Les p21-activated kinases (PAK) du sous-groupe I sont impliquées dans de nombreux processus cellulaires tels la prolifération, les mouvements cellulaires, l’adhérence et l’apoptose. Ces kinases sont des effecteurs des Rho-GTPases Rac1 et Cdc42 et participent à la régulation du cytosquelette d’actine. Les deux kinases neuronales PAK1 et PAK3, qui présentent de fortes identités de séquence, régulent le cytosquelette d’actine, contrôlant ainsi la dynamique des épines dendritiques, et la plasticité synaptique.Les mutations du gène pak3, localisé sur le chromosome X, sont responsables de déficience intellectuelle chez l’homme, et les mécanismes moléculaires et cellulaires associés aux défauts cognitifs sont mal connus. Il a été montré que PAK3 participe à la voie proneurale au cours de l’embryogénèse précoce du xénope en favorisant la sortie du cycle cellulaire et la différenciation neuronale. Cependant, le rôle de PAK3 dans la neurogenèse adulte n’a pas été étudié. Or depuis maintenant une quinzaine d’années, il est admis que la neurogenèse perdure à l’âge adulte et participe aux processus de mémorisation et d’apprentissage. Nous nous sommes donc intéressés à l’implication de PAK3 dans la régulation de la neurogenèse adulte, posant l’hypothèse qu’un défaut de neurogenèse serait responsable, au moins en partie, des défauts cognitifs chez les patients. Nous avons montré que PAK3 n’est pas exprimée dans les cellules souches neurales/progéniteurs prolifératifs mais son expression augmente fortement dès le retrait des facteurs de croissance, ex vivo, suggérant un rôle dans la neurogenèse adulte. Nous avons montré que l’invalidation de pak3 provoque une augmentation de la fréquence de neurosphères primaires formées ainsi qu’un accroissement de leur taille, ceci sans affecter la taille du réservoir de cellules souches ni les propriétés cardinales de celles-ci (multipotence, auto-renouvellement et prolifération). Toutefois, les cellules progénitrices pak3- poursuivent leur prolifération dans des conditions de culture induisant normalement la différenciation, suggérant un défaut de sortie du cycle cellulaire.Nous nous sommes ensuite demandé si les mutations de déficience intellectuelle du gène pak3 altèrent la neurogenèse adulte. Nous avons créé pour cela un modèle murin portant la mutation R67C, responsable chez l’homme de la forme la plus sévère de déficience intellectuelle associée aux mutations de ce gène. Nous mettons en évidence, dans cette souris knock-in, une forte diminution du nombre de cellules nouveau-nées dans les deux zones neurogéniques du cerveau (la zone sous-ventriculaire et le gyrus denté de l’hippocampe) et une augmentation de la proportion de neurones nouveau-nés immatures. Ces données suggèrent que la mutation R67C n’induit pas une perte de fonction de la kinase mais un changement de fonction dépendante d’une activation préférentielle par la GTPase Rac1.En conclusion, ce travail de thèse montre que PAK3 participe à la régulation de la neurogenèse adulte chez les mammifères, contrôle la sortie du cycle cellulaire des progéniteurs neuraux et que la mutation R67C impacte la maturation des neurones nouveau-nés. L’ensemble de ces données suggère que les défauts de neurogenèse adulte dus aux mutations de déficience intellectuelle du gène pak3 sont à l’origine de certains dysfonctionnements cognitifs. / The group I p21-activated kinases (PAK) are involved in many cellular processes such as proliferation, cell movement, adhesion and apoptosis. These kinases are effectors of Rho GTPases Rac1 and Cdc42, and participate in the regulation of the actin cytoskeleton. Both neuronal kinase PAK1 and PAK3, which exhibit high sequence identities, regulate the actin cytoskeleton, thereby controlling the dynamics of dendritic spines and synaptic plasticity. Mutations of the X-linked pak3 are responsible for intellectual disability (ID) in humans, and the molecular and cellular mechanisms associated with cognitive defects are poorly described. It was shown that PAK3 participates in the proneural pathway during early Xenopus embryogenic development, by promoting cell cycle exit and neuronal differentiation of neural precursors. However, the role of PAK3 in the adult neurogenesis has not been studied in mammals. It is now generally accepted that neurogenesis persists during human adulthood and is involved in learning and memory. We are therefore interested in the involvement of PAK3 in the regulation of adult neurogenesis, on the assumption that defects in neurogenesis may be responsible, at least in part, for cognitive defects in ID patients.We showed that PAK3 is not expressed in proliferative neural stem/progenitor cells but its expression increased significantly upon growth factor removal, suggesting a role in adult neurogenesis. We showed that the invalidation of pak3 gene causes an increase in the frequency and in size of primary neurospheres. However Pak3 invalidation does not affect the size of the stem cell reservoir nor the NCS cardinal properties (pluripotency, self-renewal and proliferation). However, the pak3- progenitor cells continue their proliferation in culture conditions normally inducing differentiation, suggesting a defect in cell cycle exit. We then asked whether pak3 ID mutations affect adult neurogenesis. We created a knock-in model expressing the pak3-R67C mutation responsible in humans for a severe form of intellectual impairment. We observed in the knock-in mice, a significant decrease in the number of newborn cells in both neurogenic areas of the brain (the subventricular zone inforebrain, and the dentate gyrus of the hippocampus) and an increase in the proportion of immature newborn neurons. These data suggest that the R67C mutation does not induce a loss of function of the kinase but a change of a function dependent on preferential activation by the Rac1 GTPase.In conclusion, we show that PAK3 play an important role in the regulation of adult neurogenesis in mammals by controlling the cell cycle exit of neural progenitors. The R67C ID mutation impacts both newborn cell proliferation and their maturation. Taken together, these data suggest that defects in adult neurogenesis caused by ID mutations in the pak3 gene may be involved in some cognitive dysfunctions.

No description available.

在成年的哺乳類動物大腦中有兩個區域，可以不斷的有新的神經細胞生成，一個位於大腦側腦室旁內側(Subventricular zone of anterior lateral ventricle ;SVZ)，另一個位於海馬迴(hippocampus)內的齒狀迴(Subgran- ular zone of dentate gyrus ;SGZ) ，其中海馬迴是本論文主要探討的腦區。 神經前驅細胞(Neural progenitor cells :NPC)因具有自我更新(self -renewal）、增殖(proliferative）、多能(multipotent）的能力以及遷移性(Migration)，所以可利用海馬迴內生性的神經前驅細胞(NPC)，促進其增殖以替代因損傷、老化或疾病而損失的神經細胞。神經前驅細胞經由細胞體外培養過程會形成神經球(Neurospheres)，神經球和神經前驅細胞同樣具有自我更新以及可以分化成其他神經細胞的能力。 本研究觀察到，對成年神經新生進行體外藥物的篩選中，化合物Chemical-X，有明顯的促進神經新生的能力。實驗中取健康成年雄性大鼠為實驗動物，分離出成年大鼠之海馬迴神經前驅細胞。用Chemical-X處理後，觀察神經球自我更新能力，以及再把新生成的神經球利用免疫螢光染色處理，瞭解神經前驅細胞經藥物處理後所新生成的細胞，是否仍維持在神經前驅細胞的狀態。進而評估藥物能否達到促進神經新生的目的。

Le traumatisme cérébral (TC) touche des millions de personnes chaque année dans le monde. Les premières conséquences peuvent être une perte de conscience, des hémorragies et l’apparition d’un œdème cérébral. Cependant les personnes qui subissent un TC peuvent présenter des séquelles importantes à plus long terme. Ainsi le traitement préventif des pathologies post-traumatiques est devenu un réel problème de santé publique. La dépression représente la pathologie post-traumatique dont l’occurrence est la plus fréquente. Les origines connues de son apparition s’orientent vers une altération de la neurogenèse adulte hippocampique ainsi que des changements dans la neurotransmission GABAergique, qui est dépendante de l’homéostasie des ions chlorures. Mon travail de thèse suggère que la phase critique, responsable de l’apparition des pathologies post-traumatiques, survient au cours de la première semaine suivant le TC. Pendant cette période, mes résultats montrent que l’hyperexcitabilité des réseaux neuronaux hippocampiques est due à une perturbation des transporteurs des ions chlorure entraînant notamment, une diminution de l’inhibition neuronale. J’ai aussi pu mettre en évidence une altération de la neurogenèse adulte hippocampique liée à la perte d’interneurones dans le gyrus denté. Consécutivement à ces changements, vont s’installer des troubles dépressifs majeurs. Mes travaux indiquent également que la restauration précoce, de l’homéostasie des ions chlorure par un agent pharmacologique, prévient la mort des interneurones ainsi que les changements dans la neurogenèse et permet sur le long terme de réduire très fortement les troubles dépressifs majeurs. / Traumatic brain injury (TBI) affects annually millions of people over the world. The first major consequences include loss of consciousness, haemorrhage and the appearance of cerebral edema. However, people who experience TBI may have significant long-term sequelae and in the majority of cases develop major depressive disorders. In addition, debilitating effects of TBI substantially impair health-related quality of life and are associated with high health care costs. Hence, preventive treatment against posttraumatic pathologies has become a real public health concern. Increasing evidence points to an association between depressive disorders and changes in GABAergic neurotransmission as well as alteration of adult hippocampal neurogenesis.My thesis suggests that the critical phase of posttraumatic pathology occurs over the first week following the trauma. During this period, my results show that hippocampal network hyperexcitability is induced by a disruption of the chloride ion transporters, leading notably to a decrease in neuronal inhibition. Then my work highlighted an alteration of hippocampal neurogenesis related to the loss of interneurons in the dentate gyrus. After some latency, these changes will trigger major depressive disorders. My work also indicates that the early restoration, during this first post-traumatic week, of chloride ion homeostasis by a pharmacological agent, prevents cell death of interneurons as well as changes in neurogenesis and allows significant long-term reduction of major depressive disorders. This therefore suggests the possibility of developing new therapeutic strategies to prevent the emergence of posttraumatic pathologies.

Le cerveau adulte des mammifères conserve sa capacité à générer de nouvelles cellules cérébrales à partir de cellules souches neurales (CSNs), principalement localisées dans deux régions cérébrales spécifiques, l'hippocampe et la zone sous-ventriculaire (SVZ). Ce processus, appelé neurogenèse, permet la formation de nouveaux neurones et de nouvelles cellules gliales (astrocytes et oligodendrocytes). Différents signaux contrôlent la prolifération et la différenciation des CSNs. Parmi ces signaux, les hormones thyroïdiennes (HTs) sont impliquées dans la prolifération des CSNs de la SVZ et dans la différenciation neuronale. À l’inverse des cellules différenciées, telles que les neurones ou les glies, les CSNs ont un fonctionnement – ou métabolisme – principalement basé sur la glycolyse et sur une faible respiration mitochondriale. Or l'évolution du métabolisme des CSNs peut influencer leur choix de destin cellulaire. Les HTs jouant un rôle important dans l'activation du métabolisme mitochondrial, j'ai testé l'hypothèse selon laquelle le choix du destin des CSNs de la SVZ adulte se ferait grâce à l'influence de la signalisation thyroïdienne sur l'activité mitochondriale. J'ai tout d'abord montré in vivo et in vitro que les HTs permettent la détermination des CSNs en précurseurs neuronaux dans la SVZ, tandis qu'une période d'hypothyroïdisme favorise la détermination gliale. La transthyrétine, protéine de liaison des HTs, est spécifiquement présente dans les cellules de la SVZ ayant un destin neuronal, alors que la désiodase de type 3, inactivatrice des HTs, est exprimée par les précurseurs oligodendrocytaires (OPCs), indiquant une activationdifférentielle de la signalisation thyroïdienne dans les deux lignages cellulaires. Par ailleurs j'ai pu observer que les cellules s'engageant vers un destin neuronal possèdent une plus grande activité mitochondriale que les OPCs. La présence d'HTs favorise de plus la respiration mitochondriale, ainsi que la production de dérivés réactifs de l'oxygène (ROS) issus de l'activité des mitochondries, dans les cellules de la SVZ. Un blocage des protéines de la chaîne respiratoire empêche les HTs de promouvoir la détermination neuronale, montrant la nécessité de l'activation mitochondriale pour l'engagement des CSNs en précurseurs neuronaux. On sait d'autre part que les modifications morphologiques (ou dynamiques) mitochondriales sont nécessaires à l'augmentation de la respiration. La division (ou fission) des mitochondries est en particulier essentielle à une bonne répartition intracellulaire de la production de l'énergie issue de la respiration, ainsi qu'à la migration cellulaire. Dans les cellules de la SVZ, j'ai montré que l'action des HTs permet l'activation de la protéine DRP1, médiatrice de la fission mitochondriale, et ce principalement dans les cellules du lignage neuronal. Les HTs favorisent donc la détermination des CSNs de la SVZ vers un destin neuronal grâce à l'activation de la respiration et de la fission mitochondriales. / The adult mammalian brain maintains its capacity to generate new cells from neural stem cells (NSCs), mainly localized in two specific brain regions, the hippocampus and the sub-ventricular zone (SVZ). This process, named neurogenesis, results in the production of new neurons and new glial cells (astrocytes and oligodendrocytes). Several signals control NSCs proliferation and differentiation. Among those, thyroid hormones (THs) are involved in NSCs proliferation in the SVZ and in neuronal differentiation. NSC metabolism relies mainly on glycolysis associated with a low mitochondrial activity, whereas mature cells, like neurons and glia, preferentially use oxidative phosphorylation. Changes in NSC metabolism can impact cell fate. As THs play an important part in activating mitochondrial metabolism, I hypothesized that the influence of TH signaling on mitochondrial activity triggers NSC fate choice in the adult SVZ. First, I showed in vivo and in vitro that THs allow NSC determination in neuronal precursors, whereas a short hypothyroidism favors glial determination. Transthyretine, a TH binding protein, is specifically present in the SVZ cells having a neuronal fate, while type 3 deiodinase, a TH inhibitor, is expressed by oligodendrocyte precursor cells (OPCs). These results indicate that THs signaling isdifferentially activated in neuronal and glial cell lineages. I observed that cells adopting a neuronal fate display a greater mitochondrial activity when compared to OPCs, and that TH signaling favors mitochondrial respiration and ROS production in the SVZ cells. Inhibiting the mitochondrial respiratory chain prevents TH-mediated promotion of neuronal determination, proving the need of mitochondrial activation for NSC commitment toward a neuronal phenotype. Besides, it is also known that modifications of mitochondrial morphology (or mitochondrial dynamics) are required for the respiration to increase. Among mitochondrial dynamics, fission is crucial for a good intracellular repartition of energy production, and for cell migration. In the SVZ cells, I showed that, DRP1, the main inducer of mitochondrial fission, is activated by THs mainly in cells adopting a neuronal fate. Thus, THs favor NSC fate choice toward a neuronal phenotype through the activation of mitochondrial metabolism and mitochondrial fission in the adult mouse SVZ.

Submitted by LINA MARIA DELGADO GARCIA (linadelgadomvz@gmail.com) on 2016-06-03T17:50:42Z No. of bitstreams: 1 Diss_LinaMDelgadoGarcia_PPG_MV_FMVZ.pdf: 5259973 bytes, checksum: 96b8449017ba967633f1d9ef9da7c741 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-06-06T18:25:40Z (GMT) No. of bitstreams: 1 delgado-garcia_lm_me_bot.pdf: 5259973 bytes, checksum: 96b8449017ba967633f1d9ef9da7c741 (MD5) / Made available in DSpace on 2016-06-06T18:25:40Z (GMT). No. of bitstreams: 1 delgado-garcia_lm_me_bot.pdf: 5259973 bytes, checksum: 96b8449017ba967633f1d9ef9da7c741 (MD5) Previous issue date: 2016-05-02 / No final dos anos 60, os experimentos em proliferação celular anunciaram a neurogênese adulta em mamíferos. Três décadas depois a relação entre neurogênese e células-tronco neurais (NSCs) foi estabelecida. Atualmente, as NSCs são objeto de pesquisas na medicina como modelo de estudo de múltiplos estados anormais e distúrbios orgânicos, além de se propor como uma estratégia em condições com poucas alternativas terapêuticas. Contudo o desenvolvimento destas terapias depende do entendimento dos mecanismos moleculares, celulares e biológicos que controlam a neurogênese e as NSCs. Assim, o objetivo deste trabalho foi o estudo das teorias no funcionamento dos nichos neurogênicos e as NSCs com ênfase na proliferação, migração e diferenciação, além da descrição dos aspectos celulares in vitro dos precursores neurais (NPCs) dos nichos neurogênicos dos mamíferos. Os nichos são regiões do sistema nervoso adulto que apresentam neurogênese pela presença das NSCs e um microambiente celular apropriado. Nos mamíferos existem pelo menos dois nichos, a zona subventricular (SVZ) dos ventrículos laterais e a zona subgranular (SGZ) do hipocampo. Os estudos revisados demostram que existem diferenças e semelhanças no comportamento das NSCs nos nichos neurogênicos adultos, levando a que a proliferação, migração e diferenciação seja menos efetiva quando comparada com o desenvolvimento embrionário. Para finalizar, se descreveu o protocolo para isolamento e cultivo dos NPCs e seus aspectos celulares. Os NPCs proliferaram como populações heterogêneas multipotentes. Após a diferenciação, as células migraram e apresentaram características morfológicas e imunofenotípicas de células neurais imaturas, com o predomínio de células gliais. Em conjunto, os NPCs in vitro mimetizam os aspectos gerais da neurogênese. / In the late 60`s, the experiments on cell proliferation announced adult neurogenesis in mammals. Three decades later, the link between neurogenesis and neural stem cells (NSCs) was recognized. Currently, NSCs are the matter of research in human and veterinary medicine as a model of multiple abnormal states and organic disorders, in addition to be proposed as a strategy for diseases and conditions with few therapeutic alternatives. However, the successful development of these therapies depends on the understanding of molecular, cellular and biological mechanisms that control neurogenesis and NSCs. Therefore, the aim of this work was the study of the theories on neurogenic niches and NSCs with focus in proliferation, migration and differentiation, beyond the description of the cellular aspects of in vitro neural precursors cells (NPCs) of the neurogenic niches of the mammals. The neurogenic niches are regions of the adult nervous system which display complete neurogenesis because of the presence of NSCs and an appropriate cell microenvironment. In mammals, there are at least two neurogenic niches, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. The reviewed studies showed that exists differences and similarities in the behavior of the adult NSCs in the neurogenic niches that lead to less effective proliferation, migration and differentiation; when compared with the embryonic development. Finally, was described the protocol for isolation and cultivation of NPCs and their cellular aspects. NPCs proliferated as heterogeneous multipotent populations. Differentiation analyses showed that cells migrated and showed morphological and immunophenotypical characteristics of immature cells with the predominance of glial cells. Overall, NPCs effectively reproduce the general aspects of neurogenesis.

Orientador: Rogério Martins Amorim / Resumo: No final dos anos 60, os experimentos em proliferação celular anunciaram a neurogênese adulta em mamíferos. Três décadas depois a relação entre neurogênese e células-tronco neurais (NSCs) foi estabelecida. Atualmente, as NSCs são objeto de pesquisas na medicina como modelo de estudo de múltiplos estados anormais e distúrbios orgânicos, além de se propor como uma estratégia em condições com poucas alternativas terapêuticas. Contudo o desenvolvimento destas terapias depende do entendimento dos mecanismos moleculares, celulares e biológicos que controlam a neurogênese e as NSCs. Assim, o objetivo deste trabalho foi o estudo das teorias no funcionamento dos nichos neurogênicos e as NSCs com ênfase na proliferação, migração e diferenciação, além da descrição dos aspectos celulares in vitro dos precursores neurais (NPCs) dos nichos neurogênicos dos mamíferos. Os nichos são regiões do sistema nervoso adulto que apresentam neurogênese pela presença das NSCs e um microambiente celular apropriado. Nos mamíferos existem pelo menos dois nichos, a zona subventricular (SVZ) dos ventrículos laterais e a zona subgranular (SGZ) do hipocampo. Os estudos revisados demostram que existem diferenças e semelhanças no comportamento das NSCs nos nichos neurogênicos adultos, levando a que a proliferação, migração e diferenciação seja menos efetiva quando comparada com o desenvolvimento embrionário. Para finalizar, se descreveu o protocolo para isolamento e cultivo dos NPCs e seus aspectos celulares. O... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the late 60`s, the experiments on cell proliferation announced adult neurogenesis in mammals. Three decades later, the link between neurogenesis and neural stem cells (NSCs) was recognized. Currently, NSCs are the matter of research in human and veterinary medicine as a model of multiple abnormal states and organic disorders, in addition to be proposed as a strategy for diseases and conditions with few therapeutic alternatives. However, the successful development of these therapies depends on the understanding of molecular, cellular and biological mechanisms that control neurogenesis and NSCs. Therefore, the aim of this work was the study of the theories on neurogenic niches and NSCs with focus in proliferation, migration and differentiation, beyond the description of the cellular aspects of in vitro neural precursors cells (NPCs) of the neurogenic niches of the mammals. The neurogenic niches are regions of the adult nervous system which display complete neurogenesis because of the presence of NSCs and an appropriate cell microenvironment. In mammals, there are at least two neurogenic niches, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. The reviewed studies showed that exists differences and similarities in the behavior of the adult NSCs in the neurogenic niches that lead to less effective proliferation, migration and differentiation; when compared with the embryonic development. Finally, was described the proto... (Complete abstract click electronic access below) / Mestre

Les cellules épendymaires sont des cellules multiciliées qui tapissent les parois de toutes les cavités du cerveau. Une fois différenciées, ces cellules ne se divisent plus au cours de la vie. Le battement de ces multiples cils motiles joue un rôle important pour maintenir un flux constant de liquide cérébrospinal à travers toutes les cavités cérébrales. Les cellules épendymaires assurent également des fonctions critiques d’échanges moléculaires avec le liquide cérébrospinal. Dans son ensemble, l’implication des cellules épendymaires et de leurs cils motiles s’avère d’une importance majeure dans le maintien des circuits neuraux ainsi que dans le fonctionnement plus global du cerveau. Récemment, une nouvelle caractéristique des cellules épendymaires a été identifiée ; elles font partie d’un microenvironnement appelé une « niche » centrée autour de cellules souches neurales dans le cerveau du rongeur adulte. Ces cellules souches neurales adultes sont capables de produire de nouveaux neurones qui migreront vers le bulbe olfactif des rongeurs adultes. Concernant leur origine, il a été montré que les cellules épendymaires multiciliées dérivent des cellules souches neurales durant les stades tardifs embryonnaires. Ces mêmes cellules souches peuvent d’ailleurs donner naissance à la plupart des différents types de cellules du cerveau. Cependant, les mécanismes par lesquels les cellules souches décident de leur destin cellulaire restent largement méconnus. Dans ce projet, nous étudions quel type de division donne naissance à des cellules épendymaires et nous nous intéressons également au lignage épendymaire. Nos données suggèrent que les cellules épendymaires ne migrent pas après leur dernière division et qu’elles restent à proximité de l’endroit où elles ont été produites. Chose particulièrement intéressante, nous montrons que les cellules épendymaires peuvent être générées par division symétrique ou asymétrique. Nos résultats révèlent aussi que les cellules souches neurales embryonnaires se divisent de manière asymétrique pour donner naissance à la fois à une celluleépendymaire et à une cellule souche neurale adulte. Ces données viennent s’ajouter à la connaissance actuelle que nous avons du développement du cerveau. De plus, elles pourraient contribuer à ouvrir de nouvelles perspectives et stratégies thérapeutiques pour soigner les maladies neurodégénératives à beaucoup plus long terme. / Ependymal cells are multiciliated cells lining the walls of all brain cavities. Once they are mature, they do not divide during life. Their motile ciliary beating endorses a crucial role in maintaining a proper flow of cerebrospinal fluid throughout all brain cavities. Ependymal cells also ensure critical molecular exchanges of the cerebrospinal fluid. On the whole, the involvement of ependymal cells and their multiple motile cilia in the maintenance of the neural circuits and more globally in the well-functioning of the entire brain have proven paramount. More recently, a new characteristic of ependymal cells has been brought to light. Namely, they are part of a microenvironment so called a “niche” surrounding adult neural stem cells in the adult rodent brain. Noteworthy, these adult neuralstem cells are capable of producing new neurons that will migrate to the olfactory bulb of rodents. In terms of their origin, it was shown that multiciliated ependymal cells derive from neural stem cells during late embryonic stages. Besides, the same stem cells can give rise to most cell types of the brain. However, little is known about how fate-decision is made in neural stem cells. In this project, we tackle more particularly how multiciliated ependymal cells arise from the neural stem cells. Most specifically, we address the type of celldivision and the ependymal cell lineage. We find that ependymal cells are not migrating subsequent to their last division, but rather stay where they were first produced. Most interestingly, they can be generated through both symmetric and asymmetric cell division. We also show that embryonic neural stem cells divide asymmetrically to give rise to both an ependymal cell and an adult stem cell. We are confident that these data bring major new insights in the current understanding of neural development. Additionally, these findingscould contribute in opening new therapeutic perspectives and strategies to cure neurodegenerative diseases in a much longer term.

La production de nouveaux neurones, un processus appelé neurogenèse, persiste à l’âge adulte et est assurée par les cellules souches neurales (CSN) au sein de niches spécialisées telle que la zone sous-ventriculaire (ZSV). Cependant, la neurogenèse adulte diminue à la suite de diverses atteintes cérébrales et au cours du vieillissement, provoquant des déclins cognitifs pour l’heure irréversibles. A l’aide d’une méthode de cytométrie en flux développée au laboratoire, nous avons montré que le déclin progressif de la neurogenèse de la ZSV au cours du vieillissement est lié, non pas à une diminution du nombre des CSN, mais à une forte réduction de leur prolifération due, notamment, à l’allongement spécifique de la phase G1 médiée par l’augmentation du TGFβ1. Par ailleurs, nous avons isolé les CSN quiescentes et les CSN en prolifération afin de caractériser leurs propriétés cellulaires et établir leur profil d'expression génique. L’analyse comparative de ces deux populations de CSN a révélé plusieurs niveaux de régulation de la balance entre quiescence et prolifération, telles que l’intégration de signaux en provenance du microenvironnement et l’existence de programmes de transcription distincts. L’ensemble de ces résultats ouvrent des perspectives pour l’utilisation des CSN quiescentes endogènes comme cibles thérapeutiques au cours du vieillissement ou pour régénérer les tissus cérébraux lésés. / The production of new neurons, a process called neurogenesis, persists during adulthood and is ensured by neural stem cells (NSCs) that are located in specialized niches in the mammalian brain such as the subventricular zone (SVZ). However, adult neurogenesis declines dramatically following brain damage and during aging leading to irreversible cognitive deficits. Using a flow cytometry-based cell sorting strategy, we show that the progressive age-related decline in SVZ neurogenesis is not caused by a loss of NSCs but rather by a proliferation deficit of NSCs with the lengthening of their G1 phase due to increased levels of TGFβ1. We then sorted quiescent and proliferative NSCs to characterize their functional properties and define their gene expression profiles. Comparative analysis of the two populations of NSCs reveals that the balance between quiescence and proliferation is regulated at multiple levels with the integration of external signals from the microenvironment and distinct transcriptional programs. Taken together, our results open new vistas into the potential use of endogenous quiescent NSCs as therapeutic targets to increase neurogenesis in the aged brain and to participate to the regeneration of damaged brain tissue.

Neurogenesis is the process of generation of neurons during embryonic development and adulthood. The focus of this doctoral work is the study of the cell biological aspects of neurogenesis and the mechanisms regulating the switch of neural stem cells from proliferation to differentiation. During embryonic development neurogenic divisions occur at the apical or basal side of the pseudostratified epithelium that forms the wall of the neural tube, the neuroepithelium. Apical asymmetric neurogenic divisions (AP) give rise to a neuron and a progenitor cell, while basal symmetric neurogenic divisions (BP) give rise to two neurons. The first part of this thesis is focused on the study of some cell biological aspects of BPs. We first validated the use of the Tis21-GFP knock in mouse line, previously generated in our laboratory. We found that the totality of neurogenic progenitors is marked by the expression of a nuclear GFP. We calculated the abundance of BPs overtime since the onset of neurogenesis showing that BPs overcome APs over development. We studied the loss of apical contact of the basal dividing cells. We found that both neurogenic and non-neurogenic basally dividing progenitors miss the apical contact; which is lost prior mitosis. We generated and characterized a second mouse line, the Tubb3-GFP line expressing a plasma membrane-localized GFP in neurons. These two lines were crossed to obtain a new line (TisTubb-GFP) allowing detection of neurogenic divisions and tracking daughter cells. Using this model: (i) we imaged symmetric neurogenic divisions of BPs, identifying daughter cells as neurons, during imaging; (ii) we compared the kinetics of betaIII-tubulin-GFP appearance after apical or basal mitosis, showing that daughters of BPs express betaIII-tubulin-GFP faster than daughters coming from apical divisions; (iii) we imaged neuronal migration and localization of the Golgi apparatus. Neurogenesis in the adult is confined to two specific regions in the telencephalon: the sub ependymal zone, lining the ventricle, and dentate gyrus of the hippocampus. The second part of this thesis focuses on the adult neurogenic progenitors lineage. Tis21-GFP expression was found and characterized in the two adult neurogenic regions from early postnatal to adulthood. Using a panel of markers for the adult neurogenic cell lineage and confocal imaging, we characterized Tis21-GFP expression, in the dentate gyrus. Tis21-GFP is first expressed in the neurogenic subpopulation of doublecortin positive cells. Tis21-GFP is inherited by the neurons and eventually degraded. Moreover, our data suggest that mitotic Tis21-GFP cells are an indicator of the levels of neurogenesis more accurate than doublecortin positive cells, in the early postnatal mouse. (Anlage Quick time movies 77,88 MB)