Pendant le développement du cervelet, les progéniteurs des neurones granulaires (PNG) nécessitent la présence du cil primaire pour proliférer en réponse à Sonic Hedgehog (SHH). En effet, la prolifération dérégulée des PNGs peut conduire à la formation d'une tumeur pédiatrique maligne appelée SHH-médulloblastome (MB), de ce fait comprendre comment le cil primaire est régulé dans les PNGs est crucial.Nous montrons que le facteur de transcription Atoh1 contrôle la présence du cil primaire dans les PNGs in vitro et in vivo. En particulier, la suppression du cil primaire par l’inactivation génétique de gènes impliqués dans la ciliogenèse (par exemple, Kif3a ou Ift88) empêche Atoh1 de maintenir les PNGs en prolifération, ce qui indique qu’Atoh1 favorise l’expansion des PNGs en maintenant la présence du cil primaire. D’un point de vue moléculaire, Atoh1 contrôle la formation du cil primaire en régulant le bon positionnement peri-centrosomal des satellites centriolaires (SC), complexes protéiques essentiels pour la ciliogenèse. L'inactivation de Atoh1 dans les PNGs perturbe en effet la distribution subcellulaire des SCs, altérant ainsi inévitablement la ciliogenèse. Cette nouvelle fonction de Atoh1 est gouvernée par la régulation transcriptionnelle directe d'un composant clé des SCs, Cep131. L’expression ectopique de Cep131 dans les PNGs restore les effets liés à l'inactivation d'Atoh1, rétablissant la localisation correcte du SC et comme conséquence la présence d’un cil primaire.De plus, nous avons montré que cette voie Atoh1-SC-cil primaire-SHH contrôlant la prolifération des PNGs est également conservée dans le contexte du SHH-MB, où Atoh1 est surexprimée et essentielle pour sa formation et sa maintenance.Ces données révèlent un mécanisme par lequel la ciliogenèse est régulée dans des progéniteurs de neurones, offrant de nouvelles informations sur la neurogenèse dans le cervelet et sur la pathogenèse du SHH-MB. / Cerebellar granule neuron progenitors (GNPs) require the primary cilium to proliferate in response to Sonic Hedgehog (SHH) during cerebellar development. As aberrant proliferation of GNPs may lead to SHH-type medulloblastoma (SHH-MB), a pediatric brain tumor, understanding which mechanisms control ciliogenesis in GNPs represents a major interest in the field. Here, we show that the proneural bHLH transcription factor Atoh1 controls the presence of primary cilia in GNPs both in vitro and in vivo, thus maintaining GNPs responsive to the mitogenic effects of SHH. Indeed, loss of primary cilia induced via knockdown of specific ciliary components (e.g. Kif3a and Ift88) abolishes the ability of Atoh1 to keep GNPs in proliferation in vivo. Mechanistically, Atoh1 controls ciliogenesis by regulating the proper peri-centrosomal clustering of centriolar satellites (CS), large multiprotein complexes working as essential machineries for ciliogenesis. Knockdown of Atoh1 in GNPs perturbs CS subcellular distribution, leading to impairment of ciliogenesis. Luciferase reporter assays and chromatin immunoprecipitation experiments indicate that Atoh1 can directly regulate the expression of Cep131, a key CS core component. Importantly, ectopic expression of Cep131 in GNPs depleted of Atoh1, is sufficient to restore proper CS localization and consequent primary cilia formation, indicating that the Atoh1-Cep131-CS axis is responsible for ciliogenesis in GNPs.In addition, we further demonstrated that these functions of Atoh1 are conserved in the context of SHH-MB, where Atoh1 is typically overexpressed and acts as a lineage-dependent transcription factor.These data reveal a mechanism whereby ciliogenesis is regulated in neuron progenitors providing novel insights into cerebellar neurogenesis and pathogenesis of SHH-MB.

On a longtemps pensé que la communication intercellulaire était essentiellement régie par les signalisations juxta-, endo- et paracrine, les gap junctions et, plus récemment, les exosomes. Cependant, les travaux de plusieurs groupes dont le nôtre ont révélé que les Tunneling Nanotubes (TNT), des protrusions membranaires riches en actine qui relient le cytoplasme de cellules distantes et permettent le transport intercellulaire dynamique de leur contenu biologique, fournissent également l'infrastructure et les machines pour une communication efficace entre cellules. Malgré des progrès significatifs, la caractérisation de ces nouveaux organites a été limitée par le manque d'informations moléculaires et structurelles. Combler ces lacunes à l'aide d'une série d'outils de pointe et d'approches novatrices est devenu l'objectif principal de ma thèse. Plus précisément, j'ai exploré le rôle des complexes régulateurs de l’actine dans la formation des TNT reliant les cellules neuronales. Mes analyses montrent que les voies moléculaires connues pour être impliquées dans la formation d'autres protrusions membranaires régulent différemment la génération des TNT. En utilisant la microscopie par imagerie en direct, la microscopie électronique cryocorrélative et la tomographie, j'ai également étudié la nano-architecture des TNT neuronaux. Mes découvertes ont démontré que les TNT des cellules neuronales sont composés de plusieurs TNT individuels permettant le passage de vésicules et de mitochondries. En raison des difficultés d'identification des TNT in vivo, mes travaux ont également porté sur la mise en œuvre d'une approche « Connectomic » structurelle pour détecter les TNT dans les tissus sans avoir besoin d'un marqueur spécifique de TNT. Mes résultats indiquent que des structures de type TNT relient les cellules granulaires cérébelleuses migratrices des souris nouveau-nées, ce qui suggère que la communication intercellulaire pendant des événements migratoires dans le cerveau pourrait être médiée par des processus mettant en jeu des TNT. La squelettisation des structures identifiées fournit des informations géométriques qui corroborent les observations faites dans des expériences de couplage de colorants. L'ensemble de mes travaux de thèse fait la lumière sur la formation et la structure des TNT neuronaux in vitro et sur de nouvelles approches pour l'identification des TNT in vivo. / Inter-cellular communication has long been thought to be governed by juxta-, endo-, and paracrine signaling, tight junctions, and more recently, exosomes. However, large efforts from our and other groups revealed that Tunneling Nanotubes (TNTs), actin-rich membranous protrusions that connect the cytoplasm of distant cells and allow the dynamic inter-cellular transport of biological cargo, also provide the infrastructure and machinery for effective cell-to-cell communication. Despite significant progress made to unveil TNT-mediated cell communication, the characterization of these novel organelles has been limited by unanswered questions that hail from the lack of both molecular and structural information. Exploring these gaps in the field using a series of state-of-the-art tools and novel approaches became the main focus of my dissertation. Specifically, I explored the specific role of actin-regulator complexes in the formation of TNTs connecting neuronal cells. My analyses show that molecular pathways known to be involved in the formation of other membranous protrusions behave differently in the generation of TNTs. By employing live imaging microscopy, cryo-correlative electron microscopy and tomography approaches, I also studied the nano- architecture of neuronal TNTs. My findings demonstrated that TNTs of neuronal cells are comprised of multiple individual TNTs capable of transporting vesicles and mitochondria. Owing to the difficulties of identifying TNTs in vivo, my work also focused on the implementation of a structural Connectomic approach to detect TNTs in tissue without the need for a TNT-specific marker. My findings indicate that TNT-like structures connect migratory cerebellar granule cells of neonate mice, suggesting that inter-cellular communication during migratory events in the brain could be mediated by TNT-like processes. Skeletonization of the structures identified provide my findings with geometrical information that can be compared with observations made by corroborative dye-coupling experiments. Taken together, my dissertation work sheds light on the formation and structure of neuronal TNTs in vitro, and novel approaches for the identification of TNTs in vivo.

Theory of Mind (ToM) is the ability to infer mental states of others and this skill relies on a distributed network of brain regions. A brain region that has been traditionally disregarded in relation to non-motor functions is the cerebellum. Here, we leveraged large-scale multimodal neuroimaging data to elucidate the structural and functional role of the cerebellum in ToM. We used functional activations to determine whether the cerebellum has a domain-general or domain-specific functional role. We found that the cerebellum is organized in a domain-specific way. We used effective connectivity and probabilistic tractography to map the cerebello-cerebral ToM network. We found a left cerebellar effective and structural lateralization, with more and stronger effective connections from the left cerebellar hemisphere to the contralateral cerebral ToM areas and greater cerebello-thalamo-cortical (CTC) and cortico-ponto-cerebellar (CPC) streamline counts from and to the left cerebellum. Lastly, we examined the relationship between CTC and CPC white matter and ToM speed and accuracy but found no correlation. Our study provides novel insights to the network organization of the cerebellum, an overlooked brain structure, and ToM, one of humans’ most essential abilities to navigate the social world. / Psychology

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now the role of microglia in pruning remain under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the Purkinje cell layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3CR1) signaling during postnatal development using genetic ablation of the CX3CR1 receptor and an in–depth histological analysis of the cerebellar cortex.:1 Introduction 6 1.1 Origin of microglia 6 1.2 Synaptic refinement 7 1.3 Fractalkine Receptor CX3CR1 9 1.4 Climbing fiber maturation and PCL development 10 1.5 Aim of the study 12 2 Materials and Methods 13 2.1 Materials 13 2.1.1 General material 13 2.1.1.1.1 Hardware 13 2.1.1.1.2 Consumable supplies 13 2.1.2 Chemicals 14 2.1.3 Solutions 14 2.1.4 Animals 14 2.1.5 Primary Antibodies 15 2.1.6 Secondary Antibodies 15 2.1.7 Software 15 2.2 Methods 15 2.2.1 Genotyping 15 2.2.2 Fixation and cryopreservation 16 2.2.3 Fluorescence Immunohistochemistry 16 2.2.4 Quantification of Microglia in the Cerebellum 16 2.2.5 Assessment of VGluT2 in the cerebellum 18 2.2.6 Statistical Analysis 19 3 Results 20 3.1 Postnatal enrichment of microglia cells in the cerebellar Purkinje cell layer 20 3.2 Microglial proximity to Climbing fibers 22 3.3 Population of the granular und molecular layer of CX3CR1 knock-out mice during early postnatal development 23 3.4 Influence of CX3CR1 knock-out on microglial morphology 25 3.5 Influence of CX3CR1 deletion on the VGluT2 expression during postnatal development 29 4 Discussion 31 4.1 Role of microglia in the developing cerebellum 31 4.2 CX3Cr1 Signaling and influence on microglial motility and morphology 32 4.3 CX3CR1 signaling and synaptic pruning 33 4.4 CX3CR1 signaling and formation of functional synapses 34 4.5 Correlation of immunohistological data with electrophysiological findings 35 5 Summary and conclusion 36 6 Zusammenfassung der Arbeit 38 7 References 41 8 Erklärung über die eigenständige Abfassung der Arbeit 49 9 Publications 50

How do we learn to establish associations between arbitrary visual cues (like a red light) and movements (like braking the car)? We investigated the neural correlates of visuomotor association learning in the mid-lateral cerebellum. Although cerebellum has been considered to be a motor control center involved in monitoring and correcting the motor error through supervised learning, in this thesis, we show that its role can also be extended to non-motor learning. Specifically, when primates learned to associate arbitrary visual cues with well-learned stereotypic movements, the simple spikes of the mid-lateral cerebellar Purkinje cells reported the monkey’s most recent decision’s outcome during learning. The magnitude of this reinforcement error signal changed with learning, finally disappearing when the association had been overlearned. We modeled this change in neural activity through a drift diffusion-reinforcement learning based model. The concurrent complex spikes, contrary to traditional theories, did not play the role of teaching signal, but encoded the probability of error as a function of the state of learning. They also encoded features that indicate the beginning of a trial. Inactivating the mid-lateral cerebellum significantly affected the monkey’s learning performance while it did not affect motor performance. This is because the mid-lateral cerebellum is in a loop with other cognitive processing centers of the brain including the prefrontal cortex and the basal ganglia. Finally, we verified that the features we identified in primate experiments can also be extended to humans, by studying the visuomotor association learning in humans through functional magnetic resonance imaging. In summary, through electrophysiological and causal experiments in monkeys, imaging in humans, computational models and an anatomical framework, we delineate mechanisms through which the cerebellum can be involved in reinforcement learning and specifically, learning new visuomotor associations.

Le troisième trimestre de grossesse est une période-clé du développement cérébelleux. L’extrême prématurité et les lésions cérébelleuses (LCb) associées prédisposent ces enfants à des déficits moteurs, sociaux, comportementaux et cognitifs. Nos objectifs étaient de décrire les déficits neurocomportementaux associés à long-terme aux LCb, et d’explorer le rôle des microglies cérébelleuses dans leur survenue. À 3 jours de vie, des LCb ont expérimentalement et aléatoirement été induites chez des souriceaux transgéniques (B6.129P2(Cg)-Cx3Cr1 CreERT2-EYFP/iDTR), présentant ou non, une déplétion microgliale cérébelleuse transitoire : contrôle, hémorragies cérébelleuses, état inflammatoire systémique précoce et association des deux lésions. Une évaluation neurocomportementale a été réalisée à l’aveugle en période juvénile et à l’âge adulte. En présence de microglies cérébelleuses, les souris mâles adultes ayant souffert de LCb présentaient une diminution des comportements liés à l’anxiété (test du labyrinthe en croix surélevé) et les souris femelles juvéniles ayant souffert de LCb une augmentation des comportements d’investigation sociale (test de reconnaissance sociale) en comparaison des groupes contrôles. Vis-à-vis d’un état inflammatoire systémique précoce, la déplétion microgliale cérébelleuse assurait une récupération fonctionnelle du phénotype anxieux masculin et social féminin. Ce constat suggérait l’implication d’une activation microgliale délétère à la suite de LCb. Des études complémentaires permettront de préciser les sous-domaines neurocomportementaux affectés et d’investiguer les conséquences d’une activation microgliale (altération de l’élagage synaptique et/ou de la myélinogénèse cérébelleuse). / The third trimester of pregnancy is a key period for cerebellar development. Extreme prematurity and cerebellar injuries (CBI) predispose ex-preterm infants to long-term motor, social, behavioral, and cognitive deficits. Our objectives were to describe the long-term neurobehavioral deficits associated with CBI, and to determine if cerebellar microglia play a pathological role in these induced neurobehavioral deficits. At 3 days of life, CBI were experimentally and randomly induced in transgenic mice (B6.129P2(Cg)-Cx3Cr1 CreERT2-EYFP/iDTR), with or without transient cerebellar microglial depletion: control, cerebellar hemorrhages, early systemic inflammatory state, and association of these 2 injuries. A blinded neurobehavioral assessment was performed in juvenile and adult age. In the presence of cerebellar microglial cells, CBI adult male mice exposed to CBI showed a decrease in anxiety-related behaviors (elevated plus maze test), and CBI juvenile female mice exposed to CBI showed an increase in social investigation behaviors (social recognition test) compared to control groups. Cerebellar microglial depletion ensured a functional recovery of these anxiety-related and social investigation behaviors in mice exposed to an early systemic inflammatory state. This finding was consistent with a deleterious role of microglial activation. Further studies will precise the neurobehavioral subdomains affected and investigate the consequences of microglial activation (alteration of synaptic pruning and/or cerebellar myelinogenesis).

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now, the role of microglia in pruning remains under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the PC layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3CR1) signaling during postnatal development using genetic ablation of the CX3CR1 receptor and an in-depth histological analysis of the cerebellar cortex. We found an aberrant migration of microglia into the granule and the molecular layer. By electrophysiological analysis, we show that defective fractalkine signaling and the associated migration deficits neither affect the pruning of excess CFs nor the development of functional parallel fiber and inhibitory synapses with PCs. These findings indicate that CX3CR1 signaling is not mandatory for correct cerebellar circuit formation. Main Points - Ablation of CX3CR1 results in a transient migration defect in cerebellar microglia. - CX3CR1 is not required for functional pruning of cerebellar climbing fibers. - Functional inhibitory and parallel fiber synapse development with Purkinje cells is undisturbed in CX3CR1-deficient mice.

Phenylethanolamine-N-methyltransferase (Pnmt) is the enzyme that catalyzes the conversion of noradrenaline to adrenaline. These catecholamines are synthesized in the medulla of the adrenal gland and by some neurons of the central nervous system. The precise location of Pnmt action in the brain and its physiological significance are unknown. Prior studies led by Aaron Owji, a graduate student in Dr. Ebert’s laboratory, showed that mice with selectively ablated Pnmt cells show signs of neurological defects such as abnormal gait, weakened grip strength, lack of balance, reduced movement, and defective reflexes during tail suspension tests. The cerebellum is a small section of the brain that is responsible for fine-tuning motor commands. Since the Purkinje cells of the cerebellum act as the sole source of output from the cerebellar cortex, impairment of these cells could possibly account for the motor deficits seen in the mice models. The purpose of this project is to determine if there is indeed a change in Purkinje cells between wild type mice and Pnmt-ablated mice. The first aim is to identify quantitative differences in cell count between both genotypes. The second aim is to determine any morphological changes in the Purkinje cells. The main technique used in this project is immunohistochemistry in which cerebellum tissue from mice models are stained with Calbindin (a cellular marker for Purkinje neurons) and imaged with a confocal microscope. Results showed a slight reduction in the Purkinje cells of the ablated mice compared to the control genotype, accompanied with observable differences in cell structure. Understanding catecholamine pathway mechanisms in the nervous system is imperative for elucidating and targeting key players in neurodegenerative disorders.

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