Keynote Conference - Interevent

Symp#11 Glia Club
Chairs Bernardo Castellano and Vivaldo Moura Neto
GSK3β is a Profound Negative Regulator of Oligodendrocyte Differentiation and Myelination
Arthur M. Butt, Andrea Rivera and Kasum Azim
Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, U.K.
Oligodendrocytes are the myelinating cells of the central nervous system and are the primary targets of tissue destruction in the
demyelinating disease multiple sclerosis. The enzyme GSK3β is a target of many receptor-mediated signalling pathways that regulate the
differentiation of from their precursors (OPCs). We have examined this using a range of small molecular inhibitors of GSK3β in the mouse
brain. Inhibition of GSK3β stimulates the generation of OPCs from neural precursor cells (NPCs) in the subventricular zone (SVZ), involving the
canonical Wnt-β-catenin signalling pathway. Significantly, inhibition of GSK3β also dramatically stimulates oligodendrocyte differentiation and
myelination. A key finding is that GSK3β inhibition has equivalent effects in the adult and stimulates the regeneration of oligodendrocytes and
remyelination following demyelination in the adult forebrain. Using a genome wide microarray approach, we find that GSK3β inhibition
regulates oligodendrocyte generation via multiple positive and negative regulatory signalling pathways. GSK3β inhibition significantly upregulated
Sox10 and Olig2, key positive regulators of oligodendrocyte differentiation, and down-regulated the Wnt and Notch signalling
pathways, together with helix-loop-helix ID (inhibitor of differentiation), which are dominant negative regulators of oligodendrocyte
differentiation. This study identifies novel functions for GSK3β as a profound negative regulator of oligodendrocytes at all stages of their
differentiation in vivo. Moreover, our findings indicated that GSK3β signalling pathways contributed to inefficient regeneration of
oligodendrocytes and myelin repair in demyelination.
Role of Cancer Stem Cells in Adult and Paediatric Brain Neoplasms: Hoax or Holy Grail?
Professor Geoffrey J Pilkington BSc PhD CBiol FSB FRCPath
Professor of Cellular and Molecular Neuro-oncology, University of Portsmouth, St Michael’s Building, White Swan Road Portsmouth PO1 2DT
UK
In the 1970s stem cell-like populations were described in ethylnitrosourea-induced rat glioma and hypothesised as the origin of such tumours.
These were located around blood vessels and degenerating neurones, sub-ependymally, at the lateral ventricles. More recently cancer stem
cells (CSCs), reported largely on their expression of CD133, gathered considerable interest in adult and paediatric brain tumours. Initial
observations that the CD133+ population were the initiators of brain tumours, based upon their ability to produce tumours in xenograft
models while CD133- failed to do so have, however, been challenged. The value of CD133 (Prominin-1) has therefore been questioned but the
function of CD133 or its possible link with processes underlying tumour development and progression remains obscure. Adult glioblastoma
biopsies yield only 1-5% CSCs based upon CD133 immunostaining. We described a low passage paediatric glioblastoma culture where over
40% of the cells express CD133 and, if transferred to neural basal medium under hypoxic conditions, this elevated to >95% CD133+. Magnetic
bead immunoseparated CD133+ and CD133- were used for genetic profiling, adhesion, invasion, and proliferation assays in response to
nuclear- and mitochondrially-acting pro-apoptotic agents. We also investigated the significance of CD133 glycosylation and influence of oxygen
on such glycosylation. Whether or not CD133 is a marker of CSCs in brain tumours remains controversial but it may be of significance to the
biology of paediatric brain neoplasms and requires further investigation both in vitro and in tissue sections. Hoax? certainly not, but CD133
must be put into context and, while the Holy Grail issue remains unresolved, this is an area of intense interest in unravelling the mysteries of
tumour biology.
Mesenchymal Stem Cells lower proliferation and invasion of Glioblastoma cells, exploiting the Immune Response Mediating Chemokines
Tamara T. Lah, Motaln H 1 , Gruden K 2 , Hren M 2 , Primon M 1 and Schichor Ch 3
1. Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia. 2 Department of Biotechnology and
Systems Biology, National Institute of Biology, Ljubljana, Slovenia. 3 Klinikum Großhadern, Ludwig-Maximilians-Universität, München,
Germany.
Human mesenchymal stem cells (hMSC) are gaining the forefront position in therapies for curing several diseases. The majority of them is
focused on the improvements in the regenerative medicine, whereas only limited number of studies is addressing the potential use of hMSC
for anti-cancer therapy, although the findings in several different cancer models are suggesting they could be developed into efficient cellbased
therapeutics.
Considering recent in vivo and in vitro reports on hMSCs growth inhibitory effect on most malignant brain tumor – glioblastoma multiforme
(GBM) we focused here on the cellular processes responsible for this inhibitory effect, such as cell proliferation, invasion and senescence
which we confirmed in several GBM lines. We performed whole genome mRNA analysis and cytokine profiling of both, the hMSC and the U87-
MG GBM cells grown in co-cultures. We found that several chemokines may account either for the decrease of both, proliferation and invasion
of U87-MG, as well as for induced MSCs proliferation and invasion when the two cells were grown in the indirect co-cultures. CCL2/MCP-1 was
collectively identified as one of the few most significantly up-regulated chemokine responsible for hMSC and U87-MG paracrine signaling and
we functionally confirmed its role in GBM cell invasion in vitro.
In conclusion, our results indicate the CCL2/MCP1 to be the key player of paracrine MSC/glioma cell interactions. Several other gene/protein
putative markers were identified to take part in hMSC/glioma cell communication for the first time. Together with CCL2/MCP-1 those markers
could be utilized in future, as target genes for cell-based anticancer therapy.
Effects of cns-targeted il-6 or il-10 production on microglial activation and motor neuron degeneration after facial nerve
axotomy
B. Castellano 1, N. Villacampa 1, B. Almolda 1, I.L. Campbell 2 and B. González 1
1Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences. Universitat Autònoma de Barcelona, Spain. 2School of
Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia
Interleukin-6 (IL-6) and Interleukin-10 (IL-10) are key cytokines with an important role in the regulation of the inflammatory and immune
responses. In the central nervous system (CNS), increased expression of both IL-6 and IL-10 occurs in a wide range of pathological conditions.
Meanwhile IL-6 has been usually recognized as a cytokine with a dual role acting as a pro-inflammatory or anti-inflammatory signal inducing glial
activation while IL-10 is mainly involved counteracting the inflammatory response and immune reactions. The objective of the present study was
to evaluate the effects of local production of either IL-6 or IL-10 in the microglial response, lymphocyte infiltration and neuronal degeneration
induced by transection of the facial nerve, a sterile neuronal injury model. Facial nerve axotomy (FNA) was performed in transgenic mice with
astrocyte-targeted production of either IL-6 (GFAP-IL6Tg) or IL-10 (GFAP-IL10Tg) and their corresponding wild-type (WT) littermates. The
analysis was performed by histology, immunohistochemistry and flow cytometry at different time points, ranging from 3 to 42 days post-lesion.
Our observations clearly indicate that GFAP targeted expression of either IL-6 or IL-10 exerts a direct impact on the pattern of microglial
activation and neuronal degeneration/survival or axotomized motor neurons. As will be discussed, changes observed in the expression of
different molecules such as Iba1, CD11b, CD16/32, MHC class II and some integrins like osteopontin and their receptors (CD44 and �5) may be
involved in the differential glial activation pattern and lymphocyte recruitment producing a specific outcome of facial nerve axotomy.
Supportedby Ministerio de Ciencia e Innovación (BFU2008-04407/BFI) (BFU2011-27400) and NH&MRC grant 632754
63