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

La sclérose en plaques (SEP) est une maladie démyélinisante dont les causes ne sont pas encore bien élucidées. Cependant, l’implication des réponses auto-immunes dans la mort des oligodendrocytes engendrant la destruction des gaines de myéline et le disfonctionnement axonal est bien documentée. Les thérapies actuelles utilisant des agents anti-inflammatoires et immunomodulateurs ciblent la réduction de l’inflammation et la progression de la maladie, mais leur efficacité reste limitée et décroit après un long traitement. Toutefois, une nouvelle stratégie de traitement, basée sur la capacité endogène du cerveau à réparer la myéline, commence à voir le jour.L’utilisation des hormones stéroïdes offre une grande opportunité, compte tenu de leurs actions pléiotropiques à la fois au cours du développement et chez les sujets adultes. Notre étude montre que les androgènes et les progéstagènes jouent un rôle très important dans la prolifération des oligodendrocytes et dans la réparation de la myéline. Ces stéroïdes permettent une remyélinisation au niveau des cultures organotypiques du cervelet de souris ou de rats après une démyélinisation par la lysolécithine. En outre, l’utilisation des souris knock-out pour le récepteur de la progestérone (PR-KO) montre l’importance de ce récepteur dans l’effet promyélinisant de la progestérone. De même, l’effet pro-remyélinisant des androgènes passe par l’intermédiaire de leur récepteur nucléaire (AR) puisque la Flutamide, agent antagonisant ces récepteurs, aboli complétement cette action. De même, l’utilisation des souris knock-out pour le récepteur de la progestérone montre l’importance de ce récepteur dans l’effet promyélinisant de la progestérone.L’influence de la testostérone et des ces métabolites sur la réparation de la myéline au niveau du corps calleux est aussi montrée in vivo, en traitant les souris C57Bl/6 par la cuprizone pendant 12 à 14 semaines. Le traitement de ces souris par la testostérone ou ces métabolites 5α- dihydrotestosterone (5α-DHT), 17β-estradiol ou par un agoniste synthétique fort, le 7α-methyl-19-nortestosterone (MENT) pendant 6 semaines, induit un remarquable recrutement des progéniteurs d’oligodendrocytes, suivie par une importante remyélinisation des zones démyélinisées. Le mécanisme d’action de ces androgènes implique le récepteur AR puisque aucun effet promyélinisant n’a été observé chez les souris dont l’AR est muté (tfm : testicular feminization mutation) et les souris ARNes/cre (mutation conditionnelle de l’AR dans les neurones et les cellules macrogliales). Les souris ArKO (Aromatse Knoctout) ne pouvant pas convertir la testostérone en estradiol sont aussi insensibles au traitement par la testérone.Ces travaux montrent que les stéroïdes jouent un rôle très important dans la remyélinisation in vitro et in vivo, fournissant une preuve expérimentale pour une utilisation des stéroïdes dans des essais cliniques futurs visant à réparer la myéline. / Multiple sclerosis (MS) is a very prominent demyelinating disease. The cause of demyelination in MS is not clear, however, it involves autoimmune responses and the death of oligodendrocytes accompanied by myelin destruction and axonal dysfunction. Currently available therapies including anti-inflammatory agents and immunomodulators are targeted to reduce inflammation and disease progression but their limited efficacy further decrease after prolonged treatment.However, another therapeutic strategy has gained recently much interest, is to boost the endogenous capacity of the brain to repair myelin.Steroid hormones offer an opportunity for therapeutic interventions in a wide range of tissue abnormalities because of their multiple actions during development and in adulthood. Our studies show that androgens and progestagens play pivotal role in oligodendrocyte proliferation and subsequent myelination. Androgens or progestagens promote remyelination after lysolecithin mediated myelin insult of organotypic cerebellar slices in culture. Moreover, remyelinating effects of testosterone can be blocked by flutamide, an androgen receptor (AR) inhibitor. Also, the remyelination induced by progestagens is abolished when cerebellar slices are used from progesterone receptors (PR) knockout mice.The influence of testosterone and its metabolites on myelin repair was also evident in toxininduced demyelination in vivo. Long-term cuprizone intoxication (12-14 weeks) of adult C57Bl/6 mice caused chronic and severe demyelination in the corpus callosum. Treatment of these mice with testosterone or its metabolites, particularly 5α-dihydrotestosterone (5α-DHT), estradiol-17β and potent testosterone analog 7α-methyl-19-nortestosterone (MENT) for 6 weeks results in a marked replenishment of the corpus callosum with oligodendrocytes and remyelination. Testosterone fails to stimulate remyelination in mice carrying testicular feminization mutation (Tfm) of AR in the cuprizone model. Furthermore, we demonstrate that testosterone directly targets neuronal and macroglial AR, because the specific ablation of neural AR in (ARNes/Cre) mice prevents the myelin repair in response to testosterone. Interestingly, blocking the conversion of testosterone into estrogens by knocking out the aromatase gene (ArKO mice), also impair the remyelinating effect of testosterone.In conclusion, we provide a strong evidence for a new role of progestagens and androgens in remyelination and thus present a sound experimental support for future clinical trials based on steroid hormone therapy for demyelinating disorders.

Les mutations de la protéolipide-protéine (PLP) entraînent la mort des oligodendrocytes (OL) et les pathologies de la myéline. Pour contribuer à l'élucidation des mécanismes impliqués, ce travail de thèse, qui a utilisé des lignées d'OL 158N (normale) et 158JP (porteuse de PLP mutée), démontre une mortalité élevée des cultures 158JP comparées aux témoins. Une hausse du ratio Bax (pro-)/Bcl2 (anti-apoptose) est observée chez les 158JP. La protéine BiP, marqueur de stress du réticulum endoplasmique (SRE), est surexprimée chez les 158JP. L'exposition au SRE induit par la tunicamycine a révélé que la DE50 pour les 158JP est 67 fois plus faible que la DE50 pour les 158N. Les 158JP surexpriment aussi les protéines CHOP et caspase-12 qui déterminent le basculement des processus intracellulaires vers l'apoptose. Le 4-Phénylbutyrate, inhibiteur du SRE, améliore la survie des 158JP et diminue les marqueurs du SRE et de l'apoptose. Des perspectives intéressantes sont ouvertes pour l'exploration de stratégies efficaces contre les pathologies dysmyélinisantes. / Mutations of proteolipid-protein (PLP) cause oligodendrocyte (OL) death and myelin disorders. To contribute to the elucidation of the mechanisms involved, the present PhD work has used 158N (normal) and 158JP (mutated PLP) OL lines, to show the occurrence of a high cell death percentage in 158JP OL cultures compared to the controls. An increased Bax (pro-)/Bcl2 (anti-apoptosis) ratio is evidenced in 158JP cells. Also, the endoplasmic reticulum stress marker (SRE) BiP is overexpressed in 158JP OL. Exposure of 158N and 158JP cells to tunicamycin-induced SRE revealed that the ED50 for 158JP OL is 67 times lower than the ED50 for 158N OL. Proteins CHOP and caspase-12, that pivotally determine the switching from survival to apoptotic pathways, are upregulated in 158JP cells. 4-Phenylbutyrate, a SRE inhibitor, which improves 158JP cell survival, also decreases the levels of SRE and apoptosis markers in 158JP OL. The thesis opens promising perspectives for the development of effective strategies against myelin disorders.

No description available.

This thesis contains the findings from four years of research regarding an oligodendrocyte protein named neurofascin 155. The role of this protein in maintaining adhesion between the myelin sheath of oligodendrocytes and the axons of neurons has become well established in recent years and the research presented here has revealed that while western blots have previously shown one protein/band representing neurofascin 155, there are two proteins/bands. These two proteins have been named neurofascin 155 high and neurofascin 155 low due to their previous inclusion in the single band. The work leading up to their discovery, findings, and the relevance of these two proteins will be discussed in animal models with disrupted myelin:axon adhesion and in the human disease multiple sclerosis.

During development, oligodendrocytes (OLGs), the myelinating cells of the central nervous system (CNS), undergo a stepwise progression during which OLG progenitors, specified from neural stem/progenitor cells, differentiate into fully mature myelinating OLGs. This progression along the OLG lineage is characterized by well-synchronized changes in morphology and gene expression patterns. The studies presented in this dissertation identified the extracellular factor Autotaxin (ATX) as a novel upstream signal modulating HDAC1/2 activity and gene expression in cells of the OLG lineage. Using the zebrafish as an in vivo model system, as well as rodent primary OLG cultures, this functional property of ATX was found to be mediated by its lysoPLD activity, which has been well-characterized to generate the lipid signaling molecule lysophosphatidic acid (LPA). LPA binds to Gprotein-coupled LPA receptors (LPARs) on the surface of OLGs to initiate downstream signaling events. ATX’s lysoPLD activity was found to modulate HDAC1/2 regulated gene expression during a time window coinciding with the transition from OLG progenitor to early differentiating OLG. When looking further downstream of the ATX-LPA axis, down-regulation of LPA receptor 6 (LPA6) was found to reduce the expression of OLG differentiation genes as well as the overall process network area of OLGs. Thus, LPA6 plays a role in both the gene expression and morphology changes seen in OLG differentiation. These findings prove useful for future therapeutic targets needed for demyelinating diseases of the CNS such as Multiple Sclerosis (MS), in which OLGs fail to differentiate into mature OLGs, needed for remyelination. Additionally, white matter injury has been frequently reported in HIV+ patients. Previous studies showed that HIV-1 Tat (transactivator of transcription), a viral protein that is produced and secreted by HIV-infected cells, is a toxic factor to OLGs. We show here that Tat treatment reduces the expression of OLG differentiation genes and the overall process network area of OLGs. Additionally, Tat-treated OLGs have reduced ATX lysoPLD activity and there is a physical interaction between Tat and ATX. Together, these data strongly suggest functional implications of Tat blocking ATX’s lysoPLD activities and thus the ATX-LPA signaling axis proves to play a significant role in the development of OLGs.

Production of large numbers of desirable human cell types in the laboratory is one of the major goals of stem cell research. Current experimental approaches have focused on the strategy of recapitulating the events of normal embryogenesis in culture, by treating cells - either tissue stem cells or pluripotent stem cells (iPS/ES cells) - with cocktails of growth factors, matrix proteins or pharmacological agents. This is challenging and often requires weeks or months of elaborate cell culture regimes. An alternative approach is the forced expression of master regulatory transcription factors; this can bypass developmental programs and drive conversion to the target cell type. Each of these strategies is inefficient and unreliable. Recently a new opportunity has arisen to exploit synthetic transcription factors (sTFs) to program and reprogram cell fate. To create such sTFs the CRISPR/Cas9 system is repurposed through tethering of catalytically dead Cas9 to various transcriptional regulatory effector domains (e.g. VP16, KRAB). In this thesis, we have explored sTFs as tools to reset transcriptional regulatory networks in neural stem cells and mouse embryonic fibroblasts. We tested transcriptional activation of key neural lineage target genes (e.g Olig2, Sox10 and Nkx6.2). We designed and validated a series of sTFs that could effectively activity these. We have found that activation of Sox10 by dCas9-VP160 in mouse neural stem cells can increase the amount of arising oligodendrocyte and oligodendrocyte precursors cells during the differentiation. The activity of sTFs strongly depends on cellular context: i.e. a specific sTF might work well in one cell type but not another. Importantly, these biological barriers are not easily overcome by increasing the strength of the sTF - either through levels or types of effector domains used. Our data inspecting single cells suggests that multiplex delivery of sTFs can indeed cooperate by both increasing the number of cells that activated the gene of interest and increasing the level of transcriptional activation in a given cell. To fully exploit these new technologies, we therefore developed a new construction pipeline that allows easy and efficient assembly of multiple sTFs. Using this approach, we were able to successfully activate three different target genes from a single expression plasmid (Olig2, Sox10 and Nkx6.2) in fibroblasts. These sTFs we able to force fibroblast transdifferentiation towards oligodendrocyte lineage. Future studies will explore further how to exploit these sTFs to augment or replace current reprograming strategies.

No description available.

No description available.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Genetic testing for this pathogenic mutation is challenging due to its GC rich, repetitive nature. I developed PCR based assays to detect the presence of the pathogenic variant, which were used in screening an archival cohort of Scottish ALS patients, and have also been implemented within a diagnostic setting. These PCR assays allow amplification of larger repeat expansions than have previously been reported, and can determine whether a C9orf72 expansion of greater than 100 repeats is present or not. It is not well understood how the repeat expansion leads to disease, but several potential mechanisms have been hypothesised, including reduced expression, RNA toxicity and protein toxicity via dipeptide repeat proteins produced through repeat associated non-AUG translation. Motor neurons are an understandably well studied target in amyotrophic lateral sclerosis, however the role of glia, particularly oligodendrocytes, in the pathogenesis of the disease has recently been highlighted from studies on rodent models and post mortem tissue. To investigate the effect of the C9orf72 repeat expansion on oligodendrocytes, we have applied a differentiation protocol to hiPSCs with the expansion and controls, including an isogenic control which has been generated in the lab. There was no difference in the production of neuronal and glial cell types between these cell lines. I went on to look for evidence of the main proposed pathological mechanisms of C9orf72 repeat expansions: loss of function or gain of function through either RNA or protein toxicity. hiPSC derived oligodendrocytes from both carrier and control showed low expression of C9orf72 mRNA, and there was no difference due to the presence of a repeat expansion. Carrier hiPSC derived oligodendrocytes displayed sense RNA foci, which did not appear to have an effect on cellular morphology. The detection of dipeptide repeat proteins proved challenging, and the results were inconclusive as to their presence in hiPSC derived oligodendrocytes. I went on to show there was no evidence of mislocalisation of TDP-43 in C9orf72 carrier oligodendrocytes. Finally, the study showed similar levels of cell death in basal conditions in carrier and control cells, and no clear difference in the response to endoplasmic reticulum stress. Further research will be required to elucidate the role of oligodendrocytes in C9orf72 related amyotrophic lateral sclerosis.