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A doença de Parkinson é caracterizada pela perda selectiva de neurónios dopaminérgicos na substantia nigra pars compacta. A origem desta doença não é completamente compreendida e têm sido propostas diversas hipóteses em relação aos possíveis factores envolvidos na degeneraração e perda dos neurónios dopaminérgicos nesta região. Entre elas, encontra-se a neuroinflamação, que é cada vez mais reconhecida como o principal factor na patogénese da doença de Parkinson. Inúmeras evidências têm sugerido que as células microgliais são a fonte predominante de inflamação que contribui para a neurodegeneração dopaminérgica. Os astrócitos desempenham funções importantes na manutenção da função normal do cérebro, podendo actuar como reguladores fisiológicos prevenindo as respostas microgliais inflamatórias excessivas. No entanto, pouco se sabe sobre a forma como os astrócitos modulam a activação microglial. Dada a relevância das interacções astrócitos-microglia na regulação da neuroinflamação, é importante identificar mediadores envolvidos neste processo, os quais podem actuar como agentes anti-inflamatórios naturais no cérebro. Neste trabalho, o principal objectivo foi o estudo do efeito da MT-I/II libertada pelos astrócitos na activação microglial induzida pelo agente inflamatório Zymosan A. Para a determinação do efeito da MT-I/II na actividade microglial, culturas primárias de microglia do mesencéfalo ventral foram previamente expostas, a meio condicionado de astrócitos (ou meio de cultura – controlo), e posteriormente tratadas com 0.5 μg/mL de Zymosan A. Verificou-se que a pré-incubação das células microgliais com o meio condicionado de astrócitos preveniu o aumento da actividade microglial induzida pelo Zymosan A, mantendo a actividade fagocítica bem como os níveis de produção de NO semelhantes aos níveis de controlo. Para avaliar a importância da MT-I/II libertada pelos astrócitos na prevenção da activação microglial induzida pelo Zymosan A, foi usado um anticorpo primário de forma a bloquear a acção da MT-I/II, presente no meio condicionado pelos astrócitos. Assim, este meio foi tratado com anti-MT-I/II e, adicionado às culturas de microglia posteriormente expostas a Zymosan A. Observou-se que o bloqueio da acção da MT-I/II previne completamente a activação microglial induzida pelo Zymosan A. A importância da MT-I/II na prevenção da activação microglial induzida pelo Zymosan A foi confirmada em experiências semelhantes mas utilizando anticorpo anti-MT-I/II desnaturado. De seguida avaliou-se o efeito da adição da MT-IIA na modulação da reactividade microglial. Os resultados mostraram que a MT-IIA previne eficazmente a activação microglial induzida pelo Zymosan A. O modo como estas metalotioneínas modulam a reactividade microglial e os receptores envolvidos não é ainda conhecido. Os receptores de metalotioneínas LRP1 e megalina são possíveis candidatos a testar. Assim, avaliou-se a expressão do receptor LRP1 pela microglia do mesencéfalo ventral por imunocitoquímica, verificando-se que o mesmo é expresso nestas células. Assim, trabalhos futuros deverão avaliar a sua contribuição para o efeito anti-inflamatório das MT-I/II, em particular da MT-IIA. Em resumo, os resultados obtidos neste trabalho demonstraram que as MT-I/II e, em particular a MT-IIA, libertadas pelos astrócitos, são capazes de modular as respostas inflamatórias microgliais. Estes resultados poderão contribuir para o desenvolvimento de uma possível terapia de prevenção da neuroinflamação, e indirectamente reduzir o desenvolvimento da Doença de Parkin
Parkinson's disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. The origin of this disease is not completely understood and several hypotheses have been proposed regarding the possible factors involved in the degeneration and loss of dopaminergic neurons in this area. Among them, neuroinflammation, is increasingly recognized as a major factor in the pathogenesis of Parkinson's disease. Several evidences suggest that microglial cells are the predominant source of inflammation that contributes to dopaminergic neurodegeneration. Astrocytes have an important role in the maintenance of normal brain function and they could act as physiological regulators preventing excessive inflammatory microglial responses. However, little is known about how astrocytes may modulate microglial activation. Given the importance of microglia-astrocyte interactions in the regulation of neuroinflammation, it is important to identify mediators involved in this process, which could act as natural anti-inflammatory agents in the brain. In the present work, the main goal was to study the effect of MT-I/II released by astrocytes in microglial activation induced by the inflammatory agent Zymosan A. To determine the effect of MT-I/II on microglial activity, primary cultures of microglia obtained from the ventral midbrain were previously exposed, to media conditioned by astrocytes (or culture medium - control) and then treated with 0.5 µg/mL Zymosan A. It was found that astrocytes conditioned media were able to prevent an increase in microglial activity induced by Zymosan A, keeping the levels of phagocytic activity and production of NO similar to control levels. To evaluate the role of MT-I/II released by astrocytes in the prevention of microglial activation induced by Zymosan A, an primary antibody was used to block the action of MT-I/II. Thus, astrocytes conditioned media were treated with anti-MT-I/II and added to the microglial cultures, which were then exposed to Zymosan A. It was observed that MT-I/II is able to completely prevent microglial activation induced by Zymosan A. To confirm this, we proceeded to the denaturation of the antibody anti-MT-I/II prior to its addition to the media conditioned by astrocytes, and subsequent application to the microglial cultures. The results obtained corroborated with a role of MT-I/II in the modulation of microglial reactivity by astrocytes. We further evaluated the role of MT-IIA in this process by exogenously adding this protein to the microglial culture medium prior to Zymosan A treatment. We found that MT-IIA is able to prevent the Zymosan A-induced microglial activation. The mechanism by which metallothioneins modulate microglial reactivity as well as the receptors involved are still unknown. The LRP1 and megalin metallothionein receptors are good candidates to be tested. Therefore, the expression of LRP1 receptor by microglial cells was investigated by immunocytochemistry. The results showed that the LRP1 receptor is present in microglial cells obtained from the ventral midbrain. Hence, future work should focus on its role in mediating the anti-inflammatory effect of MT-I/II and, in particular, of MT-IIA. In summary the results obtained in this work show that astrocyte-derived MT-I/II and, specifically MT-IIA, are able to modulate microglia inflammatory responses. These results may contribute to the development of a potential therapy for preventing neuroinflammation, and indirectly reduce the development of Parkinson´s Disease.
Parkinson's disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. The origin of this disease is not completely understood and several hypotheses have been proposed regarding the possible factors involved in the degeneration and loss of dopaminergic neurons in this area. Among them, neuroinflammation, is increasingly recognized as a major factor in the pathogenesis of Parkinson's disease. Several evidences suggest that microglial cells are the predominant source of inflammation that contributes to dopaminergic neurodegeneration. Astrocytes have an important role in the maintenance of normal brain function and they could act as physiological regulators preventing excessive inflammatory microglial responses. However, little is known about how astrocytes may modulate microglial activation. Given the importance of microglia-astrocyte interactions in the regulation of neuroinflammation, it is important to identify mediators involved in this process, which could act as natural anti-inflammatory agents in the brain. In the present work, the main goal was to study the effect of MT-I/II released by astrocytes in microglial activation induced by the inflammatory agent Zymosan A. To determine the effect of MT-I/II on microglial activity, primary cultures of microglia obtained from the ventral midbrain were previously exposed, to media conditioned by astrocytes (or culture medium - control) and then treated with 0.5 µg/mL Zymosan A. It was found that astrocytes conditioned media were able to prevent an increase in microglial activity induced by Zymosan A, keeping the levels of phagocytic activity and production of NO similar to control levels. To evaluate the role of MT-I/II released by astrocytes in the prevention of microglial activation induced by Zymosan A, an primary antibody was used to block the action of MT-I/II. Thus, astrocytes conditioned media were treated with anti-MT-I/II and added to the microglial cultures, which were then exposed to Zymosan A. It was observed that MT-I/II is able to completely prevent microglial activation induced by Zymosan A. To confirm this, we proceeded to the denaturation of the antibody anti-MT-I/II prior to its addition to the media conditioned by astrocytes, and subsequent application to the microglial cultures. The results obtained corroborated with a role of MT-I/II in the modulation of microglial reactivity by astrocytes. We further evaluated the role of MT-IIA in this process by exogenously adding this protein to the microglial culture medium prior to Zymosan A treatment. We found that MT-IIA is able to prevent the Zymosan A-induced microglial activation. The mechanism by which metallothioneins modulate microglial reactivity as well as the receptors involved are still unknown. The LRP1 and megalin metallothionein receptors are good candidates to be tested. Therefore, the expression of LRP1 receptor by microglial cells was investigated by immunocytochemistry. The results showed that the LRP1 receptor is present in microglial cells obtained from the ventral midbrain. Hence, future work should focus on its role in mediating the anti-inflammatory effect of MT-I/II and, in particular, of MT-IIA. In summary the results obtained in this work show that astrocyte-derived MT-I/II and, specifically MT-IIA, are able to modulate microglia inflammatory responses. These results may contribute to the development of a potential therapy for preventing neuroinflammation, and indirectly reduce the development of Parkinson´s Disease.
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Universidade da Beira Interior