungsgesellschaft des Campushat die BBBManagement GmbH im Jahr 2009 dieNetzwerkarbeit auf allen Ebenenzur Unterstützung vonUnternehmen und zur Stärkung des Standorts fortgeführtund ausgeweitet. Der Standort wurde erneutinternational und national auf Messen und Veranstaltungenpräsentiert. Für 26 Besuchergruppen mit Vertreternaus Wissenschaft, Wirtschaft, Politik und Mediensowie Besuchern aus 11 Ländern wurden Veranstaltungsprogrammeorganisiert und betreut. Zugunsteneiner nachhaltigen Campusentwicklung hat sich dieBBB erneut intensiv in die weitere Profilierung desStadtteils Berlin-Buch zu einer Gesundheitsregion eingebracht.Die BBB Management GmbH Campus Berlin-Buch isteine Gründung desMax-Delbrück-Centrums für Moleku-256 Overview

was founded by the Max Delbrück Center forMolecular Medicine (MDC) Berlin-Buch. The Institutefor Molecular Pharmacology (FMP) as well as BayerSchering Pharma are co-shareholders (20% each).Life Science Learning LabIn 2009 the Life Science Learning Lab (GläsernesLabor) celebrated its tenth anniversary. This year morethan 12,500 middle school and high school students,teachers, technical assistants and scientists as well asmembers of the general public visited lab courses andfurther education programs on topics such as methodsand applications of genetic research, cell biologyand molecular medicine. These activities are supportedsignificantly by the MDC and other campus institutions.The activities of the Life Science Learning Lab focusedon the development of new lab courses relating toresearch topics of the MDC, the establishment of newlab courses in the MDC student lab and the encouragementof socially disadvantaged students. As partof the German Children and Youth Foundation project"Schools prepare for the future," the Life ScienceLearning Lab developed school lessons and experimentsin biology, health education and nutrition forthe local Hufeland school.Central activities of the CampusPR/Life Science LearningLab team included the editing and publication ofthe newsletter “CampusNews”, the publication ofbooklets and flyers for location marketing as well asthe successful realisation of events such as the NewYear’s reception and “Long Night of the Sciences," astrong magnet for visitors.In the campus kindergarten “CampusSterne”, whichwas initiated by the CampusPR, all 30 places are completelybooked and just three years after opening thekindergarten is going to be enlarged in 2010.Leibniz-Institut für Molekulare Pharmakologie(FMP)The Leibniz-Institut für Molekulare Pharmakologie(FMP) conducts basic research in the field of molecularpharmacology. Due to the close spatial proximity tolare Medizin (MDC) Berlin-Buch. Mitgesellschafter sinddas Leibniz-Institut für Molekulare Pharmakologie(FMP) sowie die Bayer Schering Pharma AG.Das Gläserne LaborDas Gläserne Labor hat als Life Science-Bildungszentrumim Jahre 2009 mehr als 12.500 Schülerinnen undSchülern, Lehrkräften und einem breiten Laien- undFachpublikum anspruchsvolle Laborkurse und Fortbildungenzu aktuellen Methoden und Anwendungen derGenforschung, Zellbiologie und molekularen Medizinangeboten. Das MDC und andere Campuseinrichtungenhaben diese Aktivitäten maßgeblich unterstützt.Die Bildungsarbeit des Gläsernen Labors fokussierte sichu. a. dabei auf die Entwicklung neuer Laborkurse mitBezug zu Forschungsthemen des MDC, auf den Ausbauder Kursangebote im MDC-Schülerlabor sowie die Förderungvon sozial benachteiligten Schülerinnen undSchülern. Als Teil des Verbundprojekts »Die Schule machtfit für die Zukunft« der Deutschen Kinder- und Jugendstiftungentwickelte das Gläserne Labor geeigneteUnterrichtseinheiten und Experimente zum Thema Biologie,Gesundheit und Ernährung für die an den Campusangrenzende Hufeland-Hauptschule. Zu den zentralenAktivitäten der Campus-PR/Gläsernes Labor zähltewie in den Vorjahren die Gestaltung und Herausgabeder Mitarbeiter und Imagezeitung „CampusNews“, diePublikation von Broschüren zum Standortmarketingdes Campus sowie die Organisation und erfolgreicheDurchführung von Veranstaltungen wie dem Neujahrsempfangder Campus-Einrichtungen und der äußersterfolgreichen und besucherstarken Langen Nacht derWissenschaften.Die von der Öffentlichkeitsarbeit des Campus initiierteund vor drei Jahren eröffnete betriebsnahe Kindertagesstätte»CampusSterne« ist mit 30 verfügbaren Plätzenausgebucht. Weitere Kitaplätze werden seitens derBeschäftigten des Campus nachgefragt. Für 2010 isteine räumliche Erweiterung der Kita vorgesehen.Leibniz-Institut für Molekulare Pharmakologie(FMP)Das Leibniz-Institut für Molekulare Pharmakologie(FMP) betreibt Grundlagenforschung auf dem GebietOverview 257

the MDC, the existing collaborations between the twoinstitutes have been considerably intensified. Theresearch concepts of the MDC and the FMP complementeach other: while the molecular medicalresearch at the MDC is particularly dedicated to diseasesor clinical symptoms and their molecular explanations,the FMP investigates the functional andstructural characterization of proteins. As a result, newapproaches are developed toward the modulation ofprotein function, especially of small molecules. Hence,a main emphasis of research is the development ofnew drugs and pharmaceuticals. The FMP is known forits scientific work that combines the fields of chemistryand biology.The close connection between the two research establishmentsextends into the organizational level. Guestscientist contracts make it possible for scientists ofone institute to use the equipment in the other. Bothestablishments send representatives to importantcommittees of the other establishment respectively.The planning of costly and long-term research projectsas well as the appointment of leading scientiststakes place in joint agreement. The MDC and the FMParrange and finance joint events for those studying fortheir doctorates.der molekularen Pharmakologie. Begünstigt durch dieräumliche Nähe auf dem Campus Berlin-Buch bestehenintensive Forschungskooperationen zwischen MDC undFMP. Ihre Forschungskonzepte ergänzen sich: Währendsich die molekular-medizinische Forschung des MDCbesonders mit Krankheiten oder klinischen Symptomenund ihren molekularen Grundlagen beschäftigt, stehenim Mittelpunkt der FMP-Forschung der Aufbau undFunktion von Proteinen als Grundbausteine des Körpers.Ziel ist es, Wirkstoffe zu finden, die an Proteine bindenund deren Funktionen ändern können. Sie kommendann als Werkzeuge für die Forschung sowie als Bausätzefür neue Arzneimittel in Frage.Gastwissenschaftlerverträge machen es möglich, dassWissenschaftler des einen Instituts Geräte des anderenverwenden. Beide Einrichtungen entsenden Vertreter indie wichtigsten Gremien der jeweils anderen Einrichtung.Kostspielige, langfristige Forschungsprojekte werdengemeinsam geplant, führende Wissenschaftlern ingegenseitigem Einvernehmen ernannt. Auch organisierenund finanzieren MDC und FMP gemeinsam Veranstaltungenfür ihre Promotionsstudenten.258 Overview

Organizational StructureOrganisationsstrukturDhe Max Delbrück Center for MolecularMedicine (MDC) Berlin-Buch is a foundationunder public law of the State ofBerlin with the purpose of pursuing medicalresearch at the molecular and cellular levels andimplementing its clinical application.Board of TrusteesThe Board of Trustees is the supervisory body of MDCand monitors the conduct of operations with respectto legality, appropriateness, economic efficiency andfinancial viability. It decides upon general researchobjectives as well as important research policy andfinancial matters of the Foundation.Members of the Board of TrusteesMinistry Director, Dr. Peter LangeFederal Ministry of Education and Research (BMBF),Berlin (Chair)Senatsdirigent Wolfgang Eckey (to May 2008)Senate Administration for Education, Science andResearch, Berlin (Vice-Chair)Dr. Jutta Koch-Unterseher (since August 2008)Senate Administration for Education, Science andResearch, Berlin (Vice-Chair)Prof. Dr. Günter Breithardt (to September 2008)University of Münster*Prof. Dr. Magdalena Götz (since September 2008)Helmholtz Zentrum München*Prof. Dr. Roger GoodyMax Planck Institute for Molecular Biology, Dortmund*Prof. Dr. Annette Grüters-KieslichCharité – University Medicine Berlin*Prof. Dr. Reinhard JahnMax Planck Institute for Biophysical Chemistry,Göttingen*Department Head Oda KepplerFederal Ministry of Education and Research (BMBF)Prof. Dr. Maria LeptinInstitute for Genetics, University of Cologne*Das Max-Delbrück-Centrum für MolekulareMedizin (MDC) Berlin-Buch ist eine Stiftungdes öffentlichen Rechts des LandesBerlin mit dem Zweck, medizinische Forschung aufmolekularer und zellulärer Ebene und ihre klinischeAnwendung und praktische Umsetzung zubetreiben.Das KuratoriumDas Kuratorium ist das Aufsichtsgremium des MDC. Esüberwacht die Rechtmäßigkeit, Zweckmäßigkeit undWirtschaftlichkeit der Geschäftsführung. Es entscheidetüber die allgemeinen Forschungsziele und über wichtigeforschungspolitische und finanzielle Angelegenheitender Stiftung.Mitglieder des KuratoriumsMinisterialdirektor Dr. Peter LangeBundesministerium für Bildung und Forschung(BMBF), Berlin (Vorsitz)Senatsdirigent Wolfgang Eckey (bis Mai 2008)Senatsverwaltung für Bildung, Wissenschaft undForschung, Berlin (stellv. Vorsitz)Dr. Jutta Koch-Unterseher (seit August 2008)Senatsverwaltung für Bildung, Wissenschaft undForschung, Berlin (stellv. Vorsitz)Prof. Dr. Günter Breithardt (bis September 2009)Universität Münster*Prof. Dr. Magdalena Götz (seit September 2008)Helmholtz Zentrum München*Prof. Dr. Roger GoodyMax-Planck-Institut für molekulare Physiologie,Dortmund*Prof. Dr. Annette Grüters-KieslichCharité – Universitätsmedizin Berlin*Prof. Dr. Reinhard Jahn (bis September 2009)Max-Planck-Institut für biophysikalische Chemie,Göttingen*Ministerialrätin Oda KepplerBundesministerium für Bildung und Forschung(BMBF)260 Overview

Prof. Dr. Gary R. LewinMDC Berlin-BuchProf. Dr. Michael LinscheidHumboldt University BerlinProf. Dr. Renato Paro (since May 2006)Center of Biosystems, ETH Zürich, SwitzerlandProf. Dr. Martin Paul (since April 2007)Charité – University Medicine BerlinProf. Dr. Annemarie Poustka,German Cancer Research Center (DKFZ), Heidelberg*Prof. Monika Schäfer-Korting (since October 2007)Free University BerlinSenatsdirigent Martin SchmahlSenate Administration for Health, Environment, andConsumer Protection, BerlinDr. Birgit Schnieders (since March 2006)Federal Ministry of Health, BonnRegierungsdirektorin Marianne Pyrczek(since March 2009)Federal Ministry of Finance, BonnDr. Ulrike Ziebold (to April 2009)MDC Berlin-BuchProf. Michael Bader (since May 2009)MDC Berlin-BuchDr. Matthias Selbach (since May 2009)MDC Berlin-BuchProf. Dr. Mathias Hentze (since Oktober 2009)EMBL Heidelberg*Prof. Dr. Elisa Izaurralde (since August 2009)Max Planck Institut für Entwicklungsbiologie*Prof. Dr. Elisabeth Knust (seit August 2009)Max Planck Institut für Molekulare Zellbiologie undGenetik** Also members of the Scientific CommitteeProf. Dr. Maria LeptinInstitut für Genetik der Universität zu Köln*Prof. Dr. Gary R. Lewin (bis April 2009)MDC Berlin-BuchProf. Dr. Michael W. Linscheid (seit Oktober 2007)Vizepräsident der Humboldt-Universität zu BerlinProf. Dr. Renato ParoCenter of Biosystems, ETH Zürich, Schweiz*Prof. Dr. Martin Paul (bis August 2008)Charité – Universitätsmedizin BerlinProf. Dr. Annemarie Poustka (verstorben am 3.5.2008)Deutsches Krebsforschungszentrum (DKFZ), Heidelberg*Prof. Dr. Hans Jürgen Prömel (bis September 2007)Vizepräsident der Humboldt-Universität zu BerlinProf. Monika Schäfer-KortingFreie Universität BerlinSenatsdirigent Martin SchmahlSenatsverwaltung für Gesundheit, Umwelt und Verbraucherschutz,BerlinDr. Birgit SchniedersBundesministerium für Gesundheit, BonnRegierungsdirektorin Marianne Pyrczek(seit März 2009)Bundesministerium der Finanzen, BerlinDr. Ulrike Ziebold (bis April 2009)MDC Berlin-BuchProf. Michael Bader (ab Mai 2009)MDC Berlin-BuchDr. Matthias Selbach (ab Mai 2009)MDC Berlin-BuchProf. Dr. Mathias Hentze (ab Oktober 2009)EMBL Heidelberg*Prof. Dr. Elisa Izaurralde (seit August 2009)Max Planck Institut für Entwicklungsbiologie*Prof. Dr. Elisabeth Knust (seit August 2009)Max Planck Institut für Molekulare Zellbiologie undGenetik** zugleich Mitglieder des Wissenschaftlichen AusschussesOverview 261

Scientific CommitteeThe Scientific Committee of the Board of Trustees preparesthe decisions of the Board of Trustees in scientificmatters. The Scientific Committee is responsible forthe ongoing evaluation of the results of the researchwork of MDC through scientific assessment. Togetherwith the scientific members of the Board of Trustees,up to seven external specialists sit on the ScientificCommittee.Der Wissenschaftliche AusschussDer Wissenschaftliche Ausschuss des Kuratoriums bereitetdie Entscheidungen des Kuratoriums in wissenschaftlichenFragen vor. Er trägt die Verantwortung fürdie fortlaufende Ergebnisbewertung der Forschungsarbeitendes MDC durch wissenschaftliche Begutachtung.Dem Wissenschaftlichen Ausschuss gehören neben denwissenschaftlichen Mitgliedern des Kuratoriums bis zusieben externe Fachwissenschaftler an.Members of the Scientific CommitteeProf. Dr. Reinhard Jahn (to September 2009)Max-Planck-Institut für biophysikalische Chemie,Göttingen (Vorsitzender)*Prof. Dr. Maria Leptin (vice-chair)Institut für Genetik der Universität zu Köln*Prof. Dr. Corinne Antignac (since September 2007)INSERM, Hopital Necker – Enfants Malades, Paris,FrankreichProf. Dr. Rudi Balling (to Mai 2009)Helmholtz-Zentrum für Infektionsforschung)Prof. Dr. Günter Breithardt (to September 2009)Universität Münster*Prof. Dr. Johannes Carolus CleversHubrecht Laboratory, Netherlands Institute forDevelopmental Biology, Utrecht; NiederlandeProf. Dr. Anna Dominiczak (since September 2007)University of Glasgow, Cardiovascular ResearchCentre, Glasgow, United KingdomProf. Dr. Magdalena Götz (since September 2008)Helmholtz Zentrum München*Prof. Dr. Roger GoodyMax-Planck-Institut für molekulare Physiologie,Dortmund*Prof. Dr. Annette Grüters-Kieslich (to August 2008)Charité – Universitätsmedizin Berlin*Prof. Dr. Christoph HuberUniversität MainzProf. Dr. Thomas Meitinger (to May 2009)Forschungszentrum für Umwelt und Gesundheit (GSF),NeuherbergMitglieder des Wissenschaftlichen AusschussesProf. Dr. Reinhard Jahn (bis September 2009)Max-Planck-Institut für biophysikalische Chemie,Göttingen (Vorsitzender)*Prof. Dr. Maria Leptin (Stellv. Vorsitz)Institut für Genetik der Universität zu Köln*Prof. Dr. Corinne Antignac (seit September 2007)INSERM, Hopital Necker – Enfants Malades, Paris,FrankreichProf. Dr. Rudi Balling (bis Mai 2009)Helmholtz-Zentrum für Infektionsforschung)Prof. Dr. Günter Breithardt (bis September 2009)Universität Münster*Prof. Dr. Johannes Carolus CleversHubrecht Laboratory, Netherlands Institute forDevelopmental Biology, Utrecht; NiederlandeProf. Dr. Anna Dominiczak (seit September 2007)University of Glasgow, Cardiovascular Research Centre,Glasgow, United KingdomProf. Dr. Magdalena Götz (seit September 2008)Helmholtz Zentrum München*Prof. Dr. Roger GoodyMax-Planck-Institut für molekulare Physiologie,Dortmund*Prof. Dr. Annette Grüters-Kieslich (bis August 2008)Charité – Universitätsmedizin Berlin*Prof. Dr. Christoph HuberUniversität MainzProf. Dr. Thomas Meitinger (bis Mai 2009)Forschungszentrum für Umwelt und Gesundheit(GSF), Neuherberg262 Overview

Prof. Dr. Renato ParoCenter of Biosystems, ETH Zürich, Schweiz*Prof. Dr. Annemarie Poustka (died on May 3, 2008)Deutsches Krebsforschungszentrum (DKFZ),Heidelberg*Prof. Sir George K. Radda (to May 2009)University of Oxford, Great BritainProf. Dr. Mathias Hentze (since October 2009)EMBL Heidelberg*Prof. Dr. Elisa Izaurralde (since August 2009)Max Planck Institut für Entwicklungsbiologie*Prof. Dr. Elisabeth Knust (seit August 2009)Max Planck Institut für Molekulare Zellbiologie undGenetik** Also members of the Board of TrusteesExecutive BoardThe Executive Board manages the Institute and consistsof a scientific member, Prof. Walter Birchmeier,and an administrative member, Dr. Stefan Schwartze.The Chair of the Executive Board is Prof. Dr. WalterBirchmeier.Scientific CouncilThe Scientific Council advises the Executive Board inmatters of fundamental scientific importance.Members of the Scientific CouncilDr. Salim Abdelilah-SeyfriedProf. Dr. Michael BaderDr. Daniel BesserProf. Dr. Carmen Birchmeier-KohlerProf. Dr. Walter BirchmeierProf. Dr. Thomas BlankensteinDr. Oliver Daumke (Stellv. Vorsitzender)Prof. Dr. Rainer DietzProf. Dr. Bernd DörkenDr. Iduna FichtnerProf. Dr. Michael GotthardtProf. Dr. Udo HeinemannProf. Dr. Norbert HübnerDr. Inés Ibanez-TallonProf. Dr. Renato ParoCenter of Biosystems, ETH Zürich, Schweiz*Prof. Dr. Annemarie Poustka (verstorben am 3. Mai2008)Deutsches Krebsforschungszentrum (DKFZ),Heidelberg*Prof. Sir George K. Radda (bis Mai 2009)Universität Oxford, GroßbritannienProf. Dr. Mathias Hentze (ab Oktober 2009)EMBL Heidelberg*Prof. Dr. Elisa Izaurralde (seit August 2009)Max Planck Institut für Entwicklungsbiologie*Prof. Dr. Elisabeth Knust (seit August 2009)Max Planck Institut für Molekulare Zellbiologie undGenetik** zugleich Mitglieder des KuratoriumsDer StiftungsvorstandDer Stiftungsvorstand leitet das Institut und bestehtaus einem wissenschaftlichen Mitglied, Prof. WalterRosenthal, und einem administrativen Mitglied, CorneliaLanz. Vorsitzender des Stiftungsvorstands ist Prof. Dr.Walter RosenthalWissenschaftliche RatDer Wissenschaftliche Rat berät den Stiftungsvorstandin den Angelegenheiten von grundsätzlicher wissenschaftlicherBedeutung.Mitglieder des Wissenschaftlichen RatesDr. Salim Abdelilah-SeyfriedProf. Dr. Michael BaderDr. Daniel BesserProf. Dr. Carmen Birchmeier-KohlerProf. Dr. Walter BirchmeierProf. Dr. Thomas BlankensteinDr. Oliver Daumke (Stellv. Vorsitzender)Prof. Dr. Rainer DietzProf. Dr. Bernd DörkenDr. Iduna FichtnerProf. Dr. Michael GotthardtProf. Dr. Udo HeinemannOverview 263

Dr. Zsuzsanna IzsvákProf. Dr. Thomas JentschProf. Dr. Helmut KettenmannDr. Markus LandthalerDr. Stefan LechnerProf. Dr. Young-Ae LeeProf. Dr. Ferdinand le NobleProf. Dr. Achim LeutzProf. Dr. Gary R. Lewin (Vorsitzender)PD Dr. Martin LippProf. Dr. Friedrich C. LuftPD Dr. Jochen MeierProf. Dr. Ingo L. MoranoDr. Thomas MüllerProf. Dr. Thoralf NiendorfDr. Cristiane NolteDr. Matthew N. PoyProf. Dr. Nikolaus RajewskyProf. Dr. Fritz G. RathjenDr. Armin RehmDr. Frank RosenbauerProf. Dr. Claus ScheidereitProf. Dr. Peter SchlagDr. Ruth Schmidt-UllrichDr. Björn Christian SchroederDr. Anja SchützDr. Matthias SelbachDr. Jan-Erik SiemensProf. Dr. Thomas SommerDr. Francesca SpagnoliProf. Dr. Ludwig ThierfelderProf. Dr. Erich WankerProf. Dr. Thomas WillnowDr. Jana WolfDr. Ulrike ZieboldProf. Dr. Frauke ZippProf. Dr. Norbert HübnerDr. Inés Ibanez-TallonDr. Zsuzsanna IzsvákProf. Dr. Thomas JentschProf. Dr. Helmut KettenmannDr. Markus LandthalerDr. Stefan LechnerProf. Dr. Young-Ae LeeProf. Dr. Ferdinand le NobleProf. Dr. Achim LeutzProf. Dr. Gary R. Lewin (Vorsitzender)PD Dr. Martin LippProf. Dr. Friedrich C. LuftPD Dr. Jochen MeierProf. Dr. Ingo L. MoranoDr. Thomas MüllerProf. Dr. Thoralf NiendorfDr. Cristiane NolteDr. Matthew N. PoyProf. Dr. Nikolaus RajewskyProf. Dr. Fritz G. RathjenDr. Armin RehmDr. Frank RosenbauerProf. Dr. Claus ScheidereitProf. Dr. Peter SchlagDr. Ruth Schmidt-UllrichDr. Björn Christian SchroederDr. Anja SchützDr. Matthias SelbachDr. Jan-Erik SiemensProf. Dr. Thomas SommerDr. Francesca SpagnoliProf. Dr. Ludwig ThierfelderProf. Dr. Erich WankerProf. Dr. Thomas WillnowDr. Jana WolfDr. Ulrike ZieboldProf. Dr. Frauke Zipp264 Overview

Staff CouncilThe Staff Council is involved in decisions of the MDCBerlin-Buch that concern personnel and staff welfarematters.PersonalratDer Personalrat ist an Entscheidungen des MDC Berlin-Buch beteiligt, welche die personellen und sozialenBelange der Beschäftigten betreffen.Members of the Staff CouncilIngo Kahl (Chair)Carola Bernert (vice chair)Lutz Else (vice chair)Dagmar Gerhard (vice chair)Manuela AdloffGitta BlendingerDr. Oliver DaumkeRobby FechnerDaniela KeynerSiegne KnespelJan TimmBrigitta Wedekind (secr.)Mitglieder des PersonalratesIngo Kahl (Vorsitz)Carola Bernert (stellv. Vorsitz)Lutz Else (stellv. Vorsitz)Dagmar Gerhard (stellv. Vorsitz)Manuela AdloffGitta BlendingerDr. Oliver DaumkeRobby FechnerDaniela KeynerSiegne KnespelJan TimmBrigitta Wedekind (Sekr.)Women’s RepresentativeThe women’s representative is responsible for mattersand measures concerning equal opportunitiesfor women at the MDC. She advises not only in theplanning but also in the decisions of the Board andother organizational units, in particular with regardto personnel, welfare, and organizational decisions.Currently, Dr. Hannelore Haase serves as the MDCwomen’s representative.FrauenvertreterinDie Frauenvertreterin ist für Angelegenheiten und Maßnahmender Gleichstellung der Frauen am MDC zuständig.Sie wird sowohl bei der Planung als auch bei Entscheidungendes Vorstands und anderer Organisationseinheiten,insbesondere bei personellen, sozialen undorganisatorischen Entscheidungen beratend beteiligt.Derzeit nimmt Dr. Hannelore Haase die Funktion derFrauenvertreterin am MDC wahr.Overview 265

Facts and FiguresFakten und KennzahlenPersonnel / PersonalPersonnel (as of December 31, 2008) / Personalstand in Köpfen mit Stichtag 31.12.2008Total/ limited contract/ permanent/ in-house financing/ third-party financing/Gesamt befristet unbefristet grundfinanziert drittmittelfinanziertScientists (not including PhD students) / 215 182 33 122 93Wissenschaftler (ohne Doktoranden)PhD students / 211 211 0 79 132Doktoranden und studentische HilfskräfteTechnical Assistants (TAs) in the scientific sphere / 190 103 87 129 61Technische Angestellte im wissenschaftlichen BereichOther / Andere 213 52 161 197 16In training / In Ausbildung 51 51 0 51 0Total / Summe 880 599 281 578 302Other*/Andere*24%In training/In Ausbildung6%Technical Assistants/TAs22%26%Scientists (not including PhD students)/Wissenschaftler (ohne Doktoranden)24%PhD Students/DoktorandenType of Employment Contract / Art der Verträgelimited/befristet permanent/unbefristet250200limited/befristetpermanent/unbefristetScientists/Wissenschaftler 182 33PhD Students/Doktoranden. 211 0150Technical Assistants/TAs 103 87Other*/Andere* 52 161100In training/Ausbildung 51 050*Infrastructure, administration, and technology transfer*Infrastruktur, Verwaltung und Technologietransfer0Scientists/ PhD Students/ TechnicalWissenschaftler Doktoranden Assistants/TAsOther*/Andere*In training/Ausbildung266 Overview

Type of Financing/Art der Finanzierungin-house financing/grundfinanziertthird-party financing/drittmittelfinanziert250200in-house financing/grundfinanziertthird-party financing/drittmittelfinanziertScientists/Wissenschaftler 122 93PhD Students/Doktoranden 79 132Technical Assistants/TAs 129 61Other*/Andere* 197 16In training/Ausbildung 51 0150100500Scientists/ PhD TechnicalWissenschaftler Students/ Assistants/TAsDoktorandenOther*/Andere*In training/AusbildungFinancing / FinanzierungCosts of research programs in 2008 (in thousands of €) / Kosten der Forschungsprogramme 2008 in T€Programs & Categories / Programme & KategorienCosts / KostenCancer Research/Krebsforschung 13.806Cardiovascular and metabolic diseases/Herz- Kreislauf- und Stoffwechselerkrankungen 16.696Function and dysfunction of the nervous system/Funktion und Dysfunktion des Nervensystems 6.791Comparative Genomics/Vergleichende Genomforschung 259Independent Research/Programmungebundene Forschung 134Technology transfer/Technologietransfer 1.116Special work (training)/Sonderaufgaben 2.666Centre Management/ 4.427Scientific infrastructure and basic operation/ 5.745Total/Gesamt 51.640Scientific infrastructure and basic operation/Infrastruktur und BasisbetriebCentre Management/Verwaltung11%Special work (training)/Sonderaufgaben9%27%Cancer Research/KrebsforschungTechnology transfer/Technologietransfer 2%Independent Research/0,3%Programmungebundene Forschung0,5%Comparative Genomics/Vergleichende GenomforschungFunction and dysfunction of the nervous system/Funktion und Dysfunktion des Nervensystems5%13%32%Cardiovascular and metabolic diseases/Herz- Kreislauf- und StoffwechselerkrankungenOverview 267

Extramural funding in 2008 (in thousands of €) / Drittmittelfinanzierung 2008 in T €Extramural funds / DrittmittelgeberAmounts / DrittmittelerlöseGerman Research Foundation/Deutsche Forschungsgemeinschaft 6.138EU/EU 2.266Industry and other organizations/Industrie und sonstige Organisationen 4.295Federal Ministry of Education and Research/Bundesministerium für Bildung und Forschung (BMBF) 8.665State of Berlin/Land Berlin 181Total / Gesamt 21.545State of Berlin/Land Berlin1%EU/EU11%Industry and other organizations/Industrie und sonstige Organisationen20%40%Federal Ministry of Education and Research/Bundesministerium für Bildung und Forschung (BMBF)German Research Foundation/Deutsche Forschungsgemeinschaft28%268 Overview

Research Projects 2008-2009Forschungsprojekte 2008-2009Collaborative research centres (SFB) und transregional research centres (TRR) ofthe German Research Foundation (DFG)SFB 449:Structure and function of membrane receptorsSFB 594:Molecular machines in protein folding and protein transportSFB 633:SFB 650:SFB 577:SFB 618:SFB 665:SFB 740:SFB 555:SFB Transregio 19:SFB Transregio 36:SFB Transregio 43:SFB Transregio 54:Induction and modulation of T cell-impartedImmunreactions in the gastrointestinal tractCellular approaches to the suppression of undesirableimmunroactionsMolecular principles of the clinical variability of monogenicdiseasesTheoretic biology: robustness, modularity and evolutionarydesign of living systemsDevelopment disorders in the nervous systemFrom molecules to modules: Organization and dynamics offunctional units in cellsComplex non-linear processesInflammatory cardiomyopathy – molecular pathogenesisand therapyPrinciples and applications of adoptive T cell therapyThe brain as a target of inflammatory processesGrowth and survival, plasticity und cellular interactivity oflymphatic neoplasiasSFB Transregio 52: Transcriptional programming of individual T-cell populationsSFB Transregio 3: Mesial temporal lobe epilepsiesSFB Transregio 3: Mesiale Temporallappen-EpilepsienSonderforschungsbereiche (SFB) und Transregios (TRR) der DeutschenForschungsgemeinschaft (DFG)SFB 449Struktur und Funktion membranständiger RezeptorenSFB 594SFB 633SFB 650SFB 577SFB 618SFB 665SFB 740SFB 555SFB Transregio 19SFB Transregio 36SFB Transregio 43SFB Transregio 54SFB Transregio 52SFB Transregio 3Molekulare Maschinen in Proteinfaltung undProteintransportInduktion und Modulation T-zellvermittelter Immunreaktionenim GastrointestinaltraktZelluläre Ansätze zur Suppression unerwünschterImmunreaktionenMolekulare Grundlagen klinischer Variabilität monogenbedingter KrankheitenTheor. Biologie: Robustheit, Modularität und evolutionäresDesign lebender SystemeEntwicklungsstörungen im NervensystemVon Molekülen zu Modulen: Organisation und Dynamikzellulärer FunktionseinheitenKomplexe nichtlineare ProzesseInflammatorische Kardiomyopathie – MolekularePathogenese und TherapieGrundlagen und Anwendung adoptiver T-ZelltherapieDas Gehirn als Angriffsziel inflammatorischer ProzesseWachstum und Überleben, Plastizität und zelluläreInteraktivität lymphatischer NeoplasienTranskriptionelle Programmierung individueller T-Zell-PopulationenMesiale Temporallappen-EpilepsienNational Genome Research Network (NGFN-2) ProjectsGenome network cardiovascular diseases: Comparative genomics of leftventricular hypertrophy and dysfunction in hypertensionGenome network cardiovascular diseases: Functional genomics of cardiacdamage in hypertensionGenome network cardiovascular diseases: Location Berlin MDC: Prevalenceof Titin-mutations and identification of new disease genes in patients withfamilial dilative cardiomyopathySystematic-methodical platform "DNA", location MDC, Berlin"Systematic-methodical platform "DNA": National genotyping platformSystematic-methodical platform GEM: Location Berlin "Genomwide correlationanalysis and association studies with 10K and 100K arrays"Genome network neuro: Systematic gene identification und functional analysisfor frequent neurologic diseasesGenome network neuro: Molecular genetic identification of activity scheduledgene configurations of genetically determined epilepsiesCancerNet: Organ Specificity of Colorectal Cancer MetastasisSystematic-methodical platform "Protein": Verification and identification ofprotein-protein interactions and systematic analysis of target proteins via X-ray structure analysis, TP 7 – Structure determinationSystematic-methodical platform "Protein": Verification and identification ofprotein-protein interactions and systematic analysis of target proteins via X-ray structure analysis, TP 20 – Project managementSystematic-methodical platform "Protein": Verification and identification ofprotein-protein interactions and systematic analysis of target proteins via X-ray structure analysis, TP 8 Yeast two-hybrid protein interaction networksSystematic-methodical platform "Protein", Location MDC Berlin: Verificationand identification of protein-protein interactions and systematic analysis oftarget proteins via X-ray structure analysis, TP 1.1. Subcloning of full-lengthORF's and cDNA-fragments into expression plasmidsDetermination of genes underlying Williams-Beuren Syndrom by generationof an allelic series of mutations with the aid of novel transposonapproaches, TP1Projekte im Nationalen Genomforschungsnetz (NGFN-2)Genomforschungsnetz Kardiovaskuläre Krankheiten: Vergleichende Genomicsder links-ventrikulären Hypertrophie and Dysfunktion bei BluthochdruckGenomforschungsnetz Kardiovaskuläre Krankheiten: Funktionelle Genomicsder Herzschädigung bei BluthochdruckGenomforschungsnetz Kardiovaskuläre Krankheiten: Standort Berlin MDC:Prävalenz von Titin-Mutationen und Identifizierung neuer Krankheitsgene inPatienten mit Familiärer Dilativer KardiomyopathieSystematisch-Methodische Plattform "DNA", Standort MDC, Berlin"Systematisch-Methodische Plattform "DNA": NationaleGenotypisierungsplattformSystematisch-methodische Plattform GEM: Standort Berlin "GenomweiteKopplungsanalyse und Assoziationsstudien mit den 10K und 100K Chips"Genomnetz Neuro: Systematische Genidentifikation und funktionelle Analysenbei häufigen neurologischen ErkrankungenGenomnetz Neuro: Molekulargenetische Identifizierung von disponierendenGenkonfigurationen bei genetisch determinierten EpilepsienCanerNet: Organspezifität von Darmkrebs MetastasierungSystematisch-Methodische Plattform "Protein": Vertifikation und Identifikationvon Protein-Protein Interaktionen und systematische Analyse vonTargetproteinen mittels Röntgenstrukturanalyse, TP 7 – Struktur- aufklärungSystematisch-Methodische Plattform "Protein": Vertifikation und Identifikationvon Protein-Protein Interaktionen und systematische Analyse vonTargetproteinen mittels Röntgenstrukturanalyse, TP 20 – Projekt- managementSystematisch-Methodische Plattform "Protein": Vertifikation und Identifikationvon Protein-Protein Interaktionen und systematische Analyse vonTargetproteinen mittels Röntgenstrukturanalyse, TP 8 Yeast two-hybrid ProteinInteraktionsnetzwerkSystematisch-Methodische Plattform "Protein", Standort MDC Berlin:Verifikation und Identifikation und systematische Analyse von Targetproteinenmittels Röntgenstrukturanalyse", TP 1.1. Umklonierung von full-length ORF'sund cDNA-Fragmenten in Expressions-PlasmideBestimmung der Gene, die dem Williams-Beuren Syndrom zugrundeliegendurch die Generation einer allelischen Serie von Mutationen mit Hilfe von neuartigenTransposon Ansätzen, TP1Overview 269

National Genome Research Network (NGFN-Plus) ProjectsNetwork genetics of heart failure: TP3 Regulatory networks of susceptabilitygenes for cardial hypertrophy and heart failureNetwork neurodegeneration (NeuroNet): Data integration and generation ofphenotype-protein-active agent networksNetwork neurodegeneration (NeuroNet): Protein Interaction screening via quantitativemass spectroscopyNetwork neurodegeneration (NeuroNet): Protein-protein interaction networksfor neurodegenerative diseasesNetwork neurodegeneration (NeuroNet): Generation and systematic analysis ofprotein-protein interaction networks via Yeast-2-Hybrid quantitative proteomicsNetwork neurodegeneration (NeuroNet): Generation of Gene expression signaturesfor neurodegenerative disease processesNetwork neuroblastoma: Transposon based mutagenesis screen in the mouseNetwork leukemia: The role of the WNT-signal transduction pathway on thedevelopment of leukemia stem cellsNetwork Alzheimer: The physiological function of BACE1-is BACE1 a safetherapeutic target?Systems biology of genetically determined diseases: Mutanome. Generationand systematical analysis of protein-protein interaction networksNetwork Alzheimer: Identification and characterization of modulators ofAlzheimer's disease pathogenesisNetwork: Molecular cause for affective disorders and schizophrenia.MOODS (TP11)Installation of the scientific platform "Interactom" for systematic proteininteraction studiesProjekte im Nationalen Genomforschungsnety (NGFN-Plus)Verbund Genetik des Herzversagens: TP3 Regulatorische Nektzwerke vonSuszeptibilitätsgenen für kardiale Hypertrophie und HerzversagenVerbund Neurodegeneration (NeuroNet): Datenintegration und Erstellung vonPhänotyp-Protein-Wirkstoff NektzwerkenVerbund Neurodegeneration (NeuroNet): Protein Interaktionsscreening durchquantitative MassenspektroskopieVerbund Neurodegeneration (NeuroNet): Protein-Protein-Interaktionsnetzwerkebei neurodegenerativen ErkrankungenVerbund Neurodegeneration (NeuroNet): Erstellung und systematische Analysevon Protein-Protein Interaktionsnetzwerken durch Yeast-2-Hybrid quantitativeProteomicsVerbund Neurodegeneration (NeuroNet): Erstellung von Genexpressionssignaturenvon neurodegenerativen KrankheitsprozessenVerbund: Neuroblastome: Transposon-basierter Mutagenese Screen in der MausVerbund: Leukämien: Die Rolle des WNT-Signalweges bei der Entstehung vonLeukämie-StammzellenVerbund Alzheimer: Die physiologische Funktion von BACE1-ist BACE1 einsicherer therapeutischer Angriffspunkt ?Systembiologie genetisch bedingter Erkrankungen: Mutanom. Erstellung undsystematische Analyse von Protein-Protein InteraktionsnetzwerkenVerbund Alzheimer: Identifikation and Charakterisierung von Modulatoren derPathogenese von Alzheimer.Verbund: Molekulare Ursachen bei Affektiven Störungen und Schizophrenie.MOODS (TP11)Aufbau der wiss. Plattform "Interaktom" für systematische Protein-InteraktionsstudienEU ProjectsFP6MEMORIES – Development, characterisation and validation of new andoriginal models for Alzheimer's Disease'MYORES – Multi-organismic Approach to study Normal and Aberrant MuscleDevelopment, Function and RepairAXON SUPPORT – Axonuclear Communication in Health and DiseaseEuReGene – European Renal Genome Project: Funktionelle Charakterisierungneuronaler K-CI-Kotransporter mit Hilfe transgener MausmodelleEuroHear – Advances in hearing science: from functional genomics totherapiesES-TRAP – A systematic approach to find new cancer molecules: an enhancer-trapscreen to identify genes required for proliferation and differentiationin murine stem cells - Förderung einer ArbeitsgruppeTranslational and Functional Onco-Genomics: from cancer-oriented genomicscreenings to new diagnostic tools and improved cancer treatmentRUBICON – Role of Ubiquitin and Ubiquitin-like Modifiers in CellularRegulationSPINE2-COMPLEXES – From receptor to gene: structures of complexes fromsignaling pathways linking immunology, neurobiology and cancerINTACT – Identification of Novel Target Genes for Cancer TherapyATTACK – Adoptive engineered T-cell Targeting to Activate Cancer KillingINNOCHEM – Innovative Chemokine-based Therapeutic Strategies forAutoimmunity and Chronic InflammationEUROSCA – European integrated project on spinocerebellar ataxias:Pathogenesis, genetics, animal models and therapyEURATools – European Rat Tools for Functional GenomicsINTHER – Development and application of transposons and site-specificintegration technologies as non-viral gene delivery methods for ex vivogene-based therapies – Research costsCIC-5 regulation and endocytosis at the renal proximal tubule or molecularbases of Dents disease (Marie Curie Intra-European Fellowship)High throughput development of drugs for immunotherapy of(auto)immune diseases (Marie Curie – Research Training Networks)EU ProjekteFP6MEMORIES - Entwicklung, Charakterisierung und Validierung von neuen undursprünglichen Modellen fuer die Krankheit AlzheimerMYORES – Multi-Organismus Ansatz , um normale und abweichendeMuskelentwicklung, Funktion und Regeneration zu untersuchenAXON SUPPORT – Axonukleare Kommunication bei Gesundheit und KrankheitEuReGene – Europäisches Nieren-Genomprojekt: FunktionelleCharakterisierung neuronaler K-CI-Kotransporter mit Hilfe transgenerMausmodelleEuroHear – Fortschritt der Wissenschaft des Hörens: von der funktionellenGenomik zu TherapienES-TRAP – Ein systematischer Ansatz, um neue Krebsmoleküle zu finden: ein„enhancer-trap screen“ zur Identifikation von Genen, die fuer Wachstum undDifferenzierung muriner Stammzellen notwendig sind - Förderung einerArbeitsgruppeTranslationale und funktionelle Onko-Genomics: von Krebs bezogenen Genom-Screenings zu neuen diagnostischen Moeglichkeiten und verbesserter Krebs-BehandlungRUBICON – Die Rolle von Ubiquitin und Ubiquitin-ähnlichen Modifikatoren inder ZellregulationSPINE2-COMPLEXES – Vom Rezeptor zum Gen: Struktur der Komplexe derSignalwege, die Immunologie, Neurobiologie und Krebs verbindenINTACT - Identifikation neuer Zielgene fuer KrebstherapieATTACK - Adaptiver technisch veränderter T-Zell Angriff zur Zerstörung vonKrebszellenINNOCHEM - Innovative Chemokin-basierte therapeutische Strategien fürAutoimmunität and chronische EntzündungenEUROSCA – Europäisches integriertes Projekt zu Spinocerebellum Ataxien:Pathogenese, Genetik, Tiermodelle und TherapieEURATools – Europaweite Ratten-Tools für funktionelle GenomikINTHER – Entwicklung und Anwendung von Transposons und Technologienfuer spezifische Integration als nicht virale Transportmethoden fuer ex-vivoGen-basierte Therapien270 Overview

CIC-5 Regulation und Endocytose am proximalen Nierenkanal oder die molekularenGrundlagen der Dents Krankheit (Marie Curie Intra-EuropäischesStipendium)Hochdurchsatz-Entwicklung von Medikamenten fuer die Immunotherapie von(Auto-) Immunerkrankungen (Marie Curie – Wissenschaftlicher Trainings-Verbund)FP7HGF/SF and MET in metastasisEVONET – Evolution of gene regulatory networks in animal developmentSET-DEV – Science, Ethics and Technological Responsibility in Developing andEmerging CountriesStart of projects in beginning of 2009PERSIST – Persisting TransgenesisINDUSTEM – Comparative stem cell research in mouse and humansFP7HGF/SF und MET in der MetastasenbildungEVONET – Die Abstammung von gen-regulierenden Netzwerken in derEntwicklung von TierenSET-DEV – Wissenschaft, Ethik und technologische Verantwortung inEntwicklungs- und SchwellenlaendernProjektbeginn zu Anfang 2009PERSIST – Ueberdauernde TransgeneseINDUSTEM – Vergleichende Wissenschaft zu Stammzellen in Maus undMenschTechnology Transfer / TechnologietransferFigures for technology transfer in 2008 / Kennzahlen zum Technologietransfer 2008Patent applications 2008 / Patentanmeldungen 2008 8Patent rights / Schutzrechtsbestand 220License agreements / Lizenzverträge (Neuabschlüsse 2008) 4License revenues / Lizenzerträge 303 T€R&D commissions (number) / FuE-Aufträge (Anzahl) 14R&D commissions (proceeds) / FuE-Aufträge (Erträge) 283 T€R&D cooperations (number) / FuE-Kooperationen (Anzahl) 222R&D cooperations (proceeds) / FuE-Kooperationen (Erträge) 229 T€In 2007 the MDC created a new internal program to stimulate technology transfer. The aim is to support projects that are reaching the point that they can be turnedinto applications and to open new career perspectives in the direction of applied research for MDC scientists. Researchers can apply for funding for their ownposition, that of a technician, and consumables for a maximum of 30,000 Euros per year, initially to run for a period of 18 months. An extension for a total of 36months is possible. Proposals should aim to open possibilities for further funding from external sources, for example under the GO-Bio program, upon the end ofMDC funding.Zur Förderung des Technologietransfers hat das MDC im Jahr 2007 ein neues internes Förderprogramm, das Pre-GO-Bio-Programm, aufgelegt. Dieses Programm dientder Förderung von anwendungsnahen Projekten und eröffnet den Wissenschaftlern und Wissenschaftlerinnen des MDC eine Karriereperspektive in Richtung angewandterForschung. Wissenschaftler und Wissenschaftlerinnen des MDC können eine Förderung für ihre eigene Stelle, eine TA-Stelle und Sachmittel bis zu 30.000 EURpro Jahr zunächst für 18 Monate erhalten. Eine Verlängerung auf insgesamt 36 Monate ist möglich. Das beantragte Projekt sollte die Möglichkeit eröffnen, imAnschluss an die MDC-Förderung über eine Drittmittelförderung, wie z.B. GO-Bio, weiter entwickelt zu werden.Shareholdings in companies / Beteiligungen an UnternehmenCompany / Unternehmen Registered office / Sitz des Unternehmens HomepageBBB Management GmbH Campus Berlin-Buch Robert-Rössle-Straße 10, 13125 Berlin www.bbb-berlin.deRZPD Deutsches Ressourcenzentrum für Genomforschung* Heubnerweg 6, 14059 Berlin www.rzpd.deHELIOS Research Center GmbH (HRC) HELIOS Klinikum Berlin, Karower Str. 11, www.helios-kliniken.deHaus 214, 13125 Berlin* The RZPD ended operations on July 31, 2007, and is in a shutting-down phase.* Das RZPD hat zum 31.07.2007 seinen Betrieb beendet und befindet sich in der Abwicklungsphase.Overview 271

Organisation chart of the Max-Delbrück-Center for Molecular Medicine Foundation under public lawScientific CommitteeChairProf. Dr. Reinhard JahnScientific CouncilChairProf. Dr. Gary LewinBoard of TrusteesChairMinDir Dr. Peter LangeBoard of DirectorsScientific DirectorProf. Dr. Walter RosenthalAdministrative DirectorCornelia LanzM O L E C U L A R M E D I C I N ECardiovascular and Metabolic Diseases Coordinator: Prof. Dr. Thomas WillnowClinical ResearchCoordinator Prof. Dr. Friedrich C. LuftBasic conceptsof cardiovascular functionProf. Dr. Thomas WillnowProf. Dr. Ingo L. MoranoProf. Dr. Michael GotthardtProf. Dr. Thomas JentschDr. Francesca SpagnoliDr. Ferdinand le NobleDr. Salim SeyfriedDr. Kai Schmidt-OttGenetics and pathophysiology ofcardiovascular diseasesProf. Dr. Nikolaus RajewskyProf. Dr. Norbert HübnerProf. Dr. Friedrich C. LuftProf. Dr. Ludwig ThierfelderDr. Markus LandthalerDr. Matthew PoyProf. Dr. Thoralf NiendorfCancer Research Coordinator: Prof. Dr. Claus ScheidereitClinical ResearchCoordinator: Prof. Dr. Peter SchlagSignaling pathways, cell biologyand tumor biologyProf. Dr. Achim LeutzProf. Dr. Claus ScheidereitProf. Dr. Clemens SchmittProf. Dr. Harald SaumweberProf. Dr. Peter M. SchlagProf. Dr. Walter BirchmeierProf. Dr. Peter DanielProf. Dr. Thomas SommerDr. Ulrike ZieboldDr. Frank RosenbauerStructural and functional genomicsProf. Dr. Udo HeinemannDr. Oliver DaumkeBioethicsDr. Christof TannertFunction and Dysfunction of the Nervous SystemCoordinators: Prof. Dr. Carmen Birchmeier-Kohler and Prof. Dr. Helmut KettenmannClinical ResearchCoordinator: Prof. Dr. Frauke ZippSignaling pathways and mechanismsin the nervous systemProf. Dr. Carmen BirchmeierProf. Dr. Jochen MeierProf. Dr. Gary LewinProf. Dr. Fritz G. RathjenDr. Inés Ibañez-TallonDr. Björn SchröderDr. Jan Erik SiemensDr. James PouletImaging the living brainProf. Dr. Frauke Zipp272 Overview

MDCMAX-DELBRÜCK-CENTRUMFÜR MOLEKULARE MEDIZINBERLIN-BUCHStaff CouncilChairIngo KahlRepresentativesWoman’s Representative: Dr. Hannelore HaaseData protection officer: Christine Rieffel-BrauneAnti-Corruption Officer: Ina HerrmannOmbudsman: Prof. Dr. Jens ReichStaff OfficesAdministrationand InfrastructureScientific CoordinationDr. Christina QuenselPersonnel DepartmentDr. Hans-J. SeehrichProf. Dr. Rainer DietzProf. Dr. Michael BaderProf. Dr. Young-Ae LeeDr. Matthias SelbachDr. Zsuzsanna IzsvákDr. Jana WolfAdministrative CoordinationDana LafuentePress / Public RelationsBarbara BachtlerFinances / ControllingAndrea WinnefeldAnimal FacilitiesDr. Karin JacobiTumor immunologyPD Dr. Martin LippProf. Dr. Thomas BlankensteinProf. Dr. Bernd DörkenProf. Dr. Antonio PezzuttoProf. Dr. Wolfgang UckertDr. Iduna FichtnerScience WritingRussel HodgeCongress OrganizationMichaela LangerInternational AffairsPamela CohenLibraryDr. Dorothea BusjahnITBernd LemkeTechnical Facility ManagementConstruction: Ralf StreckwallOperation: Michael ArnoldEU / Third Party FundsDr. Sabine BaarsPurchasing and Central ServicesWolfgang GrollPathophysiological mechanisms ofneurological and psychiatric diseasesProf. Dr. Helmut KettenmannProf. Dr. Erich WankerPhD CoordinationDr. Oksana SeumenichtSafetyDr. Regina DietlAuditing and Legal Affairs /Technology TransferChristine Rieffel-BrauneStand: November 2007Overview 273

274 IndexIndexAAbdelilah-Seyfried, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .262Adamidi, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26, 28Adams, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .208Adloff, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .265Aebi, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .105Agarkova, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13Ahlers, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78Ahrens, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151Aische, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Alenina, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48, 50Alsamah, W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Ammar, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53An, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41Anagnostopoulos, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121Anders, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36Anders, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Anderson, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17Andrade, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VI, 193, 218Andree, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33Anistan, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .214Antignac, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .262Apostel-Krause, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Arlt, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97Arslan, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79Arumughan, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183Arunachalam, V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182Asselborn, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177Attwell, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Auberson, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154Aue, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24Auer, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .166Aumann, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97, 98, 99Aydin, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32, 33Ayoub, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27BBabila, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182Babych, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117Bachmann, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25Baddack, U. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117Bader, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .III, 25, 36, 48, 261, 262Bagola, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103Bähring, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32Baldenhofer, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130Balling, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .262Baltz, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62Balueva, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150, 151Barasch, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25Barata, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Barbosa, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .218Barby, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151Barda, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163Barros, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48Bashammakh, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48, 50Bashir, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52, 53Bauer, L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97Bauerfeind, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30Baum, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60, 61Bayer, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246Becker, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91Becker, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .230Becker, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .136, 137Begay, V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90Behrens, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .136Benary, U. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60Benedetti, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179Bengtsson, L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154Benlasfer, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .184Berger, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .174Berger, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109Bergmann, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .210Bergmann, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41, 239Bergmann, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16Bergsdorff, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155, 157Bernal-Sierra, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162Bernert, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .168, 265Bernhard, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .124Besser, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .III, 72, 82, 85, 86, 244, 262Betz, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Bhattacharya, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109Bhushan, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .194Biber, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Bick-Sander, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178Biederer, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246Bielka, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245Bieschke, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .V, 148, 186Billig, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155Birchmeier, C. . . . . . . . . . . . . . . . . . . . .V, 85, 141, 143, 147, 150, 245, 262Birchmeier, W. . . . . . . . . . . . . . . III, VIII, XIX, 67, 68, 69, 82, 84, 85, 89,97, 98, 203, 237, 244, 245, 262Bit-Avragim, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18, 19Blachut, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31Blankenburg, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .114Blankenstein, T. . . . . . . . . . . . . . . . . . . . .IV, 75, 76, 77, 122, 130, 131, 262Blaschke, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38, 40, 41, 42, 44Blendinger, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .88, 89, 265Blumenthal-Barby, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .126Böddrich, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185Bohl, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .212Böhm, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109Böhmer, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Bölling, O. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97Bontscho, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .210Borgwald, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Born, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31Borst, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246Botchan, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .196Böttger, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Bounab, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183Boyé, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48, 212Brakebusch, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152Brand, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112Brand, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Brandenburg, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163, 172Braunschweig, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151Brecht, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Breiderhoff, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Breithardt, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .260, 262Brembeck, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82Brendebach, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103Briesemeister, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Briscoe, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150Bröhl, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150, 151

Index 275Bröske, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94Buchert, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151Budczies, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82Bulaj, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246Bulavina, L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179Bunse, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125Burbach, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100Burgert, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Burock, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .98, 99Buschow, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Buttgereit, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38, 48, 50Buxbaum, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250CCaglayan, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Calegari, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246Callen, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246Cano, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84Cardoso, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48Cardoso, Cr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .234Carl, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .208Carlo, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Cerda-Esteban, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Chang, Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198Chap, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .88, 89Charo, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122Chaurasia, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183, 239Chen, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16, 17Chen, J. . . . . . . . . . . . . . . . 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. . . . . . . .97Förster, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96, 97Förstera, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .168Fortin, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Foulle, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182Frahm, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .166Frauenrath, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46Frenzel, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163, 164Freyert, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155Friedlaender, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26, 27, 28Friedrich, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91Friedrich, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182, 186Fritschy, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246Fritzmann, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82, 96Fritzsche, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183Fröhler, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198Fröhlich, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112Fuhrmann, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154Fujita, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Futschik, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183, 239GGajera, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10, 11Gan, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106Gao, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112Garcia, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155Garratt, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85, 150Gärtner, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Gebhardt, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .132Gebhardt, R. . . . . . . . . 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.127Gerull, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42Ghadge, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Ghani, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94Giang, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Giering, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56, 61, 88, 94, 135Giese, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .170, 246Gillissen, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .132Gimenez, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54Glahs, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106Glass, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178Gloy, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209Glumm, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177Gohlke, U. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .108Gojowy, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177Goldbrich, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17Gollasch, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VI, 32, 214Goncalves, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Goody, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .260, 262Görlitz, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155Gösele, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30Gossen, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .244Gotthardt, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .II, 3, 16, 263Gottschling, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151Götz, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .247, 260, 262Govindarajan, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16Grabundzija, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52, 53Grähning, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177Gramlich, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42Gratze, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36Graubmann, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94Green, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Greengard, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33Gregersen, L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62Grelle, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .186Grieben, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Gries, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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Index 277Gross, V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32, 33Große, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97Grosse, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250Grosskopf, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85Großmann, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82, 85, 151Grüger, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Grunz, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31Grüters-Kieslich, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .207, 260, 262Grzela, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52Guillemot, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .247Guimaraes, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48Gunsalus, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28Günther, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177Günther, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135Gupta, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83Gupta, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134Guzman, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250HHaag, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97Haas, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109Haase, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12, 14, 245, 265Haberland, L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .114Haensch, W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96Haink, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56, 61, 88, 94Hamati, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Hammes, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10, IIHamscho, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91Hanack, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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. . . . . . . . . . . . . . .109Knespel, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26, 27, 265Knobelsdorff-Brenkenhoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .212Knoblauch, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .208Knoblich, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91Knoke, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155Knust, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .261, 262Kobelt, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97, 98, 99Koch-Unterseher, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .260Koch, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79Krause, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117, 230Krauss, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Kreher, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .126Krek, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26, 27Kressmann, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .127Kretschel, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Kretschmer, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62Krishtal, O. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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Index 279Kur, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Kurths, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109Kurzhals, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .166Kutschbach, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177Küttner, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125Kutza, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Kvakan, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36LLalowski, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182Lamounier-Zepter, V. . . . . . . . . . . . . . . . . . . . . . . . . . . . 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130Nguyen, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36Nguyen, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .131Nickel, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43Nicotera, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .248Niendorf, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .III, XIV, 46, 205, 238, 264Niesner, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176, 239Nitschke, U. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .127Noack, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Nolte, C. . . . . . . . . . . . . . . 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Index 281Piano, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28Pichorner, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97Pietzke, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .196Pifferi, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162, 164Pisch, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185Pischon, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .251Piske, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179Plans, V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154Plaßmann, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .184Pless, O. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90, 92Pofahl, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Pohl, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Pohlmann, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46Politi, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60, 61Polzin, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213Pongrac, I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Ponomarenko, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .251Poole, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162, 163Popova, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49, 50Popovic, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Poppe, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20Porras-Millan, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183Posch, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Poulet, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .V, 142, 148, 174, 238Poustka, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .261, 262Poy, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .III, 58, 236, 264Prat, A. . . . . . . . . . . . . . . . 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.6Schmitt, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52Schmitt, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .IV, 71, 100, 204Schmolka, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130, 131Schmollinger, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122Schmude, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91Schneggenburger, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .248Schneider, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209Schneider, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151Schnieders, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .261Schnögl, S. . 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. . . . .210Schroeder, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .V, 142, 146, 235Schroeder, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .170, 264Schugardt, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .184Schultze-Motel, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .184Schulz-Menger, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VI, 212Schulz, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54Schulz, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30, 85Schulz, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137Schulze-Topphoff, U. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176Schulze, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209Schulze, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52, 53Schulze, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .108Schulze, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .116Schunck, W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32Schunk, W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36Schütz, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .102, 108, 264Schütz, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .248, 252Schwanhäusser, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56Schwartz, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195Schwartze, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .236, 262Schwarz, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54Schwede, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117, 120Schwefel, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112Schweizer, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .106Scudiero, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .98Seeger-Zografakis, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179Seeger, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177Seguel, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Seidler, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155Seifert, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .179Seifferth, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159Seipold, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18Seja, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155, 157Selbach, M. . . . . . . . . . . . . . . . . . . .III, X, 5, 28, 32, 56, 85, 193, 261, 264Selby, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .98Seliger, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .248Seumenicht, O. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245Seyfried, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4, 18, IIShah, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112Shah, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183, 186Shi, Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16, 17, 20Shmidt, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49, 50Shoemaker, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .98Shu, X. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Sibilak, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91Siegel, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .98Siegert, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12, 13Siemens, J. . . . . . . . . . . . . . . . . . . . . . . . . . .V, XIX, 142, 147, 172, 239, 264Sievers, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159Siffrin, V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176Sigrist, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .248Sihn, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48, 49, 50Simsek, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12, 13Singer, E. . . . . . . . . . . . . . . . . . . . . . . . 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Index 283Smink, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90Smith, E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162, 165Smith, J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97Socci, N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .252Sommer, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24Sommer, C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56Sommer, T. . . . . . . . . . . . . . . . . . . . . . . . . .IV, 72, 102, 236, 244, 245, 264Sommermayer, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .124, 125Sonderegger, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .248Song, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .172Spading, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185Spagnoli, F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .II, XIX, 22, 236, 239, 264Spallek, B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Specowiak, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123Spitzl, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Spitzmaul, G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154, 157Spohr, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159Sporbert, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VI, 224Spricenatu, R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .220Spuler, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VI, 204, 208Stainier, D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18Stallerow, P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151Stanick, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .222Stärck, L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .124Starcke, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134Stauber, T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154Stauss, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .125Stecklum, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137Stein, H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121, 126Stein, S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83Stein, U. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82, 96, 97, 98, 99Stein, V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .157Stelzl, U. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .239Sternjak, A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134Stilmann, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79Stock, K. . . . . . . . . . . . . . . . . . . . . . . . . . . . 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