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Le kisspeptine (kp) est un peptide, dérivé du gène kiss-1, jouant un rôle majeur dans le contrôle central de la fonction de reproduction en régulant la sécrétion du GnRH chez l’adulte mais également au cours du développement. Les neurones exprimant kiss-1 sont situés dans la région rostrale périventriculaire du 3ème ventricule (RP3V) et le noyau arqué (ARC). L’expression de kiss-1 est hautement régulée par les stéroides sexuels, positivement dans RP3V et négativement dans ARC. Ces deux populations de neurones à kp semblent avoir des rôles différents. Les neurones à kp du RP3V seraient impliqués dans la genèse du pic préovulatoire et ceux de l’ARC dans la régulation de la sécrétion pulsatile de GnRH.L’objectif de la thèse était de déterminer la période de neurogenèse des neurones à kp ainsi que les variations de l’expression de kiss-1 et de kp dans ces deux régions au cours des différentes phases du développement chez le rat mâle et femelle.Nos résultats ont permis de cibler les périodes clés de l’ontogenèse des neurones à kp en montrant 1) que les neurones à kp de l’ARC naissent sur une période étendue à partir du jour embryonnaire (E)12,5; 2) l’existence d’une sous- expression péri-natale du kp dans l’ARC indépendante du sexe; 3) la mise en place, en période néonatale, de différences sexuelles dans les niveaux d’expression et la distribution neuroanatomique du kp; 4) l’existence de régulations péri-pubertaires de kp, dépendantes du sexe et de la région ; 5) la présence de fibres à kp dans des régions hypothalamiques suggère un rôle de kp au-delà de la fonction de reproduction. / Kisspeptin (kp) is a neuropeptide, derived from the kiss-1 gene, which plays a key role in the central control of reproduction by regulating GnRH secretion in adult but also during development. Cells which express kiss-1 are localized in two distincts hypothalamic regions: the rostral peri-ventricular third ventricule area (RP3V) and the arcuate nucleus (ARC). Kiss-1 expression is highly regulated by sex steroids: positively in the RP3V and negatively in the ARC. RP3V kp neurons have been implicated in the pre-ovulatory GnRH surge whereas ARC kp neurons may predominantly act on GnRH secretion pulsatility. The aim of this PhD work was to determine the neurogenesis period of kp neurons and changes of kiss-1 and kp expression in both regions during different stages of development in rats. Our results highlight key periods of kp neurons ontogenesis and show that: 1) ARC kp neurons are born during an extended embryonic neurogenesis period starting at embryonic day (E) 12,5; 2) a sex independent down-regulation of kp occurs during peri-natal period; 3) sex difference in the expression level and neuroanatomique distribution of kp establishes during neo-natal period; 4) kp was regulated during peri-pubertal period in sex and region dependant manner; 5) kp-ir fibers are detected throughout the septo-hypothalamic continuum suggesting that kp could be implicated in other functions than reproductive function.

Microglia, the innate immune cells of the brain, are not only immune surveyors, but also play important roles in neural development and maintenance. Microglial aberrations, including changes in morphology, gene expression, and phagocytic activity, have been observed in humans and animal models of pathologies involving cognitive and behavioral consequences. However, the precise contribution of microglial biology is not well characterized. Expression profiling of microglia and neural stem cells, co-culture assays, and transgenic mice were used to identify microglial micro-RNAs and genes, and study their roles in neural development. The results show that a specific micro-RNA, miR-155, participates in the neurogenic deficits induced by inflammation, and microglia-derived Wnt5a is essential for neural differentiation and maturation. This indicates the potential involvement of abnormal microglia in neurodevelopmental disorders such as autism spectrum disorders (ASDs). ASDs are group of debilitating disorders characterized by behavioral symptoms, including social and communication deficits and repetitive or restricted behaviors. I hypothesize that aberrant microglial biology plays a role in neurogenic and behavioral deficits in a mouse model of ASD. I performed a time-course study of microglial gene expression profiling, neural and microglial morphology, neurophysiology, and behavior in the maternal immune activation (MIA) model of ASD induced by the innate immunity ligand polyinosinic:polycytidylic acid. Microglia in MIA offspring displayed altered expression of 22 genes including 14 involved in cell-cell interaction, increased complexity of branching, and increased interactions with dendritic spines of cortical layer V pyramidal neurons. Microglial abnormalities were associated with neurophysiological alterations, measured by whole-cell patch clamp recordings, increased neuronal spine density, and ASD-like behaviors. MIA offspring treated with a colony stimulating factor -1 receptor inhibitor, to deplete and replenish microglia, showed correction of specific behaviors, microglial gene expression and branching, microglia-spine interactions, and spine density, and partial correction of neurophysiology. The data presented here shed new insight into the functional effects of microglia gene and microRNA expression in neurodevelopment. Furthermore, inflammation induces microglial aberrations that lead to altered neurodevelopment; this strongly supports the idea that targeting specific microglial genes and miRNAs will be a worthwhile approach to pursue for molecular intervention in ASD and related disorders. / 2018-11-02T00:00:00Z

The recently accepted phenomenon of adult neurogenesis is important for basic biological research and, potentially, can have major implications for the treatment of age-related cognitive decline and disease. Investigation into the mechanisms of adult neurogenesis and its ability to replenish brain circuits with new functional neurons requires whole animal models.  Zebrafish, a diurnal vertebrate, has robust cell proliferation in several neurogenic niches, including the cerebellum and dorsal telencephalon, the latter bearing homology to mammalian hippocampus. Because zebrafish demonstrate rapid regeneration in all tissues, including successful repair following brain traumas, they are promising as a model for designing therapies for human brain traumas or stroke. Their long lifespan and gradual aging also makes them an interesting model for the role of neurogenesis in counteracting human neurodegenerative disorders of aging. In different models, it has been found that cell proliferation in adult brain can be significantly affected by behavioral and environmental factors. Among those is nutrition, impacting adult neurogenesis through the amount of caloric intake, meal frequency, and meal content. The study presented here addressed the effects of nutritional factors on adult neurogenesis in a zebrafish model of premature aging due to excessive caloric food intake since early development. Fish were exposed to fasting, different diets and feeding schedules, with the rate of cell proliferation documented in two largest neurogenic niches of the zebrafish brain, the cerebellum and dorsal telencephalon. Here we show that, under normal conditions, fish with premature aging demonstrate dramatic decline in adult neurogenesis in both niches, when compared to age-matched control. The present findings establish an effect of nutrition on neurogenesis in the cerebellum and dorsal telencephalon of adult zebrafish. Zebrafish maintained on HFD, subjected to fasting, or fed only in the evenings showed significant changes in neurogenesis in two distinct neurogenic niches from that of control fish. Remarkably, the two brain regions under investigation displayed partially different responses to nutrition related factors. This was reflected in the cerebellar niche in which neurogenesis was significantly increased by 24h fast/24h refeed, high fat diet, and evening feeding conditions.  Neurogenesis of the cerebellum was significantly decreased in 24h fast, 42h fast/refeed conditions.  In the dorsal telencephalon, neurogenesis was significantly amplified by high protein, and similar to the cerebellum, high fat diet and evening feeding conditions.  In contrast, neurogenesis of the dorsal telencephalon was significantly attenuated only in the 72h fasting condition. This study provides evidence that nutrition plays important role in the modulation of adult neurogenesis in zebrafish, and presence of niche-specific responses to nutritional factors. This further suggests that zebrafish can serve as a model for studying the effects of specific diets, metabolic factors and drugs that affect metabolism in search for prophylactic and therapeutic measures for age-related cognitive decline or neurodegenerative disorders.

Genetic dissection of loci important in the control of neurogenesis has improved our understanding of both the evolutionarily conserved and divergent processes in neurodevelopment. These loci include not only protein coding genes [1, 2], but also noncoding RNAs [3-5]. One important family of non-coding RNAs is miRNAs, which control gene expression fundamental in developmental regulation and mature cell maintenance [3, 5-9]. Here, we will first focus our efforts by surveying miRNA regulation in the developing brain. We hypothesize a strong regulatory role of miRNAs during proliferation, cell death, migration and differentiation in the developing mammalian forebrain that has yet to be adequately described in the literature. Second, we will assess miRNA's role in the evolutionary divergence of brain-related gene expression. We hypothesize that a human specific single nucleotide change(s) in the miRNA recognition element of transcription factors 3' untranslated regions contributes to species-specific differences in transcription factor expression and ultimately alters regulatory function.

Attention Deficit Hyperactivity Disorder, a disorder marked by a pattern of inattention and hyperactivity, is commonly treated with the drug methylphenidate (MPH), which inhibits reuptake of the neurotransmitters norepinephrine and dopamine, thereby increasing the levels of these catecholamines in the synaptic cleft. In addition, MPH is abused by students studying for exams to increase focus and wakefulness. Despite the extensive use of MPH, little is known its long-term effects on the brain. In this study, we examined the impact of 4 weeks of MPH treatment on neurogenesis or the “birth” of new brain cells in the hippocampus of male adolescent mice. Neurogenesis was measured using 5’-ethinyldeoxyuridine (EdU), a thymidine analog that gets incorporated into DNA before cell division, and total neuron numbers were estimated using the neuronal marker, NeuN. Interestingly, low (1 mg/kg) and high (10 mg/kg) doses of MPH delivered twice daily, increased the rate of neurogenesis after 4 weeks. We also examined the survival of the new cells 4 weeks after EdU injection, both with and without continued MPH treatment. Cell counts were performed, and ratios of EdU+/NeuN+ cells were compared. Although both 1 mg/kg and 10 mg/kg MPH increased the ratio of EdU+/NeuN+ cells, the EdU+/NeuN+ ratios were no different from control if MPH was not continued. If low dose of MPH was continued for an extra 4 weeks, survival of newly generated cells was enhanced; this was not the case for the high dose of MPH. To investigate the mechanism for MPH-induced changes in hippocampal neurogenesis, we examined the levels of proteins linked to cell growth and survival in the hippocampus, including brain derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), tropomyosin receptor kinase B (TrkB, the receptor for BDNF) and beta-catenin. Levels of BDNF or GDNF were examined using enzyme-linked immunosorbent assays (ELISAs), and VEGF, TrkB, and beta-catenin expression was investigated using simple western. Interestingly, 1 mg/kg MPH appears to increase VEGF, TrkB, and beta catenin after 4 weeks. In animals treated with 10 mg/kg MPH, despite the increases in neurogenesis after 4 weeks of treatment, beta catenin levels decreased compared to control at 4 weeks, and VEGF, TrkB and beta catenin levels were decreased at 8 weeks. Thus, long-term exposure to MPH increases neurogenesis rate in the hippocampus, and the effect of low doses of MPH may be related to the increased expression of VEGF, TrkB and beta catenin.

Le projet de thèse s’inscrit dans la continuité de l’étude pilote développée dans le cadre du programme Doremi (Consortium européen de programmes de recherche portant sur les effets des faibles doses). En partant des résultats préliminaires déjà obtenus sur ce projet, l’objectif est d’approfondir les études sur les effets d’une contamination chronique à l’uranium via l’eau de boisson sur le processus de neurogénèse au cours du développement cérébral mais également au stade adulte. La première partie du projet de thèse consiste à comparer la prolifération, la survie et le potentiel de différentiation des cellules des zones neurogéniques (dans l’hippocampe principalement) à l’aide de marqueurs spécifiques de chaque stade de différentiation chez des rats contaminés ou non à l’uranium dès le stade in utero. Cette étude in vivo sera entreprise à différents stades : pendant le développement cérébral embryonnaire et post natal et à l’âge adulte. Cette première partie donnera des pistes pour étudier plus en détails les mécanismes d’action. La deuxième partie du projet de thèse vise donc à étudier comment l’uranium agit sur la neurogénèse à l’aide de modèles in vitro et ex vivo. Des cultures primaires de neurosphères seront utilisées afin d’étudier l’effet de l’uranium sur les capacités de multipotentialité des cellules souches neurales. En parallèle, un modèle de culture organotypique d’hippocampe sera développé. Ce modèle est particulièrement intéressant car il permet de réaliser des expositions aux radionucléides « à façon », d’en étudier les mécanismes d’action dans des aires cérébrales ayant une cytoarchitecture préservée et mettant en jeu différents types cellulaires, tout en combinant des méthodes d’analyse en histologie et en biologie moléculaire. / The PhD project is a continuity of the Doremi program (European Consortium of research programs on low doses effects). The objective is to assess the effects a chronic uranium contamination via drinking water on neurogenesis during brain development and in adult rats. The first part of the project will evaluate proliferation, survival and cell differentiation in neurogenic zones (in particular in the hippocampus) using specific markers for each differentiation stage in control and contaminated rats from the in utero life. This in vivo study will be performed at different stages: during embryonic and postnatal brain development and at the adult age. This part of the project will provide some clues on the potential mechanisms of action that we aim to study more in details. For this purpose, the second part of the project will be performed on in vitro and ex vivo model. Neurosphere primary cultures will be performed to assess uranium effects on the multipotential properties of neural stem cells. We also plan to use a model of hippocampal organotypic culture which will allow the study of the mechanisms of action in a preserved ex vivo structure in terms of cytoarchitecture, cell interactions, and being able to test different uranium concentrations and combine multiple analyses methods (histology, molecular biology…).

Following stroke there is a robust increase in the proliferation of neural stem and progenitor cells (NSPCs) that ectopically migrate from the subventricular zone (SVZ) to surround the site of damage induced by stroke (infarct). Previous in vivo studies by our lab and others have shown that a majority of migrating NSPCs when labelled prior to stroke become astrocytes surrounding the infarct. In contrast, our lab has shown that the majority of NSPCs when labelled after stroke become neurons surrounding the infarct. This thesis aims to elucidate the contributions of intrinsic changes that can alter the temporal fate of the NSPCs. The NSPCs were fate mapped in this study using the nestin-CreERT2 mouse model and strokes were induced using the photothrombosis model within the cortex. In alignment with our previous findings, fate-mapping the NSPCs using a single injection of tamoxifen treatment revealed a temporal-specific switch in neuronal fate when NSPCs were labeled at timepoints greater than 7 days following stroke. Single cell RNA sequencing and histological analysis identified significant differences in the proportion of populations of NSPCs and their progeny labeled at the SVZ in the absence or presence of a stroke. NSPCs labelled after stroke were comprised of a reduced proportion of quiescent neural stem cells alongside an accompanied increase in doublecortin-expressing neuroblasts. The RNA transcriptional profile of the NSPCs labelled also revealed NSPCs and their progeny labeled after stroke had an overall enrichment for a neuronal transcription profile in all of the labeled cells with a reduction in astrocytic gene expression in quiescent and activated neural stem cells. Furthermore, we highlight the presence of perturbed transcriptional dynamics of neuronal genes, such as doublecortin following stroke. Altogether, our study reveals following a stroke there is a sustained intrinsic regulated neuronal-fated response in the NSPCs that reside in the SVZ that may not be exclusive from extrinsic regulation. This work raises the challenge to learn how to harness the potential of this response to improve recovery following stroke through examining their contributions to recovery.

Great strides have been made with regard to neurogenesis in the enteric nervous system (ENS), rapidly following in the wake of recent revelations about the neurogenic properties of the central nervous system (CNS). As the ENS is more exposed, highly complex, and vulnerable to a variety of developmental, acquired, and multisystemic disorders, there is a sense of urgency for studies to address the potential within the gut to restore neurons that are injured or lost. It is the intricacies of the ENS and yet unclear relationships between agonists and embryonic precursors that have made demonstrating the arrival of new neurons in mature gut difficult under steady-state conditions. This thesis demonstrates that inducible models of a wide range of insults to the gut have yielded crucial information about ENS neurogenesis, while in vivo experimentation under steady-state conditions has proven inconsistent. Specifically, the signaling pathways of Ret and PTEN have revealed the existence of a ‘natural block’ that normally regulates neurogenesis and keeps proliferation well controlled. Furthermore, the overwhelming effects of serotonin agonism on neuron density in response to injury have uncovered an essential role of neuronal transdifferentiation by enteric glial cells that extends beyond what was once understood to be a largely homeostatic role. The influence of extrinsic innervation of the gut will also be explored, physiology of which is important for both the utility of gut microbiota and the role of Schwann cell progenitors in the development of the ENS. Therefore, this thesis will outline ENS organization and function, as well as describe common pathologies that serve as the foundation upon which neurogenesis is investigated. Critical inducible models to which chemical and molecular agonists are applied will be examined in detail, as it is through these models that therapeutics and biomedical engineering can be optimized in order to correct the pathophysiology of enteric neuropathies that as of now are still treated with surgical intervention.

The olfactory system is a great model to ask questions related to neuronal regeneration, axon guidance and synapse formation. Processing of smell begins in the olfactory epithelium where sensory neurons are present and the olfactory bulb is the first stop in processing odor information in the central nervous system. While the olfactory bulb has neurons that regenerate as well, we are interested in the regeneration that occurs in the olfactory epithelium after being injured because it possesses a source of neural stem cells – something unique to the rest of the body. Earlier studies have proven that the introduction of methimazole will effectively damage the olfactory sensory neurons while keeping the neural stem cells intact. By using a fate mapping technique involving Cre-ERT2 mice, we are able to track the regeneration of these sensory neurons after a methimazole induced injury. Using immunohistochemistry in combination with ImageJ software analysis, we are able to pinpoint the colocalization of markers of new olfactory sensory neurons (green fluorescent protein (GFP)) with markers of neuron maturation (olfactory marker protein (OMP)) and synapse formation (tyrosine hydroxylase (TH) and synaptophysin). Analysis of maturation was done in the olfactory epithelium by studying the colocalization of the protein OMP and GFP. Data shows that after regeneration, neurons coexpress both markers 11 days after lesion. In the olfactory bulb, we characterized the recovery of synaptic markers TH and synaptophysin after axons reached the olfactory bulb, where olfactory sensory neuron axons make synaptic contacts with dendrites of projection neurons. Overall, these data are the first one to establish a timeline for axonal regeneration and synapse formation after injury in the olfactory system.