L’objectif de la thèse: À ce jour, il est admis que la TSPO joue un rôle important dans le processus inflammatoire, et qu’il est possible de suivre sa présence à l’aide d’une variété de radiotraceurs adaptés. Les impacts de l’inflammation touchent un grand nombre de personnes à travers le monde pour diverses raisons ; c’est pourquoi, quoique le [ ¹ ⁸F]DPA-714 est très prometteur, il est nécessaire d’aller plus loin pour explorer ses capacités et ses applications possibles. L’inflammation a une forte incidence sur différentes maladies, par conséquent, à impact social élevé (comme la maladie inflammatoire de l’intestin (IBD), la neuroinflammation, et le choc septique). Dans ces modèles nous analyserons et quantifierons les niveaux de d’expression de TSPO 18kDa par imagerie TEP que nous comparerons au niveau exprimé trouvés chez des sujets contrôles. L’objectif étant de déterminer si la TSPO peut constituer une cible biologique d’intérêt pour l’évaluation et la quantification d’un état inflammatoire chez l’individu en utilisant l’imagerie TEP avec le radioligand [ ¹ ⁸F]DPA-714.Aperçu sur le travail de recherche : L’étude entreprise dans cette thèse a fourni des informations conduisant à la conclusion suivante : la TSPO 18kDa peut en effet être utile comme biomarqueur pour l’évaluation d’un état inflammatoire dans plusieurs maladies. Nous avons pu illustrer par l’intermédiaire de deux modèles de la maladie inflammatoire de l’intestin, un modèle de la neuroinflammation et un modèle de choc septique, que la TSPO est un indicateur du niveau de l’inflammation dans la zone affectée. De plus, nous avons pu suivre, mesurer et quantifier l’évolution d’une zone inflammée en fonction du temps.Bien que le [ ¹ ⁸F]DPA-714 est le traceur utilisé pour déterminer la présence et le niveau de l’inflammation, d’autres traceurs sont constamment en cours de développement. Cela est démontré par le travail de collaboration effectuée avec l’équipe de radiochimie, dans lequel nous avons illustré le potentiel d’un nouveau radioligand de TSPO, le [ ¹ ⁸F]DPA-C5yne. / Purpose : The purpose of this study was to determine the in vivo potential of the TSPO 18 kDa as a biomarker of inflammation, with the use of its radioligand [ ¹ ⁸F]DPA-714, to non-invasively quantify the inflammatory state within the scope of various pathologies. Procedure : Multiple animal models of various inflammatory diseases, to include : inflammatory bowel disease, neuroinflammation, and septic shock, were developed and put in place by adapted measures. The animals well-being and the subsequent inflammation was evaluated. The inflammatory state was measured using quantitative PET imaging with the TSPO radioligand [ ¹ ⁸F]DPA-714 and correlated to the expression of conventional inflammatory markers using microscopy. Results : Based on the observed data, we were able to distinguish control groups from treated groups when using [ ¹ ⁸F]DPA-714. This TSPO radioligand permitted us to quantify the inflammatory level and to observe evolutionary changes in the inflammatory state of the disease in multiple models. The PET results, using the [ ¹ ⁸F]DPA-714 signal was correlated with an increased TSPO expression at cellular level. Conclusion : Results indicate that [ ¹ ⁸F]DPA-714 is a suitable tracer for studying inflammation of multiple diseases.[ ¹ ⁸F]DPA-714 could be a good molecular probe to non-invasively evaluate the level and localization of inflammation. Moreover, in vivo imaging using this TSPO ligand is potentially a powerful tool to stage and certainly to follow the evolution and therapeutic efficiency at molecular level in inflammatory diseases.

Les lésions nerveuses périphériques (LNP) ont des origines variées. Elles peuvent être causées par un traumatisme, une inflammation, une ischémie mais également par la compression de nerfs par une tumeur. Ces lésions traumatiques entrainent une perte sensorielle mais aussi une atteinte motrice pouvant avoir une incidence importante sur la vie des patients et leurs familles. De nombreux progrès ont été réalisés dans le traitement des LNP ces dernières années, cependant, la récupération des patients reste incomplète. Ainsi, l’amélioration du pronostic clinique demeure un défi pour les chercheurs et les cliniciens avec l’utilisation de nouvelles techniques et produits thérapeutiques. Ceci m’a conduit pour ma thèse à utiliser une nouvelle approche avec deux types de greffons combinés avec plusieurs stades de lésion du nerf donneur, mais également à tester le potentiel thérapeutique de petites molécules de types neurostéroïdes, ligands TSPO et activateurs de canaux potassiques afin d’étudier les effets sur la neurorégénération et la neuroprotection lors d’une lésion traumatique. Ainsi, ma thèse se compose de deux parties, faisant appel à deux modèles expérimentaux de rats. 1) Afin de déterminer les meilleures conditions de lésion du nerf donneur lors de la greffe, une neurorrhaphie entre le nerf donneur et la greffe a été réalisée avec deux types de greffons (frais ou pré-dégénérés). Un marquage des neurones par un traceur rétrograde, l’analyse des axones myélinisés et l’évaluation des potentiels d’actions ont été réalisés pour étudier la neuroprotection et la neurorégénération. Cette approche a permis de montrer une régénération axonale efficace à condition que la partie axonale fournie par le nerf donneur atteigne 50 % du nombre total d’axones. De plus, l’utilisation du greffon pré-dégénéré a montré un effet bénéfique en potentialisant le processus de neurorégénération. 2) Dans le but d’étudier l’effet de l’administration de petites molécules telles que la progestérone, la Nestorone, l’étifoxine et les activateurs des canaux potassiques Kv7.2/7.3 sur la neuroprotection, la régénération axonale et les fonctions de récupération, on a utilisé un modèle de lésion par compression du nerf spinal cervical. Les analyses histologiques mentionnées ci dessus, la mesure des potentiels évoqués et des tests de comportements sensori-moteurs ont été effectués pour étudier la neuroprotection, la neurorégénération et les fonctions de récupération. La progestérone et la Nestorone ont montré des effets bénéfiques sur la neuroprotection à court terme, mais également sur la neurorégénération et la récupération fonctionnelle à long terme après une lésion nerveuse. D’une manière intéressante, l’action seule de l’étifoxine améliore la récupération fonctionnelle motrice tandis que le GRT12 seul a plutôt une action bénéfique sur la récupération sensitive. Les composés de synthèse associant les propriétés de ligands TSPO d'activateurs des canaux Kv7.2/7.3 ont montré des effets sur la récupération fonctionnelle à la fois motrice et sensitive. Notre étude met en évidence qu’une neurorrhaphy réalisée avec une lésion de 50% du nerf donneur est la meilleure condition pour la neurorégénération. Elle a également permis de vérifier l’avantage de l’utilisation d'un greffon nerf pré-dégénéré pour améliorer le processus de neurorégénération. Nous avons démontré l’action thérapeutique de la progestérone et de la Nestorone sur la neuroprotection et neurorégénération, mais également celle des nouveaux composés synthétiques (ligand TSPO combiné à l’activateur Kv7.2/7.3) sur la récupération sensori-motrice. / Peripheral nerve injury (PNI) results from multiple causes, including trauma, inflammation, ischemia and tumor compression. Dysfunction of affected muscles and sensory deprivation caused by PNI result in huge obstacles for suffering patients and their families. Even though many achievements for PNI treatment have been realized during the last decades, poor clinical prognosis remains a challenge for researchers and clinicians. This prompted me to explore new approaches for nerve repair by using two different types of nerve grafts associated with several nerve injuries of donor nerve, but also investigate the therapeutic potential of several small molecules including neurosteroids, TSPO agonist ligands and potassium channel activators for promoting neuroprotection and neuroregeneration. My thesis is divided into two parts and makes use of two experimental rat models. The major aims were : 1) To determine the optimal injury condition of a donor nerve for end-to-side neurorrhaphy by using a sciatic end-to-side neurorrhaphy model with different types of autologous nerve grafts (fresh or predegenerated). Neuronal retrograde labeling, analysis of myelinated axons and the evaluation of action potentials were performed to evaluate neuroprotection and neuroregeneration. This approach showed that effective axonal regeneration into a nerve graft occurred only when axonal resources supplied by the donor nerve reached 50% of its total number of axons. In addition, the use of a predegenerated nerve graft was beneficial for neuroregeneration by improving the environment of the nerve pathway. 2) To investigate the efficacy of the administration of small molecules including progesterone, Nestorone, etifoxine and Kv7.2/7.3 channel activators for neuroprotection, axonal regeneration and functional recovery. For these studies, we developed a cervical spinal nerve crush injury model. The same histological analysis mentioned above, measures of evoked potentials and motor or sensory behavioral tests were performed to evaluate neuroprotection, neuroregeneration and functional recovery. Progesterone and Nestorone showed benefits on neuroprotection at early stages and on neuroregeneration and functional recovery at later stages after nerve injury. Interestingly, the mono-therapy of etifoxine promoted motor functional recovery while the mono-therapy of GRT12 was beneficial for sensory functional recovery. The synthetic compounds combining TSPO ligand and Kv7.2/7.3 channel activator properties promoted both motor and sensory functional recovery. In conclusion, our study revealed that 50% axotomy of a donor nerve in the end-to-side neurorrhaphy paradigm is the optimal condition for neuroregeneration. We also verified the advantage of using predegenerated instead of a fresh nerve graft to improve the regenerative environment. We also demonstrated the effectiveness of progesterone and Nestorone in neuroprotection and neuroregeneration, and that new synthetic compounds with TSPO ligand and Kv7.2/7.3 channel activator properties promoted motor and sensory recovery.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13378号 / 論医博第2212号 / 新制||医||1047(附属図書館) / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 渡邉 大 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

O aumento da expectativa de vida da população mundial tem se associado com uma maior prevalência de doenças neurodegenerativas. A Doença de Alzheimer (DA) é a doença neurodegenerativa mais comum e a principal causa de demência em indivíduos com mais de 60 anos, sendo caracterizada por um declínio progressivo na memória e função mental dos pacientes. Esses sintomas são acompanhados por alterações histopatológicas no cérebro desses indivíduos, incluindo a presença de uma grande quantidade de placas senis, formadas pela deposição do peptídeo beta-amiloide (Aβ), e de emaranhados neurofibrilares formados pela hiperfosforilação da proteína Tau. Estudos indicam que a deposição do Aβ é uma das principais responsáveis pelo desenvolvimento da DA, causando dano neuronal através da ativação de várias vias pró-apoptóticas e dando origem aos sintomas de demência típicos dessa doença. Até o momento, não existem tratamentos eficazes para o combate à DA, de forma que a maior parte das intervenções farmacológicas é destinada apenas ao tratamento de alguns de seus sintomas. A proteína translocadora (TSPO) se localiza em pontos de contato entre as membranas mitocondriais interna e externa e está relacionada com o transporte de colesterol para o interior da mitocôndria e com a regulação da esteroidogênese e da apoptose. Estudos mostram que ligantes da TSPO apresentam efeitos neuroprotetores em diferentes modelos experimentais de lesão cerebral e doenças neurodegenerativas. Especificamente em relação à DA, um estudo indicou que o 4’-clorodiazepam (4’-CD), um ligante da TSPO, apresenta efeitos neuroprotetores em um modelo animal dessa doença, sendo um possível candidato para o seu tratamento. Dessa forma, o objetivo desse estudo foi verificar o efeito neuroprotetor do 4’-CD em diferentes modelos in vitro de toxicidade induzida pelo Aβ, além de seus efeitos sobre o desenvolvimento de neurônios hipocampais. Inicialmente, demonstramos que o 4’-CD reduziu a morte celular de células SH-SY5Y expostas a um modelo de toxicidade induzida pela administração de Aβ. Esses efeitos estiveram associados com a redução da expressão da proteína pró-apoptótica Bax e com um aumento da expressão da survivina, uma proteína anti-apoptótica. A expressão das proteínas Bcl-xl e procaspase-3, por outro lado, não foi alterada pelos tratamentos. Posteriormente, estudamos os efeitos neuroprotetores do 4’-CD contra a toxicidade induzida pela administração do Aβ em culturas organotípicas de hipocampo. Nesses experimentos, foi demonstrado que o 4’-CD reduz a morte celular de culturas organotípicas de hipocampo expostas ao Aβ através de um aumento na expressão da enzima SOD, sem alterar, no entanto, a expressão das proteínas Akt e procaspase-3. Por fim, foi avaliado o efeito do 4’-CD sobre o desenvolvimento de culturas primárias de neurônios hipocampais de camundongos machos e fêmeas. Foi observado que as culturas de neurônios hipocampais das fêmeas apresentaram um desenvolvimento mais rápido do que as dos machos. O 4’-CD acelerou a maturação e aumentou a ramificação neurítica dos neurônios hipocampais dos machos, mas não exerceu qualquer efeito sobre os neurônios das fêmeas. Em suma, foi observado que o 4’-CD apresenta efeitos neuroprotetores contra o Aβ em células SH-SY5Y e em culturas organotípicas do hipocampo, apresentando-se como um fármaco em potencial para o tratamento da DA. Além disso, foi observado que o 4’-CD exerceu um efeito dependente do sexo sobre o desenvolvimento de culturas primárias de neurônios hipocampais, estimulando o desenvolvimento e a ramificação neurítica de neurônios hipocampais de machos, mas não de fêmeas. / The increase in life expectancy of the world population has been associated with a higher prevalence of neurodegenerative diseases. The Alzheimer’s Disease (AD) is the most common neurodegenerative disorder and the main cause of dementia among people over 60 years, being characterized by a progressive decline in the memory and mental function of the patients. These symptoms are associated with histopathological changes in the brain of these patients, including the presence of senile plaques, formed by the deposition of amyloid-beta (Aβ), and neurofibrillary tangles, which are related to the hyperphosphorylation of Tau protein. Studies indicate that Aβ deposition is a major contributor to AD progression, promoting neuronal damage through the activation of different pro-apoptotic pathways and giving rise to the typical dementia symptoms of this disease. To date, there are no effective treatments for AD, so that most of the pharmacological intervention is intended for the treatment of some of its symptoms. The translocator protein (TSPO) is located in contact sites between the outer and the inner mitochondrial membranes and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and apoptosis. Studies show that TSPO ligands present neuroprotective effects in different experimental models of brain injury and neurodegenerative diseases. Specifically regarding AD, a study indicated that 4’-chlorodiazepam (4’-CD), a TSPO ligand, is neuroprotective in an animal model of this disease, being a possible candidate for its treatment. Therefore, the aim of this study was to evaluate the neuroprotective effect of 4’-CD in different experimental models of Aβ- induced neurotoxicity in vitro, as well as its effects on the development of hipocampal neurons. First, it was demonstrated that 4’-CD decreased the cell death of SH-SY5Y cells exposed to the Aβ. This effect was associated with the inhibition of the Aβ-induced upregulation of Bax, a pro-apoptotic protein, and downregulation of survivin, a prosurvival protein. On the other hand, the expression of Bcl-xl and procaspase-3 was not change by the treatments. After, it was studied the neuroprotective effects of 4’-CD against Aβ in organotypic hipocampal cultures. In these experiments, it was shown that 4’-CD decreases the cell death of organotypic hippocampal slices exposed to the Aβ by increasing the protein expression of SOD, but without changing the expression of Akt and procaspase-3. Finally, due to the importance of the processes of neuronal development and maturation in the regeneration of CNS after injury, it was evaluated the effect of 4’-CD on the development of primary hippocampal neurons of male and female mice. It was observed that female primary hippocampal neurons presented an increased rate of development than male neurons. 4’-CD stimulated the development and increased the neuritic branching of male but not from female neurons. In summary, it was observed that 4’-CD presented a neuroprotective effect against Aβ in SH-SY5Y cells and in rat organotypical hippocampal slices, presenting itself as a promising agent for the treatment of AD. Also, it was observed that 4’-CD modulates the development of hippocampal neurons in a sex-dependent manner, stimulating the development of male but not from female cells.

O aumento da expectativa de vida da população mundial tem se associado com uma maior prevalência de doenças neurodegenerativas. A Doença de Alzheimer (DA) é a doença neurodegenerativa mais comum e a principal causa de demência em indivíduos com mais de 60 anos, sendo caracterizada por um declínio progressivo na memória e função mental dos pacientes. Esses sintomas são acompanhados por alterações histopatológicas no cérebro desses indivíduos, incluindo a presença de uma grande quantidade de placas senis, formadas pela deposição do peptídeo beta-amiloide (Aβ), e de emaranhados neurofibrilares formados pela hiperfosforilação da proteína Tau. Estudos indicam que a deposição do Aβ é uma das principais responsáveis pelo desenvolvimento da DA, causando dano neuronal através da ativação de várias vias pró-apoptóticas e dando origem aos sintomas de demência típicos dessa doença. Até o momento, não existem tratamentos eficazes para o combate à DA, de forma que a maior parte das intervenções farmacológicas é destinada apenas ao tratamento de alguns de seus sintomas. A proteína translocadora (TSPO) se localiza em pontos de contato entre as membranas mitocondriais interna e externa e está relacionada com o transporte de colesterol para o interior da mitocôndria e com a regulação da esteroidogênese e da apoptose. Estudos mostram que ligantes da TSPO apresentam efeitos neuroprotetores em diferentes modelos experimentais de lesão cerebral e doenças neurodegenerativas. Especificamente em relação à DA, um estudo indicou que o 4’-clorodiazepam (4’-CD), um ligante da TSPO, apresenta efeitos neuroprotetores em um modelo animal dessa doença, sendo um possível candidato para o seu tratamento. Dessa forma, o objetivo desse estudo foi verificar o efeito neuroprotetor do 4’-CD em diferentes modelos in vitro de toxicidade induzida pelo Aβ, além de seus efeitos sobre o desenvolvimento de neurônios hipocampais. Inicialmente, demonstramos que o 4’-CD reduziu a morte celular de células SH-SY5Y expostas a um modelo de toxicidade induzida pela administração de Aβ. Esses efeitos estiveram associados com a redução da expressão da proteína pró-apoptótica Bax e com um aumento da expressão da survivina, uma proteína anti-apoptótica. A expressão das proteínas Bcl-xl e procaspase-3, por outro lado, não foi alterada pelos tratamentos. Posteriormente, estudamos os efeitos neuroprotetores do 4’-CD contra a toxicidade induzida pela administração do Aβ em culturas organotípicas de hipocampo. Nesses experimentos, foi demonstrado que o 4’-CD reduz a morte celular de culturas organotípicas de hipocampo expostas ao Aβ através de um aumento na expressão da enzima SOD, sem alterar, no entanto, a expressão das proteínas Akt e procaspase-3. Por fim, foi avaliado o efeito do 4’-CD sobre o desenvolvimento de culturas primárias de neurônios hipocampais de camundongos machos e fêmeas. Foi observado que as culturas de neurônios hipocampais das fêmeas apresentaram um desenvolvimento mais rápido do que as dos machos. O 4’-CD acelerou a maturação e aumentou a ramificação neurítica dos neurônios hipocampais dos machos, mas não exerceu qualquer efeito sobre os neurônios das fêmeas. Em suma, foi observado que o 4’-CD apresenta efeitos neuroprotetores contra o Aβ em células SH-SY5Y e em culturas organotípicas do hipocampo, apresentando-se como um fármaco em potencial para o tratamento da DA. Além disso, foi observado que o 4’-CD exerceu um efeito dependente do sexo sobre o desenvolvimento de culturas primárias de neurônios hipocampais, estimulando o desenvolvimento e a ramificação neurítica de neurônios hipocampais de machos, mas não de fêmeas. / The increase in life expectancy of the world population has been associated with a higher prevalence of neurodegenerative diseases. The Alzheimer’s Disease (AD) is the most common neurodegenerative disorder and the main cause of dementia among people over 60 years, being characterized by a progressive decline in the memory and mental function of the patients. These symptoms are associated with histopathological changes in the brain of these patients, including the presence of senile plaques, formed by the deposition of amyloid-beta (Aβ), and neurofibrillary tangles, which are related to the hyperphosphorylation of Tau protein. Studies indicate that Aβ deposition is a major contributor to AD progression, promoting neuronal damage through the activation of different pro-apoptotic pathways and giving rise to the typical dementia symptoms of this disease. To date, there are no effective treatments for AD, so that most of the pharmacological intervention is intended for the treatment of some of its symptoms. The translocator protein (TSPO) is located in contact sites between the outer and the inner mitochondrial membranes and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and apoptosis. Studies show that TSPO ligands present neuroprotective effects in different experimental models of brain injury and neurodegenerative diseases. Specifically regarding AD, a study indicated that 4’-chlorodiazepam (4’-CD), a TSPO ligand, is neuroprotective in an animal model of this disease, being a possible candidate for its treatment. Therefore, the aim of this study was to evaluate the neuroprotective effect of 4’-CD in different experimental models of Aβ- induced neurotoxicity in vitro, as well as its effects on the development of hipocampal neurons. First, it was demonstrated that 4’-CD decreased the cell death of SH-SY5Y cells exposed to the Aβ. This effect was associated with the inhibition of the Aβ-induced upregulation of Bax, a pro-apoptotic protein, and downregulation of survivin, a prosurvival protein. On the other hand, the expression of Bcl-xl and procaspase-3 was not change by the treatments. After, it was studied the neuroprotective effects of 4’-CD against Aβ in organotypic hipocampal cultures. In these experiments, it was shown that 4’-CD decreases the cell death of organotypic hippocampal slices exposed to the Aβ by increasing the protein expression of SOD, but without changing the expression of Akt and procaspase-3. Finally, due to the importance of the processes of neuronal development and maturation in the regeneration of CNS after injury, it was evaluated the effect of 4’-CD on the development of primary hippocampal neurons of male and female mice. It was observed that female primary hippocampal neurons presented an increased rate of development than male neurons. 4’-CD stimulated the development and increased the neuritic branching of male but not from female neurons. In summary, it was observed that 4’-CD presented a neuroprotective effect against Aβ in SH-SY5Y cells and in rat organotypical hippocampal slices, presenting itself as a promising agent for the treatment of AD. Also, it was observed that 4’-CD modulates the development of hippocampal neurons in a sex-dependent manner, stimulating the development of male but not from female cells.

O aumento da expectativa de vida da população mundial tem se associado com uma maior prevalência de doenças neurodegenerativas. A Doença de Alzheimer (DA) é a doença neurodegenerativa mais comum e a principal causa de demência em indivíduos com mais de 60 anos, sendo caracterizada por um declínio progressivo na memória e função mental dos pacientes. Esses sintomas são acompanhados por alterações histopatológicas no cérebro desses indivíduos, incluindo a presença de uma grande quantidade de placas senis, formadas pela deposição do peptídeo beta-amiloide (Aβ), e de emaranhados neurofibrilares formados pela hiperfosforilação da proteína Tau. Estudos indicam que a deposição do Aβ é uma das principais responsáveis pelo desenvolvimento da DA, causando dano neuronal através da ativação de várias vias pró-apoptóticas e dando origem aos sintomas de demência típicos dessa doença. Até o momento, não existem tratamentos eficazes para o combate à DA, de forma que a maior parte das intervenções farmacológicas é destinada apenas ao tratamento de alguns de seus sintomas. A proteína translocadora (TSPO) se localiza em pontos de contato entre as membranas mitocondriais interna e externa e está relacionada com o transporte de colesterol para o interior da mitocôndria e com a regulação da esteroidogênese e da apoptose. Estudos mostram que ligantes da TSPO apresentam efeitos neuroprotetores em diferentes modelos experimentais de lesão cerebral e doenças neurodegenerativas. Especificamente em relação à DA, um estudo indicou que o 4’-clorodiazepam (4’-CD), um ligante da TSPO, apresenta efeitos neuroprotetores em um modelo animal dessa doença, sendo um possível candidato para o seu tratamento. Dessa forma, o objetivo desse estudo foi verificar o efeito neuroprotetor do 4’-CD em diferentes modelos in vitro de toxicidade induzida pelo Aβ, além de seus efeitos sobre o desenvolvimento de neurônios hipocampais. Inicialmente, demonstramos que o 4’-CD reduziu a morte celular de células SH-SY5Y expostas a um modelo de toxicidade induzida pela administração de Aβ. Esses efeitos estiveram associados com a redução da expressão da proteína pró-apoptótica Bax e com um aumento da expressão da survivina, uma proteína anti-apoptótica. A expressão das proteínas Bcl-xl e procaspase-3, por outro lado, não foi alterada pelos tratamentos. Posteriormente, estudamos os efeitos neuroprotetores do 4’-CD contra a toxicidade induzida pela administração do Aβ em culturas organotípicas de hipocampo. Nesses experimentos, foi demonstrado que o 4’-CD reduz a morte celular de culturas organotípicas de hipocampo expostas ao Aβ através de um aumento na expressão da enzima SOD, sem alterar, no entanto, a expressão das proteínas Akt e procaspase-3. Por fim, foi avaliado o efeito do 4’-CD sobre o desenvolvimento de culturas primárias de neurônios hipocampais de camundongos machos e fêmeas. Foi observado que as culturas de neurônios hipocampais das fêmeas apresentaram um desenvolvimento mais rápido do que as dos machos. O 4’-CD acelerou a maturação e aumentou a ramificação neurítica dos neurônios hipocampais dos machos, mas não exerceu qualquer efeito sobre os neurônios das fêmeas. Em suma, foi observado que o 4’-CD apresenta efeitos neuroprotetores contra o Aβ em células SH-SY5Y e em culturas organotípicas do hipocampo, apresentando-se como um fármaco em potencial para o tratamento da DA. Além disso, foi observado que o 4’-CD exerceu um efeito dependente do sexo sobre o desenvolvimento de culturas primárias de neurônios hipocampais, estimulando o desenvolvimento e a ramificação neurítica de neurônios hipocampais de machos, mas não de fêmeas. / The increase in life expectancy of the world population has been associated with a higher prevalence of neurodegenerative diseases. The Alzheimer’s Disease (AD) is the most common neurodegenerative disorder and the main cause of dementia among people over 60 years, being characterized by a progressive decline in the memory and mental function of the patients. These symptoms are associated with histopathological changes in the brain of these patients, including the presence of senile plaques, formed by the deposition of amyloid-beta (Aβ), and neurofibrillary tangles, which are related to the hyperphosphorylation of Tau protein. Studies indicate that Aβ deposition is a major contributor to AD progression, promoting neuronal damage through the activation of different pro-apoptotic pathways and giving rise to the typical dementia symptoms of this disease. To date, there are no effective treatments for AD, so that most of the pharmacological intervention is intended for the treatment of some of its symptoms. The translocator protein (TSPO) is located in contact sites between the outer and the inner mitochondrial membranes and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and apoptosis. Studies show that TSPO ligands present neuroprotective effects in different experimental models of brain injury and neurodegenerative diseases. Specifically regarding AD, a study indicated that 4’-chlorodiazepam (4’-CD), a TSPO ligand, is neuroprotective in an animal model of this disease, being a possible candidate for its treatment. Therefore, the aim of this study was to evaluate the neuroprotective effect of 4’-CD in different experimental models of Aβ- induced neurotoxicity in vitro, as well as its effects on the development of hipocampal neurons. First, it was demonstrated that 4’-CD decreased the cell death of SH-SY5Y cells exposed to the Aβ. This effect was associated with the inhibition of the Aβ-induced upregulation of Bax, a pro-apoptotic protein, and downregulation of survivin, a prosurvival protein. On the other hand, the expression of Bcl-xl and procaspase-3 was not change by the treatments. After, it was studied the neuroprotective effects of 4’-CD against Aβ in organotypic hipocampal cultures. In these experiments, it was shown that 4’-CD decreases the cell death of organotypic hippocampal slices exposed to the Aβ by increasing the protein expression of SOD, but without changing the expression of Akt and procaspase-3. Finally, due to the importance of the processes of neuronal development and maturation in the regeneration of CNS after injury, it was evaluated the effect of 4’-CD on the development of primary hippocampal neurons of male and female mice. It was observed that female primary hippocampal neurons presented an increased rate of development than male neurons. 4’-CD stimulated the development and increased the neuritic branching of male but not from female neurons. In summary, it was observed that 4’-CD presented a neuroprotective effect against Aβ in SH-SY5Y cells and in rat organotypical hippocampal slices, presenting itself as a promising agent for the treatment of AD. Also, it was observed that 4’-CD modulates the development of hippocampal neurons in a sex-dependent manner, stimulating the development of male but not from female cells.

Les neurostéroïdes sont des composés endogènes qui peuvent moduler la transmission synaptique et avoir un effet neuroprotecteur dans les maladies neurodégénératives. Les systèmes régulant leur biosynthèse ne sont pas connus mais la première étape de celle-ci peut être régulée par la protéine TSPO. Cette protéine mitochondriale facilite le transport du cholestérol vers l’intérieur de la mitochondrie pour y être métabolisé en prégnénolone. Ce stéroïde est le précurseur principal de la biosynthèse des neurostéroïdes et l’utilisation in vitro de ligands du TSPO permet d’augmenter sa sécrétion. Dans ce travail de thèse, nous avons ainsi cherché à développer de nouvelles familles de ligands solubles du TSPO augmentant la sécrétion de prégnénolone. Le développement de ces nouvelles familles a nécessité la réalisation d’une méthodologie de synthèse faisant intervenir une réaction de cyclisation pallado-catalysée de type Buchwald-Hartwig. Une étude de solubilité des composés synthétisés a été effectuée expérimentalement, leur activité a été évaluée par des méthodes fonctionnelles et leur effet neuroprotecteur a été testé sur un modèle cellulaire de la maladie d’Alzheimer. / Neurosteroids are endogenous compounds which can alter the synaptic transmission and enhance neuroprotection in neurodegenerative diseases. The systems that regulates their biosynthesis are not described but its first step ca be regulated by the TSPO. This mitochondrial protein facilitates the transport of cholesterol to the mitochondrial matrix to be metabolized in pregnenolone. This steroid is the precursor of neurosteroid biosynthesis and in vitro use of TSPO ligands induces its secretion. For this project, we looked forward to develop new families of soluble TSPO ligands that can increase pregnenolone production. The access to 3-amino-3,4-dihydroquinolin-2-ones required the establishment of a synthesis methodology of a palladium-catalyzed cyclization following Buchwald-Hartwig amination. A solubility study of synthesized compound was performed, their activity was established based on functional assays and their neuroprotective effect was evaluated on a cellular model of Alzheimer disease.