La voie de signalisation activée par la protéine Sonic Hedgehog (Shh) est connue pour son rôle majeur au cours de l’embryogenèse et en particulier dans la prolifération et la spécification cellulaire ou encore le guidage axonal au cours de l’établissement des structures du système nerveux. Depuis quelques années, ce morphogène a aussi été identifié comme un régulateur important de plusieurs processus physiologiques du cerveau adulte comme le maintien de la neurogenèse ou la régulation de l’activité électrique de certains neurones (Traiffort et al., 2010). La suractivation de la voie Shh dans un cerveau sain entraine une augmentation significative de la prolifération des cellules progénitrices des oligodendrocytes (OPCs), la source des oligodendrocytes matures, les cellules responsables de la formation des gaines de myéline (Loulier et al., 2006). Au cours de ma thèse, j’ai étudié le potentiel que représente l’activation de la voie Shh dans la régulation de ces progéniteurs dans un contexte de démyélinisation. Pour cela, j’ai utilisé une souris transgénique plp-GFP, chez laquelle la protéine fluorescente verte est exprimée par les cellules du lignage oligodendrocytaire. Après avoir caractérisé le profil d’expression de la GFP dans le cerveau mature de ces souris, j’ai mis au point un modèle de démyélinisation focale par injection stéréotaxique d’un détergent spécifique de la myéline, la lysolécithine (LPC). J’ai identifié les cellules du lignage oligodendrocytaire comme source directe de protéines Shh au sein de la lésion à un temps très précoce après l’injection de LPC. Les gènes cibles de la voie Shh sont aussi fortement induits dans cette population cellulaire à une période plus tardive, correspondant à la différenciation des OPCs en cellules matures. L’utilisation d’adénovirus codant soit pour Shh lui-même soit pour son antagoniste physiologique Hip, m’a permis de réaliser des expériences de gain et de perte de fonction et ainsi d’analyser comment la modulation de la voie Shh peut influencer sur le processus de régénération des oligodendrocytes suite à une lésion. La surexpression de Shh permet d’augmenter la prolifération des OPCs mais aussi d’accélérer leur différenciation, aboutissant à un nombre plus élevé d’oligodendrocytes matures plus précocement au cours du processus de remyélinisation. De plus, il est intéressant de constater que la densité des cellules astrocytaires et microgliales, notamment associées au processus inflammatoire, diminue dans la lésion chez les animaux ayant reçu l’adénovirus Shh comparés au animaux contrôles. A l’inverse, le blocage de la voie induit l’arrêt complet de la production de nouveaux oligodendrocytes. Au-delà de l’amélioration de notre compréhension de la physiologie et de la régulation du lignage oligodendrocytaire dans le cerveau adulte, l’ensemble de ce travail montre de quelle manière la voie Shh peut représenter une nouvelle piste dans la recherche de cibles thérapeutiques dans les affections de la myéline telles que la sclérose en plaques. / The Sonic Hedgehog (Shh) signaling pathway is known for its role during embryogenesis and in particular for controlling cell proliferation and specification, as well as axon guidance. In recent years, this morphogen has also been identified as an important regulator of several physiological processes in the adult brain such as the maintenance of neurogenesis or the regulation of the electrophysiological propreties of mature neurons (Traiffort et al., 2010). Overactivation of the Shh pathway in a healthy brain causes a significant increase in the proliferation of oligodendrocyte progenitor cells (OPCs), the source of mature oligodendrocytes, the cells responsible for the formation of myelin sheaths (Loulier et al., 2006).In my thesis, I studied the effects of the Shh pathway activation on OPC regulation in the context of demyelination. To that purpose, I used a plp-GFP transgenic mouse, in which the green fluorescent protein (GFP) is expressed by cells belonging to the oligodendrocyte lineage. After characterization of the expression pattern of GFP in the mature brain of these mice, I developed a model of focal demyelination by stereotaxic injection of lysolecithin (LPC). I identified the oligodendrocyte lineage cells as a source of Shh protein within the lesion, soon after the LPC injection. Target genes of the Shh pathway are also strongly induced in this cell population, at a time corresponding to the differentiation of OPCs into mature cells. The use of adenoviral vectors encoding either Shh itself or its physiological antagonist Hip allowed me to conduct gain- and loss-of-function experiments. This way I could analyze how the modulation of Shh pathway may influence the regeneration ofoligodendrocytes after injury. Shh overexpression increases the survival and proliferation of OPCs but also accelerates their differentiation, resulting in a higher number of mature oligodendrocytes earlier during the remyelination process. In addition, the density of astrocytes and microglia, associated with the inflammatory process, is decreased in animalsreceiving the Shh adenoviral vector compared to control animals. Altogether these effects are associated with a reduction of the lesion. Conversely, blocking the pathway induced a complete arrest of new oligodendrocyte production. Besides the fundamental knowledge gained about the molecular mechanism involved in the oligodendroglial precursor cells survival, proliferation, differentiation and myelin repair in vivo, this project should also give valuable insights concerning the potential use of pharmacological modulators of Shh signaling as a novel therapeutic approach for the treatment of multiple sclerosis and other myelin diseases.

Chitosan is widely investigated for biomedical applications due to its excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial, etc. In the field of neural engineering, it has been extensively studied in forms of film and hydrogel, and has been used as scaffolds for nerve regeneration in the peripheral nervous system and spinal cord. One of the main issues in neural engineering is the incapability of neuron to attach on biomaterials. The present study, from a new aspect, aims to take advantage of the bio-adhesive property of chitosan to develop chitosan-based materials for neural engineering, specifically in the fields of brain repair and neuroprosthetics. Neuronal responses to the developed biomaterials will also be investigated and discussed. In the first part of this study (Chapter II), chitosan was blended with a well-studied hydrogel material (agarose) to form a simply prepared hydrogel system. The stiffness of the agarose gel was maintained despite the inclusion of chitosan. The structure of the blended hydrogels was characterized by light microscopy and scanning electron microscopy. In vitro cell studies revealed the capability of chitosan to promote neuron adhesion. The concentration of chitosan in the hydrogel had great influence on neurite extension. An optimum range of chitosan concentration in agarose hydrogel, to enhance neuron attachment and neurite extension, was identified based on the results. A “steric hindrance” effect of chitosan was proposed, which explains the origin of the morphological differences of neurons in the blended gels as well as the influence of the physical environment on neuron adhesion and neurite outgrowth. This chitosan-agarose (C-A) hydrogel system and its multi-functionality allow for applications of simply prepared agarose-based hydrogels for brain tissue repair. In the second part of this study (Chapter III), chitosan was blended with graphene to form a series of graphene-chitosan (G-C) nanocomposites for potential neural interface applications. Both substrate-supported coatings and free standing films could be prepared by air evaporation of precursor solutions. The electrical conductivity of graphene was maintained after the addition of chitosan, which is non-conductive. The surface characteristic of the films was sensitively dependent on film composition, and in turn, influenced neuron adhesion and neurite extension. Biological studies showed good cytocompatibility of graphene for both fibroblast and neuron. Good cell-substrate interactions between neurons and G-C nanocomposites were found on samples with appropriate compositions. The results suggest this unique nanocomposite system may be a promising substrate material used for the fabrication of implantable neural electrodes. Overall, these studies confirmed the bio-adhesive property of chitosan. More importantly, the developed chitosan-based materials also have great potential in the fields of neural tissue engineering and neuroprosthetics.

Stroke causes CNS injury associated with strong fast microglial activation as part of the inflammatory response. Fast activation of microglia in response to neuronal damage requires the rapid availability of a large amount of energy to trigger diverse cytotoxic or neuroprotective signals. ATP-dependent potassium (K(ATP)) channels play important roles in many cellular functions by coupling cell metabolism to electrical activity. K(ATP) channels were first detected in cardiac myocytes and later found in beta-cells of the pancreas, skeletal muscle, neurons, smooth muscle, heart, pituitary, and tubular cells of the kidney. Our group and others have also demonstrated its expression in reactive microglia after brain injury. In rat models of stroke, blockade of the sulfonylurea receptor (SUR), with glibenclamide (Gbc) reduced cerebral edema and infarct volume. Furthermore, clinical data suggest the effectiveness of Gbc to treat stroke. Gbc close the K(ATP) channel by interaction with two drug-binding sites on SUR subunits, as well as, the astroglial NC(Ca-ATP) channel, which mediates the Gbc-induced prevention of edema after cerebral ischemia. In these studies however, the function of the K(ATP) channel remained unclear. Therefore, as Gbc may bind to constitute functional K(ATP) channels after ischemic stroke, other possible effects of Gbc might explain the effectiveness of this drug in the treatment of stroke. Giving the fact that, SUR1-regulated channels are exquisitely sensitive to changes in the metabolic state of the cell, and that microglia are sensing the environment, the expression of K(ATP) channels in activated microglia, will couple cell energy to membrane potential. We herein postulate, that the effectiveness of Gbc to treat stoke, at least in part, is caused by the KATP channel closure expressed by activated microglia, which may then be critical in determining, their participation in the pathogenic process. Given the analogy with beta-cells, K(ATP) channel blockade in microglia would response faster and more efficiently to the external signals released after brain injury. If true, blockade of microglial K(ATP) channel with low doses of Gbc during the early stages of stroke might foster neuroprotective microglial activity, could enhance ischemia-induced neurogenesis in the SVZ, and consequently will lead to an improved functional outcome. The work presented in this thesis demonstrates that, Gbc improves functional neurological outcome in stroke, accompanied by neuron preservation in the core of the ischemic brain. In this region, reactive microglia from tMCAO rats upregulate the K(ATP) channel, which makes microglia a target to Gbc actions in the early stages of stroke. Furthermore, Gbc also strengthens the neuroprotective role of microglia in the acute phase after focal cerebral ischemia, enhance long-term neurogenesis and brain repair processes. As such, identify microglial K(ATP) channels as a key target for stroke treatment. Overall, these results provide new therapeutic avenues for the treatment of other neurological disorders that involve microglia.

Chitosan is widely investigated for biomedical applications due to its excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial, etc. In the field of neural engineering, it has been extensively studied in forms of film and hydrogel, and has been used as scaffolds for nerve regeneration in the peripheral nervous system and spinal cord. One of the main issues in neural engineering is the incapability of neuron to attach on biomaterials. The present study, from a new aspect, aims to take advantage of the bio-adhesive property of chitosan to develop chitosan-based materials for neural engineering, specifically in the fields of brain repair and neuroprosthetics. Neuronal responses to the developed biomaterials will also be investigated and discussed.In the first part of this study (Chapter II), chitosan was blended with a well-studied hydrogel material (agarose) to form a simply prepared hydrogel system. The stiffness of the agarose gel was maintained despite the inclusion of chitosan. The structure of the blended hydrogels was characterized by light microscopy and scanning electron microscopy. In vitro cell studies revealed the capability of chitosan to promote neuron adhesion. The concentration of chitosan in the hydrogel had great influence on neurite extension. An optimum range of chitosan concentration in agarose hydrogel, to enhance neuron attachment and neurite extension, was identified based on the results. A “steric hindrance” effect of chitosan was proposed, which explains the origin of the morphological differences of neurons in the blended gels as well as the influence of the physical environment on neuron adhesion and neurite outgrowth. This chitosan-agarose (C-A) hydrogel system and its multi-functionality allow for applications of simply prepared agarose-based hydrogels for brain tissue repair.In the second part of this study (Chapter III), chitosan was blended with graphene to form a series of graphene-chitosan (G-C) nanocomposites for potential neural interface applications. Both substrate-supported coatings and free standing films could be prepared by air evaporation of precursor solutions. The electrical conductivity of graphene was maintained after the addition of chitosan, which is non-conductive. The surface characteristic of the films was sensitively dependent on film composition, and in turn, influenced neuron adhesion and neurite extension. Biological studies showed good cytocompatibility of graphene for both fibroblast and neuron. Good cell-substrate interactions between neurons and G-C nanocomposites were found on samples with appropriate compositions. The results suggest this unique nanocomposite system may be a promising substrate material used for the fabrication of implantable neural electrodes. Overall, these studies confirmed the bio-adhesive property of chitosan. More importantly, the developed chitosan-based materials also have great potential in the fields of neural tissue engineering and neuroprosthetics.

Le diabète de type 2 est une pathologie complexe et multifactorielle caractérisée par une hyperglycémie chronique et une résistance à l’insuline. Actuellement, le diabète de type 2 est une préoccupation majeure et mondiale de santé publique. Les objectifs de cette thèse étaient d’étudier l’impact de l’hyperglycémie (aiguë, récurrente et chronique) sur des modèles complémentaires in vivo (poisson zèbre et souris) à un niveau périphérique et central. L’hyperglycémie aiguë et/ou chronique chez le poisson zèbre a altéré l’expression génique des cytokines pro-inflammatoires du cerveau, a modulé l’expression des gènes impliqués dans l’établissement de la barrière hémato-encéphalique, a diminué la prolifération des progéniteurs neuronaux de la plupart des niches neurogéniques et a impacté les mécanismes de réparation du télencéphale. L’hyperglycémie récurrente a induit un prédiabète caractéristique chez la souris avec des altérations périphériques et centrales que la glycine a atténuées en partie. L’ensemble des résultats indique que l’hyperglycémie contribue aux dysfonctionnements observés dans le diabète au niveau de l’inflammation, du stress oxydatif, de la barrière hémato-encéphalique, de la plasticité neuronale et des processus neurogéniques, ainsi que des fonctions cognitives, et souligne le potentiel antidiabétique intéressant de la glycine. En complément de ces résultats de recherche, une mission d’enseignement orientée santé a été menée auprès d’étudiants universitaires. Cette approche holistique de la pathologie diabétique pourrait contribuer, à terme, à l’élaboration de programmes de prévention adaptés, de dépistages précoces et de thérapies efficaces. / Type 2 diabetes is a complex and multifactorial disease characterized by chronic hyperglycemia and insulin resistance. Type 2 diabetes is currently a major and worldwide public health issue. The main objectives of this thesis were to investigate the peripheral and central impact of hyperglycemia (acute, recurrent and chronic) in vivo on complementary models (zebrafish and mouse). In zebrafish, acute and/or chronic hyperglycemia modulated cerebral expression of pro-inflammaty cytokines, as well as expression of genes involved in blood-brain barrier establishment. It also reduced neural progenitor proliferation in main neurogenic niches and impaired brain repair mechanisms. In the mouse, recurrent hyperglycemia induced a characterized prediabetes with central and peripheral alterations partially alleviated by glycine. All results suggest that, hyperglycemia contributes to related diabetes dysfunctions, through inflammation and oxydative stress, on blood-brain barrier integrity, neural plasticity including neurogenesis, and cognitive functions, and highlights the antidiabetic potential of glycine. In addition, a health-oriented teaching mission has been undertaken on university students. This holistic approach on diabetic disease could help in establishing effective prevention programs, early screenings and efficient therapies.