京都大学 / 新制・課程博士 / 博士(医学) / 甲第25006号 / 医博第5040号 / 新制||医||1070(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 荒川 芳輝, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumor that mainly affects adults. GBM displays remarkable intra- and inter-tumoral heterogeneity and contains a subpopulation of cells named glioma stem cells that is believed to be responsible for tumor maintenance, progression and recurrence. We have established and characterized a biobank of 48 cell lines derived from GBM patients. The cells were explanted and maintained as adherent cultures in serum-free, defined neural stem cell medium. These GBM cells (GCs) displayed NSC marker expression in vitro, had orthotopic tumor initiating capability in vivo, harboured genomic alterations characteristic of GBM and represented all four TCGA molecular subtypes. Our newly established biobank is also connected with a database (www.hgcc.se) that provides all molecular and clinical data. This resource provides a valuable platform of valid in vitro and in vivo models for basic GBM research and drug discovery. By using RCAS/tv-a mouse models for glioma, we found that GBMs originating from a putative NSC origin caused more tumorigenic GCs that had higher self-renewal abilities than those originating from putative glial precursor cell origin. By transcriptome analysis a mouse cell origin (MCO) gene signature was generated to cluster human GCs and GBM tissue samples and a functional relationship between the differentiation state of the initially transformed cell and the phenotype of GCs was discovered, which provides the basis for a new predictive MCO-based patient classification. LGR5 was found to be highly expressed in the most malignant mouse GC lines of putative NSC origin and also enriched in proneural GBMs characterized by PDGFRA alterations and OLIG2 up-regulation. By overexpressing or depleting LGR5 we discovered that high LGR5 expression in proneural GC lines increased the tumorigenicity, self-renewal and invasive capacities of the cells and could potentiate WNT signalling through its ligand RSPO1. Through transcriptome analysis we identified the candidate genes CCND2, PDGFRA, OLIG2, DKK1 that were found to be regulated by LGR5. In the last study, we found that mouse OPCs could initiate both astrocytic and oligdendroglial gliomas, which indicated that oncogenic signalling is dominant to cell of origin in affecting the histology of gliomas.

Calcium-influx through ionotropic glutamate receptors expressed on non-excitable cells, such as CNS glia, may regulate important cell events via intracellular signaling mechanisms. Oligodendrocytes and oligodendrocyte progenitors (OPCs), two glial populations supporting CNS myelination and myelin repair, express AMPA and NMDA receptors. Although calcium-influx through these receptors is thought to cause glutamate excitotoxicity to oligodendrocytes in CNS injuries, more recent studies suggest that AMPA or NMDA receptor-mediated synaptic transmission between neurons and OPCs plays a positive role in neuronal activity-dependent oligodendrocyte development and regeneration. Given the opposing roles of glutamate receptors in oligodendrocyte death and repair, the clinical relevance of these receptors in white matter injuries remain unclear. Another major challenge for exploring the role of these receptors in white matter injuries is that OPCs and neurons express a similar complement of AMPA and NMDA receptor subunits, which has complicated the interpretation of pharmacological manipulations and global genetic deletion approaches. To define the cell autonomous role of AMPA and NMDA receptor-mediated calcium signaling in oligodendroglia, I abolished the calcium influx through glutamate receptors using two different genetic approaches, and examined their impacts on oligodendrocyte development, injury-induced cell death, and regeneration. First, I employed a new mouse line which allows overexpression of GluA2, the calcium-impermeable AMPA receptor subunit, in a Cre activity-dependent manner. After crossing these mice with OPC- or oligodendrocyte-lineage-specific Cre mice, I applied hypoxic-ischemic injury to these multiple transgenic mice. Surprisingly, even though AMPA receptor-mediated calcium influx was blocked in OPCs, oligodendrogenesis or myelin integrity was not affected. However, GluA2 overexpression significantly promoted oligodendrocyte regeneration and OPC proliferation after injury, while the same manipulation in oligodendrocytes did not protect them from the initial cell loss. Moreover, GluA2 overexpression also stimulated transcriptional activities linked to myelinogenesis, even without injury. Second, I used conditional knockout mice for Grin1, the gene encoding an essential subunit of NMDA receptor complexes. As with GluA2 overexpressing mice, the removal of NMDA receptors from OPCs or all oligodendroglia did not significantly change normal oligodendrocyte development. However, the ablation of NMDA receptor in OPCs exacerbated oligodendrocyte loss by impairing new oligodendrogenesis in hypoxic-ischemic injury. These results suggest that neither AMPA receptors nor NMDA receptors mediate glutamate excitotoxicity in oligodendrocytes in neonatal hypoxic-ischemic injury. Instead, these receptors play distinct roles in post-injury oligodendrocyte development: AMPA receptor-mediated calcium suppresses oligodendrocyte regeneration, and NMDA receptor signaling supports oligodendrocyte regeneration after injury. / Biomedical Sciences

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Methyl-CpG-binding protein 2 (MeCP2 is) is an epigenetic regulator that binds to methylated DNA. Initially identified as transcriptional repressor, MeCP2 also binds to different proteins functioning as gene activator. Importantly, MecCP2 gene mutations and changes in MeCP2 levels are associated to several forms of mental retardation and autism-related disorders; including Rett, a neurodevelopmental disorder affecting primarily girls. While brain MeCP2 was considered to be exclusively neuronal, this regulator is also present in glia. We found that oligodendrocytes, the myelinating cells of the central nervous system (CNS), express particularly high MeCP2 levels at a developmental stage that precedes their final maturation. Moreover, downregulation of MeCP2 levels by treatment of immature oligodendrocytes with small interference RNA (siRNA), reduced the expression of 14 kDa myelin basic protein (MBP) and MOG, two markers of mature oligodendrocytes. These observations raise the possibility that oligodendrocytes have a direct participation in Rett syndrome and other autism-related disorders.

No description available.