Oligodendrocytes are the myelinating cells of the central nervous system (CNS), whose function is to optimise neuronal transmission and preserving axonal integrity. Oligodendrocytes are derived from a stem cell population, called oligodendrocyte progenitor cells (OPCs). Oligodendrocyte lineage cells (OLCs) have been implicated in the pathophysiology of various diseases including not only demyelinating diseases (eg. Multiple Sclerosis (MS) or Pelizaeus-Merzbacher disease (PMD)), but also psychiatric disorders (eg. schizophrenia or Rett syndrome (RTT)). Regardless of the type of disease, understanding the underlying fundamental biology of the oligodendrocyte lineage cells is pivotal to develop therapeutic strategies. In the mouse embryonic forebrain OPCs are generated in consecutive waves from distinct brain regions along a spatiotemporal gradient; with ventral OPCs emerging before dorsal OPCs. The developmentally distinct OPCs, and their progenies, persist in the brain throughout life. To investigate whether ventrally and dorsally derived OLCs fulfil different functions in the adult brain, dorsally derived OPCs were ablated in development using a \textit{Sox10}-driven diphtheria toxin fragment A (DTA) mouse model. As dorsally derived OPCs populate the cortex, locomotor coordination and cognition were investigated following dorsal OPC ablation. Mice ablated of the dorsal OPC population do not show a significant deficit in learning and attentional function. In contrast, ablated mice show an impaired locomotor coordination, while general vigilance, gait, balance and sensation are comparable to control groups. The locomotor coordination disabilities are a result of alterations of brain, not spinal cord homeostasis, as only a neglect able number of OLCs in the spinal cord are affected by the ablation model. In addition, no signs of neuronal cell death or chronic inflammatory response was detected in response to the ablation. As the oligodendrocyte numbers are similar between control and ablated animals, the locomotor coordination phenotype can also not be explained by reduced number of oligodendrocytes. However, clustering analysis following single-cell Drop-sequencing uncovered a heterogeneity of oligodendrocyte (OL) subpopulations in the motor cortex. Whilst some OL subpopulations are of mixed developmental origin, others are exclusively formed by either ventrally or dorsally derived OLs, arguing that dorsal oligodendrocyte subpopulations are crucial for homeostatic brain function. In the absence of dorsal OPCs, ventral OPCs are not capable of forming dorsal oligodendrocyte subpopulations in response to dorsal OPC ablation. In conclusion, my results indicate a functional heterogeneity of developmentally-distinct oligodendrocytes in physiological brain function.

The interplay between oligodendrocyte (OL) and extracellular matrix (ECM) is critical to the proper maturation of this unique cell type. Recent work has established the β1 integrin-signaling pathway, a mediator for ECM/OL interactions, as an essential component of myelin sheath formation in the central nervous system (CNS). A major downstream effector of β1 integrin is integrin-linked kinase (ILK), an adaptor and structural platform protein. Herein, we (1) generated a model system to study ILK in vivo and (2) employed the model to elucidate ILK’s role in regulating OL biology. To assess the importance of ILK in OL-mediated myelination, we ablated ILK in primary OLs. ILK loss delayed morphological maturation and led to filamentous actin accumulation in the processes and cell body. Further, we noted an upregulation in RhoA activity, with pathway inhibition rescuing an OL subset. We next moved our studies in vivo. First, we assessed the proteolipid protein promoter’s utility as OL-specific Cre driver. Protocols established, we generated an ILK conditional knockout line (Ilk cKO). Ultrastructural analysis of Ilk cKO optic nerves revealed increased number of amyelinated nerve fibers at P14 with subsequent recovery by P28. The observed transient defects were due neither to a loss nor a gain in total number of mature or progenitor OLs. To rationalize recovery, we grew ILK-depleted OLs on an “inert” substrate. Here, while morphology improved, ILK-depleted OLs were characterized by enlarged and sluggish growth cones as well as microtubule disorganization. Taken together, our data suggests a role for ILK in regulating the morphological maturation of OLs both in vitro and in vivo, the loss of which results in defective OL branching and membrane formation with phenotype and subsequent recovery dependent upon niche complexity.

Multiple sclerosis (MS) is a debilitating disease in which demyelinated lesions form in the central nervous system (CNS). A specific microRNA, miR-145-5p, is dysregulated both in blood samples from RRMS patients and in chronic lesions from progressive MS patients. In the context of remyelination, miR-145-5p regulation may be important as it exhibits strong differential regulation in oligodendrocytes (OLs), the myelinating cells of the CNS, and is also expressed in other CNS glial cell types. Dysregulation of miR-145-5p may therefore play into pathologies observed in both relapsing-remitting (RRMS) and progressive MS. Using pre-clinical rodent models, we aimed to determine how altering normal expression of miR-145-5p specifically affects OL maturation, and how the dysregulation observed in MS may affect various aspects of disease. First using a miR-145 knockdown model in primary rat OLs, we found in vitro that miR-145-5p plays a role both in maintaining oligodendrocyte progenitor cells (OPCs) in their proliferative state and preventing premature differentiation to OLs and that knockdown of miR-145 in OLs enhanced their differentiation. These effects were due at least in part to miR-145-5p regulation of a critical myelin gene transcription factor. The effects of miR-145-5p were further assessed in a miR-145 knockout mouse model in vivo. Contrary to in vitro assays, enhanced myelination was not detectable during development in these animals, nor when remyelination was assessed using the cuprizone toxic model of acute demyelination. However, chronic cuprizone exposure resulted in striking remyelination and functional recovery in miR-145 deficient animals. Sparse remyelination in wild-type animals with chronic cuprizone exposure was concomitant with upregulation of miR-145-5p, which was not the case with acute exposure, identifying miR-145-5p dysregulation as a unique feature of chronic demyelination. Specific assessment of miR-145-5p overexpression in OLs in vitro resulted in severe differentiation deficits and eventual apoptosis, driven molecularly by altered expression of multiple pathways critical to successful OL differentiation and subsequent myelination. Finally, we induced an inflammatory model of demyelination, experimental autoimmune encephalomyelitis (EAE), in our miR-145 knockout mouse to assess the role of miR-145-5p in autoimmune-mediated myelin damage. The clinical severity of EAE in miR-145 deficient animals was reduced, and this was accompanied by reduced loss of myelin and lessened immune cell infiltration in miR-145 knockout spinal cords. Alterations in both astrocytic and microglial activation were detected with loss of miR-145, suggesting that improved clinical outcomes in this model may be underpinned by changes in EAE-mediated neuroinflammation. Collectively, these data suggest that miR-145-5p plays differing roles in both progressive and inflammatory MS, affecting multiple glial cell types in the CNS. Excitingly, loss of miR-145 expression in our mouse model of chronic demyelination allowed extensive remyelination and functional recovery following chronic demyelination, and in EAE improved clinical outcomes driven by underlying improvements in myelin retention and altered neuroinflammatory reactions. Thus, miR-145-5p merits further investigation as a potential therapeutic target to help overcome both remyelination failure in all forms of progressive MS and inflammation-driven demyelination in RRMS and early secondary progressive MS (SPMS).

Each year, aging and age-related deficits in cognitive function affect larger population worldwide. Research on aging has focused on changes in gray matter and white matter with age. A quantitative analysis of magnetic resonance images from healthy subjects of 16-79 years showed a significant negative correlation between gray matter volume and age (Taki et al., 2004). In addition, age-related cognitive decline is reported to be associated with white matter changes such as myelin damage, a result of both the inability of microglia to clear out damaged myelin debris and oligodendrocyte to support remyelination. Eotaxin-1 (CCL11) belongs to a group of eosinophil-specific chemoattractant originally found in peripheral immune system mediating allergic inflammation, asthma and atopic dermatitis (Garcia-Zepeda et al., 1996; Spergel, Mizoguchi, Oettgen, Bhan, & Geha, 1999). Recently it has been reported to have endogenous sources in the CNS and to increase with age in cerebral spinal fluid (CSF) as well as periphery in blood plasma. While CCL11 has been identified to increase with age, injection of CCL11 inhibit neurogenesis in young mice, which is likely to be mediated by C-C chemokine receptor type 3 (CCR3). CCR3 is also the only receptor for CCL11 that is expressed by oligodendrocyte precursor cells (OPCs) and by activated microglia in mice, which means it may participate in the process of microglial phagocytosis and oligodendrocyte myelination. To investigate if CCR3 is an important factor in the normal aging brain and its potential role in these existing findings, immunohistochemistry, stereology and densitometry were performed in the anterior cingulate cortex and cingulum from brain tissue of 4 young adults and 6 aged rhesus monkeys that were behaviorally tested previously to 1) demonstrate any association between CCR3 expression level and age 2) characterize changes in CCR3 level in relation to cognitive impairment 3) identify cellular localization of CCR3. We found a significant increase in amount of CCR3 cingulate cortex with age, which suggests its pro-disease effect in other pathways such as the interaction between CNS and T cell immune system. Although for aged group increase in CCR3+ cell density in white matter appeared insignificant, we found that CCR3 was expressed exclusively in OPCs but was absent in mature oligodendrocytes. indicating its role in OPC proliferation, oligodendrocyte maturation and myelination.

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La voie de signalisation activée par la protéine Sonic Hedgehog (Shh) est connue pour son rôle majeur au cours de l’embryogenèse et en particulier dans la prolifération et la spécification cellulaire ou encore le guidage axonal au cours de l’établissement des structures du système nerveux. Depuis quelques années, ce morphogène a aussi été identifié comme un régulateur important de plusieurs processus physiologiques du cerveau adulte comme le maintien de la neurogenèse ou la régulation de l’activité électrique de certains neurones (Traiffort et al., 2010). La suractivation de la voie Shh dans un cerveau sain entraine une augmentation significative de la prolifération des cellules progénitrices des oligodendrocytes (OPCs), la source des oligodendrocytes matures, les cellules responsables de la formation des gaines de myéline (Loulier et al., 2006). Au cours de ma thèse, j’ai étudié le potentiel que représente l’activation de la voie Shh dans la régulation de ces progéniteurs dans un contexte de démyélinisation. Pour cela, j’ai utilisé une souris transgénique plp-GFP, chez laquelle la protéine fluorescente verte est exprimée par les cellules du lignage oligodendrocytaire. Après avoir caractérisé le profil d’expression de la GFP dans le cerveau mature de ces souris, j’ai mis au point un modèle de démyélinisation focale par injection stéréotaxique d’un détergent spécifique de la myéline, la lysolécithine (LPC). J’ai identifié les cellules du lignage oligodendrocytaire comme source directe de protéines Shh au sein de la lésion à un temps très précoce après l’injection de LPC. Les gènes cibles de la voie Shh sont aussi fortement induits dans cette population cellulaire à une période plus tardive, correspondant à la différenciation des OPCs en cellules matures. L’utilisation d’adénovirus codant soit pour Shh lui-même soit pour son antagoniste physiologique Hip, m’a permis de réaliser des expériences de gain et de perte de fonction et ainsi d’analyser comment la modulation de la voie Shh peut influencer sur le processus de régénération des oligodendrocytes suite à une lésion. La surexpression de Shh permet d’augmenter la prolifération des OPCs mais aussi d’accélérer leur différenciation, aboutissant à un nombre plus élevé d’oligodendrocytes matures plus précocement au cours du processus de remyélinisation. De plus, il est intéressant de constater que la densité des cellules astrocytaires et microgliales, notamment associées au processus inflammatoire, diminue dans la lésion chez les animaux ayant reçu l’adénovirus Shh comparés au animaux contrôles. A l’inverse, le blocage de la voie induit l’arrêt complet de la production de nouveaux oligodendrocytes. Au-delà de l’amélioration de notre compréhension de la physiologie et de la régulation du lignage oligodendrocytaire dans le cerveau adulte, l’ensemble de ce travail montre de quelle manière la voie Shh peut représenter une nouvelle piste dans la recherche de cibles thérapeutiques dans les affections de la myéline telles que la sclérose en plaques. / The Sonic Hedgehog (Shh) signaling pathway is known for its role during embryogenesis and in particular for controlling cell proliferation and specification, as well as axon guidance. In recent years, this morphogen has also been identified as an important regulator of several physiological processes in the adult brain such as the maintenance of neurogenesis or the regulation of the electrophysiological propreties of mature neurons (Traiffort et al., 2010). Overactivation of the Shh pathway in a healthy brain causes a significant increase in the proliferation of oligodendrocyte progenitor cells (OPCs), the source of mature oligodendrocytes, the cells responsible for the formation of myelin sheaths (Loulier et al., 2006).In my thesis, I studied the effects of the Shh pathway activation on OPC regulation in the context of demyelination. To that purpose, I used a plp-GFP transgenic mouse, in which the green fluorescent protein (GFP) is expressed by cells belonging to the oligodendrocyte lineage. After characterization of the expression pattern of GFP in the mature brain of these mice, I developed a model of focal demyelination by stereotaxic injection of lysolecithin (LPC). I identified the oligodendrocyte lineage cells as a source of Shh protein within the lesion, soon after the LPC injection. Target genes of the Shh pathway are also strongly induced in this cell population, at a time corresponding to the differentiation of OPCs into mature cells. The use of adenoviral vectors encoding either Shh itself or its physiological antagonist Hip allowed me to conduct gain- and loss-of-function experiments. This way I could analyze how the modulation of Shh pathway may influence the regeneration ofoligodendrocytes after injury. Shh overexpression increases the survival and proliferation of OPCs but also accelerates their differentiation, resulting in a higher number of mature oligodendrocytes earlier during the remyelination process. In addition, the density of astrocytes and microglia, associated with the inflammatory process, is decreased in animalsreceiving the Shh adenoviral vector compared to control animals. Altogether these effects are associated with a reduction of the lesion. Conversely, blocking the pathway induced a complete arrest of new oligodendrocyte production. Besides the fundamental knowledge gained about the molecular mechanism involved in the oligodendroglial precursor cells survival, proliferation, differentiation and myelin repair in vivo, this project should also give valuable insights concerning the potential use of pharmacological modulators of Shh signaling as a novel therapeutic approach for the treatment of multiple sclerosis and other myelin diseases.

Ce projet vise à fournir une nouvelle compréhension moléculaire du programme de transcription impliqué dans la différenciation des cellules souches neurales en oligodendrocytes myélinisant. La logique de ce travail repose sur des études antérieures ayant montré le rôle des facteurs de transcription bHLH Olig2 et Ascl1, opérant en synergie dans la spécification des OPCs, les cellules progénitrices d‘oligodendrocytes . L‘objectif central de ce travail était de comprendre au niveau génomique et transcriptomique les mécanismes par lesquels Ascl1 et Olig2 agissent pour spécifier les OPCs. Nous avons suivi une stratégie utilisant l'analyse du transcriptome et des profils de fixation des facteurs de transcription par immuno- précipitation de la chromatine. Nous avons pu identifier les cibles directes de Ascl1 et Olig2 dans les OPC et lors de la différenciation des oligodendrocytes. Nous avons également identifié de nouveaux marqueurs spécifiques des différents stades des lignées oligodendrocyte et nous sommes concentrés sur Chd7 et Tns3, deux gènes régulé par Ascl1 etOlig2 et enrichis dans la lignée oligodendrogliale à deux stades intéressants, la phase de spécification précoce et la transition entre la migration et la différenciation des oligodendrocytes, respectivement. De plus, nous avons porté notre attention sur le rôle spécifique des oligodendrocyte dans la synthèse de la créatine et son rôle possible de support métabolique dans la synthèse de myéline et de support axonal. Nous avons également initié une approche de repositionnement toxicogénomique pour identifier de nouvelles molécules à tester dans le cadre de maladie demyélinisantesLa plupart des traitements disponibles pour traiter les maladies démyélinisantes sont basées sur une approche immune modulatrice et anti-inflammatoire. A ce jour, aucun n'est en mesure de promouvoir directement la réparation de la myéline de manière efficace. Nous espérons que les gènes dont l'expression est régulée dans les lésions de démyélinisation identifiés lors de cette étude permettront de mieux comprendre le mécanisme de remyelinisation et le développement de nouvelles stratégies dans les maladies démyélinisantes telles que la sclérose en plaques ou dans les leucodystrophies. / Our project aims to provide a new molecular understanding of the transcription program involved in neural stem cells differentiation into oligodendrocytes. The rational of this work relies on previous studies demonstrating that the bHLH transcription factors Olig2 and Ascl1 work in synergy to specify OPCs, the oligodendrocyte progenitor cells. One central goal of this work was to understand at a genomic and transcriptomic level, how Ascl1 and Olig2 work together to specify OPCs. We followed a strategy using genome-wide transcriptome analysis and chromatin immuno-precipitation to characterize Ascl1 and Olig2 directly regulated genes in OPCs and during oligodendrocyte differentiation.We identified new specific markers of different stage of the neural lineages and new important genes correlated to OPCs differentiation. We focused on Chd7 and Tns3, two genes which expressions are driven by Ascl1 and Olig2 and enriched in the oligodendroglial lineage at two interesting stage, the early specification stage and the transition between migrating and differentiating oligodendrocytes, respectively. Moreover, we identified the myelinating oligodendrocyte as the cell in charge of the creatine synthesis in the brain and potentially driving axonal metabolic support. We also used an approach a toxicogenomic and drug repositioning approach to identify new molecules known to modify OPCs and myelin genes but untested in the context of demyelinating diseases. As currently, most of the available treatments for demyelinating diseases are based on immuno-modulatory and anti-inflammatory drugs but none are able to directly promote myelin repair, we expect that these identified genes involved in oligodendrogenesis and whose expression are regulated in demyelinated lesions will allow the development of new therapeutic strategies promoting an efficient remyelination in demyelinating diseases such as Multiple sclerosis or leukodystrophies.

Les Glioblastomes (Gb) sont des tumeurs primaires du SNC les plus fréquentes et sont particulièrement agressives car résistantes à la radio/chimiothérapie. Elles présentent généralement une composante solide et infiltrante. Cette dernière étant difficile à éliminer par la chirurgie sera en partie responsable de la récurrence de la tumeur. Une des avancées majeures du domaine est la mise en évidence dans les Gb de sous populations présentant des caractéristiques de précurseurs neuraux. Ces cellules cancéreuses utilisent des réseaux de gènes spécifiques pour maintenir leur prolifération et leur état indifférencié. Une approche possible pour éliminer ces cellules cancéreuses serait de cibler les facteurs de transcription impliqués dans la prolifération ou encore de forcer leur différenciation. Dans ce but, j'ai étudié le rôle de NKX2.2 et NGN2 à partir de 3 cultures primaires multipotentes. Les résultats montrent que l'expression de NKX2.2 dans ces cultures est nécessaire pour la survie, la prolifération et la capacité à former des neurosphères. A l'inverse, la surexpression de NGN2 conduit à une apoptose massive, à un arrêt de la prolifération avec formation de neurones dont certains sont électrophysiologiquement actifs. Une approche différente consisterait à cibler une des protéines impliquées dans la migration pour limiter la composante infiltrante. Des études antérieures ont montrées un rôle clef de DCX dans la migration des jeunes neurones au cours du développement. La forte expression de DCX dans certains Gb m'a conduit à étudier la régulation et le rôle de ce gène. In vitro, les résultats obtenus montrent que DCX est exprimé par une sous population de cellules. La purification des cellules Dcx+ ainsi qu'une étude clonale a permis de montrer qu'elles se comportent comme des progéniteurs multipotents avec une capacité d'autorenouvellement restreinte. Par ailleurs, j'ai montré que les cellules Dcx+ peuvent réverter vers un état Dcx- et que le gène Dcx est régulé par les voies NOTCH et SHH. / Glioblastomas (GB) are the most common primary tumors of the CNS and are particularly resistant to radio/chemotherapy. They generally have a solid and infiltrative component. The latter being difficult to remove by surgery will be partly responsible for tumor recurrence. One of the major advances in the field is highlighted in the Gb of subpopulations with features of neural precursors. Cancer cells use specific gene networks to maintain their proliferation and undifferentiated state. One approach to eliminate these cancer cells would be to target transcription factors involved in the proliferation or to force their differentiation. To this end, I studied the role of NKX2.2 and NGN2 from 3 primary multipotent cultures. The results show that NKX2.2 expression in these cultures is necessary for survival, proliferation and ability to form neurospheres. Conversely, overexpression of NGN2 led to massive apoptosis, proliferation arrest with formation of neurons, some of which are electrophysiologically active. A different approach would be to target proteins involved in migration to limit the invasive component. Previous studies have shown a key role of DCX in the migration of young neurons during development. The strong expression of DCX in some Gb led me to study the regulation and the role of this gene. In vitro, the results show that DCX is expressed by a subpopulation of cells. Purification of Dcx+ cells and clonal study has shown that they behave as multipotent progenitors with limited self-renewal capacity. I also found that Dcx+ cells can revert back to a Dcx- state and that DCX is regulated by SHH and NOTCH pathways.