A tentativa de compreender as consequências do tratamento precoce crônico com metilfenidato é muito importante uma vez que este psicoestimulante tem sido amplamente utilizado em crianças de idade pré-escolar. Além disso, pouco se sabe sobre os mecanismos envolvidos nas alterações persistentes no comportamento e no funcionamento neuronal associada à sua utilização. Neste estudo, nós inicialmente investigamos o efeito do tratamento precoce crônico com metilfenidato sobre o perfil de aminoácidos no líquido cefalorraquidiano. Além disso, foram também avaliados a homeostase glutamatérgica, a Na+,K+-ATPase e o equilíbrio redox no córtex pré-frontal de ratos jovens. Ratos Wistar receberam injeções intraperitoneais de metilfenidato (2,0 mg/kg) ou um volume equivalente de solução salina 0,9% (controles), uma vez por dia, do 15º ao 45º dia de vida. Vinte e quatro horas após a última administração de metilfenidato, os animais foram decapitados e o líquido cefalorraquidiano e o córtex pré-frontal foram obtidos e processados conforme o protocolo para cada uma das análises. Os resultados mostraram que o metilfenidato alterou o perfil de aminoácidos no líquido cefalorraquidiano, aumentando os níveis de glutamato. A captação de glutamato foi diminuída pelo tratamento crônico com metilfenidato, mas o conteúdo dos transportadores, GLAST e GLT-1, não foram alterados por esse tratamento. A atividade e o imunoconteúdo das subunidades catalíticas (α1, α2 e α3) da Na+,K+-ATPase foram diminuídos em córtex pré-frontal de ratos submetidos ao metilfenidato. Alterações na expressão gênica das subunidades α1 e α2 da Na+,K+-ATPase também foram observadas. O conteúdo de sulfidrilas, um marcador inversamente correlacionado com dano proteíco, foi diminuído. A atividade da CAT foi aumentada e a razão SOD/CAT foi diminuída em córtex pré-frontal de ratos. Os demais parâmetros avaliados não apresentaram diferenças significativas quando comparado aos controles. Os nossos resultados, tomados em conjunto, sugerem que o tratamento precoce crônico com metilfenidato promove excitotoxicidade devido, pelo menos em parte, à inibição da captação de glutamato provavelmente causada por perturbações na função da Na+,K+-ATPase e/ou pelo dano à proteína observados no córtex pré-frontal. Esses achados podem contribuir, pelo menos em parte, para uma melhor compreensão dos mecanismos envolvidos nas alterações bioquímicas e comportamentais associadas ao uso crônico de metilfenidato durante o desenvolvimento do sistema nervoso central. / Understanding the consequences of chronic treatment with methylphenidate is very important since this psychostimulant is extensively in preschool age children. Additionaly to this, little is known about the mechanisms involved in persistent changes in behavior and neuronal function related with use of methylphenidate. In this study, we initially investigate the effect of chronic treatment with methylphenidate in juvenile rats on the amino acids profile in cerebrospinal fluid, as well as on glutamatergic homeostasis, Na+,K+-ATPase function and redox balance in prefrontal cortex. Wistar rats at early age received intraperitoneal injections of methylphenidate (2.0 mg/kg) or an equivalent volume of 0.9% saline solution (controls), once a day, from the 15th to the 45th day of life. Twenty-four hours after the last administration of methylphenidate, the animals were decapitated and the cerebrospinal fluid and the prefrontal cortex were obtained and processed according to the protocol for each analysis. Our results showed that methylphenidate altered amino acid profile in cerebrospinal fluid, increasing the levels of glutamate. In the prefrontal cortex, methylphenidate administration was able to decrease the glutamate uptake, with no changes in GLAST and GLT-1; and the activity and immunocontent of catalytic subunits (α1, α2 and α3) of Na+,K+-ATPase. We also observe changes in α1 and α2 gene expression of catalytic α subunits of Na+,K+-ATPase, decrease in sulfhydryl content, CAT activity and SOD/CAT ratio in juvenile rat prefrontal cortex treated with methylphenidate. Taken together, our results suggest that chronic treatment with methylphenidate at early age induces excitotoxicity, at least in part, due to inhibition of glutamate uptake probably caused by disturbances in the Na+,K+-ATPase function and/or protein damage observed in the prefrontal cortex. These findings may contribute, at least in part, to a better understanding of mechanisms involved in the biochemical and behavioral changes associated with chronic use of methylphenidate during the development of the central nervous system.

A tentativa de compreender as consequências do tratamento precoce crônico com metilfenidato é muito importante uma vez que este psicoestimulante tem sido amplamente utilizado em crianças de idade pré-escolar. Além disso, pouco se sabe sobre os mecanismos envolvidos nas alterações persistentes no comportamento e no funcionamento neuronal associada à sua utilização. Neste estudo, nós inicialmente investigamos o efeito do tratamento precoce crônico com metilfenidato sobre o perfil de aminoácidos no líquido cefalorraquidiano. Além disso, foram também avaliados a homeostase glutamatérgica, a Na+,K+-ATPase e o equilíbrio redox no córtex pré-frontal de ratos jovens. Ratos Wistar receberam injeções intraperitoneais de metilfenidato (2,0 mg/kg) ou um volume equivalente de solução salina 0,9% (controles), uma vez por dia, do 15º ao 45º dia de vida. Vinte e quatro horas após a última administração de metilfenidato, os animais foram decapitados e o líquido cefalorraquidiano e o córtex pré-frontal foram obtidos e processados conforme o protocolo para cada uma das análises. Os resultados mostraram que o metilfenidato alterou o perfil de aminoácidos no líquido cefalorraquidiano, aumentando os níveis de glutamato. A captação de glutamato foi diminuída pelo tratamento crônico com metilfenidato, mas o conteúdo dos transportadores, GLAST e GLT-1, não foram alterados por esse tratamento. A atividade e o imunoconteúdo das subunidades catalíticas (α1, α2 e α3) da Na+,K+-ATPase foram diminuídos em córtex pré-frontal de ratos submetidos ao metilfenidato. Alterações na expressão gênica das subunidades α1 e α2 da Na+,K+-ATPase também foram observadas. O conteúdo de sulfidrilas, um marcador inversamente correlacionado com dano proteíco, foi diminuído. A atividade da CAT foi aumentada e a razão SOD/CAT foi diminuída em córtex pré-frontal de ratos. Os demais parâmetros avaliados não apresentaram diferenças significativas quando comparado aos controles. Os nossos resultados, tomados em conjunto, sugerem que o tratamento precoce crônico com metilfenidato promove excitotoxicidade devido, pelo menos em parte, à inibição da captação de glutamato provavelmente causada por perturbações na função da Na+,K+-ATPase e/ou pelo dano à proteína observados no córtex pré-frontal. Esses achados podem contribuir, pelo menos em parte, para uma melhor compreensão dos mecanismos envolvidos nas alterações bioquímicas e comportamentais associadas ao uso crônico de metilfenidato durante o desenvolvimento do sistema nervoso central. / Understanding the consequences of chronic treatment with methylphenidate is very important since this psychostimulant is extensively in preschool age children. Additionaly to this, little is known about the mechanisms involved in persistent changes in behavior and neuronal function related with use of methylphenidate. In this study, we initially investigate the effect of chronic treatment with methylphenidate in juvenile rats on the amino acids profile in cerebrospinal fluid, as well as on glutamatergic homeostasis, Na+,K+-ATPase function and redox balance in prefrontal cortex. Wistar rats at early age received intraperitoneal injections of methylphenidate (2.0 mg/kg) or an equivalent volume of 0.9% saline solution (controls), once a day, from the 15th to the 45th day of life. Twenty-four hours after the last administration of methylphenidate, the animals were decapitated and the cerebrospinal fluid and the prefrontal cortex were obtained and processed according to the protocol for each analysis. Our results showed that methylphenidate altered amino acid profile in cerebrospinal fluid, increasing the levels of glutamate. In the prefrontal cortex, methylphenidate administration was able to decrease the glutamate uptake, with no changes in GLAST and GLT-1; and the activity and immunocontent of catalytic subunits (α1, α2 and α3) of Na+,K+-ATPase. We also observe changes in α1 and α2 gene expression of catalytic α subunits of Na+,K+-ATPase, decrease in sulfhydryl content, CAT activity and SOD/CAT ratio in juvenile rat prefrontal cortex treated with methylphenidate. Taken together, our results suggest that chronic treatment with methylphenidate at early age induces excitotoxicity, at least in part, due to inhibition of glutamate uptake probably caused by disturbances in the Na+,K+-ATPase function and/or protein damage observed in the prefrontal cortex. These findings may contribute, at least in part, to a better understanding of mechanisms involved in the biochemical and behavioral changes associated with chronic use of methylphenidate during the development of the central nervous system.

O L-glutamato (L-Glu) é o principal neurotransmissor excitatório em vertebrados e é fundamental para funções primordiais do sistema nervoso central (SNC), tais como aprendizagem e memória. Entretanto, quando este neurotransmissor está em excesso na fenda sináptica, pode provocar uma série de eventos excitotóxicos, que por sua vez, estão associados a muitas neuropatologias. A terapia da maioria dessas doenças é ineficiente e provoca sérios efeitos colaterais. Portanto, é necessário desenvolver fármacos mais efetivos e com menos efeitos colaterais. Assim, peçonhas de artrópodes como a da aranha P. bistriata, se apresentam como fontes alternativas de compostos neuroativos, pois já demonstraram efeitos neuroprotetores in vitro e in vivo, bem como anticonvulsivos. Destarte, o objetivo deste estudo foi investigar um possível efeito neuroprotetor da fração RT10, isolada da peçonha de P.bistriata, em cultura primária de neurônios e glia (CPNGs), do tecido nervoso de ratos recém-nascidos, expostos a concentrações tóxicas de L-Glu (5 mM). As CPNGs foram tratadas durante 3 horas, previamente à lesão, que foi feita por um período de 12h. Ambas as exposições (tratamento e lesão) foram conduzidas no 7.º dia in vitro (DIV). Para analisar quantitativamente e qualitativamente os efeitos dos tratamentos, bem como demonstrar a composição das CPNGs foram realizados ensaios de viabilidade celular, com o sal sódico de resazurina (SSR) e, imunomarcações com anticorpos primários para MAP2, NeuN e GFAP. A fração RT10 foi neuroprotetora, pois diminuiu a perda celular nos testes com o SSR em 10%, nas CPNGs, expostas ao L-Glu, além de apresentarem efeito maior (5%), que o do fármaco Riluzol (RIL). A neuroproteção da RT10 também foi observada nos ensaios de imunocitoquimica. Os neurônios tratados com RT10 e RIL, que foram marcados com anti-MAP2 tiveram maior prolongamentos dos dendritos em relação aos neurônios não tratados. Portanto, a intensidade da fluorescência de anti-MAP2 para os neurônios tratados com esta fração foi 38% maior em relação aos não tratados; e 21% maior quando comparados ao grupo RIL. Deste modo, podemos considerar a RT10, como uma ferramenta para a prospecção de novos fármacos contra neurodegenerações, in vitro e principalmente estudos de mecanismo de ação, cujas variáveis podem ser mais bem controladas. / L-Glutamate (L-Glu), the major excitatory neurotransmitter in the central nervous system of vertebrates, is essential to the occurrence of cognitive functions. However, when L-Glu is over-accumulated in a synaptic cleft it can provoke excitotoxicity (EXT), which has been implicated in many neurological disorders (NDs). The current therapies against NDs are undereffective and can provoke side effects, so it is necessary to develop new treatments. In this regard, neuroactive compounds obtained from Parawixia bistriata spider venom are an alternative source of neuroactive compounds, because they showed neuroprotective effects in vitro and in vivo. Thus, the main aim of this work was to evaluate a possible neuroprotective effect of RT10 fraction obtained from P. bistriata venom in primary culture of neuron and glial cells (PCNGCs) from cerebral tissue of newborn Wistar rats, after the exposition to L-glu toxic concentration (5mM). The PCNGCs were submitted to the neuroprotection treatments for 3 hours and previously to the neurotoxic treatment, which the L-glu stayed for 12h in the PNGCs. The both expositions were conducted on the 7th day in vitro (DIV). The Resazurin sodium salt (RSS) and immunocytochemistry (MAP2, NeuN e GFAP primary antibodies) trials were utilized to measure quantitatively and qualitatively the treatments, as well as to prove the culture composition. In the RSS trial, the RT10 was neuroprotector, since avoided the cell death in 10%, under the PCNGCs which were exposed to L-Glu. in addition, RT10 demonstrated higher effect than rilozole (5%). RT10 attenuated the toxic effects of L-Glu under the neuromorphology, consequently the fluorescence intensity of MAP2 at PCNGC treated with RT10 was 38% higher than untreated group and it was 21% higher than riluzole group. Thus, we can consider that RT10 compounds are valuable tools to the prospection of new drugs against NDs.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutaric acidemia type I (GA-I) is an inborn error of metabolism (EIM) biochemically characterized by the main accumulation of glutaric acid (GA) and 3- hydroxyglutaric acid (3-OH-GA), and pathologically by a characteristic striatal degeneration. Due the absence of effective therapeutic strategies for this acidemia, several studies have investigated new therapies, since that one in three children subject to current treatments show striatal degeneration. In this context, the present work aimed in the first part, to investigate the effects of acute treatment with creatine (Cr), an endogenous compound guanidine which has shown neuroprotective effects in a variety of experimental models of neurodegenerative diseases and also in organic acidemias, on the GA-induced behavioral and neurochemical changes in vivo. Our results demonstrated that acute administration of Cr prevented the GA-induced behavioral and electrographic seizures, the carbonyl protein content increased and the Na+,K+-ATPase enzyme activity reduction in rats. Moreover, the Cr also protected the GA-induced sinaptossomal L-[3H]glutamate uptake reduction in vitro. As was observed in this first part, that the GA in a low concentration (10 nM) was able to reduce the L-[3H]glutamate uptake in striatal sinaptossomas of rats, and since that responsible mechanisms for striatal degeneration observed in patients are still poorly understood, we decided to evaluate in a second step, a primary action mechanism for the neurotoxic effects this low concentration of GA, which possibly may be present at the beginning of GA-I. We find that the GA reduced the L-[3H]glutamate uptake and increased the reactive species (ER) formation in sinaptossomas the striatum of rats in all times tested. Furthermore, we observed for the first time that the GA reduced the efficacy (VMax), but not the affinity (KD) of L-[3H]glutamate uptake in striatal sinaptossomas, suggesting a non-competitive inhibition. The addition of both the L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a glutamate transporters inhibitor, with the GA did not alter the inhibitory effect on the of L-[3H]glutamate uptake induced by organic acid, indicating the involvement of glutamate transporters in the GA-induced uptake reduction. Since the glutamate transporters activity can be inhibited by oxidation, we show that although the antioxidant trolox protects against GA-induced ER formation increase, it did not protect against GA-induced glutamate uptake reduction in the synaptosomes of cerebral structure studied, suggesting that ER formation may be a late event in the neurotoxicity observed in GA-I. Moreover, we can not exclude the possibility that the GA may also directly stimulate the glutamate receptors, since the GA-induced ER formation was reduced by the non-NMDA glutamate receptor antagonist, the CNQX, but not by MK-801, suggesting that these receptors contributed, at least partly, to the GA-induced oxidative stress. Also determined GA, in this low concentration, did not show oxidant activity per se. Therefore, from results in this study it was observed the protective effect of Cr administration in the deleterious actions caused by the GA. Furthermore, we show for the first time that a GA low concentration cause primary excitotoxicity, and oxidative stress in brain structure predominantly affected in this disease. Therefore, we believe that this work may help explain the genesis of GA-I, as well as the development of effective adjuvant therapeutic strategies in the treatment this acidemia. / A acidemia glutárica tipo I (GA-I) é um erro inato do metabolismo (EIM) caracterizada bioquimicamente pelo acúmulo principal de ácido glutárico (GA) e ácido 3-hidroxiglutárico (3-OH-GA), e patologicamente por uma característica degeneração estriatal. Devido à escassez de medidas terapêuticas efetivas para essa acidemia, vários estudos têm investigado novas terapias, já que uma em cada três crianças submetidas aos atuais tratamentos sofre degeneração estriatal. Nesse contexto, o presente trabalho teve por objetivo em sua primeira parte, investigar os efeitos do tratamento agudo com creatina (Cr), um composto guanidínico endógeno que tem mostrado efeitos neuroprotetores em uma variedade de modelos experimentais de doenças neurodegenerativas e também em acidemias orgânicas, sobre as alterações comportamentais e neuroquímicas induzidas pelo GA in vivo. Nossos resultados demonstraram que a administração aguda de Cr preveniu as convulsões comportamentais e eletrográficas, o aumento do conteúdo de proteína carbonil e a redução da atividade da enzima Na+,K+-ATPase induzidos pelo GA em ratos. Além disso, a Cr também protegeu da redução da captação de L-[3H]glutamato sinaptossomal induzida pelo GA in vitro. Como foi observado nesta primeira parte, que o GA em uma baixa concentração (10 nM) foi capaz de reduzir a captação de L-[3H]glutamato em sinaptossomas estriatais de ratos, e desde que os mecanismos responsáveis pela degeneração estriatal observada nos pacientes glutaricoacidêmicos ainda não estão bem esclarecidos, decidimos avaliar em uma segunda etapa, um provável mecanismo de ação primário para os efeitos neurotóxicos desta baixa concentração de GA, que possivelmente pode estar presente no início da GA-I. Verificamos que o GA reduziu a captação de L-[3H]glutamato e aumentou a formação de espécies reativas (ER) em sinaptossomas de estriado de ratos em todos os tempos testados. Além disso, observamos pela primeira vez que o GA reduziu a eficácia (VMax), mas não a afinidade (KD) da captação de L-[3H]glutamato em sinaptossomas estriatais, sugerindo uma inibição do tipo não competitiva. A adição simultânea do L-trans-pirrolidina-2,4-dicarboxilato (PDC), um inibidor dos transportadores de glutamato, com o GA não alterou o efeito inibitório sobre a captação de L-[3H]glutamato induzido pelo ácido orgânico, indicando a participação dos transportadores de glutamato na redução da captação desse neurotransmissor induzida pelo GA. Desde que a atividade dos transportadores de glutamato pode ser inibida por oxidação, evidenciamos que embora o antioxidante trolox proteja do aumento da formação de ER induzidas pelo GA, ele não protege da redução da captação de L-[3H]glutamato induzida por este ácido orgânico em sinaptossomas de estriado. Estes achados sugerem que a formação de ER pode ser um evento tardio na neurotoxicidade observada na GA-I. Além disso, não podemos excluir a possibilidade de que o GA também possa estimular diretamente os receptores de glutamato, desde que a formação de ER induzidas pelo GA foi atenuada pelo antagonista de receptor de glutamato não-NMDA, o CNQX, mas não pelo MK-801 e o GA, nessa baixa concentração, não apresentou atividade oxidante per se. Portanto, os resultados apresentados no presente estudo demonstram que a administração previa de creatina protege das ações deletérias ocasionadas pelo GA. Além disso, evidenciamos, pela primeira vez, que uma baixa concentração de GA causa ações excitotóxicas primárias, bem como, estresse oxidativo na estrutura cerebral predominantemente afetada nesta doença. Assim, acreditamos que este trabalho possa auxiliar na elucidação da gênese da GA-I, bem como no desenvolvimento de estratégias terapêuticas adjuvantes eficazes no tratamento desta acidemia.

Les interfaces sang-cerveau comme la barrière hémato-encéphalique (BHE), les plexus choroïdes (PC) ou les organes circumventriculaires (OCV), constituent des barrières physiologiques nécessaires au fonctionnement du système nerveux central. Ces barrières sont à la fois « physiques », constituées de jonctions serrées, et « enzymatiques ». Longtemps considérées comme immatures chez le fœtus, ces barrières sont en réalité présentes précocement au cours du développement. Leurs caractéristiques et leurs propriétés restent peu connues chez l'homme. Nos travaux montrent que les PC expriment, précocement au cours du développement, des protéines de jonction serrée, les claudines (CLDN) 1, 2 et 3 chez le rat et chez l'homme. Cette expression est dynamique au cours du développement avec une apparition progressive de la CLDN2 pouvant avoir un lien avec la sécrétion du liquide céphalo-rachidien. Les CLDN 1 et 3 sont identifiées chez le fœtus humain au niveau de l'organe sous-commissural (OSC), un des OCV. La CLDN5 est exprimée précocement au niveau de la BHE chez le rat et chez l'homme et son expression est altérée dans un modèle d'excitotoxicité néonatale. Nos travaux montrent également que l'analyse du profil des CLDN est utile en pathologie tumorale notamment dans la compréhension et le diagnostic de tumeurs développées à partir des PC ou de l'OSC. Enfin, diverses enzymes antioxydantes et de détoxification dont l'époxyde hydrolase microsomale sont exprimées à 22 semaines d'aménorrhée principalement au niveau des PC du fœtus humain. Ces données suggèrent des capacités de détoxification des PC, d'installation précoce au cours du développement chez l'homme / Blood-brain interfaces including blood-brain barrier (BBB), choroid plexuses (CP) or circumventricular organs (CVO) are physiological barriers required for brain homeostasis. These barriers are “physical”, with tight junctions, and “enzymatic”. Though long considered immature in fetuses, these barriers are present from an early stage of development. Their characteristics and their properties are largely unknown in humans. Our work demonstrates that CP express tight junction-associated proteins claudins (CLDN) 1, 2, and 3 at early stages of development in rat and human. This expression is dynamic during development as shown by the progressive increase of CLDN2 immunopositivity that may follow increase in cerebrospinal fluid secretion. CLDN 1 and 3 are identified in human fetal subcommissural organ (SCO), one of the CVO. CLDN5 is early expressed in rat and human BBB and its expression is disrupted by excitotoxic injury. Our work also shows that CLDN immunohistochemical profile is useful in tumoral pathology, notably to better understand and diagnose tumors arising from CP or the SCO. Finally, various antioxidant and detoxifying enzymes such as the microsomal epoxide hydrolase are expressed at 22 weeks of gestation in the human fetus, mainly in CP. These results suggest a high detoxifying capacity for the CP during development in humans

Les motoneurones tiennent une place remarquable dans l'organisme : ils constituent l'interface entre le système nerveux central et le système musculaire. Leur excitabilité est une caractéristique primordiale dans le comportement moteur puisqu'elle définit la force musculaire développée en réponse à la commande motrice. Chez la souris, la décharge des motoneurones est marquée par la présence d'oscillations de mode mixte (MMOs) entre les potentiels d'action. Ces MMOs permettent la décharge des motoneurones à basse fréquence et sont responsables d'un régime de décharge particulier nommé zone sous-Primaire, pendant lequel la fréquence de décharge est très variable et le gain de la relation courant-Fréquence élevé. Nous avons étudié les mécanismes responsables de l'apparition de ces MMOs à la fois de manière expérimentale, dans une préparation in vivo de souris anesthésié, incluant l'utilisation du Dynamic Clamp, et théorique, au moyen d'un modèle mono-Compartimental de motoneurone. Nos résultats ont montré que ces MMOs étaient causées par les courants sodiques et potassiques responsables des potentiels d'action et qu'elles émergeaient d'un état de faible excitabilité de la membrane, dû à l'inactivation lente des courants sodiques. Nous avons également montré que le courant de post-Hyperpolarisation pouvait paradoxalement augmenter l’excitabilité des motoneurones et réduire les MMOs en dé-Inactivant le courant sodique. La Sclérose Latérale Amyotrophique (SLA) conduit à la dégénérescence spécifique de ces motoneurones qui s'accompagne d'une vacuolisation de leur arborisation dendritique. L'augmentation précoce de l'excitabilité des motoneurones dans la maladie a largement été évoquée pour rendre compte de leur atteinte. Une hyperexcitabilité, aussi bien d'origine intrinsèque qu'extrinsèque pourrait en effet produire une excitotoxicité délétère pour la cellule. Si une telle modification de l'excitabilité est en cause dans la maladie, elle devrait persister jusqu'aux âges auxquels se produisent les premières dénervations des jonctions neuromusculaires. Nous avons enregistré les propriétés électrophysiologiques des motoneurones dans une préparation in vivo de souris adultes SOD1-G93A, modèle de la SLA. Nos résultats ont montré que leur conductance d'entrée était augmentée dans les jours qui précèdent les premières dénervations de leurs jonctions neuromusculaires. Malgré cela, leur excitabilité n'était pas modifiée. Loin d'être intrinsèquement hyperexcitables, une fraction d'entre eux perdaient même leur capacité à décharger de manière répétée. Nous avons finalement étudié la vacuolisation qui prend place dans les dendrites des motoneurones au cours de la maladie et son lien avec la couverture synaptique. Nous avons montré que la vacuolisation dendritique prenait place avant les dénervations et que la taille des vacuoles augmentait avec l'âge des souris SOD1-G93A. De manière intéressante, cette progression semblait plus rapide dans les motoneurones les plus sensibles à la maladie. Bien que la couverture synaptique n'était pas modifiée au cours de la maladie, nous avons mis en évidence une densité de synapses excitatrices et inhibitrices plus importante sur les régions dendritiques qui se vacuolisent. Ces résultats suggèrent un lien entre l'activité synaptique et la formation de vacuoles dans les motoneurones au cours de la SLA. Les motoneurones ne présentant pas d'hyperexcitabilité intrinsèque, une excitotoxicité d'origine synaptique pourrait alors être responsable de leur dégénérescence. / Motoneurones hold a remarkable position in the organism: they are the interface between the central nervous system and the muscular system. Their excitability is a crucial characteristic in motor behavior since it determines the muscular force produced in response to motor command. In mice, motoneurone discharge is marked by the presence of sub-Threshold oscillations between action potentials which create a behavior of mixed mode oscillations (MMOs). These MMOs allow the motoneurones to fire at low frequency and are responsible for a sub-Primary range of discharge during which the firing frequency is irregular and the slope of current-Frequency relation is steep. We investigated the mechanisms responsible for these MMOs by in vivo recordings in anesthetized mice, using Dynamic Clamp, and by theoretical modelization in a monocompartimental model of motoneurone. Our results showed that MMOs were caused by sodium and potasium currents responsible for action potentials and that they emerged from a state of low membrane excitability caused by a slow inactivation of the sodium current. Paradoxically, we also showed that the after-Hyperpolarization current was able to increase the membrane excitability and to reduce MMOs by de-Inactivating the sodium current. Amyotrophic Lateral Sclerosis (ALS) leads to the specific degeneration of these motoneurones and is accompanied by a vacuolation of their dendritic trees. An early increase in motoneurons excitability during the disease has been widely proposed to account for their degeneration. Indeed, a motoneuron hyperexcitability of intrinsic or extrinsic origin could produce a deleterious excitotoxicity. If such a change of excitability is involved in the disease, it should last until the ages where the first denervation of neuromuscular junctions occurs. We recorded the electrophysiological properties of motoneurones in an in vivo preparation of adult SOD1-G93A mice, model of ALS. Our results showed that their input conductance was increased before the first denervation of their neuromuscular junctions. Nevertheless, their excitability was not modified. Far from being intrinsically hyperexcitable, a fraction of them even lost their ability to discharge repeatedly. We finally studied the vacuolation that takes place in dendrites of motoneurones during the disease and its relation with synaptic coverage. We have shown that the dendritic vacuolation takes place before the denervation and that the size of these vacuoles increases with age in SOD1-G93A mice. Interestingly, this increase was faster in the most vulnerable motoneurones. Although synaptic coverage was not altered in the disease, we ¬revealed higher densities of excitatory and inhibitory synapses on dendritic regions that vacuolate. These results suggest a link between synaptic activity and vacuoles formation in motoneurones during ALS. Motoneurones were not intrinsically hyperexcitable, instead, an excitotoxicity from a synaptic origin may be responsible for their degeneration.

>Magister Scientiae - MSc / This study focused on the synthesis of a series of novel tricycloundecane derivatives and evaluation of these compounds for neuroprotection using the fluorescent ratiometric calcium assay that indicates the ability of the test compounds to inhibit NMDA receptors and VGCC. The cycloaddition reaction between p-benzoquinone and monomerised dicyclopentadiene yielded tricycloundeca- 4,9-diene-3,6-dione which was used as the base structure and further derivatised. These derivatives were conjugated with benzylamine to form a series of imines and amines. A total of 10 compounds were synthesised for evaluation of inhibition of calcium influx through NMDA receptor channels and voltage-gated calcium channels. The structures were confirmed using NMR, IR and MS. On the proton NMR, the characteristic AB-quartet system was observed in the region of 1-2 ppm for all the compounds and the aromatic moiety was observed between 6.5-7.5 ppm for the novel polycyclic amines. These, with other functional groups, were used to confirm the individual structures

Ischemic stroke occurs as a result of a lack or severe reduction of blood supply to the brain. Presently therapeutic interventions are limited and there is a need to develop new and efficacious stroke treatments. To this end, a great deal of research effort has been devoted to studying the potential molecular mechanisms involved in ischemic neuronal death. Correlative evidence demonstrated a paradoxical activation of the cell cycle machinery in ischemic neurons. The levels and activity of key cell cycle regulators including cyclin D1, Cdk2 and Cdk4 are upregulated following ischemic insults. However, the functional relevance of these various signals following ischemic injury was unclear. Accordingly, the research described in this thesis address the functional relevance of the activation of the cell cycle machinery in ischemic neuronal death. The data indicate that the inhibition of Cdk4 protects neurons from ischemia-induced delayed death, whereas abrogation of Cdk5 activity prevents excitotoxicity-induced damage in vitro and in vivo. Examination of upstream activators of mitotic-Cdks showed that Cdc25A is a critical mediator of delayed ischemic neuronal death. Investigation of the potential molecular mechanism by which cell cycle regulators induced neuronal death revealed perturbations in the levels and activity of key downstream targets of Cdk4. The retinoblastoma protein family members, pRb and p130 are increasingly phosphorylated following ischemic stresses. Importantly, p130 and E2F4 proteins are drastically reduced following ischemic insults. Additionally, E2F1 association with promoters of pro-apoptotic genes are induced while that of E2F4 is reduced. These changes appear to be important determinants in ischemic neuronal death. Cumulatively, the data supports the activation of the cell cycle machinery as a pathogenic signal contributing to ischemic neuronal death. The development of neuroprotectant strategies for stroke has been hampered in part by its complex pathophysiology. Previous research indicated that flavopiridol, a general CDK-inhibitor, is unable to provide sustained neuroprotection beyond one week following cerebral ischemia. The potential benefit of combining flavopiridol with another neuroprotectant, minocycline, was explored. The data indicate that while this approach provided histological protection 10 weeks after insult, the protected neurons are not functional due to progressive dendritic degeneration. This evidence indicates that targeting cell cycle pathways in stroke while important must be combined with other therapeutic modalities to fully treat stroke-induced damage.

Accidents Vasculaire Cérébraux (parfois appelés AVC ou attaques cérébrales) sont les 3èmes causes principales de mortalité et les causes de handicap dans les pays industrialisés. Ils représentent un problème de santé publique en raison de leurs fréquences, de leurs mortalités et des handicaps physiques et cognitifs qu'ils entraînent. Malgré d’importants progrès réalisés dans le domaine de la physiopathogénie de l’ischémie cérébrale, on ne dispose pas encore, aujourd’hui, de thérapeutiques efficaces pour traiter un accident vasculaire cérébral lors de sa phase aiguë. Les gangliosides GM1 sont des composants majeurs du feuillet externe de la membrane cellulaire au niveau du système nerveux central. Ces gangliosides ont des effets antineurotoxiques, neuroprotecteurs et les propriétés neurorestoratrices sur les divers neurotransmetteurs du SNC mais pour le moment ils n’ont pas des valeurs thérapeutiques en raison de leurs manques de biodisponibilités et de leurs manques de perméabilité de la barrière hématoencéphalique (BHE). Nous avons synthétisé les structures simplifiées de ces gangliosides GM1, L’idée qui a prévalu pour la conception, des nouvelles structures a été : d’introduire des chaînes grasses saturées ou insaturées sur un motif sérine, tyrosine ou cystéine, de remplacer la fonction carboxylique par des groupements reconnus comme bioisostères classiques ou non classiques de cette fonction ; comme par exemple les fonctions phosphonique, tétrazolique, 2,4-oxadiazolidine-3,5-dione. Nous avons élaboré un nouveau modèle de composé neuroprotecteur dans lequel une chaîne grasse est couplée à une entité acide ascorbique pour améliorer le passage de la BHE. Pour étudier l’activité neuroprotectrice des composés synthétisés nous avons utilisé deux modèles biologiques in vitro : un modèle cellulaire (cellules HT22) et un modèle tissulaire (tranches de cerveaux). / Stroke is the third leading cause of mortality and the primary cause of disability in adults. Therefore, it is critical to identify new, efficacious pharmacological treatments. One pharmacological approach for treatment of stroke is called neuroprotective therapy. It has been reported that the amphiphilic, monosialotetrahexosylganglioside (GM1) (II3 NeuAc-GgOsc4Cer) has antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. Since GM1 is not of therapeutic value because of its lack of bioavailability and its low blood-brain barrier (BBB) permeation. Thus, molecules that mimic GM1 represent a novel approach to neuroprotection. We have synthesized the smalls GM1-like analogues whose simplified structure includes various lipophilic saturated, unsaturated, or cyclic polyunsaturated moieties have been introduced, while in the second series, thecarboxylic acid function was replaced by different hydrophilic groups including bioisosters of the carboxylate, such as a phosphonic acid, a tetrazole, the 1,2,4-oxadiazolidine-3,5-dione or an ascorbic acid moiety. Introduction of ascorbic acid was supported by several reports that showed that ascorbic acid conjugates can improve BBB permeation properties. We report their neuroprotective effects in two distinct models of nerve cell death using hippocampus-derived HT22 cells and an additional neuroprotective assays using cortical slices injured by glutamate confirmed these results.

Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.