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A Doença de Alzheimer (DA) tem sido relacionada com danos oxidativos. O objetivo desse trabalho foi investigar o envolvimento NADPH oxidase 2 (Nox2), uma enzima que produz espécies reativas de oxigênio, na memória, na expressão de proteínas relacionadas à DA, na inflamação e morte neuronal no hipocampo na patologia Alzheimer-símile induzida por estreptozotocina (STZ), comparando os efeitos dessa droga em camundongos nocautes para Nox2 e camundongos selvagens (WT). A expressão gênica de Nox2 foi aumentada em animais WT após a injeção de STZ, além disso, esses animais apresentaram déficit de memória, aumento na fosforilação de TAU, na expressão de beta-amilóide, neurofilamentos, caspase-3 e marcadores de astrócitos e microglia, além de aumento na liberação de citocinas inflamatórias, após o tratamento. Estes efeitos não foram observados após a deleção de Nox2. A deleção de Nox2 aumentou a produção basal de IL-10, sugerindo que este pode ser um mecanismo pelo qual os camundongos nocautes são protegidos contra a patologia Alzheimer-símile induzida por STZ. / Alzheimer\'s disease (AD) has been linked to oxidative stress. The goal of this study was to investigate the involvement of NADPH oxidase 2 (nox2), an enzyme that produces reactive oxygen species, in memory, in AD-related proteins expression, inflammation and neuronal death in the hippocampus in the streptozotocin (STZ)-induced AD-like pathology by comparing the effects of that drug on mice lacking Nox2 and wild type (Wt) mice. Nox2 gene expression was increased in Wt mice after STZ injection. Moreover, these animals presented impairment in memory, increased phosphorylation of Tau and increased amyloid-β protein, neurofilaments, caspase-3 and astrocyte and microglial markers expression, in addition to increased inflammatory cytokines release after treatment. Nox2 depletion prevented these effects. The baseline IL-10 levels were found increased following Nox2 deletion, suggesting that this is one mechanism by which mice lacking Nox2 are protected against STZ-induced AD-like pathology.

Linhagens secretoras de insulina, como a BRIN-BD11, são utilizadas para o estudo do mecanismo de secreção de insulina estimulado pela glicose (GSIS). Essas células expressam o GPR40, que quando ativado por ácidos graxos (AG), potencializam a GSIS. Considerando que as espécies reativas de oxigênio (EROs) podem estar relacionadas com a secreção de insulina mediada por AG, este estudo tem como foco avaliar se há envolvimento do GPR40 na GSIS e na produção de EROs, via NADPH oxidase (NADPHox), em células BRIN-DB11. A ativação do GPR40 foi feita com agonista GW9508 (GW). A secreção de insulina (SI), o conteúdo de superóxido (CS) e ativação da NADPHox foram analisados em resposta a glicose na presença ou ausência de GW (20mM). O envolvimento da NADPHox no CS induzido pelo GW foi analisado após inibição da NADPHox. O aumento da concentração de glicose paralela com aumento da SI, causa redução no CS. A presença do GW potencializa a SI em altas concentrações de glicose, aumenta o CS, e aumenta a translocação da subunidade p47phox do plasma para a membrana. Com a inibição da NADPHox o efeito do GW não ocorre. Os dados confirmam um efeito positivo do GPR40 na secreção de insulina estimulada pela glicose. O GW ativa a NADPHox resultando em um aumento do CS nas células BRIN-BD11. / BRIN-BD11, an Insulin secreting cell line, are used to study the mechanisms of glucose-stimulated insulin secretion (GSIS). These cells express GPR40, which is activated by fatty acids (FA) and potentiates GSIS. Considering that reactive oxygen species (ROS) is a signal that modulates insulin secretion (IS) induced by FA, this study aimed to investigate the involvement of GPR40 in ROS production, via NADPH oxidase (NADPHox), and in GSIS. To activate the GPR40 its agonist, GW9508 (GW), was used. IS and superoxide production (SP) were analyzed in response to increasing glucose concentrations (IGC), with or without GW (20mM). The activation of NADPHox was analyzed. The involvement of NADPHox on SP induced by GW was also evaluated using a small interference RNA or using the NADPHox inhibitor, VAS2870 (12mM). The presence of GW potentiated the IS at high glucose concentrations, together with an increased effect on SC. This increase was paralleled by an increase in the translocation of p47phox to the plasma membrane. Moreover, the inhibition of NADPHox abolished the GW9508 effect. These results show a positive effect of the GPR40 activation in GSIS. Additionally, GW9508 activates NADPHox resulting in an increase in the SC in BRIN-BD11 cells.

Os genes relógio são responsáveis pelo ritmo circadiano e homeostase de diversos sistemas biológicos, incluindo o pâncreas endócrino. Nas células β são de grande importância para a regulação do metabolismo e da secreção de insulina (SI), e sua ausência pode levar ao desenvolvimento do diabetes. A NADPH oxidase (NOX) é um complexo enzimático responsável pela produção do ânion superóxido através da redução do oxigênio molecular. Em ilhotas pancreáticas, a NOX participa da regulação do metabolismo da glicose e da SI, através da modulação do estado redox intracelular. O objetivo do nosso estudo foi verificar se a desregulação dos genes relógio mediada pela ausência de Bmal1 seria capaz de modular a NOX e o estado redox nas células β pancreáticas, influenciando assim a SI. Observamos que a ausência de Bmal1 alterou a atividade e expressão da NOX, desregulando o estado redox intracelular. Essas alterações levaram à redução da viabilidade celular e mudanças na resposta à estimulação com glicose, resultando em uma deficiência na principal função da célula β a SI. / Clock genes are responsible for homeostasis and circadian rhythm in various biological systems, including endocrine pancreas. In β -cells, they are important for the regulation of metabolism and insulin secretion (IS), and its absence can lead to development of diabetes. NADPH oxidase (NOX) is an enzymatic complex responsible for production of superoxide anion by reducing molecular oxygen. In pancreatic islets, NOX regulates glucose metabolism and IS through modulation of the intracellular redox state. The aim of our study was to investigate whether dysregulation of clock genes mediated by Bmal1 suppression would be able to modulate NOX activity and redox state in pancreatic β cells, thus influencing the SI. In this work, the lack of Bmal1 altered the activity and expression of NOX, deregulating the intracellular redox state. These changes led to reduced cell viability and changes in cell response after stimulation with glucose, resulting in a deficiency in β cell main function, GSIS.

Avaliamos importância da NADPH oxidase 2 (NOX2) na produção de espécies reativas de oxigênio (EROs) em ilhotas de ratas alimentadas ou em jejum, incubadas na presença de 2,8 mM ou 16,7 mM de glicose, associada ou não a leucina, com ou sem inibição da NOX2. As ilhotas dos animais alimentados ou em jejum apresentaram reduzida secreção de insulina e altas concentrações de EROs na presença de 2,8 mM de glicose. Esses parâmetros foram invertidos pela adição de inibidores da NOX2. A leucina, que é metabolizada no Ciclo dos Ácidos Tricarboxílicos, também aumentou a secreção de insulina por aumento de ATP, e diminuiu as EROs, devido ao aumento de NADPH, um substrato do sistema antioxidante. Desse modo, quando as ilhotas são submetidas ao jejum, a diminuição da atividade secretória é fundamental para impedir que quantidades maiores de hormônio sejam secretadas, podendo levar a uma hipoglicemia. Porém, na presença de alta concentração de glicose, a ativação das defesas antioxidantes da célula b atenua o excesso de EROS, liberando a secreção de insulina. / We sought to evaluate the importance of NADPH oxidase 2 (NOX2) in the production of reactive oxygen species (ROS) in islets from rats fed or fasted, incubated in the presence of glucose 2.8 mM or 16.7 mM, with or without leucine or inhibition of NOX2. Islets of fed or fasted animals showed reduced insulin secretion and high concentration of ROS in the presence of 2.8 mM glucose. These parameters were reversed by addition of inhibitors NOX2. Leucine that is metabolized in the cycle of Tricarboxylic Acids (TCA) also increased insulin secretion, by increasing ATP, and ROS decreased due to the increase of NADPH, a substrate of the antioxidant system. Thus, when the islets are subjected to fasting, decreased secretory activity is essential to prevent amounts of the hormone be secreted and may lead to hypoglycaemia. However, in the presence of high glucose levels, activation of antioxidant defenses of b cell attenuates the excess of ROS, releasing insulin secretion.

Lors d’une infection, les polynucléaires neutrophiles phagocytent l’agent pathogène et le détruisent grâce à la production de formes réactives de l’oxygène (FRO) par la NADPH oxydase. Cette enzyme est constituée de sous-unités membranaires (Nox2, p22phox) et cytosoliques (p67phox, p47phox,p40phox, Rac) qui s’assemblent soit à la membrane plasmique, lors de l’activation des cellules par un stimulus soluble comme le fMLF, soit à la membrane du phagosome, lors de la phagocytose de particules. La régulation de la NADPH oxydase implique divers facteurs comme la signalisation calcique et les lipides, notamment les phospholipides anioniques. En effet, il a été montré que l’activation et la translocation de la petite protéine G Rac peuvent être dépendantes du calcium. D’autre part les sous unités Rac et p47phox peuvent interagir avec les phospholipides anioniques tels que la phosphatidylsérine,grâce à des interactions stéréosélectives et/ou électrostatiques.L’objectif de ce travail est donc d’évaluer le rôle du calcium et de la phosphatidylsérine dans le recrutement de p47phox et/ou Rac lors de l’assemblage de NADPH oxydase. Pour suivre la dynamique des deux protéines, nous avons exprimé ces sous-unités marquées avec des protéines fluorescentes dans des lignées phagocytaires mimant les neutrophiles (HL-60 et PLB-985). Nous avons ainsi pu suivre, par vidéomicroscopie, le déplacement des sous-unités marquées lors d’une stimulation par fMLF ou PMA etdurant la phagocytose de particules opsonisées. Après stimulation par fMLF, Rac1 transloque du cytosol à la membrane plasmique, alors que le mutant constitutivement actif de Rac1 est constamment localisé àla membrane plasmique, indépendamment de la stimulation par fMLF. De plus après stimulation parPMA, le mutant constitutivement actif de Rac2 transloque à la membrane plasmique, suggérant que sa translocation pourrait être possible en absence d’un influx de calcium extracellulaire. Lors de la phagocytose, en masquant la phosphatidylsérine avec le domaine C2 discoïdine de la lactadhérine qui lie spécifiquement ce phospholipide, nous avons pu montrer que la phosphatidylsérine régule la production initiale des FRO en favorisant le recrutement de p47phox et de Rac2 au phagosome. De plus, ces deux sous-unités se détachent du phagosome alors que la production intraphagosomale de FRO continue,suggérant que leur départ n’est pas un signal de terminaison pour l’activité oxydase. Plus précisément,p47phox et Rac2 sont recrutées de manière transitoire, pendant seulement 1 à 3 minutes après la fermeture du phagosome. Ceci est en accord avec le modèle qui propose que p47phox servirait principalement à transporter p67phox au phagosome, et que les deux sous-unités p47phox et Rac2 faciliteraient le positionnement de p67phox dans le complexe membranaire. / During phagocytosis, neutrophils internalize pathogens in a phagosome and kill them through the production of reactive oxygen species (ROS) by the NADPH oxidase enzyme. The cytosolic NADPHoxidase subunits (p67phox p47phox, p40phox, Rac2) and the membranous subunits (Nox2, p22phox) assemble either at the plasma membrane after stimulation with soluble agonist, or at the phagosomal membrane during particle phagocytosis. The regulation of this enzyme involves several actors like calciumsignalling and anionic phospholipids. Actually Rac activation and translocation was found to be calciumdependent and, on the other hand, p47phox and Rac can interact with anionic phospholipids such asphosphatidylserine through stereoselective and/or electrostatic interactions.Therefore we wanted to investigate the role of calcium and phosphatidylserine in p47phox and Rac membrane recruitment. To study this dynamic, we expressed the subunits tagged with a fluorescent protein in neutrophil-like cells (HL-60 and PLB-985), and followed them by videomicroscopy duringfMLF or PMA stimulation or during phagocytosis of opsonised particles. After fMLF stimulation, Rac1translocated from the cytosol to the plasma membrane whereas constitutively active form of Rac1 was permanently located at the plasma membrane, independently of fMLF stimulation. In addition, afterPMA treatment, constitutively active form of Rac2 translocated to the plasma membrane, suggesting that its translocation could occur without extracellular calcium entry. By using the specific phosphatidylserine binding discoïdine C2 domain of lactadherin, we could mask this phospholipid and found that phosphatidylserine is involved in NADPH oxidase activity by participating in the phagosomal recruitment of p47phox and Rac2. In addition we show that these two subunits detached from the phagosome while ROS production continued for a longer period, suggesting that their dissociation from the complex is not a termination signal for oxidase activity. More precisely, p47phox and Rac2 were briefly recruited to the phagosomal membrane, for 1 to 3 minutes after the phagosome closure. These results support the model in which p47phox serves as a carrier for p67phox and both p47phox and Rac2 are adapters that correctly position p67phox in the complex.

Le polynucléaire neutrophile (PN) humain est une cellule phagocytaire qui constitue une des premières barrières de défense de l’organisme contre les agents pathogènes. Sa stimulation par des facteurs chimioattractants, provoque sa migration de la circulation sanguine vers le foyer inflammatoire. Dans le site inflammatoire, les PN reconnaissent l’agent pathogène par l'intermédiaire d'opsonines, des fractions résultant de l'activation du complément et par l’intermédiaire de motifs de reconnaissance conservés au cours de l’évolution des agents pathogènes qui se lient à des récepteurs de la famille Toll (Toll-like receptors ; TLR). Le contact du pathogène avec le PN va provoquer sa phagocytose et sa destruction par la libération de molécules contenues dans les granules du PN et par la production de formes réactives de l’oxygène (FRO) par un complexe enzymatique la NADPH phagocytaire composée au repos de de protéines cytosoliques (p40phox, p47phox, p67phox et Rac 2) et membranaires (gp91phox et p22phox formant le cytochrome b558). Un des événements majeur de l’activation de la NADPH oxydase est la phosphorylation de certains composants cytosoliques comme la p47phox ou la p67phox ce qui conduit à la translocation de ces protéines vers le cytochrome b558 membranaire et permet d’activer l’enzyme pour la production de FRO. L’hyperactivation de cette enzyme ou son « priming » consiste en une pré-activation du PN par des agents dit « primants » tels que des cytokines (TNFα, GM-CSF, IL-1), des chimiokines comme l’IL-8, des molécules lipidiques (PAF et LTB4), ou encore des endotoxines bactériennes LPS, agoniste de TLR4. Les TLR sont des récepteurs exprimés à la surface de nombreuses cellules dont les cellules immunitaires ; ils détectent des motifs conservés au cours de l’évolution des agents pathogènes appelés PAMPs pour "pathogen-associated molecular patterns", des protéines modifiées reconnues comme étrangères, des lipides oxydés, des ligands endogènes. Quelques agonistes des TLR comme le LPS ont été décrits pour induire un priming de la production des FRO par les PN. D’autres ont été connus par leur pouvoir activateur de la NADPH oxydase des PN. Le CL097 (Imidazoquinoline : agoniste des TLR7/8) était l’agoniste des TLR induisant le plus fort effet de « priming » par les PN stimulés par le fMLP. Le CL097 induit la phosphorylation de la p47phox sur la sérine 345. Cette phosphorylation implique des MAPKinases ERK1/2 et de la p38MAPK. La phosphorylation de ce site induit le changement de conformation de la p47phox sous l’action d’une proline isomérase Pin1. Ce changement de conformation favorise la phosphorylation des autres sites (Ser-315, Ser-328) et par conséquent l’activation de la NADPH oxydase. La comparaison de l’effet du CL097 à deux agonistes reconnaissant l’un le TLR7, l’autre le TLR 8 a montré que l’action du CL097 dépendait du TLR8. Le zymosan non opsonisé (agoniste de TLR2) stimule l’activation de la NADPH oxydase des neutrophiles. IL induit la phosphorylation de la p47phox au niveau des Ser-345, -315 et -328. Ces phosphorylations font intervenir respectivement les MAPK ERK1/2 et p38, une protéine tyrosine kinase et les PKC. En plus cet agoniste active la petite protéine cytosolique Rac2, nécessaire à l’activation de la NADPH oxydase des PN. Ces données permettraient d’identifier de nouvelles cibles thérapeutiques de première importance afin de moduler les réponses inflammatoires pathologiques. / Superoxide anion production by the neutrophil NADPH oxidase plays a key role in host defense; however, excessive superoxide production is believed to participate to inflammatory reactions. Neutrophils express several TLR that recognize a variety of microbial motifs or agonists. The interaction between TLR and their agonists is believed to help neutrophils to recognize and to eliminate the pathogen. However, the effects of some TLR agonists on the NADPH oxidase activation and the mechanisms controlling these effects have not been elucidated. In this study, we show that the TLR7/8 agonist CL097 by itself did not induce NADPH oxidase activation in human neutrophils, but induced a dramatic increase of fMLF-stimulated activation. Interestingly, CL097 induced cytochrome b558 translocation to the plasma membrane and the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328 and Ser315. Phosphorylations of Ser328 and Ser315 were significantly increased in CL097-primed and fMLF-stimulated neutrophils. Phosphorylation of Ser345, Ser328 and Ser315 was decreased by inhibitors of p38MAPK and the ERK1/2-pathway. Phosphorylation of Ser328 was decreased by a PKC inhibitor. Genistein, a braod range protein tyrosine kinase inhibitor, inhibited the phosphorylation of these serines. Our results also show that CL097 induced proline isomerase (Pin1) activation and that juglone, a Pin1 inhibitor, inhibited CL097-mediated priming of fMLF-induced p47phox phosphorylation and superoxide production. These results show that activation of TLR7/8 in human neutrophils induces hyper-activation of the NADPH oxidase by stimulating the phosphorylation of p47phox on selective sites, and suggest that p38MAPK, ERK1/2, PKC and Pin1 control this process.Zymosan a cell-wall preparation from saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan induced ROS production by human neutrophils. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328 and Ser315; and activation of the GTPase Rac2. Phosphorylation of p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor. Wortmannin a PI3Kinase inhibitor, inhibited phosphorylation of p47phox on Ser328 and Ser315 and Rac2 activation. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway respectively, inhibited phosphorylation of p47phox on Ser345. GF109203X a PKC inhibitor inhibited phosphorylation on Ser328 and Ser315. Zymosan-induced ROS production was inhibited by genistein, wortmannin, SB203580, UO126 and GF109203X. These results show that zymosan induced ROS production by NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation. Our results also suggest that a protein tyrosine kinase and PI3Kinase control p47phox phosphorylation and Rac2 activation while p38MAPK, ERK1/2 and PKC are involved in zymosan-induced p47phox phosphorylation.

La production d’anion superoxyde (O2-) par le complexe NADPH oxydase 2 (NOX2) du polynucléaire neutrophile (explosion oxydative, EO) contribue à l’élimination d’agents pathogènes. Cette fonction de défense est stimulée par divers agents pro-inflammatoires, notamment par des peptides bactériens (fMLP), qui déclenchent une cascade de signalisation impliquant différentes protéines kinases (PKC, AKT, MAP-Kinases) et aboutissant à l’activation de la NOX2 (également nommée gp91phox), le coeur catalytique du complexe. Dans cette thèse, j’ai identifié la protéine kinase mTOR comme un nouvel effecteur majeur de l’EO dans les neutrophiles sains et j’ai comparé son mode d’action transductionnel dans les neutrophiles de patients ayant une cirrhose alcoolique décompensée en vue de comprendre leur grande susceptibilité aux infections bactériennes.Une contribution majeure de mTOR dans la production d’O2- induite par le fMLP a pu être démontrée à l’aide de son antagoniste spécifique, le médicament Rapamycine et par une approche antisense. mTOR agit en amont de la p38-MAPK qui phosphoryle la p47phox, un composant majeur du complexe NADPH oxydase. Dans les neutrophiles de patients cirrhotiques, l’EO est fortement défaillante, associée à un défaut d’activation de la voie p38-MAPK/p47phox(S345). Ce déficit d’EO est aggravé par la Rapamycine. Les neutrophiles de patients cirrhotiques présentent également un déficit d’expression de la gp91phox (NOX2), p22phox, p47phox et mTOR. Un déficit d’expression de la NOX2 a pu être reproduit en traitant les neutrophiles sains par le fMLP ou du plasma des patients. De plus, ce phénomène met en jeu une dégradation protéolytique insoupçonnée de la gp91phox impliquant l’élastase. Enfin, la déficience fonctionnelle des neutrophiles de patients a pu être corrigée dans des neutrophiles isolés et dans du sang total des patients cirrhotiques à l’aide d’un agoniste de récepteurs « Toll-like receptor (TLR) » qui agit en favorisant la transcription du gène de la gp91phox et sa synthèse protéique.En conclusion, mTOR émerge comme un nouvel effecteur transductionnel majeur de l’EO des neutrophiles, favorisant l’activation de la voie p47phox/gp91phox via les MAPK. Cette nouvelle voie de signalisation est fortement impactée au cours de la cirrhose alcoolique, ce qui favorise la susceptibilité des patients aux infections bactériennes. Bien que notre étude soulève ainsi des inquiétudes quant à l’utilisation d’inhibiteurs de mTOR chez les patients immunodéprimés, elle suscite par ailleurs la perspective de pouvoir corriger les déficits fonctionnels des neutrophiles à l’aide d’agents capables de stimuler des TLR intracellulaires. / Superoxide anion (O2-) production by NADPH oxidase 2 (NOX2) complex of polymorphonuclear neutrophil (i.e respiratory burst, RB) contributes to efficient elimination of pathogens. This defense function is stimulated by various pro-inflammatory agents, especially by bacterial peptides (fMLP) which trigger a signaling cascade involving many protein kinases (PKC, AKT, MAP-Kinases) resulting in activation of NOX2, also called gp91phox, the catalytic core of the complex. In this thesis, I identified the protein kinase mTOR as a novel major RB effector of healthy neutrophils and I compared its transductional activity in neutrophils from patients suffering from alcoholic decompensated liver cirrhosis, aiming at understanding their high susceptibility to bacterial infections.A major contribution of mTOR to fMLP-induced neutrophil superoxide production was demonstrated using its specific drug antagonist Rapamycin, and by an antisens strategy. mTOR is activated upstream of p38-MAPK which phosphorylates p47phox, a major component of the NADPH oxidase complex. In neutrophils from cirrhotic patients, the RB is dramatically impaired and this was associated with a deficient activation of the p38-MAPK/p47phox(S345) signaling pathway. This RB deficiency was aggravated by Rapamycin. Neutrophils from cirrhotic patients also exhibited a deficient expression of gp91phox (NOX2), p22phox, p47phox and mTOR. A deficient NOX2 expression can be reproduced by treating healthy neutrophils with fMLP or plasma from cirrhotic patients. Furthermore, this phenomenon involved an unexpected proteolytic degradation of gp91phox mediated by elastase. Finally, this deficient superoxide production by neutrophil from cirrhotic patients can be corrected ex vivo in isolated neutrophils and in patients’ whole blood, using a Toll-like receptor agonist that acts by promoting the transcription and traduction of gp91phox .In conclusion, mTOR emerges as a novel and major signaling effector of neutrophil RB, promoting the activation of p47phox/gp91phox through MAPKs. This novel signaling pathway is strongly impaired during alcoholic liver cirrhosis, which increases patients’ susceptibility to bacterial infections. Although our study raises concerns about the use of mTOR inhibitors in immunocompromised patients, it also provides therapeutic propects for correcting neutrophil functional deficiencies using agents capable of stimulating intracellular TLR .

A doença de Parkinson (DP) é considerada a segunda doença neurodegenerativa mais comum em idosos e é caracterizada pela presença de disfunções motoras decorrentes da redução de neurônios dopaminérgicos na substância negra pars compacta (SNpc). Uma das principais neurotoxinas utilizadas para o estudo de DP em modelos animais é a 6-hidroxidopamina (6-OHDA), que possui como mecanismo de neurotoxicidade a formação de espécies reativas de oxigênio (EROs). A liberação de EROs pela NADPH oxidase (Nox) e a ativação microglial constituem os eventos iniciais da neurodegeneração induzida pela 6-OHDA. A Nox2 está relacionada com a modulação dos fenótipos microgliais e encontra-se superativada na DP, levando a um desequilíbrio redox e a danos celulares. A microglia é o principal componente da defesa imune no sistema nervoso central (SNC) e é dependente do receptor fator 1 estimulador de colônias (CSF1R) para a sobrevivência. O inibidor de CSF1R é utilizado como método para depletar microglia em modelos de neurodegeneração e, consequentemente, compreender o impacto da sua eliminação no processo da doença. Baseado nisso, foram propostas duas abordagens para compreender o papel da microglia na progressão da DP induzida pela 6-OHDA, sendo que o primeiro investigou a modulação da ativação microglial pela Nox2 em camundongos nocautes para a subunidade gp91phox, enquanto que o segundo avaliou o impacto da depleção por meio do tratamento com 1200mg/Kg de PLX5622, um tipo de inibidor de CSF1R. Os animais naïves gp91phox-/- apresentaram aumento da proliferação de microglias e da expressão da enzima iNOS. Quando submetidos à 6-OHDA, os nocautes não apresentaram declínio motor em função do tempo avaliado pelo teste do cilindro, devido à maior sobrevivência de neurônios dopaminérgicos na SN em relação aos selvagens (WT). Um dos possíveis fatores relacionados à perda expressiva de neurônios nos WT foi a indução precoce da enzima iNOS, sugerindo que a ativação de Nox2 conjuntamente com iNOS na microglia aumenta os níveis de EROs tornando os neurônios mais susceptíveis à morte celular. Também foram observadas modulações temporais distintas entre os grupos com relação à expressão de CD86 e Arginase-1, além dos nocautes apresentarem baixos níveis de lesões oxidativas no DNA mitocondrial quando comparado com os WT. Assim, a ativação da Nox2 e da iNOS parecem atuar em sinergia no processo neurodegenerativo causado pela 6-OHDA. Por outro lado, os resultados com PLX5622 mostraram que a depleção microglial acelera o comprometimento da coordenação motora e da bradicinesia avaliado pelo teste do poste, bem como reduz o número de neurônios dopaminérgicos na SNpc. Além disso, a redução de células postivas para GFAP, do marcador CD68 e da regulação de genes ligados a microglia foram observados. O gene MeCP2, que está relacionado à via nigroestriatal e que consiste em um importante modulador da expressão de TH, está pouco expresso no grupo submetido à depleção microglial e a 6-OHDA. Esses achados sugerem que a deficiência na comunicação entre microglias e astrócitos, bem como a redução do nível de transcritos do gene MeCP2 causados pela depleção microglial, podem contribuir para a aceleração do processo neurodegenerativo causado pela 6-OHDA. / Parkinson\'s disease (PD) is considered the second most common neurodegenerative disease in elderly people and is characterized by the presence of motor impairment, which is a consequence of dopaminergic neuron loss in the substantia nigra pars compacta (SN). A major neurotoxin used for PD study in animal models is 6-hydroxydopamine (6-OHDA), which exerts neurotoxic effects through the production of reactive oxygen species (ROS). ROS release by the enzyme NADPH oxidase (Nox) and microglial activation are early events of neurodegeneration induced by 6-OHDA. Nox2 is related to modulation of microglia phenotypes and is overactivated in PD, which can lead to redox imbalance and cellular damage. Microglial cells are the main components of immune defense in the central nervous system and are dependent upon Colony-stimulating factor 1 receptor (CSF1R) for survival. The CSF1R inhibitor is used with a tool to deplete microglia in models of neurodegenerative diseases and, consequently, to understand the impact of its elimination in the disease process. Based on that, we proposed two approaches to evaluate the microglial role in the PD progression induced by 6-OHDA, in which the first one investigated the modulation of microglial activation by Nox2 in gp91phox knockout mice, whereas the second one evaluated the impact of microglial depletion through a treatment with 1200 mg/Kg of PLX5622, one type of CSF1R inhibitor. The gp91phox-/- naïve mice showed an increase of microglia proliferation and in the iNOS expression. When the knockout mice were submitted to 6-OHDA, they did not have motor impairments as a function of toxin exposure as evaluated by cylinder test. This result is due to the increase number of dopaminergic neurons survival in SNpc compared to wild type (WT). One of the possible factors involved in neurons death in WT was the early iNOS induction, which indicates that Nox2 and iNOS simultaneous activation in microglial cells enhance ROS levels, leading to neurons more vulnerable to cell death. Moreover, the expression of CD86 and Arginase-1 have differences in temporal modulation between both groups, as well as knockout mice showed lower levels of oxidative damage in mitochondrial DNA when compared with WT. Thus, the activation of Nox2 and iNOS could act sinergically in the neurodegenerative process caused by 6-OHDA.On the other hand, results with PLX5622 indicated that the microglial depletion aggravates the impairment of bradykinesia and motor coordination evaluated by pole test, as well as the reduction of tyrosine hydroxylase (TH) positive neurons in the SNpc. Moreover, the decrease of GFAP positive cells, of CD68 marker and regulation of genes linked to microglia were observed. The MeCP2 gene, related to the nigrostriatal pathway and an important modulator of TH expression, is down-regulated in the group with microglial elimination and submitted to 6-OHDA. These findings suggest that the deficiency of crosstalk between astrocytes and microglia, as well as the reduced MeCP2 transcript levels may contribute to the acceleration of neurodegenerative process initiated by 6-OHDA.

Doctor en ciencias Biomédicas / Physical activity plays a protective role in the development of chronic non-communicable diseases. Molecular adaptations explain the beneficial effects of exercise in diverse tissues such as skeletal muscle, adipose tissue, and heart. One of the multiple signals involved in the benefits of exercise are the oxidation-reduction reactions called redox signaling. Reversible and non-reversible posttranslational modifications of cysteine residues are capable of changing the function, localization, or stability of diverse proteins. In skeletal muscle, reactive oxygen species (ROS) are continuously produced and cleared during resting and contracting conditions. There is substantial evidence indicating that redox signaling plays a role in some of the health-benefits elicited by endurance training, however, the precise mechanism has been long unknown. The aim of the current Ph.D. thesis was therefore to study the involvement of NOX2 and redox signals in the regulation of exercise-stimulated glucose transport and adaptive gene expression in mature skeletal muscle. A combination of pharmacological inhibitors and murine NOX2-deficient models were used to address the necessity of NOX2 for glucose transport and adaptive signals induced by acute exercise. The current Ph.D. thesis demonstrated for the first time that NOX2 is activated during moderateintensity endurance exercise in skeletal muscle and it is a major source of ROS under those conditions. Furthermore, the analyses of genetic mouse models lacking the regulatory NOX2 subunits p47phox and Rac1 revealed striking phenotypic similarities, including severely impaired exercise-stimulated glucose uptake and GLUT4 translocation, indicating that NOX2 is a requirement for this classic acute myocellular adaptation to exercise. Overall, NOX2 is thus a major ROS source regulating adaptive responses to exercise in skeletal muscle. / La actividad física juega un papel protector en el desarrollo de enfermedades crónicas no transmisibles. Las respuestas moleculares explican los efectos beneficiosos del ejercicio en diversos tejidos como el músculo esquelético, el tejido adiposo y el corazón. Una de las múltiples señales involucradas en los beneficios del ejercicio son las reacciones de oxidación-reducción llamadas señalización redox. Las modificaciones postraduccionales reversibles e irreversibles de residuos de cisteína son capaces de cambiar la función, localización o estabilidad de diversas proteínas. En el músculo esquelético, las especies de oxígeno reactivo (ROS) se producen y eliminan continuamente durante las condiciones de reposo y contracción. Existe evidencia sustancial que indica que la señalización redox juega un papel en algunos de los beneficios para la salud provocados por el entrenamiento de resistencia, sin embargo, el mecanismo preciso ha sido desconocido durante mucho tiempo. El objetivo de la presente la tesis fue estudiar la participación de NOX2 en la regulación del transporte de glucosa durante el ejercicio y la expresión de genes adaptativos en el músculo esquelético adulto. Se utilizó una combinación de inhibidores farmacológicos y modelos deficientes en NOX2 ratón para abordar la necesidad de NOX2 para el transporte de glucosa y las señales adaptativas inducidas por el ejercicio agudo. Esta tesis demostró por primera vez que el NOX2 se activa durante el ejercicio de resistencia de intensidad moderada en el músculo esquelético y es una fuente importante de ROS en esa condición. Además, los análisis de modelos de ratones genéticos que carecen de las subunidades reguladoras NOX2 p47phox y Rac1 revelaron sorprendentes similitudes fenotípicas, incluida la captación de glucosa estimulada por el ejercicio y la translocación de GLUT4, lo que indica que NOX2 necesaria para esta respuesta fisiológica durante el ejercicio. En resumen, NOX2 es, por lo tanto, una importante fuente de ROS que regula las respuestas de adaptación al ejercicio en el músculo esquelético. / 22/03/2020