La neuropathie sensitive et motrice héréditaire avec agénésie du corps calleux (NSMH/ACC) se traduit par une atteinte neurodégénérative sévère associée à des anomalies développementales dans le système nerveux central et du retard mental. Bien que rare dans le monde, ce désordre autosomique récessif est particulièrement fréquent dans la population Québécoise du Canada Français du fait d’un effet fondateur. L’unique étude réalisée sur la mutation québécoise du gène qui code pour le co-transporteur de potassiumchlore 3 (KCC3) a montré qu’il y a une perte de fonction de la protéine. Cependant, la maladie est également retrouvée hors du Québec et il reste encore à élucider les pathomécanismes mis en jeu. Nous avons donc séquencé les 26 exons du gène KCC3 chez des individus recrutés dans le monde entier et suspectés d’être atteints de la maladie. Nous avons ainsi identifié trois nouvelles mutations. L’étude fonctionnelle de ces mutations nous a confirmé la perte de fonction systématique des co-transporteurs mutés. Puisque l’inactivation de KCC3 se produit majoritairement via l’élimination de segments peptidiques en C-terminus, nous avons concentré notre attention sur l’identification des interactions qui s’y produisent. À l’aide d’approches double hybride, pull-down et immunomarquage, nous avons déterminé que KCC3 interagit avec la créatine kinase CK-B et que cette interaction est perturbée par les mutations tronquantes. De plus, l’utilisation d’un inhibiteur de créatine kinase inactive KCC3, ce qui démontre qu’il existe bien un lien fonctionnel et pathologique entre KCC3 et ses partenaires C-terminaux. Nous avons aussi identifié des anomalies majeures de localisation membranaire des KCC3 mutés. Que KCC3 soit tronqué ou pleine longueur, sa distribution subcellulaire est affectée dans des cellules en culture, dans les ovocytes de Xenopes et dans des échantillons de cerveau de patients. La perte d’interaction entre KCC3 et CK-B et/ou les défauts de transit intracellulaire de KCC3 sont donc les mécanismes pathologiques majeurs de la NSMH/ACC. / Heredirary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC) is a severe neurodegenerative disease associated with developmental anomalies in the central nervous system and mental retardation. Although rare worldwide, this autosomal-recessive disorder is frequent in the French-Canadian population of Quebec because of a founder effect. Different mutations in the gene coding for the potassiumchloride co-transporter 3 (KCC3) have been associated with the disease; however, little is known about the mechanisms leading to the inactivation of the co-transporter. We sequenced 26 exons of the KCC3 gene in individuals recruited worldwide and suspected to be affected by the disease. We identified three new mutations. The functional study of these mutations gave confirmation of a systematic loss-of-function of the mutant co-transporters. As the loss of function occurs mainly via the elimination of C-terminal peptide fragments, we focused on the identification of C-terminal interacting partners. Using different biochemical approaches, such as yeast two-hydbrid, pull-down, and immunostaining, we established that KCC3 interacts with the brain-type creatine kinase CK-B and that this interaction is disrupted by the HMSN/ACC truncation mutations. In addition, a specific creatine kinase inhibitor inactivates KCC3 and shows for the first time the functional link between KCC3 and its C-terminal partners. In addition, we found that anomalies in KCC3 transit—as seen in cultured cells, in Xenopus oocytes, and in human brain samples—is a major pathogenic mechanism that also leads to the disease manifestations.

Les expansions du codon CAG (polyQ) sont impliquées dans neuf maladies neurodégénératives. Notre groupe a démontré que, lors de la traduction de la protéine ataxine-3 (Atx3) mutée qui est impliquée dans l’ataxie spinocérébelleuse de type 3 (SCA3), un changement du cadre de lecture vers un cadre décalé -1 (GCA) se produit. La traduction dans ce nouveau cadre de lecture entraine la production de polyalanine et ceci amplifierait la toxicité des polyQ. Le changement de cadre de lecture (ccl) ribosomique peut se produire des virus aux mammifères mais peu de choses sont connues sur son impact chez l’humain. Afin d’étudier ce phénomène dans la protéine Atx3 avec expansion de polyQ, nous avons établi un modèle de Drosophile transgénique et testé si c’était l’ARNm ou la protéine mutée qui était toxique. Nous avons aussi employé un essai de toeprinting (TP) afin d’identifier l’emplacement précis où les ribosomes changent de cadre de lecture sur l’ARNm. Nos résultats indiquent que la toxicité est due à la présence de polyalanines faisant suite au ccl et que l’ARNm en soi n’est pas la cause directe de la toxicité. De plus, nous avons observé que les ribosomes s’arrêtent au 48ième codon glutamine et que cet arrêt est spécifique aux polyQ. L’arrêt des ribosomes a d’ailleurs aussi été observé dans d’autres maladies avec expansions de polyQ. Puisque ces maladies ont des caractéristiques communes, un blocage de ce ccl pourrait atténuer les symptômes des patients SCA3 et d’autres maladies à expansions de polyQ / Coding CAG repeat disorders have been associated with nine neurodegenerative disorders. Our group has previously shown that during the translation of mutant ataxine-3 (Atx3), the protein involved in Spinocerebellar Ataxia type 3 (SCA3), a ribosomal frameshift occurs and leads to the reading of a GCA frame rather than a CAG frame. This new reading frame causes the production of polyalanine in the polyglutamine peptide which increases its toxicity. Ribosomal frameshifts are known to occur in all organisms but little is known about this phenomenon in human. To study ribosomal frameshift along the ATXN3 transcript, we generated a transgenic Drosophila model in which we looked at the toxicity of the mRNA. Also, we developed a toeprinting assay to precisely evaluate where the change of reading frame occurs along the mRNA. Our results suggest that the toxicity observed in our Drosophila model results from the production of polyalanine and not from the presence of the mRNA per se. Moreover, the change in reading frame seems to occur at the 48th CAG codon and this pausing of the ribosome also occurs in other polyQ tracts. Because CAG repeat disorders share many characteristics, an alteration of the frameshift could alleviate symptoms of SCA3 patients as well as of many other diseases with coding CAG repeats.

La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative qui affecte les neurones moteurs. 10% des cas sont des cas familiaux et l’étude de ces familles a mené à la découverte de plusieurs gènes pouvant causer la SLA, incluant SOD1, TARDBP et FUS. L’expansion de la répétition GGGGCC dans le gène C9orf72 est, à ce jour, la cause la plus connue de SLA. L’impact de cette expansion est encore méconnu et il reste à déterminer si la toxicité est causée par un gain de fonction, une perte de fonction ou les deux. Plusieurs gènes impliqués dans la SLA sont conservés entre le nématode Caenorhabditis elegans et l’humain. C. elegans est un vers transparent fréquemment utilisé pour des études anatomiques, comportementales et génétiques. Il possède une lignée cellulaire invariable qui inclue 302 neurones. Aussi, les mécanismes de réponse au stress ainsi que les mécanismes de vieillissement sont très bien conservés entre ce nématode et l’humain. Donc, notre groupe, et plusieurs autres, ont utilisé C. elegans pour étudier plusieurs aspects de la SLA. Pour mieux comprendre la toxicité causée par l’expansion GGGGCC de C9orf72, nous avons développé deux modèles de vers pour étudier l’impact d’une perte de fonction ainsi que d’un gain de toxicité de l’ARN. Pour voir les conséquences d’une perte de fonction, nous avons étudié l’orthologue de C9orf72 dans C. elegans, alfa-1 (ALS/FTD associated gene homolog). Les vers mutants alfa-1(ok3062) développent des problèmes moteurs causant une paralysie et une dégénérescence spécifique des neurones moteurs GABAergiques. De plus, les mutants sont sensibles au stress osmotique qui provoque une dégénérescence. D’autre part, l’expression de la séquence d’ARN contenant une répétition pathogénique GGGGCC cause aussi des problèmes moteurs et de la dégénérescence affectant les neurones moteurs. Nos résultats suggèrent donc qu’un gain de toxicité de l’ARN ainsi qu’une perte de fonction de C9orf72 sont donc toxiques pour les neurones. Puisque le mouvement du vers peut être rapidement évalué en cultivant les vers dans un milieu liquide, nous avons développé un criblage de molécules pouvant affecter le mouvement des vers mutants alfa-1 en culture liquide. Plus de 4 000 composés ont été évalués et 80 ameliore la mobilité des vers alfa-1. Onze molécules ont aussi été testées dans les vers exprimant l’expansion GGGGCC et huit diminuent aussi le phénotype moteur de ces vers. Finalement, des huit molécules qui diminent la toxicité causée par la perte de fonction de C9orf72 et la toxicité de l’ARN, deux restaurent aussi l’expression anormale de plusieurs transcrits d’ARN observée dans des cellules dérivées de patient C9orf72. Avec ce projet, nous voulons identifier des molécules pouvant affecter tous les modes de toxicité de C9orf72 et possiblement ouvrir de nouvelles avenues thérapeutiques / Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting the motor neurons. 10% of the cases are familial and using those families, many genes were shown to be involved in ALS pathogenesis, including SOD1, TARDBP and FUS. The GGGGCC repeat found in the first intron of C9orf72 is, to this day, the most common genetic cause of ALS. Many hypotheses have been speculated to explain the toxicity of the pathogenic GGGGCC repeat, including loss and gain of function mechanisms. Many proteins involved in amyotrophic lateral sclerosis (ALS) are evolutionarily conserved in the worm Caenorhabditis elegans. C. elegans is a transparent nematode widely used for anatomical, behavioural and genetic studies. It possesses an invariant cell lineage that includes 302 neurons in the adult nematode. Also, cellular stress responses and survival mechanisms are genetically regulated and conserved from the nematode and human. Therefore, our group, and others, have used C. elegans to model different aspects of neurodegenerative diseases including ALS. To better understand the toxicity caused by the GGGGCC repeat expansion in C9orf72, we have developed two C. elegans models to understand either the impact of the loss of function of C9orf72 or the gain of toxicity of the RNA containing the GGGGCC repeat. To understand the loss of function, we have characterized the orthologue of C9orf72 in C. elegans, alfa-1 (ALS/FTD associated gene homolog). Mutant alfa-1 worms exhibit motor impairments leading to paralysis and neurodegenereation of the GABAergic neurons. Mutant worms are also sensitive to osmotic stress which can lead to increased neurodegeneration. On the other part, exposure of C. elegans neurons to the RNA containing the GGGGCC repeat causes also motor problem and degeneration affecting the motor neurons. Therefore, our data suggest that both loss of function of C9orf72 and toxic gain of function are detrimental to neurons. Since motor dysfunctions in worms can be easily accessed in liquid culture, we have screened more than 4,000 FDA approved compounds in the alfa-1(ok3062) worms. 80 molecules were shown to improve alfa-1 impaired function and eleven of those were also tested for their effect to reduce the neurotoxicity caused by the GGGGCC repeat RNA. Eight molecules were shown to affect both types of neurotoxicity. Finally, from these eight molecules that can improveboth types of toxicity, two were shown to restore the abnormal RNA expression observed in C9orf72 patient-derive cells. With this project, we aimed to identify molecules that can affect the loss of C9orf72 toxicity and the toxic gain of RNA function containing the GGGGCC repeat to hopefully open new therapeutic avenues for ALS patients.

No description available.

O uso da cocaína na forma de crack vem crescendo nos últimos anos quando comparado às demais vias de administração. Contribuem para esse fato a obtenção quase imediata de efeitos e a maior facilidade de uso, que dispensa a necessidade de material injetável. O usuário de crack sofre os efeitos não só da cocaína, mas também de seu produto de pirólise, a metilecgonidina (AEME). Existem evidências de que a cocaína leva à neurodegeneração, entretanto a participação da AEME nesse processo ainda não foi estudada. A proposta deste estudo foi investigar a participação da AEME no processo neurodegenerativo utilizando cultura primária de hipocampo realizada a partir de fetos de ratos. Foram realizados ensaios de viabilidade celular (MTT) e da atividade da lactato desidrogenase (LDH), além da avaliação morfológica por microscopia de fluorescência. Foi estudada também a participação do dano oxidativo no processo de neurodegeneração, como a formação de aduto de DNA; atividade das enzimas antioxidantes glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa S-transferase (GST); e a produção de malonaldeído (MDA), um biomarcador de peroxidação lipídica. Tanto a cocaína quanto a AEME mostraram-se neurotóxicas. A partir dos ensaios de viabilidade e da avaliação morfológica foi possível inferir que, em células hipocampais, a cocaína leva à morte celular tanto por necrose quanto por apoptose e que a provável via envolvida na neurodegeneração da AEME é a apoptose. A AEME não causou lesão direta ao DNA, uma vez que não foi observada a formação de adutos nem com a desoxiguanosina (d-G), a base nitrogenada mais reativa, nem com DNA comercial. Mais ainda, nossos resultados mostraram que a AEME e a cocaína, nas concentrações de 1 e 2 mM, respectivamente, foram equipotentes e a incubação concomitante das duas substâncias nessas concentrações apresentou efeito aditivo após 48 horas de exposição. A morte de células hopocampais evidenciada a partir de 24 horas de exposição foi precedida pela diminuição da atividade da GPx após 3 horas de incubação tanto com a AEME e a cocaína, quanto com a associação entre essas substâncias. A atividade da GST também diminuiu, no entanto, somente após 6 horas de exposição, antecedendo a morte celular. Não foi observada alteração na atividade da GR. Houve um aumento, porém, não estatisticamente significativo, de MDA após 48 horas de incubação. Nossos resultados sugerem uma maior susceptibilidade à neurodegeneração com o uso de crack do que com a cocaína isoladamente. / Smoking crack has increased in the last years when compared to the other routes of cocaine administration. Its advantage is the quicker and stronger high effects and its ease of use without the need of needles. Smoking crack involves inhaling not only cocaine, but also its pyrolysis product, methylecgonidine (AEME). There are evidences that cocaine causes neurodegeneration, but AEME involvement in this process has not been studied yet. The aim of this study was to investigate AEME participation in neurodegeneration using a primary hippocampus culture made from rat fetuses. Cellular viability assays (MTT), lactate dehydrogenase activity (LDH) and morphological evaluations with fluorescence microscopy were performed. The involvement of oxidative injury in the neurodegeneration process was also studied through DNA adduct formation; the evaluation of antioxidants enzymes activities as glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST); and the production of malonaldehyde (MDA), a lipoperoxidation biomarker. Both cocaine and AEME showed neurotoxic effects. Through viability assays and morphologic evaluations we can suggest that, in hippocampal cells, cocaine cell death mechanism involves not only necrosis, but also apoptosis and that AEME pathway involved in neurodegeneration is only apoptosis. AEME did not produce a direct DNA injury, as no DNA adduct was observed with desoxyguanosine (d-G), the most reactive nitrogenous base, nor with commercial DNA. Moreover, our results showed that 1 and 2 mM of AEME and cocaine, respectively, were equipotent and the concomitant incubation of both compounds in those concentrations showed additive effect after 48 hours of exposure. Hippocampal cell death at 24 hours was preceded by a decrease in GPx activity after 3 hours of incubation with AEME, cocaine and association between these two compounds. GST activity also decreased but only after 6 hours of exposure, also before cell death. There was no alteration in GR activity. There was an increase, although not statistically significant, in MDA after 48 hours of exposure. As smoking crack abusers are exposed to both cocaine and AEME, our results suggest a higher susceptibility to neurodegeneration in smoking crack than with cocaine alone.

A inflamação é fundamental para a manutenção da homeostasia e para a resposta do organismo à injúria. A resposta inflamatória deve ser adequada aos estímulos agressores; no sistema nervoso central, sua inadequação conduz à gênese de diferentes doenças neurodegenerativas. A proteína anexina A1 (ANXA1) e os receptores ativados por proliferadores de peroxissomo (PPAR) controlam a inflamação, pois ambos inibem o desenvolvimento da inflamação e aceleram sua resolução. Nosso grupo de pesquisa tem mostrado que a ANXA1 modula a expressão de PPARγ em macrófagos. Assim, o presente trabalho investigou a modulação da expressão do PPARγ e das suas funções em células da microglia pela ANXA1. Foram empregadas células imortalizadas da linhagem BV2 (microglia murina), inalteradas ou transfectadas para redução da expressão de ANXA1, tratadas com ANXA1 exógena (recombinante - rANXA1) ou com agonista ou antagonista de PPARγ (pioglitazona e GW9662, respectivamente). Os resultados obtidos mostraram que: 1) tratamento com rANXA1 aumenta as expressões gênica (RT-PCR) e proteica (Western Blotting) de PPARγ, e ambas as expressões estão reduzidas em células com deficiência endógena de ANXA1, sendo que tal efeito foi revertido pela ação da rANXA1; 2) tratamento com rANXA1 não induz a expressão dos fatores de transcrição ligados a expressão de PPARγ: proteínas ligantes de elementos de resposta ao cAMP - CREB - e transdutores de sinais e ativadores de transcrição - STAT6 - (Western Blotting), mas os níveis de ambos os fatores estão reduzidos em células transfectadas, e tal efeito não foi revertido pelo tratamento com rANXA1; 3) tratamento com pioglitazona ou com rANXA1 individualmente aumenta a fagocitose de células PC12 apoptóticas (citometria de fluxo), mas o tratamento simultâneo não altera a fagocitose induzida por pioglitazona ou rANXA1; no entanto, tratamento com GW9662 inibiu a fagocitose induzida pelo tratamento com rANXA1; 4) o tratamento com rANXA1 aumenta a expressão de CD36 (citometria de fluxo); a expressão de CD36 está reduzida em células transfectadas e tal expressão não é revertida pelo tratamento com rANXA1. Em conjunto, os dados obtidos mostram a modulação da ANXA1 sobre PPARγ em células da micróglia, com possível ação sobre a fagocitose de células apoptóticas, e que a redução da expressão de ANXA1 reduz acentuadamente a expressão dos fatores de transcrição STAT6 e CREB, bem como a expressão de CD36. A elucidação dos efeitos resultantes destas alterações desencadeadas pela deficiência de ANXA1 endógena poderá contribuir para compreensão da fisiopatologia da neuroinflamação. / Inflammation is a key process in maintaining homeostasis and is essential for the body\'s response to injury. The inflammatory response must be proportional to the aggressor stimuli; in the central nervous system, a failed proper modulation leads to the development of different neurodegenerative diseases. Protein annexin A1 (ANXA1) and peroxisome proliferated-activated receptors (PPAR) control inflammation, as both inhibit development of inflammation and accelerate its resolution. Our research group has demonstrated that ANXA1 modulates PPARγ expression in macrophages. Thus, the present work investigated the modulation of PPARγ expression and its functions in microglia cells by ANXA1. In order to assess such, immortalized cells from cell line BV2 (murine microglia), either unadulterated or transfected for reduced expression of ANXA1, were treated with exogenous ANXA1 (recombinant protein - rANXA1) or either with PPARγ agonist or antagonist (pioglitazone and GW9662, respectively). The obtained results demonstrated that: 1) treatment with rANXA1 increases both gene (RT-PCR) and protein (Western Blotting) expressions of PPARγ, and also that both expressions are reduced in cells with endogenous deficiency of ANXA1, and such effect was reversed by the actions of rANXA1; 2) treatment with rANXA1 does not promote the expression of transcription factors associated with PPARγ expression: cAMP response element binding protein - CREB - and signal transductor and activator of transcription 6 - STAT6 (Western Blotting), but the expression levels of both factors are reduced in transfected cells, and such effect was not reversed by treatment with rANXA1; 3) individual treatment with pioglitazone or rANXA1 increases phagocytosis of apoptotic PC12 cells (flow cytometry), but simultaneous treatment does not affect pioglitazone/rANXA1-induced phagocytosis; however, treatment with GW9662 inhibited rANXA1-induced phagocytosis; 4) treatment with rANXA1 increases CD36 expression (flow cytometry); the expression of CD36 is reduced in transfected cells, and such expression is not reversed by treatment with rANXA1. The obtained data demonstrate the modulation ANXA1 exerts upon PPARγ in microglia cells, with a possible action upon phagocytosis of apoptotic cells, and that reduction of ANXA1 expression greatly reduces the expression of transcription factors STAT6 and CREB, as well as the expression of CD36. Elucidation of such effects that arise from a deficiency of endogenous ANXA1 will contribute to a better comprehension of the pathophysiology of neuroinflammation.

No description available.

No description available.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-12-03T17:13:22Z No. of bitstreams: 1 MICHELLE ANTUNES.pdf: 2333470 bytes, checksum: 8ad88d537e1a1c446e2d18440cf12514 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-12-03T17:13:34Z (GMT) No. of bitstreams: 1 MICHELLE ANTUNES.pdf: 2333470 bytes, checksum: 8ad88d537e1a1c446e2d18440cf12514 (MD5) / Made available in DSpace on 2018-12-03T17:13:34Z (GMT). No. of bitstreams: 1 MICHELLE ANTUNES.pdf: 2333470 bytes, checksum: 8ad88d537e1a1c446e2d18440cf12514 (MD5) Previous issue date: 2017 / A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais prevalente na população idosa, tendo como principal característica neuropatológica a depleção de dopamina (DA) estriatal, condição esta, que conduz ao aparecimento dos sintomas motores da doença. Além de sintomas motores, os pacientes acometidos com essa doença apresentam ainda sintomas não motores e neuropsiquiátricos onde destaca-se a depressão. Atualmente, nenhuma das drogas utilizadas é totalmente eficaz para a DP, nesse contexto, terapias promissoras ainda precisam ser exploradas. Alguns estudos demonstraram que o consumo de flavonoides podem diminuir a incidência e os sintomas de doenças neurodegenerativas e de transtornos depressivos. Nosso estudo anterior já constatou que o flavonoide hesperidina (4'-methoxy-7-O-rutinosyl-3',5-dihydroxyflavanone), intervém na DP e no comportamento tipo depressivo via estresse oxidativo, em um modelo induzido por 6-hidroxidopamina (6-OHDA) em camundongos. Baseando-se nas evidências, se torna interessante investigar o efeito da hesperidina em outros parâmetros da DP no modelo da 6-OHDA, além de seu efeito antidepressivo no modelo da bulbectomia olfatória (BO). Para avaliar a DP, a hesperidina ou o veículo (50 mg/kg) foram administrados via oral (gavagem) durante 28 dias. Após este período, os camundongos foram submetidos a testes comportamentais. Para avaliar a depressão no modelo OB, a hesperidina ou o veículo (50 mg/kg) foram administrados via oral (gavagem) durante 13 dias. Após este período, os camundongos foram submetidos aos testes comportamentais e neuroquímicos. No modelo induzido pela 6-OHDA, o tratamento com a hesperidina protegeu contra a inibição das enzimas mitocondriais dos complexos I, II, IV e V, da enzima Na+/K+ ATPase, diminuição do potencial de membrana mitocondrial, e aumento das atividades das caspases 3 e 9 e níveis de marcadores de inflamação. Além disso, a administração do flavonoide culminou na restauração dos níveis de fatores neurotroficos, assim como dos neurônios positivos para tirosina hidroxilase (TH), e níveis de DA e seus metabolitos no estriado. O tratamento com hesperidina também atenuou as alterações comportamentais de rotação, ansiedade, anedonia, memória e nos danos olfatórios induzidos pela 6-OHDA. No modelo de depressão induzido pela OB, o tratamento oral com hesperidina foi efetivo em reverter o declínio dos fatores neurotróficos, o aumento das citocinas pro-inflamatórias e da atividade da enzima acetilcolinesterase (AchE) no hipocampo, bem como o aumento da atividade locomotora, os prejuízos na memória, o comportamento tipo depressivo e a anedonia decorrentes da remoção dos bulbos. No modelo de DP induzido pela 6-OHDA pode-se propor que a hesperidina atua na restauração dos neurônios dopaminérgicos através do restabelecimento dos fatores neurotróficos, controle das citocinas, e modulação da atividade mitocondrial e apoptótica no estriado, o que reflete na recuperação dos prejuízos comportamentais. No modelo da depressão, indica-se que a reparação hipocampal sobre os fatores neurotróficos e marcadores inflamatórios são responsáveis pelo reestabelecimento comportamental no tratamento com hesperidina. Estes resultados certificam o efeito potencial da hesperidina no tratamento da DP e dos distúrbios depressivos, indicando que esse flavonoide pode atuar como terapia multialvo, associando DP e os transtornos mentais. Isso se deve à 4 regulação de fatores neurotróficos, inflamatórios e apoptóticos, atividades enzimáticas (AchE e Na+/K+ ATPase), disfunção mitocondrial e recuperação neuronal dopaminérgica. / Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the elderly population. Its main neuropathological characteristic is the striatal dopamine (DA) depletion, which leads to the appearance of the motor symptoms of the disease. In addition to motor symptoms, the patients with this disease also present non-motor and neuropsychiatric symptoms where depression stands out. Currently, none of the drugs used is fully effective for PD, in this context, promising therapies still need to be explored. Some studies have shown that consumption of flavonoids may decrease the incidence and symptoms of neurodegenerative diseases and depressive disorders. Our previous study has found that the flavonoid hesperidin (4'-methoxy-7-O-rutinosyl-3 ', 5-dihydroxyflavanone) plays a role in PD and in the depressive behavior via oxidative stress in mice induced by 6-hydroxydopamine (6 -OHDA). Based on the evidence, is interesting to investigate the effect of hesperidin in other parameters of PD in the 6-OHDA model, besides the its antidepressant effect in olfactory bulbulometric (BO) model. To evaluate the PD, hesperidin or vehicle (50 mg / kg) were administered orally (gavage) for 28 days. After this period, the mice were submitted to behavioral tests. To assess depression in the OB model, hesperidin or vehicle (50 mg / kg) were administered orally (gavage) for 13 days. After this period, the mice were submitted to behavioral and neurochemical tests. In the 6-OHDA-induced model, treatment with hesperidin protected against inhibition of mitochondrial enzymes of complexes I, II, IV and V, Na + / K + ATPase enzyme, against the decrease in mitochondrial membrane potential, and against the increase of caspases 3 and 9 activities and inflammatory markers levels. In addition, flavonoid administration culminated in restoration of neurotrophic factors levels, as well as tyrosine hydroxylase (TH) positive neurons, DA and its metabolites levels in the striatum. The treatment with hesperidin also attenuated the behavioral changes of rotation, anxiety, anhedonia, memory and olfactory damage induced by 6-OHDA. In addition, in depression model OB-induced, oral hesperidin treatment was effective in reversing the decline of neurotrophic factors, increase of proinflammatory cytokines levels and acetylcholinesterase (AchE) activity in the hippocampus, as well as increase of locomotor activity, memory impairment, depressive behavior and anhedonia due to removal of bulbs. In DP model induced by 6-OHDA it can be proposed that hesperidin acts in the restoration of dopaminergic neurons through the reestablishment of neurotrophic factors, cytokine control, and modulation of mitochondrial and apoptotic activity in striatum, which reflects in the recovery of damages behavioral. In depression model, it is indicated that hippocampal repair on neurotrophic factors and inflammatory markers are responsible for behavioral reestablishment by hesperidin treatment. These results confirm the potential effect of hesperidin in the treatment of PD and depressive disorders, indicating that this flavonoid can act as a multi-target therapy, associating PD and mental disorders. This is due to the regulation of neurotrophic, inflammatory and apoptotic factors, enzymatic activities (AchE and Na+/K+ ATPase), mitochondrial dysfunction and dopaminergic neuronal recovery.

O complexo da esclerose tuberosa (TSC) é uma doença genética que pode afetar órgãos específicos de qualquer sistema do organismo humano. Em geral, as lesões surgem pela inativação bialélica de um dos genes supressores tumorais Tuberous Sclerosis Complex 1 (TSC1) ou 2 (TSC2). Por outro lado, nas regiões corticais e subcorticais do cérebro, as lesões decorrentes de falhas de migração neuronal e sua arborização podem ser explicadas pela haploinsuficiência de TSC1 ou TSC2. As lesões do córtex cerebral apresentam-se comumente com epilepsia refratária, a qual, por sua vez, pode se associar a deficiência intelectual e transtornos do comportamento. Estes quadros clínicos podem estar presentes em pacientes com TSC sem lesão anatômica detectável à ressonância nuclear magnética do crânio. As proteínas hamartina ou tuberina, conhecidas também como TSC1 e TSC2, são codificadas respectivamente pelos genes TSC1 e TSC2. Elas agem juntas em um complexo molecular citosólico que inativa a pequena GTPase Rheb, a qual tem ação ativadora da cinase alvo da rapamicina em mamíferos (mTOR), regulando diversos processos celulares, como proliferação, diferenciação, crescimento, migração e metabolismo. Com a hipótese de que a quantidade de TSC1 ou TSC2 no neurônio pode alterar suas funções de forma dependente do estado metabólico, tivemos, neste trabalho, o objetivo geral de caracterizar os padrões de expressão e atividade de TSC1 e TSC2 em dois modelos de neurodegeneração induzida no camundongo adulto e verificar se a redução de quantidade de TSC1 tem efeito sobre a extensão da lesão de neurônios dopaminérgicos em modelo de hemiparkinsonismo. No primeiro modelo empregado, cinco estruturas encefálicas de camundongos submetidos a dieta hiperlipídica mostraram alteração da quantidade de RNAm de Tsc1 e/ou Tsc2 ou sinais de estresse oxidativo. A redução de transcritos de Tsc1 e Tsc2 no córtex cerebral foi dependente de jejum realizado imediatamente antes da eutanásia. No córtex cingulado, houve evidência de estresse oxidativo. O aumento específico de RNAm foi observado no hipocampo (Tsc1 e Tsc2) e no estriado e hipotálamo (Tsc1), embora de forma independente do jejum, sugerindo se tratar de alterações relacionadas à dieta hiperlipídica. No modelo de hemiparkinsonismo, camundongos adultos submetidos a injeção intracerebral de 6-hidroxidopamina apresentaram redução da quantidade total de proteína S6 no lado encefálico tratado quando comparado ao segmento contralateral (p =0,004, r=0,8795; teste de Pearson, IC: 95%), sem alteração de TSC1 ou TSC2. Em análises de imunoperoxidase do encéfalo, descrevemos, de forma independente da lesão, a expressão de TSC1 no estriado, núcleos entopeduncular e arqueado e de TSC2 no tálamo e hipotálamo. Com o objetivo de obter um modelo de camundongo sem expressão pós-natal de Tsc1 em várias regiões encefálicas, de forma independente do tipo celular, realizamos cruzamentos entre uma linhagem de camundongo transgênico em que o gene Tsc1 contém sequências lox nos íntrons 16 e 18 e outra linhagem com Tsc1 tipo-selvagem (WT) em homozigose e o transgene para expressão da recombinase Cre em fusão ao domínio de ligação ao ligante do receptor de estrógeno humano (ESR1) sob o controle de expressão do promotor de ubiquitina C (UBC). Em F1, obtivemos camundongos portadores do transgene UBC-CreESR1 e heterozigotos para Tsc1 (Tsc1WT/Flox). Em F2, entre os animais homozigotos Tsc1Flox/Flox (N = 153) gerados por retrocruzamento, nenhum era portador do transgene (Nesperado = 85; Nobservado = 0; X2 = 348,185; p < 0,0001) É possível que o segmento genômico em que houve inserção do vetor lentiviral que carrega o transgene UBC-CreESR1 esteja ligado ao loco de Tsc1 no cromossomo 2 do camundongo, segregando juntos. O tratamento com 4-hidroxitamoxifeno de animais heterozigotos e portadores do transgene aumentou a quantidade de TSC1 no estriado (p < 0,05) e o cerebelo não apresentou alteração. É possível que mecanismos transcricionais ou traducionais, funcionais no estriado, tenham favorecido o aumento de TSC1 de forma dependente de 4-hidroxitamoxifeno / Tuberous sclerosis complex (TSC) is a genetic disorder that can affect any specific organs. In general, lesions are caused by biallelic inactivation of the tumor suppressor genes Tuberous Sclerosis Complex 1 (TSC1) or 2 (TSC2). On the other hand, in cortical and subcortical brain regions, lesions associated with neuronal migration and arborization failures can be explained by TSC1 or TSC2 haploinsufficiency. Brain cortical lesions commonly cause refractory epilepsy, which, in turn, may be associated with intellectual disabilities and behavioral disorders. These medical conditions may be present in TSC patients without detectable anatomic lesion on magnetic resonance images. TSC1 and TSC2 genes encode hamartin and tuberin, also known as TSC1 and TSC2, respectively. They act together in a cytosolic molecular complex that inactivates small GTPase Rheb, which is a mammalian target of rapamycin (mTOR) activator, regulating diverse cellular processes such as proliferation, differentiation, growth, migration and metabolism. With the hypothesis that the amount of TSC1 or TSC2 in the neuron can change its function depending on the metabolic state, the overall objective of this study was to characterize TSC1 and TSC2 expression patterns and activity in two mice models of induced neurodegeneration; and check whether TSC1 reduction changes dopaminergic neurons damage extent in a hemiparkinsonins model. For the first model, five brain structures from mice fed with high fat diet showed alterations in Tsc1 and/or Tsc2 mRNA, or oxidative stress signals. Reduction of Tsc1 and Tsc2 transcripts in the cerebral cortex was dependent on fasting performed immediately prior to euthanasiaThere was evidence of oxidative stress in the cingulate cortex. Increase in mRNA was observed in the hippocampus (Tsc1 and Tsc2) and striatum and hypothalamus (Tsc1), although independent of the fasting, suggesting that this effect is related to the high fat diet. In hemiparkinsonism model, adult mice subjected to intracerebral injection of 6-hydroxydopamine had decreased levels of S6 in the brain treated side compared to the contralateral segment (p = 0.004, r = 0.8795; Pearson test, CI: 95 %), without alterations in TSC1 nor TSC2. Using imunoperoxidase analysis, we described TSC1 expression in the striatum, entopeduncular and arcuate nuclei, and TSC2 in the thalamus and hypothalamus, independently from the 6-OHDA lesion. To obtain a mouse model without TSC1 postnatal expression in different brain regions, independently of the cell type, we performed crosses between transgenic mouse strain in which the Tsc1 gene contains lox sequences in introns 16 and 18 and strain with Tsc1 wild-type (WT) and the transgene for expression of Cre recombinase fused to the binding domain of the human estrogen receptor (ESR1) ligand, controlled by ubiquitin C (UBC) promoter expression. In F1, we obtained mice carrying the transgene UBC-CreESR1 and heterozygous for Tsc1 (Tsc1WT/flox). In F2, among animals homozygous Tsc1Flox/Flox (N=153) generated by backcrossing, none was carrying the transgene (Nexpected = 85; Nobserved = 0; X2= 348.185, p <0.0001) It is possible that the genomic segment containing the lentiviral vector insertion bearing UBC-CreESR1 transgene is linked to the TSC1 region on mouse chromosome 2, and they segregate together. Treatment with 4-hydroxytamoxifen in animals heterozygous and positive for the transgene showed increased TSC1 in the striatum (p <0.05), while there was no change in the cerebellum. It is possible that transcriptional or translational functional striatum mechanisms favored TSC1 increasing, in a 4-hydroxytamoxifen-dependent manner