Annual Report of Activities CNC 2008 - Center for Neuroscience and ...

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Glutamatergic synapses | Head: Ana Luisa CarvalhoObjectivesWe are interested in understanding the connectionsbetween nerve cells in the brain, and how they aremodified with experience. The ability of synapses tochange their strength is thought to be the cellularcorrelate of learning and memory, and synapticdysfunction is correlated with several neurodegenerativediseases. We focus on excitatory glutamatergic synapses,and study their regulation from a cellular and molecularbiology viewpoint. We use a combination of primaryneuronal cultures, molecular cell biology andbiochemistry to address these questions.1. Proteomic analysis of the interactome of AMPAtypeglutamate receptors (AMPAR). AMPARbinding partners regulate their synaptic targeting. Weidentified several novel interactors for AMPARsubunits in a proteomic screening, and are currentlyaddressing their function in regulating AMPARs.2. Regulation of the mRNA stability of the mRNAfor GluR1 AMPAR subunit. We found evidence forpost‐transcriptional regulation of the mRNA levels forGluR1, and are addressing its mechanisms.3. Mechanisms of synaptic traffic of NMDAreceptors (NMDAR). NMDARs play a role in theinduction of synaptic plasticity. We use culturedneurons from knock‐out mice for NMDARsubunits to understand subunit‐specific rules thatgovern synaptic targeting of NMDARs.4. Regulation of glutamatergic transmission byghrelin in the hippocampus. Ghrelin is anappetite‐stimulating hormone which was shown toenhance memory processes and synaptic plasticityin the hippocampus. We are characterizing thesubcellular localization of ghrelin receptors in thehippocampus, and evaluating how ghrelinreceptors affect glutamatergic transmission.5. Synaptic dysfunction in a mouse model ofMachado‐Joseph disease (MJD). MJD is aspinocerebellar ataxia caused by a glutamineexpansion in ataxin‐3, an ubiquitin protease. Weare using a transgenic mouse model of MJD toaddress the role of excitotoxicity and synapticdysfunction in the pathogenesis of this disease.Main AchievementsProteins that interact with AMPA‐type glutamatereceptors (AMPAR) are crucial for appropriatetargeting of receptors to synapses (Santos et al. 2009).We have performed a proteomic screening forbinding partners of Ca 2+ ‐permeable AMPARs. Afterre‐isolating known AMPAR interactors, we identifiednovel interactors, such as motor proteins and proteinsof the neuronal RNA granules. Moreover, we isolatedthe cell adhesion molecule Contactin associatedprotein 1 (Caspr1). Functional analyses revealed thatCaspr1 affects the cell surface and synaptic expressionof AMPAR subunits in cultured hippocampalneurons (Manuscripts in preparation).NMDA receptors (NMDAR) are the coincidencedetector in the induction of synaptic plasticity. We areaddressing the mechanism of synaptic accumulationof NMDARs, and found that the NR2B subunit ofNMDARs is necessary to establish the synapticscaffold for NMDARs, since synaptic clusters ofNMDARs are dramatically reduced in primaryhippocampal neuronal cultures from NR2B knock‐outmice. Engineered subunits of the NMDAR complexare being reintroduced into neuron cultures fromknockout mice, to dissect molecular signals involvedin NMDAR trafficking.The number of dendritic spines, where excitatorysynapses are located, is regulated by molecules thatorganize their actin cytoskeleton, e.g. cortactin. Weare studying how cortactin acetylation influences itsrole in the morphogenesis of spines, and found thatoverexpression of the acetylation and deacetylationmimetics of cortactin changes the density of synapsesin hippocampal neurons in culture (Fig.1).Fig.1. Hippocampal neurons in culture transfected at 7 DIVwith GFP and cortactin, and labeled at 15 DIV for a marker ofexcitatory synapses (PSD95), and a somatodendritic marker(MAP2). Expression of cortactin changes the density ofsynapses in cultured hippocampal neurons.Another interest in the group is to study dysfunctionof glutamatergic transmission in pathology. We areinterested in ataxin‐3, the polyQ‐containing proteaseinvolved in Machado‐Joseph disease (MJD). We havecharacterized its nucleocytoplasmic shuttling activity(Macedo‐Ribeiro et al. 2009), and will now test thehypothesis that there are alterations in glutamatergicsynapses in MJD.22
Neuroprotection and Neurogenesis in Brain Repair | Head: João MalvaObjectivesThe research activity of the “Neuroprotection andNeurogenesis in Brain Repair” group is foundedon four main research pillars: 1‐ Neuromodulation;2‐ Neuroprotection; 3‐ Neuroinflammation; 4‐Neural stem cells and brain repair. This researcheffort aims at finding novel molecular and cellulartargets to prevent the progression of brain damageand to treat brain diseases.proven to be a valuable tool for drug discovery and tofunctionally evaluate the quality of differentiatedstem cell cultures for pre‐transplantation procedures.With this method we are able to functionallycharacterize a diversity of cells in the same culturesincluding: immature cell, astrocytes,oligodendrocytes, progenitors and neurons. Twopatents were produced (WO2008100168‐A1,PAT2008100089504/0198).In 2008 we focused particularly on the impact ofneuroinflammation in neural stem cell fate. Themain objective of the research group has been thedevelopment of new competences and new tools tostudy neuroprotective and neuroregenerativepathways for brain repair purposes.Specific objectives included:1) To establish a novel single cell calcium imaging‐basedtool to functionally identify different neural phenotypesdifferentiating from neural stem cell cultures2) To identify novel proneurogenic and prooligodendrogenicfactors as well as new drugsaffecting neural stem cellphysiology/differentiation useful for stem cellbasedbrain repair strategies223) To develop new pharmacological procedures to treatneural stem cell cultures in order to boostdifferentiation of specific neural stem cell phenotypesin central nervous system disease models. Thesemodels included: temporal lobe epilepsy;methamphetamine‐induced neural damage;demyelinating diseases; retinal excitotoxic injury.At the long‐term, we aim to contribute with uniquetools to discover new molecular and cellular targets tobetter understand neural stem cell biology.Ultimately, these tools will allow the identification ofnew targets for drug discovery and new pretransplantationprocedures of neural stem cells into tothe diseased brain.Main AchievementsWe developed a novel robust method useful toidentify the phenotype of living cells differentiatingfrom neural stem cell cultures. This method is basedin single cell calcium imaging technology and allowsthe simultaneous identification of more than 100 cellsin neural stem cell populations, matching uniquefunctional profile of responses with the phenotypicmarkers of neural cell lineages. This method hasWe could identify the proneurogenic effect ofneuropeptide Y (NPY) and tumor necrosis factoralpha (TNFalpha) in neural stem cell cultures derivedfrom the subventricular zone of the mice (SVZ).Interestingly, according to previous findings of thegroup in organotypic brain slice cultures, we foundthat TNFalpha exerts ambiguous effects in SVZcultures. At low concentrations TNFalpha inducesproliferation and differentiation of neurons (involvingJNK activation and axonogenesis) whereas at highconcentrations this cytokine is mainly toxic. Thesedata reinforces the double effect ofneuroinflammation in brain repair, depending on themagnitude of the inflammatory response to braininjury. Moreover, we also determined that NPY is apotent proliferative and proneurogenic peptide in theSVZ stem cell niche. NPY Y1 receptor is expressedand functionally active in the adult mice SVZ and itsactivation causes phenotypic and functional neuronaldifferentiation. These projects resulted in thepublication of three major manuscripts (one inRejuvenation Research; two in Stem Cells).
Page 7 and 8: General ObjectivesThe CNC major mis
Page 11 and 12: OrganizationThe Center for Neurosci
Page 13: Microbiology | Milton CostaMicrobio
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Page 28 and 29: PublicationsAgasse F, Bernardino L,
Page 30 and 31: Santiago AR, Carvalho, CM, Carvalho
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Page 40 and 41: PublicationsAlves S, Nascimento‐F
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Page 45 and 46: Mitochondrial Toxicology and Pharma
Page 47: Pharmacometrics GroupAmílcar Falc
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Page 52 and 53: Pharmacometrics | Head: Amílcar Fa
Page 54 and 55: Correia S, Carvalho C, Santos MS, P
Page 57 and 58: Area D | MicrobiologyCoordinator |
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Page 65 and 66: Area E | Biophysics and Biomedical
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Page 71 and 72: Cell Biophysics |Head: Luís Martin
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Sobral AJFN, Justino LLG, Santos AC
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Future PlansThere is an enormous we
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Paula MotaSara M. Diniz Martins Lop
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Biology of Reproduction and Human F
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Insulin Resistance and Adipocyte |
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Biomedical Inter‐Institutional Re
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3. Pediatric Research: metabolic di
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PublicationsSantos MJ, Cleto S, Men
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7. Research in brain cancer: geneti
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Grafting SVZ neural stem cell cultu
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Structure‐function analysis of th
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Anticancer Effects of of Phytochemi
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Investigaciones Biomédicas “Albe
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Participation in the organization o
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May 2008Member of the organizing co
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Genome BiologyFebruary 25 ‐ 27Isa
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Seminars2008 Series | CNC Audithori
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13.6.2008Cells caught in the act: M
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5.12.2008Unexpected fate and functi
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Elisabete Ferreiro“Cross‐talk b
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Ana Isabel Vicente Rafael“Estudos
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Rosete Pais“Papilomavirus humano
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FLOW CYTOMETRY UNITHead of Unit: Is
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MASS SPECTROMETRY UNITHead of Unit:
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Amino Acid AnalysisOur laboratory r
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2. Areas of Expertise / Research /
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Multiple SclerosisAn extension of t
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2.2. Centre for Bioavailability Stu
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StaffCoordinatorTice Macedo, MD, Ph
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Compatible solutes from extremophil
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Proteases aspárticas secretadas em
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Mecanismos de plasticidade sinápti
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Clivagem dos transportadores vesicu
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ʺBIOINK ‐ Aprendizagem increment
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Henrique Faneca (Auxiliar Inv., CNC
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Liliana Bernardino 100Luis Miguel E
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João Teixeira 100João Teodoro 100
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Patricia Henriques Domingues 100Pat
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ADMINISTRATIVE STAFFTime % at CNCAr
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Sandra Isabel M. Cardoso (Auxiliar
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Molecular Biotechnology and HealthE
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Cell and Molecular ToxicologyLeonor
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MicrobiologyMilton Costa, PhD, Coor
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Paulo Gameiro Guerreiro 100Pedro Co
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Marta Isabel Rodrigues Baptista 100
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Url: http://www.cnbc.pt | Email: in
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