Au cours des dernières années, des progrès significatifs ont été réalisés dans la prévention des épisodes de démyélinisation qui surviennent au cours de la sclérose en plaques, mais beaucoup moins dans le domaine de la régénération de la myéline détruite. Notre équipe a précédemment démontré que la protéine Sonic Hedgehog (Shh) est un régulateur positif des progéniteurs oligodendrocytaires favorisant la réparation de la myéline dans un modèle de démyélinisation induite par injection focale de lysolécithine dans le corps calleux chez la souris. Cependant, les mécanismes moléculaires mis en jeu restent encore largement méconnus. Mon projet de thèse consiste à explorer le rôle d'un co-récepteur de Shh, Boc, une glycoprotéine reliée aux molécules d'adhésion cellulaire (CAM) et fortement induite à l’issue d’une démyélinisation. Dans un premier temps, nous avons focalisé nos investigations sur la caractérisation du mutant Boc knockout au cours de la myélinisation développementale. Nos résultats révèlent des activités région et temps spécifiques de Boc sur les mécanismes de prolifération et de maturation des progéniteurs oligodendrocytaires. De plus, les souris Boc knockout présentent un phénotype d’hypermyélinisation qui apparaît au cours des premières semaines post-natales et persiste à l’âge adulte.Dans un second temps, nous avons déterminé l'effet de l'absence de Boc sur les processus de démyélinisation et de réparation de la myéline. En utilisant le modèle d’injection focale de lysolécithine dans le corps calleux, nous avons démontré un retard dans la maturation des progéniteurs oligodendrocytaires et une réduction de la régression des lésions chez les souris dépourvues de Boc.Nos résultats indiquent ainsi un rôle positif de Boc dans le processus de régénération faisant suite à une démyélinisation. Par ailleurs, l’absence de Boc diminue la réaction gliale inflammatoire étroitement associée au mécanisme de régénération de la myéline. L’ensemble de nos résultats indique une activité différente du récepteur au cours des processus de myélinisation et de remyélinisation du système nerveux central. Ces données originales contribuent à la caractérisation d’une composante de la signalisation Hedgehog impliquée dans le processus régénératif associé aux pathologies démyélinisantes. Ces résultats seront importants à considérer dans le cadre de l’approche thérapeutique des maladies démyélinisantes. / During the last years, significant progress have been made in preventing demyelinating events that occur during multiple sclerosis, but much less in the field of regeneration of the lost myelin. Our team has previously demonstrated that the Sonic Hedgehog (Shh) protein is a positive regulator of oligodendrocyte progenitors enhancing myelin repair in a demyelination model relying on the focal injection of lysolecithin into the corpus callosum. However, the molecular mechanisms that are involved are still largely unknown. My thesis project was aimed at exploring the role of a co-receptor of Shh, Boc, a glycoprotein linked to the cell adhesion molecules (CAM) and strongly induced during myelin repair. First, we focused our investigations on the characterization of the Boc knockout mutant during developmental myelination. Our results reveal region and time specific activities for Boc in the mechanisms of proliferation and maturation of oligodendrocyte progenitors. In addition, Boc knockout mice exhibit hypermyelination that occurs in the firstpostnatal weeks and persists until adulthood In a second step, we determined the effect of the absence of Boc in the processes of demyelination and myelin repair. Using the model of focal injection of lysolecithin into the corpus callosum, we demonstrated a delay in oligodendrocyte maturation and a decrease in lesion regression in the Boc knockout mice. Thus, our results indicate a positive role for Boc in the regeneration process following demyelination.Furthermore, the absence of Boc decreases the inflammatory glial reaction closely associated with myelin repair. Altogether, our results indicate a different activity of the receptor during the processes of myelination and remyelination in the central nervous system. These original data contribute to the characterization of a component of the Hedgehog signaling pathway involved in the regenerative process associated with demyelinating pathologies.Those results will be important to consider in the context of the therapeutic approach of the demyelinating diseases.

Au cours des maladies démyélinisantes du système nerveux central (SNC), les gaines de myéline qui entourent les axones et contribuent à la rapidité de la conduction nerveuse, mais aussi les oligodendrocytes qui synthétisent la myéline sont détruits. C’est le cas dans la plus commune de ces pathologies, la sclérose en plaques, qui se caractérise par des épisodes inflammatoires et démyélinisants récurrents évoluant après quelques années vers une forme progressive secondaire. A l’heure actuelle, les traitements disponibles pour les patients sont essentiellement de type immunomodulateurs. Ces molécules sont efficaces pour réduire la fréquence des épisodes démyélinisants, mais elles sont dépourvues d’activité sur la forme progressive de la maladie. Juste avant que ne débute mon travail de thèse, les voies de signalisation respectivement induites par les protéines Hedgehog et les stéroïdes de type androgènes se sont révélées être des régulateurs positifs de la réparation de la myéline vraisemblablement en utilisant des mécanismes différents. A partir de ces découvertes, nous avons émis l’hypothèse que la modulation pharmacologique simultanée des deux voies pourrait être intéressante dans une perspective thérapeutique. En utilisant des cultures de cellules gliales mixtes ou en administrant ces modulateurs à des souriceaux nouveau-nés, nous avons d’abord montré l’existence d’une interaction fonctionnelle entre les deux voies au cours du développement physiologique de la myéline à la période post-natale précoce. De façon intéressante, le blocage de la signalisation Hedgehog est requis pour que les androgènes puissent jouer leur rôle dans le processus de myélinisation. Les mécanismes moléculaires impliqués dans la communication entre les deux voies sont apparemment indépendants de la régulation de la transcription du principal effecteur de la signalisation Hedgehog (Gli1), ainsi que de celle du récepteur nucléaire des androgènes (AR). Par ailleurs, l’administration de ces mêmes modulateurs à des animaux ayant subi une démyélinisation du SNC nous a permis de mettre en évidence un effet synergique des deux voies sur la régénération de la myéline, la résolution de l’inflammation et la récupération fonctionnelle. Ces résultats seront importants à considérer dans le contexte d’une nouvelle approche thérapeutique des maladies démyélinisantes. / During demyelinating diseases of the central nervous system (CNS), the myelin sheaths that surround the axons and contribute to nervous conduction velocity, but also the oligodendrocytes that synthesize myelin are lost. This is particularly true in multiple sclerosis, the most common of those pathologies, which is characterized by recurrent inflammatory and demyelinating attacks evolving after several years into a secondary progressive form. Presently, the treatment of patients mostly relies on the use of immunomodulators, which are successful in decreasing the frequency of the attacks. However, these molecules lead to poor results in the progressive form of the disease. Just before the beginning of my PhD, the Hedgehog and androgen signaling pathways have been identified as positive regulators of myelin repair likely by using different mechanisms. On the basis of these findings, we hypothesized that a combination therapy using pharmacological modulators of each of these pathways could be interesting from a therapeutic point of view. By using primary mixed glial cell cultures and in vivo administration of the modulators to early postnatal mice, we have shown that the Hedgehog and androgen signaling pathways functionally interact during the physiological development of myelin at the early postnatal period. Importantly, the blockade of the Hedgehog signaling is required to allow androgen to play their role in the myelination process. The molecular mechanisms implicated in the pathway crosstalk do not involve the transcriptional regulation of the main effector of Hedgehog signaling (Gli1) or the nuclear androgen receptor (AR). Moreover, the same modulators administered into demyelinated animals led us to demonstrate a synergetic effect both on myelin repair, inflammation resolution and functional recovery. These results should be considered in the context of a novel therapeutic approach of demyelinating diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22336号 / 医博第4577号 / 新制||医||1041(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 渡邉 大, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

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Quaking (QKI) is an RNA binding protein involved in the post-transcriptional regulation of gene expression. Originally identified as the cause of hypomyelination in a mouse mutant, it has since been consistently implicated in a wide range of neurological diseases. As a gene exclusively expressed in glial cells of the central nervous system, such associations emphasise the importance of an indirect, or non-neuronal link to aberrant neural function. A role in early neural development has also been suggested from the viable and embryonic lethal mouse mutants, yet detailed and in vivo study has been precluded thus far by the murine uterine gestation, and mutant lethality prior to oligodendrogenesis. This thesis examines the role of QKI in human neurological disease, and explores the use of the zebrafish as a model organism to allow the unimpeded study of neural development. We first examined the expression of QKI in human post-mortem brain samples, in separate studies of Alzheimer’s disease (AD) and schizophrenia. In AD we found that QKI and the splice variants QKI5, QKI6, and QKI7 were all significantly upregulated, and were additionally implicated in the regulation of genes related to AD pathogenesis. Within schizophrenic samples, we explored the expression of QKI6B, a newly identified splice variant of QKI, alongside GFAP. We found that both were significantly upregulated, and a previously implicated regulation of GFAP by QKI was supported. In order to advance investigations of the potential of QKI to disturb neural development, we established the suitability of zebrafish for studying qki. This was achieved through phylogenetic and syntenic analysis, coupled with examination of the qki genes expression patterns. We found that qkib and qki2 are orthologues of human QKI, and both have distinct, yet overlapping expression patterns in neural progenitors, and are not found in differentiated neurons. Following from this, we explored the effects of knockdown to qkib and qki2, finding that qkib exclusively led to aberrant motor neuron development, cerebellar abnormalities, and alterations to the progenitor domain. This clearly demonstrated the crucial role of qki in early neural development, and confirms a previously speculated, yet occluded, function prior to oligodendrogenesis.

No description available.

In order for oligodendrocyte progenitor cells (OPCs) to differentiate into fully mature, myelinating oligodendrocytes, they must be specified at the correct times and undergo coordinated changes in both gene expression and morphology. As oligodendrocytes differentiate, they transition from a bipolar morphology into a morphology characterized by a complex network of multiple processes, which will eventually generate membranous structures necessary for myelination of axonal segments. As changes are observed in cellular morphology, oligodendrocytes also undergo changes in their gene expression profile and express genes necessary for both early and later stages of development such as olig1 and myelin basic protein (mbp), respectively. Data from our laboratory demonstrate that autotaxin (ATX), also referred to as phosphodiesterase Iα/autotaxin (PD-Iα/ATX), is involved in all of these processes as a multifunctional protein by regulating lysophospholipid signaling and cell-extracellular matrix interactions. Previously, our laboratory has identified ATX as an oligodendrocyte-secreted factor present in the extracellular environment that via a newly-identified functional domain, named the MORFO domain (modulator of oligodendrocyte remodeling and focal adhesion organization), can regulate adhesion of oligodendrocytes to naturally occurring extracellular matrix (ECM) proteins and ultimately the establishment of the oligodendrocyte’s complex process network. In vitro data presented in this dissertation suggest that lysophosphatidic acid (LPA), via production from ATX’s well characterized lysophospholipase D (lysoPLD) domain, can induce the expression of myelin basic protein (mbp) and the establishment of membranous structures by differentiating oligodendrocytes, both necessary for the initial stages of myelination. Interested in relating these functions to an in vivo model and due to the early embryonic lethality of atx-null mice, we utilized the zebrafish as an in vivo model. The in vivo data presented in this dissertation demonstrate that atx expression in the zebrafish is evolutionarily conserved within vertebrates. Interestingly, in both mouse and the zebrafish, atx was found expressed by cells of the cephalic floor plate in addition to differentiating oligodendrocytes. Functionally the in vivo data presented in this dissertation confirmed ATX’s role in stimulating mbp expression during later stages of oligodendrocyte development. In addition, a novel function for ATX was revealed by the studies undertaken as part of this dissertation that has never been described before. More specifically, based on the timing of atx expression and the phenotype seen upon atx knock-down, the data presented here suggest that ATX, released by the cephalic floor plate, regulates early oligodendrocyte development and/or specification. Taken together, these data identify ATX as a major regulator for early as well as late developmental stages of the oligodendrocyte lineage.

The formation of the myelin sheath is a crucial step during development because it enables fast and efficient propagation of signals within the limited space of the mammalian central nervous system. During the process of myelination, oligodendrocytes actively interact with the extracellular matrix (ECM). These interactions are considered crucial for proper and timely completion of the myelin sheath. However, the exact regulatory circuits involved in the signaling events that occur between the ECM and oligodendrocytes are currently not fully understood. Therefore, in this dissertation we investigated the role of a known integrator of cell-ECM signaling, namely, focal adhesion kinase (FAK), during oligodendrocyte morphological maturation in vivo and in vitro. Conditional and inducible FAK-knockout mice (Fakflox/flox: PLP/CreERT mice) were generated to observe the effect of FAK loss on myelination in vivo. The role of FAK during post-migratory premyelinating oligodendrocyte morphological maturation was explored in vitro using primary rat oligodendrocyte cultures in combination with siRNA or inhibitor treatment. When inducing FAK knockout just prior to and during active myelination in vivo, we observed a significant reduction in the number of myelinated fibers following initial myelination. In addition, our data revealed a decreased number of primary processes extending from oligodendrocyte cell bodies at this stage of development under induction of FAK knockout. In contrast, myelination appeared normal on postnatal day 28. Our in vitro data demonstrated that reduction of FAK expression or activity affected the oligodendrocyte process network maturation in a way that is unique to the ECM substrate present. More specifically, our data suggest that FAK restrains the expansion of the oligodendrocyte process network in the presence of fibronectin and promotes expansion of the oligodendrocyte process network in the presence of laminin-2. We showed that the FAK-mediated restriction of oligodendrocyte morphological maturation is tightly developmentally regulated; being highly active during early stages of post-migratory premyelinating oligodendrocyte morphological maturation but greatly diminished at the later stages of oligodendrocyte development. Thus, our data suggest that FAK controls the efficiency and timing of CNS myelination during its initial stages, at least in part, by regulating oligodendrocyte process network morphological maturation.