The discovery of neural stem cells within the adult CNS has indicated an enormous potential in facilitating neuronal regeneration after injury. Studies from our laboratory have suggested that manipulation of the Rb/E2f pathway can directly impact embryonic and adult neurogenesis, thereby having tremendous potential for neuronal regeneration therapies. Recently, two new members of the Rb/E2f pathway have been discovered, the atypical E2fs: E2f7 and E2f8. Initial studies have suggested that atypical E2fs regulate diverse processes such as cell proliferation, DNA-stress response, apoptosis, however, their importance in the brain are unknown. To analyze their function during brain development, we crossed Nestin-Cre mice with mice bearing a conditional E2f7/E2f8 allele to delete both E2f7 and E2f8 in neural precursor cells. Whereas cortical development was largely unaffected by E2f7/E2f8 deficiency, we observed an enlargement of the lateral ventricles occurring postnatally, a brain condition named ventriculomegaly. We then looked at the ependymal cells, which are the cells lining the wall of the lateral ventricles, to determine if these cells were affected by the absence of atypical E2fs. We found progressive denaturation of the ependymal cell layer, distortion of the ependymal motile cilia and reactive astrocytes within the layer. We identified Gli3, a component of the Sonic hedgehog pathway (Shh), as a target for E2fs, including atypical E2fs. We unravelled a novel mechanism by which atypical E2fs regulate the expression of Gli3, leading to up-regulation of Numb/NumbL, which in consequence destabilizes cadherins organization within the ependymal cell layer. In conclusion, our work suggests that E2f7 and E2f8 transcription factors play an essential role in maintaining the ependymal cell barrier.

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The prevalence of milder forms of HIV-associated neurocognitive disorders (HAND) is rising despite combination antiretroviral therapy (cART). Efavirenz (EFV) is among the most commonly used antiretroviral drugs globally, but causes neurological symptoms that may interfere with adherence and reduce tolerability, and may have central nervous system (CNS) effects that contribute in part to HAND in patients on cART. Thus we evaluated a commonly used EFV containing regimen: EFV/zidovudine (AZT)/lamivudine (3TC) in murine N2a cells transfected with the human “Swedish” mutant form of amyloid precursor protein (SweAPP N2a cells) to assess for promotion of amyloid-beta (Aβ) production (Chapter 3). Treatment with EFV or the EFV containing regimen generated significantly increased soluble Aβ, and promoted increased β-secretase-1 (BACE-1) expression while 3TC, AZT, or, vehicle control did not significantly alter these endpoints. Further, EFV or the EFV containing regimen promoted significantly more mitochondrial stress in SweAPP N2a cells as compared to 3TC, AZT, or vehicle control. We next tested the EFV containing regimen in Aβ - producing Tg2576 mice combined or singly using clinically relevant doses. EFV or the EFV containing regimen promoted significantly more BACE-1 expression and soluble Aβ generation while 3TC, AZT, or vehicle control did not. Finally, microglial Aβ phagocytosis was significantly reduced by EFV or the EFV containing regimen but not by AZT, 3TC, or vehicle control alone. These data suggest the majority of Aβ promoting effects of this cART regimen are dependent upon EFV as it promotes both increased production, and decreased clearance of Aβ peptide. Further (Chapter 4), there is evidence that neural stem cells (NSCs) can migrate to sites of brain injury such as those caused by inflammation and oxidative stress, which are pathological features of HAND. Thus, reductions in NSCs may contribute to HAND pathogenesis. Since the HIV non-nucleoside reverse transcriptase inhibitor EFV has previously been associated with cognitive deficits and promotion of oxidative stress pathways, we examined its effect on NSCs in vitro as well as in C57BL/6J mice. Here we report that EFV induced a decrease in NSC proliferation in vitro as indicated by MTT assay, as well as BrdU and nestin immunocytochemistry. In addition, EFV decreased intracellular NSC adenosine triphosphate (ATP) stores and NSC mitochondrial membrane potential (MMP). Further, we found that EFV promoted increased lactate dehydrogenase (LDH) release, activation of p38 mitogen-activated protein kinase (MAPK), and increased Bax expression in cultured NSCs. Moreover, EFV reduced the quantity of proliferating NSCs in the subventricular zone (SVZ) of C57BL/6 mice as suggested by BrdU, and increased apoptosis as measured by active caspase-3 immunohistochemistry. If these in vitro and in vivo models translate to the clinical syndrome, then a pharmacological or cell-based therapy aimed at opposing EFV-mediated reductions in NSC proliferation may be beneficial to prevent or treat HAND in patients receiving EFV. 1 Portions of this abstract have been previously published (Brown LAM, et al., 2014; Jin, J, et al, 2016) and are utilized with permission of the publisher.1

The classical cell cycle regulatory pathway is well appreciated as a key regulator of cell fate determination during neurogenesis; however, the extent of pRB/E2F function in neural stem and progenitor cells is not fully understood, and insight into the mechanisms underlying its connection with cell fate regulation are lacking. The E2F3 transcription factor has emerged as an important regulator of neural precursor cell (NPC) proliferation in the embryonic and adult forebrain, and we demonstrate here that it also influences the self-renewal potential of NPCs. Using knockout mouse models of individual E2F3 isoforms, we demonstrate the surprising result that the classical transcriptional activator E2F3a represses NPC self-renewal and promotes neuronal differentiation, while E2F3b promotes the expansion of the NPC pool and inhibits differentiation. We attribute these opposing activities to a unique mechanism of transcriptional regulation at the Sox2 locus, a key regulator of stem cell pluripotency, whereby E2F3a recruits transcriptional repressors to this site, and E2F3b promotes Sox2 activation. Importantly, E2F3a-mediated Sox2 regulation is necessary for cognitive function in the adult. Additionally, through the determination of genome-wide promoter binding sites for E2f3 isoforms as well as E2F4, another key regulator of NPC self-renewal, we determined that E2Fs are poised to regulate an extensive set of target genes with key roles in regulating diverse cell fate choices in NPCs, including self-renewal, cell death, progenitor expansion, maintenance of the precursor state, and differentiation. Together, these results reveal a diversity of function for E2Fs in the control of neural precursor cell fate, and identify E2F3 isoforms as important regulators of the pluripotency and stem cell maintenance gene Sox2.

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.

Depuis la découverte fondatrice de la présence de cellules souches neurales (NSCs) multipotentes dans le cerveau des mammifères adultes, plusieurs études ont révélé l'importance de ces cellules pour le maintien de l'homéostasie du cerveau. Notamment, des perturbations dans l'équilibre des NSCs ont été associées au vieillissement et à diverses pathologies neurologiques, ce qui suscite un intérêt croissant pour ces cellules. Les NSCs résident dans des zones germinatives restreintes; dans le rongeur adulte les NSCs sont localisées principalement dans deux niches neurogéniques bien établies dans le télencéphale, ce qui contraste avec la situation chez le poisson zèbre adulte où des niches de NSCs actives ont été identifiées dans tout le cerveau, y compris dans le télencéphale dorsal (pallium). Aussi bien chez les rongeurs que le poisson zèbre, les NSCs adultes présentent les deux propriétés fondamentales des cellules souches: elles sont multipotentes, c’est-à-dire capables de générer de nouveaux neurones et cellules gliales, et ont la capacité d'auto-renouvellement à long terme, permettant leur maintien au long de la vie adulte. A la différence des progéniteurs neuronaux embryonnaires (NPCs), une caractéristique de ces NSCs adultes est qu’elles résident la plupart du temps dans un état d’arrêt réversible du cycle cellulaire appelé quiescence. Cet état, activement maintenu, est censé protéger la réserve de NSCs d’un épuisement prématuré, d’où l'importance de déchiffrer les mécanismes moléculaires de régulation de l’équilibre entre la quiescence et l’activation de ces cellules vers la neurogenèse.Les microARNs constituent une classe de petits ARN régulateurs, qui jouent un rôle crucial dans le contrôle d’états cellulaires et des transitions entre ces états. Ils sont capables de réagir rapidement à des signaux à la fois intra- et extracellulaires, qui peuvent moduler aussi bien leur niveau d’expression que leur impact fonctionnel, leur donnant ainsi la capacité de coordonner diverses signaux pour induire des transitions d'état cellulaire. Un microARN en particulier, miR-9, a été montré comme jouant un rôle clé et conservé au cours de la neurogenèse embryonnaire. L'objectif principal de cette étude était d'étudier, pour la première fois, un rôle potentiel de miR-9 dans le contrôle des NSCs, dans un contexte physiologique dans lequel la majorité des NSCs sont quiescentes - le pallium adulte du poisson zèbre. Nous avons constaté que miR-9 est exclusivement exprimé dans une sous-partie des NSCs, met vraisemblablement en évidence un « sous-état » de quiescence. De plus, nous avons pu montrer que miR-9 ancre les NSCs dans un état de quiescence, en partie via le maintien d’un niveau élevé d’activation de la voie de signalisation Notch. De façon surprenante, nous avons également identifié une modification de la localisation subcellulaire de miR-9 au cours du temps: alors que miR-9 est localisé dans le cytoplasme de tous les NPCs chez l’embryon ou le juvenile, chez le poisson adulte miR-9 est localisé dans le noyau des NSCs en quiescence. En outre, la localisation nucléaire de miR-9 dans ces NSCs quiescentes est fortement corrélée avec la localisation nucléaire des protéines effectrices des microARNs, les protéines Argonaute (Agos), ce qui suggère un rôle fonctionnel de miR-9 dans le noyau. De fait, l'élucidation du mécanisme de transport nucléo-cytoplasmique de miR-9/Agos nous a permis de manipuler leur localisation, et d’observer un impact de cette localisation sur l’état de quiescence vs activation des NSCs. L’ensemble des résultats de cette étude identifient ainsi miR-9 comme un régulateur essentiel de la quiescence des NSCs, fournissent pour la première fois un marqueur moléculaire d’un sous-état de quiescence spécifique du cerveau adulte et suggèrent l'implication d'un mécanisme inédit de régulation par les microARNs dans le maintien de l'homéostasie des réserves de NSCs. / Since the seminal discovery of multipotent neural stem cells (NSCs) in the adult mammalian brain, multiple studies have unravelled the importance of these cells for maintaining brain homeostasis. Notably, disturbances in NSC equilibrium have been linked to physiological aging and various neurological pathologies thus sparkling interest in harnessing them for use in regenerative medicine. NSCs reside in distinct germinal zones; in the adult rodent brain NSCs are found mainly in two well-established neurogenic niches in the telencephalon which contrasts with the situation in the adult zebrafish where NSC niches are widespread throughout the brain, including in the dorsal telencephalon or pallium. In both the rodent and zebrafish brains, adult NSCs display fundamental stem cell properties: they are multipotent, e.g. capable of generating new neurons and glia throughout adult life, and have the capacity for long-term self-renewal. Similar to stem cells in other adult tissues, and in contrast to embryonic neural progenitors, a hallmark of these adult NSCs is their relative proliferative quiescence. Quiescence is an actively maintained, reversible state of cell-cycle arrest and generally thought to protect against exhaustion of the stem cell pool. In line with this, disrupting the balance between quiescent and activated NSCs leads to a premature depletion or permanent cell-cycle exit of these cells highlighting the importance of fully deciphering the mechanisms regulating this equilibrium. microRNAs, a major class of small pleiotropic regulatory RNAs, play crucial roles in reinforcing developmental and transitional states. They are capable of reacting to environmental cues, both cell-intrinsic and -extrinsic, with varying outputs such as changing their regulatory functions and expression levels, thus enabling them to coordinating diverse cues to induce cell-state transitions. One microRNA in particular, miR-9, is a highly conserved master regulator of embryonic neurogenesis and in the embryonic zebrafish brain, it establishes a primed neural progenitor state enabling them to quickly respond to cues to differentiate or proliferate. The primary goal of this study was to investigate, for the first time, a potential role for miR-9 in influencing NSC state in a physiological context in which the majority of NSCs are quiescent – the adult zebrafish pallium. We found that miR-9 is exclusively expressed in quiescent NSCs and highlights a “sub-state” within quiescence. In part by maintaining high levels of Notch signalling, a known quiescence promoting pathway, miR-9 anchors NSCs in the quiescent state. Strikingly, we identified a conserved age-associated change in the subcellular localization of the mature miR-9 from the cytoplasm of all embryonic/juvenile neural progenitors to the nucleus of a subset of quiescent NSCs in the adult brain. Moreover, the nuclear expression of miR-9 in these quiescent NSCs is highly correlated with nuclear localization of the microRNAs effector proteins Argonaute (Agos), suggestive of a functional role for nuclear miR-9. Indeed, the elucidation of the nuclear-cytoplasmic transport mechanism of miR-9/Agos enabled us to manipulate their nuclear to cytoplasmic ratios which directly impacted NSC state. Altogether, these results identify miR-9 as a crucial regulator of NSC quiescence, provide for the first time a molecular marker for an age-associated sub-state of quiescence and suggest the involvement of a novel and unconventional microRNA-mediated mechanism to maintain homeostasis of NSC pools.

Neural stem cells are capable of differentiating into three major neural cell types, but the underlying molecular mechanisms remain unclear. Here, we investigated the mechanism by which integrin β4 modulates mouse neural stem cell differentiation in vitro. Inhibition of endogenous integrin β4 by RNA interference inhibited the cell differentiation and the expression of fibroblast growth factor receptor 2 but not fibroblast growth factor receptor 1 or fibroblast growth factor receptor 3. Overexpression of integrin β4 in neural stem cells promoted neural stem cell differentiation. Furthermore, integrin β4-induced differentiation of neural stem cells was attenuated by SU5402, the inhibitor of fibroblast growth factor receptors. Finally, we investigated the role of integrin β4 in neural stem cell survival: knockdown of integrin β4 did not affect survival or apoptosis of neural stem cells. These data provide evidence that integrin β4 promotes differentiation of mouse neural stem cells in vitro possibly through fibroblast growth factor receptor 2.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19009号 / 工博第4051号 / 新制||工||1623(附属図書館) / 31960 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 田畑 泰彦, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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