Protein arginine methyltransferase 1 (PRMT1), -4 (also known as coactivator-associated arginine methyltransferase 1; CARM1), and -5 catalyze the methylation of arginine residues on target proteins. In turn, these marked proteins mediate a variety of biological functions. By regulating molecules that are critical to the remodelling of skeletal muscle phenotype, PRMTs may influence skeletal muscle plasticity. Our study tests the hypothesis that the intracellular signals required for muscle adaptation to exercise will be associated with the induction of PRMT expression and activity. C57BL/6 mice were assigned to one of three experimental groups: sedentary (SED), acute bout of exercise (0PE), or acute exercise followed by 3 hours of recovery (3PE). The mice in the exercise groups performed a single bout of treadmill running at 15 m/min for 90 minutes. We observed that PRMT gene expression and global enzyme activity are muscle- specific, generally being higher in slow, oxidative muscle, as compared to faster, more glycolytic tissue. Despite the activation of canonical exercise-induced signalling involving AMPK and PGC-1α, PRMT expression and activity at the whole muscle level were unchanged. However, subcellular analysis revealed the exercise-evoked myonuclear translocation of PRMT1 prior to the nuclear translocation of PGC-1α, which colocalizes the proteins within the organelle after exercise. Acute physical activity also augmented the targeted methyltransferase activities of CARM1, PRMT1, and -5 in the myonuclear compartment, suggesting that PRMT-mediated histone arginine methylation is an integral part of the early signals that drive skeletal muscle plasticity. In summary, our data supports the emergence of PRMTs as important players in the regulation of skeletal muscle plasticity. / Thesis / Master of Science (MSc) / Skeletal muscle is a plastic tissue that can adapt to various physiological demands. Previous work suggests that protein arginine methyltransferases (PRMTs) are important in the regulation of skeletal muscle remodeling. However, their role in exercise-induced skeletal muscle plasticity is unknown. Therefore, the purpose of this study was to investigate the association between the intracellular signals required for muscle adaption and various metrics of PRMT biology. Our data demonstrate that PRMTs exhibit muscle-specific expression and function in mice. The movement of PRMT1 into myonuclei increased following exercise, while the specific methylation status of PRMT targets were also elevated. Overall, our data suggests that muscle-specific PRMT expression may be important for the determination and/or maintenance of different fiber type characteristics. Moreover, distinct PRMT cellular localization and methyltransferase activity may be key signals that contribute to skeletal muscle phenotypic plasticity.

Despite the emerging importance of protein arginine methyltransferases (PRMTs) in regulating skeletal muscle plasticity, the biology of these enzymes during muscle development remains poorly understood. Therefore, our purpose was to investigate PRMT1, -4, and -5 expression and function in skeletal muscle cells during the phenotypic remodeling elicited by myogenesis. C2C12 muscle cell maturation, assessed during the myoblast stage, and during days 1, 3, 5, and 7 of differentiation, was employed as an in vitro model of myogenesis. We observed PRMT-specific patterns of expression and activity during myogenesis. PRMT4 and -5 gene expression was unchanged, while PRMT1 mRNA and protein content were significantly induced. Cellular monomethylarginines and symmetric dimethylarginines, indicative of global and type II PRMT activities, respectively, remained steady during development, while type I PRMT activity indicator asymmetric dimethylarginines increased through myogenesis. Histone 4 arginine 3 (H4R3) and H3R17 contents were elevated coincident with the myonuclear accumulation of PRMT1 and -4. Collectively, this suggests that PRMTs are methyl donors throughout myogenesis and demonstrate specificity for their protein targets. Cells were then treated with TC-E 5003 (TC-E), a selective inhibitor of PRMT1 in order to specifically examine the enzymes role during myogenic differentiation. TC-E treated cells exhibited decrements in muscle differentiation, which were consistent with attenuated mitochondrial biogenesis and respiratory function. In summary, this study increases our understanding of PRMT1, -4, and -5 biology during the plasticity of skeletal muscle development. Our results provide evidence for a role of PRMT1, via a mitochondrially-mediated mechanism, in driving the muscle differentiation program. / Thesis / Master of Science (MSc) / Protein arginine methyltransferases (PRMTs) are responsible for many important functions in skeletal muscle. However, significant knowledge gaps exist with respect to PRMT expression and activity during conditions of muscle remodeling. Therefore, the purpose of this Thesis was to investigate PRMT biology throughout skeletal muscle development. Mouse muscle cells were employed to examine characteristics of PRMT1, -4, and -5 at numerous timepoints during myogenesis. PRMTs exhibited distinct patterns of gene expression and activity during muscle maturation. A PRMT1 inhibitor (TC-E) was utilized to investigate the role of this enzyme during myogenesis. Muscle differentiation was impaired in TC-E-treated cells, which coincided with reduced mitochondrial biogenesis and respiratory function. Altogether, these results suggest a PRMT-specific pattern of expression and activity during myogenesis. Furthermore, PRMT1 plays a crucial role in skeletal muscle differentiation via a mitochondrially-mediated mechanism. Our study provides a more comprehensive view on the role of PRMTs in governing skeletal muscle plasticity.

Essentielle à l'équilibre énergétique de la cellule, la mitochondrie, véritable sentinelle, joue, un rôle majeur dans le destin de la cellule, en modulant les voies de signalisation de mort cellulaire mis en jeu dans l'atrophie musculaire. L'objectif de cette thèse est de proposer des cibles thérapeutiques centrées sur la mitochondrie dans deux modèles murins dont la physiopathologie est caractérisée par une dysfonction mitochondriale associée à une atrophie musculaire : le vieillissement et la dystrophie musculaire de Duchenne (DMD). Pour lutter contre la perte de masse musculaire liée à l'âge, la déficience en myostatine (mstn), associée à un phénotype hypermusculé, est une stratégie thérapeutique prometteuse. Mais, l'altération du métabolisme mitochondrial et oxydatif induite par cette déficience réduit les effets bénéfiques d'une telle stratégie. Nous avons donc testé l'intérêt de l'utilisation de la molécule pharmacologique AICAR, activateur connu de l'AMPK, afin de « booster » la fonction mitochondriale chez la souris âgée KO mstn. Les résultats montrent chez la souris KO mstn, une amélioration du temps d'endurance de course. Au niveau signalétique, le traitement induit des effets bénéfiques mais limités sur la fonction mitochondriale. Les mécanismes restent à préciser mais tendent vers l'hypothèse d'un effet bénéfique de l'AICAR sur le stress du réticulum endoplasmique (RE). Le dysfonctionnement mitochondrial a été également largement impliqué dans la physiopathologie de la DMD. Dans notre seconde étude, ce même traitement à l'AICAR chez le modèle murin de la DMD, la souris mdx atténue le phénotype dystrophique et améliore la fonction contractile du diaphragme. Nous montrons que ces effets bénéfiques sont associés à une induction de mécanisme de survie, l'autophagie, et une limitation des phénomènes d'apoptose induit par la mitochondrie, mettant en évidence une amélioration de l'intégrité mitochondriale par stimulation de leur renouvellement dans des fibres musculaires dystrophiques. Enfin, ce travail a mis en avant pour la première fois la présence à l'état basal de stress du RE chez la mdx, propsant une nouvelle cible thérapeutique. L'impact de ce stress dans la fibre musculaire normal et pathologique est très mal connu. Nos résultats montrent que le stress du RE modifie les liens entre le réticulum sarcoplasmique et la mitochondrie, perturbe l'homéostasie calcique et active les voies de mort cellulaire associées à une dysfonction contractile. Ces résultats ouvrent une perspective de stratégie thérapeutique dans les pathologies musculaire impliquant un stress du RE, comme la DMD. Ce travail de thèse a mis en avant l'importance de développer des thérapies pharmacologiques dans les pathologies musculaires, permettant d'améliorer la fonction à la fois métabolique et de sentinelle de la mitochondrie. / Fundamental for the energetic balance of the cell, mitochondria play a key role for modulation of cell death pathway related to muscular atrophy. Thus, the purpose of this PhD is to find therapeutic strategy focus on mitochondria in two different murine models where the physiopathology is characterized by a mitochondria dysfunction associated with muscle atrophy: Aging process and Duchenne Muscular Dystrophy (DMD).To prevent loss of muscle mass associated with aging, the lack of myostatin, inducing a hypermuscular phenotype, is a promising therapeutic strategy. However, loss of myostatin is associated with a strong reduction of mitochondrial and oxidative metabolism in skeletal muscle, and this strategy need to be potentiated. In this context, we explore if mitochondrial alteration in aged wild-type mice or in aged mstn KO mice are rescued by chronic AMPK-activating treatment, using the synthetic agonist AICAR, considered as “an mimetic of exercise”. Our results show an improvement of aerobic running performance in mstn KO mice. Concerning to signaling pathways, AICAR treatment induces beneficial but limited effects on mitochondrial metabolism. Mechanisms are still under investigation but our results suggest a reduction in ER stress. Moreover, mitochondria dysfunction has been widely implicated in DMD physiopathology. This same treatment of AICAR, in the murine model of DMD, improves the diaphragm histopathology as well as maximal force generating capacity. These beneficial effects were linked with autophagy activation and apoptosis limitation, without inducing muscle fiber atrophy, and promoting the elimination of defective mitochondria.Finally, the last part of this study highlight for the first time, an increase of ER stress at basal level, suggesting a new therapeutic target. Nevertheless, ER stress impact in skeletal muscle fibers is sparsely known. The preliminary results show that ER stress decrease the link between RE and mitochondria, which have an impact on calcium homeostasis and stimulate cell death pathway with a decrease of contractile function.This study highlights the importance to develop pharmacological therapies in muscular pathology, focus on metabolic and sentinel mitochondria function.

A doença hepática gordurosa não-alcoólica (NAFLD, do inglês) é a manifestação clínica hepática da síndrome metabólica, cuja incidência aumenta consideravelmente em todo o mundo. A NAFLD pode progredir para um estado de esteatohepatite não-alcoólica (NASH, do inglês), caracterizado por inflamação hepatocelular, com ou sem fibrose. Dados na literatura mostram que o coativador-1 alfa do receptor ativado por proliferadores de peroxissoma gama (PGC-1alfa), além de estar envolvido em diversos processos metabólicos, representa uma estratégia terapêutica promissora na modulação da inflamação. Neste projeto investigamos as alterações inflamatórias no fígado induzida por dieta hiperlipídica e o papel do PGC-1alfa nesse processo. Camundongos C57black/6 receberam dieta hiperlipídica contendo 30% de gordura por 10 semanas. O peso dos animais foi avaliado semanalmente. Após a eutanásia, o tecido adiposo intra-abdominal (retroperitoneal e periepididimal) foi coletado e pesado. Analisamos o perfil glicêmico e lipídico sérico e expressão de genes envolvidos no metabolismo glicêmico e lipídico. Avaliou-se também o aspecto histológico e a inflamação do tecido hepático por quantificação das citocinas IL-6, TNF-alfa e IL-1beta. A dieta rica em gordura conduziu a um aumento dos depósitos de gordura intra-abdominal, hiperglicemia e hiperlipidemia. Os animais também apresentavam esteatohepatite, com aumento de citocinas pró-inflamatórias e diminuição na expressão de PGC-1alfa no tecido hepático. O envolvimento do PGC-1alfa na produção de mediadores inflamatórios por hepatócitos foi avaliado em células HepG2 utilizando RNA de interferência (RNAi). O knockdown da expressão de PGC-1alfa causou aumento na expressão e liberação de IL-6 em hepatócitos via aumento na fosforilação de IkBalfa e consequente ativação do NFkB. Portanto, nossos dados mostram que o PGC-1alfa inibe a produção de mediadores inflamatórios (IL-6) em hepatócitos, e fornecem novas evidências das conexões existentes entre as vias metabólicas e imunes / Nonalcoholic fatty liver disease (NAFLD) is the liver clinical manifestation of the metabolic syndrome, whose incidence increases considerably around the world. NAFLD may progress to a state of non-alcoholic steatohepatitis, characterized by hepatocellular inflammation, with or without fibrosis. Data in the literature show that the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alfa), a protein involved in various metabolic processes, represents a promising therapeutic strategy in the modulation of inflammation. In this project, we investigate the inflammatory changes in the liver induced by high fat diet and the role of PGC-1alfa in this process. C57black/6 mice were fed a high fat diet containing 30% fat for 10 weeks. The weight of the animals was measured weekly. After euthanasia, the intra-abdominal adipose tissue (retroperitoneal and periepididymal) was collected and weighed. We have analyzed the glycemic and serum lipid profile and expression of genes involved in glucose and lipid metabolism. We also assessed liver morphology by HE staining and inflammation by quantification of the cytokines IL-6, TNF-alfa and IL-1beta. The high fat diet led to an increase in intra-abdominal fat deposits, hyperglycemia and hyperlipidemia. Animals also presented steatohepatitis, an increased proinflammatory cytokines and decreased PGC-1alfa expression in the hepatic tissue. The involvement of PGC-1alfa on inflammatory mediators production by hepatocytes was evaluated in HepG2 cells using RNA interference (RNAi). The knockdown of PGC-1alfa expression caused an increase in IL6 expression and release by hepatocytes via the increase in the IkBalfa phosphorylation and subsequent activation of NFkB. Therefore, our data show that PGC-1alfa inhibits the production of inflammatory mediators (IL-6) in hepatocytes and provide further evidence of the connections between the metabolic and immune pathways

A doença hepática gordurosa não-alcoólica (NAFLD, do inglês) é a manifestação clínica hepática da síndrome metabólica, cuja incidência aumenta consideravelmente em todo o mundo. A NAFLD pode progredir para um estado de esteatohepatite não-alcoólica (NASH, do inglês), caracterizado por inflamação hepatocelular, com ou sem fibrose. Dados na literatura mostram que o coativador-1 alfa do receptor ativado por proliferadores de peroxissoma gama (PGC-1alfa), além de estar envolvido em diversos processos metabólicos, representa uma estratégia terapêutica promissora na modulação da inflamação. Neste projeto investigamos as alterações inflamatórias no fígado induzida por dieta hiperlipídica e o papel do PGC-1alfa nesse processo. Camundongos C57black/6 receberam dieta hiperlipídica contendo 30% de gordura por 10 semanas. O peso dos animais foi avaliado semanalmente. Após a eutanásia, o tecido adiposo intra-abdominal (retroperitoneal e periepididimal) foi coletado e pesado. Analisamos o perfil glicêmico e lipídico sérico e expressão de genes envolvidos no metabolismo glicêmico e lipídico. Avaliou-se também o aspecto histológico e a inflamação do tecido hepático por quantificação das citocinas IL-6, TNF-alfa e IL-1beta. A dieta rica em gordura conduziu a um aumento dos depósitos de gordura intra-abdominal, hiperglicemia e hiperlipidemia. Os animais também apresentavam esteatohepatite, com aumento de citocinas pró-inflamatórias e diminuição na expressão de PGC-1alfa no tecido hepático. O envolvimento do PGC-1alfa na produção de mediadores inflamatórios por hepatócitos foi avaliado em células HepG2 utilizando RNA de interferência (RNAi). O knockdown da expressão de PGC-1alfa causou aumento na expressão e liberação de IL-6 em hepatócitos via aumento na fosforilação de IkBalfa e consequente ativação do NFkB. Portanto, nossos dados mostram que o PGC-1alfa inibe a produção de mediadores inflamatórios (IL-6) em hepatócitos, e fornecem novas evidências das conexões existentes entre as vias metabólicas e imunes / Nonalcoholic fatty liver disease (NAFLD) is the liver clinical manifestation of the metabolic syndrome, whose incidence increases considerably around the world. NAFLD may progress to a state of non-alcoholic steatohepatitis, characterized by hepatocellular inflammation, with or without fibrosis. Data in the literature show that the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alfa), a protein involved in various metabolic processes, represents a promising therapeutic strategy in the modulation of inflammation. In this project, we investigate the inflammatory changes in the liver induced by high fat diet and the role of PGC-1alfa in this process. C57black/6 mice were fed a high fat diet containing 30% fat for 10 weeks. The weight of the animals was measured weekly. After euthanasia, the intra-abdominal adipose tissue (retroperitoneal and periepididymal) was collected and weighed. We have analyzed the glycemic and serum lipid profile and expression of genes involved in glucose and lipid metabolism. We also assessed liver morphology by HE staining and inflammation by quantification of the cytokines IL-6, TNF-alfa and IL-1beta. The high fat diet led to an increase in intra-abdominal fat deposits, hyperglycemia and hyperlipidemia. Animals also presented steatohepatitis, an increased proinflammatory cytokines and decreased PGC-1alfa expression in the hepatic tissue. The involvement of PGC-1alfa on inflammatory mediators production by hepatocytes was evaluated in HepG2 cells using RNA interference (RNAi). The knockdown of PGC-1alfa expression caused an increase in IL6 expression and release by hepatocytes via the increase in the IkBalfa phosphorylation and subsequent activation of NFkB. Therefore, our data show that PGC-1alfa inhibits the production of inflammatory mediators (IL-6) in hepatocytes and provide further evidence of the connections between the metabolic and immune pathways

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A insuficiência cardíaca (IC) é a evolução final das várias formas de doenças cardiovascular, sendo resultado de modificações estruturais, metabólicas e de contratilidade miocárdica. A fim de compreender o papel na dinâmica do metabolismo cardíaco no estado basal e na sobrecarga de pressão, utilizamos os modelos de cre-lox com deleção específica no coração para substrato do receptor de insulina (IRS) e co-ativador do PPAR (PGC-1β) e analisamos a estrutura cardíaca (histologia e estereologia), função cardíaca (ecocardiograma e técnica de Working heart), o metabolismo (isolamento de cardiomiócito e captação de glicose), ação hormonal (Western Blotting), expressão gênica (PCR-RT) de enzimas do metabolismo (lipídico, glicídico, da cadeia respiratória fatores transcricionais e hipertróficos) e a função mitocondrial. Verificamos, nos CIRS12KO, disfunção cardíaca grave, disfunção mitocondrial e prejuízo na expressão gênica das enzimas do metabolismo energético. Nos PGC-1βKO observamos disfunção mitocondrial e alteração de expressão gênica das enzimas do metabolismo energético quando submetidos à sobrecarga de pressão. Através do estudo do metabolismo cardíaco e da expressão gênica nestes diferentes modelos conseguimos explorar as vias metabólicas que levam a hipertrofia compensada à IC. Sugerimos que o mecanismo responsável pela descompensação seja a disfunção mitocondrial em conseqüência à alteração da expressão gênica. E que IRS e o PGC-1β são fatores chaves da dinâmica cardíaca, e que são indispensáveis para a estrutura e funcionamento cardíaco. Além de representar alvo promissor para limitar a transição da hipertrofia cardíaca compensada a insuficiência cardíaca. / Heart failure (HF) is the end stage of different types of cardiovascular diseases and it is characterized by changes in the metabolic and myocardial contractility. We use the models cre-lox with specific knockout for insulin receptor substrate (IRS) and co-activator of PPAR (PGC-1b) (basal and pressure overload). The objective was understood the role in the dynamics of cardiac metabolism. We analyzed cardiac structure (histology and stereology), cardiac function (echocardiography and the working heart technique), metabolism (glucose uptake), hormonal action (Western Blotting), gene expression (RT-PCR) from enzyme metabolism (lipid, carbohydrates, respiratory chain, transcriptional and hypertrophic factors) and mitochondrial function. We found in CIRS12KO, severe cardiac dysfunction, mitochondrial dysfunction and reduction of gene expression. And in the PGC-1bKO when subjected to pressure overload, the progression to heart failure, with mitochondrial dysfunction, and alteration of gene expression from enzyme metabolism. The data show that changes on cardiac metabolism and gene expression in both models explain the metabolic pathways that lead to compensated hypertrophy to HF. We suggest that the mitochondrial dysfunction and the gene expression was possible mechanisms for HF. We conclude that IRS and PGC-1b are key factors of cardiac dynamics, which are essential to the structure and heart function. IRS and PGC-1b represent a promising target for limiting the transition from compensated cardiac hypertrophy to heart failure.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A insuficiência cardíaca (IC) é a evolução final das várias formas de doenças cardiovascular, sendo resultado de modificações estruturais, metabólicas e de contratilidade miocárdica. A fim de compreender o papel na dinâmica do metabolismo cardíaco no estado basal e na sobrecarga de pressão, utilizamos os modelos de cre-lox com deleção específica no coração para substrato do receptor de insulina (IRS) e co-ativador do PPAR (PGC-1β) e analisamos a estrutura cardíaca (histologia e estereologia), função cardíaca (ecocardiograma e técnica de Working heart), o metabolismo (isolamento de cardiomiócito e captação de glicose), ação hormonal (Western Blotting), expressão gênica (PCR-RT) de enzimas do metabolismo (lipídico, glicídico, da cadeia respiratória fatores transcricionais e hipertróficos) e a função mitocondrial. Verificamos, nos CIRS12KO, disfunção cardíaca grave, disfunção mitocondrial e prejuízo na expressão gênica das enzimas do metabolismo energético. Nos PGC-1βKO observamos disfunção mitocondrial e alteração de expressão gênica das enzimas do metabolismo energético quando submetidos à sobrecarga de pressão. Através do estudo do metabolismo cardíaco e da expressão gênica nestes diferentes modelos conseguimos explorar as vias metabólicas que levam a hipertrofia compensada à IC. Sugerimos que o mecanismo responsável pela descompensação seja a disfunção mitocondrial em conseqüência à alteração da expressão gênica. E que IRS e o PGC-1β são fatores chaves da dinâmica cardíaca, e que são indispensáveis para a estrutura e funcionamento cardíaco. Além de representar alvo promissor para limitar a transição da hipertrofia cardíaca compensada a insuficiência cardíaca. / Heart failure (HF) is the end stage of different types of cardiovascular diseases and it is characterized by changes in the metabolic and myocardial contractility. We use the models cre-lox with specific knockout for insulin receptor substrate (IRS) and co-activator of PPAR (PGC-1b) (basal and pressure overload). The objective was understood the role in the dynamics of cardiac metabolism. We analyzed cardiac structure (histology and stereology), cardiac function (echocardiography and the working heart technique), metabolism (glucose uptake), hormonal action (Western Blotting), gene expression (RT-PCR) from enzyme metabolism (lipid, carbohydrates, respiratory chain, transcriptional and hypertrophic factors) and mitochondrial function. We found in CIRS12KO, severe cardiac dysfunction, mitochondrial dysfunction and reduction of gene expression. And in the PGC-1bKO when subjected to pressure overload, the progression to heart failure, with mitochondrial dysfunction, and alteration of gene expression from enzyme metabolism. The data show that changes on cardiac metabolism and gene expression in both models explain the metabolic pathways that lead to compensated hypertrophy to HF. We suggest that the mitochondrial dysfunction and the gene expression was possible mechanisms for HF. We conclude that IRS and PGC-1b are key factors of cardiac dynamics, which are essential to the structure and heart function. IRS and PGC-1b represent a promising target for limiting the transition from compensated cardiac hypertrophy to heart failure.

La sarcopénie est considérée comme un syndrome gériatrique se caractérisant par une diminution de la masse musculaire qui en s’aggravant sera à l’origine d’une détérioration de la force musculaire et des performances physiques. La sarcopénie est évidemment imputable au processus de vieillissement, mais son développement peut être accéléré sous l’effet de facteurs pathologiques et comportementaux. Depuis un siècle à peine, le comportement de l’homme moderne, en matière d’activité physique, a radicalement changé avec un mode de vie de plus en plus inactif. Cette inactivité chronique est apparue trop soudainement pour permettre à notre génotype de s’adapter, et contribue ainsi à accélérer ledéveloppement de la sarcopénie. Néanmoins, des interrogations subsistent concernant les mécanismes cellulaires et moléculaires par lesquels l’inactivité physique favoriserait ce syndrome gériatrique. L’objectif de ce travail de thèse était donc de déterminer certains de ces mécanismes en se centrant tout particulièrement sur le rôle des espèces dérivées del’oxygène (ERDO). En s’appuyant sur différents modèles expérimentaux d’activité (entraînement en endurance) et d’inactivité (suspension par la queue) chez le rongeur, nos travaux ont mis en évidence le rôle essentiel de la surproduction chronique d’ERDO (qu’elle soit liée à l’âge et/ou l’inactivité) dans l’activation de certains facteurs de transcription et kinases redox-sensibles impliqués dans la sarcopénie (i.e. NF-κB, p38 MAPK). Nos travaux démontrent également que l’avance en âge (et probablement l’inactivité chronique) induit une perte de réactivité de PGC-1α, un facteur de transcription redoxsensible régulant un certain nombre de mécanismes cellulaires impliqués dans la sarcopénie. Cette perte deréactivité pourrait s’expliquer par la surproduction chronique d’ERDO dans le muscle âgé / Aging causes a progressive decline in skeletal muscle mass that may lead to decreased strength and functionality. The term sarcopenia is especially used to characterize this geriatric syndrome. Numerous conditions and behaviors are considered to accelerate the progression of sarcopenia such as chronic diseases, malnutrition and physical inactivity. In millennia past, and until recently, among hunter-gatherers and like populations, down through the ages, all people werephysically very active during early life and later in their everyday occupations. In contrast, nowadays, in Western populations, with relative abundance of food, a sedentary lifestyle is the rule. This radical change in lifestyle counteracts our active phenotype, and thus promotes the development of sarcopenia. Despite the recent advances in the etiology of sarcopenia, some questions remain concerning the cellular and molecular mechanisms by which the physical inactivity promotes sarcopenia. Consequently, the aim of this thesis was to determine some of these mechanisms, and more especially the role played by reactive oxygen species (ROS). We used different experimental rodent models of activity (physical training) and inactivity (hindlimb suspension) to achieve these objectives. Our research underlines the essential role of age or/and inactivity related chronic ROS overproduction in the activation of redox-sensitive transcription factors and kinases involved in sarcopenia (i.e. NF-κB and p38 MAPK). We also demonstrated that aging (and probably lifelong inactivity) induces a loss of PGC-1α reactivity, a key redox-sensitive transcription factor regulating some cellular mechanisms involved in sarcopenia. Chronic ROS overproduction in aged skeletal muscles may explain this loss of PGC-1α reactivity

La reprogrammation du métabolisme est maintenant considérée comme des caractéristiques des cellules cancéreuses et une conséquence de leur adaptation à un microenvironnement hostile se traduisant par une baisse de la concentration d’oxygène et de la disponibilité des nutriments. Donc, les cellules cancéreuses sont capables d’adapter leur métabolisme pour survivre et proliférer. Des avancées récentes dans la connaissance de ces modifications permettent l’émergence de nouvelles approches thérapeutiques ciblant spécifiquement ces changements métaboliques. Un des principaux régulateurs du métabolisme cellulaire est le coactivateur transcriptionnel PGC-1α (PPARgamma coactivator1-alpha). PGC-1α contrôle, entre autres, la biogénèse mitochondriale, la phosphorylation oxydative et l’oxydation des acides gras. Récemment, il a été montré que PGC-1α facilite la biogénèse mitochondriale dans les cellules cancéreuses du sein et augmentent significativement leurs potentiels métastatiques. Au contraire, il a été montré que la surexpression de PGC-1α diminue la formation de métastases dans le mélanome et l’adénocarcinome prostatique. Cependant, les modifications métaboliques et moléculaires conduisant à l’agressivité du cancer de la prostate sont, à l’heure actuelle, peu connues. Dans ce contexte, le but de ma thèse était d’étudier le rôle de PGC-1α sur le métabolisme et l’agressivité des cellules cancéreuses de prostate. Au cours de ma thèse, nous avons démontré que la diminution de l’expression de PGC-1α augmente les trois caractéristiques fondamentales de l’agressivité tumorale : la prolifération, la migration et l’invasion. Afin de déterminer les modifications métaboliques impliquées dans ce phénotype, nous avons réalisé des expériences de métabolomiques en comparant les cellules contrôles aux cellules dont l’expression de PGC-1α est diminuée (shPGC-1α). Nous avons montré que la baisse de PGC-1α augmente significativement la biosynthèse des polyamines. Les polyamines sont impliquées dans de nombreuses fonctions cellulaires, en particulier la prolifération et la migration cellulaire. Ainsi, nous avons inhibé la synthèse des polyamines avec le DFMO, l’inhibiteur de l’enzyme limitante de la voie : ODC, ou bien des siRNA dirigés contre ODC. Nous avons montré que les effets pro-migratoires et pro-invasifs dus à l’invalidation de PGC-1α sont bloqués par le DFMO et les siRNA ODC. De façon intéressante, l’ajout de polyamines exogènes restaure partiellement l’agressivité des cellules. En accord avec ces résultats, nous montrons que ODC est surexprimée quand PGC-1α est diminué et que l’expression de ODC est régulée positivement par l’oncogène c-MYC. En s’intéressant plus en détail à cet oncogène, nous observons que son niveau d’expression augmente dans les cellules invalidées pour PGC-1α et que l’inhibition de c-MYC bloque les effets pro-migratoires et pro-invasifs dus à l’invalidation de PGC-1α. Donc c-MYC participe au phénotype agressif lié à l’augmentation de la voie de biosynthèse des polyamines. Ces résultats in vitro ont été confirmés in vivo par l’analyse des micro-métastases, ils démontrent que les cellules shPGC-1α forment plus de métastases et le traitement par le DFMO inhibe la formation de micro-métastases. Finalement, les données cliniques démontrent que l’expression de PGC-1α est diminuée chez des patients atteints de cancer de la prostate, et cette diminution est corrélée avec une augmentation de c-MYC et ODC. En conclusion, nous avons démontré que PGC-1α est le régulateur majeur d’une voie onco-métabolique par c-MYC et qui promeut l’agressivité du cancer de la prostate par l’intermédiaire de la voie de biosynthèse des polyamines. Ce nouveau circuit métabolique représente une cible thérapeutique intéressante pouvant aider à freiner les formes avancées du cancer de la prostate. / Metabolism reprogramming are now considered to be characteristic of cancer cells and a consequence of their adaptations to a hostile microenvironment resulting in a decrease in oxygen concentration (hypoxia) and the availability of nutrients, particularly glucose and glutamine. Thus, cancer cells can adapt their metabolism to survive and proliferate. Recent advances in the knowledge of these modifications allow the emergence of new therapeutic approaches targeting these metabolic changes. One of the main regulators of cellular metabolism is the transcriptional coactivator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha). PGC-1α controls mitochondrial biogenesis, oxidative phosphorylation and fatty acid oxidation. Recently, it has been shown that PGC-1α promotes mitochondrial biogenesis in cancer cells and dramatically increases their metastatic potential. On the contrary, it appears that overexpression of PGC-1α decreases the formation of metastases in melanoma and prostatic adenocarcinoma. However, the metabolic and molecular changes leading to the aggressiveness of prostate cancer are unclear. Oncogenes and tumor suppressor genes are known to be able to regulate the metabolic adaptations of cancer cells. Several studies show that the number of copies of the gene is increased in 30% of prostate cancers. Transgenic mice overexpressing c-MYC in the prostate develop prostatic intraepithelial neoplasia followed by prostatic adenocarcinoma. At the cellular level, c-MYC expression has been shown to stimulate glycolysis and glutaminolysis in tumor cells, by controlling the expression of genes involved in these metabolic pathways. In addition, c-MYC is also able to increase the polyamine synthesis pathway by inducing the expression of the limiting enzyme of this pathway, ornithine decarboxylase (ODC). In this context, the purpose of my thesis was to study the role of PGC-1α on the metabolism and aggressiveness of prostate cancer cells. During my thesis, we have shown that the decrease of PGC-1α expression increases the three fundamental characteristics of tumor aggressiveness: proliferation, migration and invasion. To determine the metabolic changes involved in this phenotype, we performed metabolic experiments and compared control cells to cells where PGC-1α expression is decreased. We show that the decrease of PGC-1α significantly increases the biosynthesis of polyamines. Polyamines are involved in many cellular functions, particularly in proliferation and cell migration. Thus, we inhibit the synthesis of polyamines with DFMO, an inhibitor ODC which is the rate limiting enzyme of this pathway. We have shown that pro-migratory and pro-invasive effects due to PGC-1α knockout are blocked by DFMO and ODC siRNA. Interestingly, the addition of exogenous polyamine partially restores the aggressiveness of the cells. Consistent with these results, we show that ODC is overexpressed when PGC-1α is decreased and that ODC expression is upregulated by the c-MYC oncogene. In addition, we observe that c-MYC expression increases in cells invalidated for PGC-1α and that the inhibition of c-MYC blocks the pro-migratory and pro-invasive effects due to the invalidation of PGC-1α. Therefore, c-MYC participates in the aggressive phenotype related to the increase of the polyamine biosynthesis pathway. These in vitro results were confirmed in vivo by micro-metastasis analysis, we demonstrate that shPGC-1α cells form more metastases and treatment with DFMO inhibits the formation of micro-metastases. Clinical data indicate that PGC-1α expression is decreased in patients with prostate cancer, and this decrease correlates with an increase in c-MYC and ODC. In conclusion, we show that PGC-1α is the major regulator of an onco-metabolic which promotes prostate cancer aggressiveness via the polyamine biosynthesis pathway.

Il est maintenant clairement établi que la vitamine B12 (cobalamine) joue un rôle important dans la formation des globules rouges, et joue un rôle essentiel dans l'efficacité du fonctionnement du système nerveux, comme les fonctions cognitives. La carence en vitamine B12 est répandue dans le monde et provoque une anémie mégaloblastique et des déficits neurologiques. La carence en vitamine B12 et l'hyperactivité glucocorticoïde (GC) contribuent à la détérioration de la plasticité et des fonctions de l'hippocampe. Pour comprendre comment la carence en cobalamine dans le système nerveux central génère des déficits neurologiques fonctionnels, nous avons utilisé un modèle de souris génétiquement modifiées, dont le gène codant le récepteur de la transcobalamine (souris CD320 KO) est invalidé exclusivement dans le cerveau. Nos analyses comportementales indiquent des déficits dans l'apprentissage visuo-spatial hippocampo-dépendant chez les souris KO. Les résultats ont montré qu'une dose journalière physiologique d'hydroxycortisone (8 mg / kg / jour I.P.) a un effet positif dans la restauration dans les performances d'apprentissage, chez les souris KO par rapport aux contrôles. Conformément aux déficits comportementaux, ce modèle knock-out montre une diminution de l'expression de protéines clés impliquées dans la plasticité cérébrale. Les résultats de western blot des extraits d'hippocampe ont révélé que les souris KO femelles montrent une diminution de l'expression des récepteurs des glucocorticoïdes (GR) et du coactivateur Proliférateur de péroxysome 1 (PGC-1), la protéine de réponse de croissance précoce -1 (EGR-1), Synapsines (Syn I, II), protéines clés connues pour être impliquées dans l'activité synaptique. L'étude du modèle CD320 KO pourrait permettre de mieux comprendre les effets de la carence en vitamine B12 observés chez l'homme, afin d’identifier des réponses potentielles aux différents troubles neurologiques associés ; notamment peut être un traitement palliatif basé sur les corticoïdes / It is now clearly established that vitamin B12 (or cobalamin), plays an important role in the formation of red blood cells, as well as it has a vital role in the efficient functioning of the nervous system such as cognitive functions. Vitamin B12 deficiency is widespread worldwide and causes megaloblastic anemia and neurological deficits. Vitamin B12 deficiency and the hyperactivity of the glucocorticoid (GC) contribute to the deterioration of hippocampal plasticity and functions. To understand whether cobalamin deficiency in the central nervous system produced functional neurologic deficits, we used a transcobalamin receptor / CD320 knockout mouse that lacks the receptor for the cellular uptake of cobalamin in the brain. Our behavioral analyses indicate deficits in hippocampo-dependant visuo-spatial learning capacities in KO mice. However, a daily physiological dose of hydroxycortisone (8 mg/kg/day I.P.) has a positive effect in improving learning performances in KO mice compared to controls. Consistent with the behavioral deficits, the knockout mouse shows impaired expression of key proteins implicated in synaptic plasticity. The results of western blot analyses of hippocampus extracts revealed that the knockout female mice showed decreased expressions of glucocorticoids receptors (GR), Peroxisome proliferator-activated receptor coactivator 1 (PGC-1), early growth response protein -1 (EGR-1), Synapsines (Syn I, II), Protein arginine N-methyltransferase -1 (PRMT-1), key proteins known to be implicated in the synaptic activity. The study of the CD320 knock out model could help to better understand the effects of vitamin B12 deficiency observed in humans, in order to identify potential responses to various neurological associated disorders; with a putative palliative treatment approach using corticoids