The aim of this study was to investigate whether exercise could improve motor behavioral deficits and alter expression of glial fibrillary acidic protein (GFAP) in dorsal striatum in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease (PD). To this end, animals were randomly divided into 4 groups: sham sedentary (SS, n = 7); sham trained (ST, n=8); lesioned sedentary (LS, n=8) and lesioned trained (LT, n = 8). Rats were unilaterally lesioned with 6-OHDA (10 μg/3 μg) injected into the left medial forebrain bundle and sham groups were only injected with vehicle solution. The treadmill training protocol consisted of running with progressive increase in velocity, 5 days/week, during 4 weeks. Behavioral tasks were applied to asses the motor abilities of all animals prior to 6-OHDA injection and at 8th and 29th days post-injection. The tyrosine hydroxylase (TH - in substantia nigra pars compacta) and GFAP (in dorsal striatum) immunostaining was evaluated by semiquantitative analysis of the intensity (optical density - OD). The 6-OHDA lesion decreased the OD of TH and increased the OD of GFAP. In addition, the 6-OHDA lesion increased the number of ipsilateral rotations induced by methylphenidate (40 mg/kg, i.p., 30 min) and caused motor behavioral deficits. On the other hand, the treadmill training resulted in an increase in maximal exercise capacity in both trained groups (ST and LT). The training was able to reduce the number of ipsilateral rotations and ameliorated the motor behavioral deficits on 8th and 29th days postlesion. Interestingly, the exercise led to a significant increase in OD of GFAP in the LT group while there was no such effect in ST group. Our results indicate that treadmill training can improve motor behavioral deficits and suggest that the effects of exercise may be directly or, indirectly, mediated by astrocytes, as an increase in GFAP was observed in the dorsal striatum. Nevertheless, these are the first data showing an increase in GFAP expression post-exercise in this model and further research is needed to determine the precise action of exercise on astrocytes in Parkinson’s disease.

In Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), aggregated α-synuclein deposit inside cells within the brain. Smaller soluble α-synuclein aggregates, oligomers, are present both intra- and extracellularly. The α-synuclein oligomers are known to be particularly harmful, although the underlying neurotoxic mechanisms are not fully understood. The aim of this thesis was to investigate the pathogenic roles of α-synuclein oligomers and the possibility to target such species with antibody treatment. Passive immunotherapy with α-synuclein antibodies can lead to reduced pathology and ameliorated symptoms in transgenic mice. However, it remains unknown whether the antibodies are taken up by cells or whether they act extracellularly. In Paper I, we assessed cellular internalization of various α-synuclein monoclonal antibodies. The oligomer selective mAb47 displayed the highest uptake, which was promoted by the extracellular presence of α-synuclein. Alpha-synuclein aggregates can be found in both neurons and glial cells, but the pathogenic role of glial deposits has only been sparsely investigated. In Paper II, co-cultures of neurons and glia were exposed to α-synuclein oligomers. The astrocytes in the cultures rapidly accumulated oligomers, which were only partially degraded by lysosomes. The sustained intracellular α-synuclein deposits were associated with mitochondrial stress reactions in the astrocytes.  In Paper III, we sought to explore whether the astrocytic pathology induced by α-synuclein oligomers could be ameliorated by antibody treatment. Pre-incubation of oligomers with mAb47 promoted α-synuclein clearance, reduced astrocytic accumulation and rescued cells from mitochondrial stress. We could demonstrate that binding of the antibody to its antigen in the extracellular space was crucial for these effects to occur. The progressive pathology in PD is believed to be driven by cell-to-cell spreading of α-synuclein aggregates, potentially via exosomes and other extracellular vesicles (EVs). In Paper IV, we found that either fusing α-synuclein to a non-physiological protein tag or introducing the PD-causing A53T mutation directed α-synuclein towards EV secretion. Also, EV-associated α-synuclein was particularly prone to induce toxicity in recipient cells. In conclusion, this thesis sheds new light on the cellular dysfunction related to α-synuclein pathology and on how the underlying pathogenic processes may be targeted by antibody treatment.

Le glutamate est le principal neurotransmetteur excitateur du système nerveux central des vertébrés, et le codage de l’information cérébrale repose en partie sur des modulations de l’amplitude et de la fréquence des transmissions synaptiques glutamatergiques. De ce fait, la résolution spatiale et temporelle de ces transmissions nécessite un contrôle fin de la présence de glutamate dans la fente synaptique. Cette durée de vie du glutamate dans les synapses dépend directement de l’action de transporteurs spécifiques exprimés à la surface des astrocytes, en particulier les transporteurs de type GLT-1, qui retirent le neurotransmetteur et permettent ainsi de « nettoyer » la fente synaptique avant la survenue d’un nouvel épisode de neurotransmission. / A classic understanding of neurotransmitter clearance at glutamatergic synapses is that, in order to ensure sufficient glutamate uptake on a fast timescale, it is necessary to have high numbers of glutamate transporters in the vicinity of release sites to compensate for their slow transport kinetics. Using a combination of single molecule imaging and electrophysiological approaches, we now challenge this view by first demonstrating that GLT-1 transporters are not static but highly mobile at the surface of astrocytes, and that their surface diffusion is dependent upon both neuronal and glial cell activities. In the vicinity of glutamate synapses, GLT-1 dynamics are strongly reduced favoring their retention within this strategic location. Remarkably, glutamate uncaging at synaptic sites instantaneously increases GLT-1 diffusion, displacing the glutamate-bound transporter away from this compartment. Functionally, impairment of the transporter lateral diffusion through an antibody-based surface cross linking, both in vitro and in vivo, significantly slows the kinetics of excitatory postsynaptic currents. Taken together, these data reveal the unexpected and major role of the astrocytic surface GLT-1 fast dynamics in shaping glutamatergic synaptic transmission.Keywords:

Glutamate is the main excitatory neurotransmitter in the mammalian brain and its transmission is responsible for higher brain functions, such as learning, memory and cognition. Glutamate action is mediated by a variety of glutamate receptors, though their properties were until now studied predominantly in neurons. Glutamate receptors are expressed also in NG2-glia, however their role under physiological conditions as well as in pathological states of the central nervous system is not fully understood. The aim of this work is to elucidate the presence, composition and function of these receptors in NG2-glia under physiological conditions and following focal cerebral ischemia. For this purpose we used transgenic mice, in which NG2-glia are labeled by a fluorescent protein for their precise identification. To analyze the expression pattern of glutamate receptors in NG2-glia we employed single-cell RT-qPCR. Furthermore, we used calcium imaging to characterize their functional properties.

La manière dont la diversité neuronale du système nerveux central est établie au cours du développement est une question d'un grand intérêt depuis plus d'un siècle. Les progéniteurs neuronaux sont contrôlés dans l'espace et dans le temps afin de générer un ensemble diversifié de neurones. Les composants moléculaires du contrôle spatial chez les vertébrés soient bien compris, mais le cadre moléculaire du contrôle temporel chez les vertébrés n’a été que peu exploré. Les travaux réalisés, au cours de la dernière décennie, sur le développement de la rétine chez la souris ont révélé l'existence de deux importants facteurs de transcription temporels (tTF). Ikzf1 confère une compétence temporelle précoce alors que Casz1 confère une compétence temporelle tardive aux cellules progénitrices de la rétine (RPC) des mammifères. Cependant, sur les sept types cellulaires présents dans la rétine, ces tTFs ne régulent pas la capacité de générer les cônes nés précocement ni pour les cellules gliales de Müller nées tardivement, suggérant la présence d'autres tTFs non identifiés. L'objectif principal de cette thèse a été de découvrir les mécanismes moléculaires contrôlant la production des cônes au cours de la rétinogenèse précoce ainsi que la production tardive des cellules gliales de Müller. Dans cette thèse, Pou2f1 a été découvert comme un tTF qui confère aux RPCs une compétence temporelle à générer des cônes. Pou2f1 active l'expression de Pou2f2, qui permet ensuite de favoriser la production de cônes en réprimant l’expression du facteur de transcription Nrl. Ikzf1 active l'expression de Pou2f1 dans les RPC alors que Pou2f1 réprime le tTF tardif Casz1, suggérant une boucle d'autorégulation transcriptionnelle entre ces différents tTF. De plus, Ikzf4, un autre membre de la famille Ikzf, s'est avéré important pour la spécification des cônes et des cellules gliales de Müller. Ikzf4 active l’expression de Pou2f1/Pou2f2 lorsqu'il est surexprimé dans des RPC tardifs, suggérant qu’il permet la production de cônes en activant directement l’expression de Pou2f1/Pou2f2. Ikzf4 était également lié à de nombreux gènes de la voie de signalisation de Notch, qui sont impliqués dans la production de cellules gliales de Müller au cours de la rétinogenèse. Ce travail établi des bases pouvant inspirer de futures études dans d'autres parties du SNC, où des tTF similaires pourraient aussi contrôler la production de différents types cellulaires en fonction du temps. Enfin, cette thèse propose de nouveaux tTF comme outils thérapeutiques pour améliorer l’efficacité de production des cônes à partir de cellules souches. / How neural diversity is established in the developing central nervous system has been a question of great interest for more than a century. Neural progenitors are spatially and temporally patterned to generate a diverse set of neurons. Although molecular components of spatial patterning in vertebrates are well understood, the molecular framework behind temporal patterning in vertebrates has been largely unexplored. Work done in the past decade on mouse retinal development has revealed insights on how temporal patterning could be established in the CNS. Retinal neurons and glia are born in a sequential but overlapping manner from a pool of multipotent retinal progenitor cells (RPCs), consisting of an early embryonic and late post-natal window of cell birth. Two temporal transcription factors (tTFs), Ikzf1 and Casz1, confer early and late temporal competence to retinal progenitor cells (RPCs), respectively. However, out of the seven cell types in the retina, these tTFs do not regulate the competence for early born cones and late born Müller glia, suggesting the presence of other unidentified tTFs. The prime goal of this thesis was to uncover the molecular mechanisms controlling cone specification during early, and Müller glia specification during late retinal development. In this thesis, Pou2f1 was discovered as a novel tTF that confers temporal competence to RPCs to generate cones. Pou2f1 activates Pou2f2, which binds to the promoter of Nrl, a key rod specification gene, and represses its expression. Early tTF Ikzf1 activates Pou2f1, whereas Pou2f1 represses late tTF Casz1 in RPCs. Additionally, Ikzf4 was discovered as an important regulator of cone specification early, and Müller glia specification late during retinal development. Ikzf4 binds to genomic regions near Pou2f1/Pou2f2 gene bodies and activates their expression during early retinogenesis. During late retinogenesis, Ikzf4 binds to promoters of Notch signaling genes and activates their expression to promote Müller glia differentiation. Taken together, these results reveal important insights on how cone and Müller glia production is temporally controlled during early and late retinogenesis. This work lays the groundwork for future studies in other parts of the developing CNS that could employ similar tTFs for temporal patterning. Finally, this thesis proposes novel tTFs as therapeutic tools to efficiently generate cones from ESC-derived retinal organoids and sheets.

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Les altérations de la barrière hémato-rétinienne (BRB) sont associées à des maladies rétiniennes telles que la rétinopathie diabétique et la dégénérescence maculaire liée à l’âge (DMLA). L’intégrité de la BRB est cruciale pour maintenir un microenvironnement rétinien en homéostasie et est étroitement régulé grâce à la régulation du transport transcellulaire et paracellulaire. Ici, nous décrirons brièvement la BRB interne, en mettant davantage l’accent sur la structure et la fonction de la BRB externe dans les états sains et malades. Nous avons hypothétisé que le dysfonctionnement des aquaporines exprimées dans des composantes de la BRB est suffisant pour générer un oedème diabétique dans une rétine diabétique. Bien que les résultats indiquent qu’aucune des aquaporines ciblées n’est suffisante pour générer un oedème cystoïde dans un modèle murin - et ce même en l’exacerbant en augmentant la perméabilité vasculaire rétinienne, nous mettons à l’avant un outil de segmentation semi-automatisé ainsi que deux modèles de recherches engageants. Le modèle murin de DT1 induit via STZ a été étudié de façon longitudinale sur un intervalle de temps modéré et pourra être ajusté pour des expériences futures en mettant à profit un outil de quantification semi-automatique performant. D’autre part, le modèle in vitro de la sénescence de l’ÉPR est conceptuellement établi et partiellement mis sur pied. Finalement, les résultats sont prometteurs et incitent à approfondir des cibles alternatives dans un modèle conceptuellement similaire, mais soit exacerbé ou plus anatomiquement près de l’humain. / Alterations in the blood-retina barrier (BRB) are associated with retinal diseases such as diabetic retinopathy and age-related macular degeneration (AMD). The integrity of the BRB is crucial for maintaining a tightly regulated and retinal microenvironment in homeostasis through the regulation of transcellular and paracellular transport. Here, we will briefly describe the inner BRB, with a greater emphasis on the structure and function of the outer BRB in both healthy and diseased states. We hypothesized that dysfunction of aquaporins expressed in components of the BRB is sufficient to generate diabetic edema in a diabetic retina. Although the results indicate that none of the targeted aquaporin are sufficient to generate a cystoid edema in a murine model, even when exacerbated by increasing retinal vascular permeability, we present a semi-automated segmentation tool and two engaging research models. A murine model and a programmation tool were developped to efficiently assess semi-automated quantitation of retinal edema. On the other hand, the in vitro model of RPE senescence is conceptually established and partially implemented. Ultimately, the results are promising and encourage further exploration of alternative targets in a conceptually similar model, either exacerbated or more anatomically close to humans.

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