DJ‐1 neuronal rescue under oxidative stress: implications for Parkinson's disease

Abstract

Mutações em vários genes, incluindo o gene DJ-1, têm sido referidas como causadores de formas hereditárias da Doença de Parkinson (DP), sendo que a partir do estudo das proteínas codificadas por esses genes já foram obtidas informações relevantes sobre os mecanismos moleculares da degeneração dopaminérgica que caracteriza esta patologia. De entre esses mecanismos o stress oxidativo tem-se evidenciado, sendo apontado como a principal causa da morte dos neurónios dopaminérgicos independentemente dos estímulos iniciais que levam à doença. A proteína DJ-1, associada a uma das formas hereditárias da DP, tem sido considerada como uma proteína de resposta ao stress oxidativo que apresenta um papel importante na protecção contra este estímulo. Para além disso, diversas funções têm-lhe sido propostas, as quais têm contribuído para aumentar o conhecimento sobre os mecanismos envolvidos na neurodegeneração associada a stress oxidativo, observada em doentes com DP. A proteína DJ-1 é considerada uma proteína multifuncional, contudo os mecanismos pelos quais esta exerce a sua função, assim como os mecanismos responsáveis pela sua regulação, não estão totalmente compreendidos. Assim, será espectável que ao se elucidar a função fisiológica da DJ-1 sejam também alcançados importantes conhecimentos sobre a DP. Além disso, o stress oxidativo é crucial para a actividade da DJ-1, encontrando-se associado à regulação da maioria dos mecanismos de acção e das interacções estabelecidas por esta. Contudo, os mechanismos exactos responsáveis pela regulação da proteína DJ-1 pelo stress oxidativo, assim como muitos dos seus interactores continuam por identificar. Nesse sentido, com este projecto pretendeu-se elucidar os mecanismos de acção da DJ-1. Mais precisamente pretendeu clarificar os mecanismos pelos quais esta protéina exerce a sua capacidade neuroprotectora a curto e longo prazo, através da identificação dos seus interactores e da dinâmica das interacções durante o stress oxidativo. Para alcançar os objectivos pretendidos, foi realizado um estudo exaustivo de interactómica que permitiu identificar e quantificar 881 proteínas envolvidas na rede de interacções estabelecidas por intermédio da actividade da DJ-1 em condições de stress oxidativo. Este estudo foi realizado em condições bem definidas associadas à activação de duas importantes vias de sobrevivência centrais para a capacidade neuroprotectora da DJ-1 (as vias da ERK1/2 e PI3-K/Akt), e com importantes implicações para o estudo da DJ-1 no contexto da DP dado que foi observado um impacto evidente na actividade mitocondrial causado pelas condições experimentais usadas. Com vista a uma melhor caracterização da natureza dinâmica das interacções estabelecidas foi desenvolvida uma estratégia baseada na optimização da análise SWATH-MS que consistiu no uso de um método de digestão em gel com elevada reprodutibilidade e eficiência, designado Short-GeLC, combinado com o uso de proteínas recombinantes como padrão interno. Esta análise permitiu traçar perfis de interacção revelando grupos de proteínas com o mesmo tipo de modulação que também partilhavam actividades biológicas similares, apontado para a implicação da DJ-1 na modulação desses mecanismos e contribuindo assim para clarificar a sua actividade neuroprotectora. A rede de interacções identificada neste trabalho contribuiu em grande parte para aumentar o conhecimento associado às vias envolvidas na resposta da DJ-1 ao stress oxidativo, que podem ser divididas de um modo geral em dois tipos de resposta: um correspondendo a uma resposta mais imediata que está principalmente associada à identificação de proteínas de resposta ao stress tais como oxireductases e fosfatases; e outro mecanismo associado a uma resposta adaptativa e de longo prazo que está associada com a forte representação de proteínas envolvidas em expressão de genes. Para além disso, foi possível confirmar que: i) a proteína DJ-1 para além de actuar em diferentes vias, também controla o mesmo mecanismo a diversos níveis, tal como no caso da regulação da expressão de proteínas e das vias intrínsecas e extrínsecas de apoptose, e ii) apontar pela primeira vez para o envolvimento da DJ-1 com grânulos de stress e na resposta a danos no ADN, cuja importância para a viabilidade celular e implicação nos mecanismos de neurodegeneração, incluindo os envolvidos em DP, tem vindo a aumentar entre a comunidade científica. Em conclusão, este trabalho resultou na identificação de várias novas proteínas envolvidas na rede de interacções da DJ-1 contribuíndo para a elucidação dos mecanismos de protecção neuronal mediados pela DJ-1 contra o stress oxidativo, e apontando novos mecanismos. Para além disso, muitas da proteínas identificadas estão bem estabelecidas em várias funções celulares implicadas na DP. Deste modo, estes resultados contribuem também para um melhor entendimento da DP e dos vários mecanismos envolvidos no estabelecimento e progressão da doença. Mutations in several genes, including DJ‐1 gene, have been reported to cause hereditary forms of Parkinson’s Disease (PD), and in fact the study of these genes has already provided valuable insights into the molecular mechanisms of dopaminergic degeneration characteristic of this pathology, from which oxidative stress has been considered the cause of dopaminergic neurons death in PD independently of the trigger of the disease. The PD‐ associated protein DJ‐1 has been recognized as a redox response protein with an important role in the protection against the oxidative stress insults. Moreover, several functions have been reported for DJ‐1 which have contributed with significant insights into the mechanisms of oxidative stress‐related neurodegeneration observed in PD patients. Considering all these evidences, DJ‐1 was considered as a multifunctional protein although its exact mechanism of action and regulation remains largely unknown. It is therefore expected that by elucidating DJ‐1 physiological function important insights into PD will also be achieved. Moreover, oxidative stress conditions seem to be crucial for DJ‐1 activity, being associated with the regulation of the majority of its mechanisms of action and the interactions established by it. However, all the exact mechanisms behind oxidative stress regulation of DJ‐1 and many of the interaction partners of DJ‐1 remain unidentified. Therefore, this project proposed to elucidate the mechanisms of action of DJ‐1. More specifically, aimed to clarify the mechanisms by which DJ‐1 modulate short‐term and long‐term neuroprotection, by identifying the binding partners of DJ‐1 during oxidative stress and the dynamics of these interactions under oxidative stress. To achieve these goals, a comprehensive interactomic study of DJ‐1 under oxidative stress conditions was conducted, allowing the identification and quantification of 881 proteins involved in the network of interactions established via DJ‐1 activity. Moreover, this study was performed in a well‐defined model associated with the activation of two key survival pathways central for DJ‐1 neuroprotective function (ERK1/2 and PI3‐K/Akt pathways), and with important implications for the study of DJ‐1 role in Parkinson’s Disease context, since an evident impact in the mitochondrial activity was also reflected in the experimental conditions used. More importantly, the optimized SWATH‐MS based pipeline developed consisting in the use of a high reproducible and efficient in‐gel digestion methods, designed as short‐GeLC, combined with the use of recombinant proteins as internal standard, was applied to characterize the dynamic nature of the interactions. The SWATH‐MS analysis allowed to trace the profiles of interaction revealing clusters of proteins with the same type of modulation that also shared similar biological activities, pointing out for an implication of DJ‐1 in those mechanisms and largely contributing to clarify the DJ‐1 neuroprotective activity. In fact, the network of interactors identified in this work largely contributes to extend the knowledge regarding the pathways involved in DJ‐1 response to oxidative stress, which can be generically divided in two different types: one corresponding with a more immediate response, associated with the identification of stress responsive proteins such as oxireductases and phosphatases; and the other corresponding to an adaptive and long term response associated with the strong representation of proteins involved in gene expression. Moreover, it was possible to confirmed that: i) DJ‐1 not only acts at different pathways but also controls the same mechanisms at different stages, such as in the case of the regulation of protein expression and of the intrinsic and extrinsic apoptotic pathways, and ii) to point out for the first time, the involvement of DJ‐1 with stress granules and with the response to DNA damage, which relevance for cell survival and implication in neurodegeneration (including in PD) is increasing in importance within the scientific community. In conclusion, this study resulted in the identification of several novel proteins involved in DJ‐1 network contributing to the elucidation of DJ‐1‐mediated neuronal protection against oxidative stress, and pointing out some new mechanisms. In addition, many of the proteins identified are well established in distinct cellular functions implicated in PD. Thus, these results may also contribute to a better understanding of PD and the distinct pathways involved in the establishment and progression of the disease.

Mutations in several genes, including DJ‐1 gene, have been reported to cause hereditary forms of Parkinson’s Disease (PD), and in fact the study of these genes has already provided valuable insights into the molecular mechanisms of dopaminergic degeneration characteristic of this pathology, from which oxidative stress has been considered the cause of dopaminergic neurons death in PD independently of the trigger of the disease. The PD‐ associated protein DJ‐1 has been recognized as a redox response protein with an important role in the protection against the oxidative stress insults. Moreover, several functions have been reported for DJ‐1 which have contributed with significant insights into the mechanisms of oxidative stress‐related neurodegeneration observed in PD patients. Considering all these evidences, DJ‐1 was considered as a multifunctional protein although its exact mechanism of action and regulation remains largely unknown. It is therefore expected that by elucidating DJ‐1 physiological function important insights into PD will also be achieved. Moreover, oxidative stress conditions seem to be crucial for DJ‐1 activity, being associated with the regulation of the majority of its mechanisms of action and the interactions established by it. However, all the exact mechanisms behind oxidative stress regulation of DJ‐1 and many of the interaction partners of DJ‐1 remain unidentified. Therefore, this project proposed to elucidate the mechanisms of action of DJ‐1. More specifically, aimed to clarify the mechanisms by which DJ‐1 modulate short‐term and long‐term neuroprotection, by identifying the binding partners of DJ‐1 during oxidative stress and the dynamics of these interactions under oxidative stress. To achieve these goals, a comprehensive interactomic study of DJ‐1 under oxidative stress conditions was conducted, allowing the identification and quantification of 881 proteins involved in the network of interactions established via DJ‐1 activity. Moreover, this study was performed in a well‐defined model associated with the activation of two key survival pathways central for DJ‐1 neuroprotective function (ERK1/2 and PI3‐K/Akt pathways), and with important implications for the study of DJ‐1 role in Parkinson’s Disease context, since an evident impact in the mitochondrial activity was also reflected in the experimental conditions used. More importantly, the optimized SWATH‐MS based pipeline developed consisting in the use of a high reproducible and efficient in‐gel digestion methods, designed as short‐GeLC, combined with the use of recombinant proteins as internal standard, was applied to characterize the dynamic nature of the interactions. The SWATH‐MS analysis allowed to trace the profiles of interaction revealing clusters of proteins with the same type of modulation that also shared similar biological activities, pointing out for an implication of DJ‐1 in those mechanisms and largely contributing to clarify the DJ‐1 neuroprotective activity. In fact, the network of interactors identified in this work largely contributes to extend the knowledge regarding the pathways involved in DJ‐1 response to oxidative stress, which can be generically divided in two different types: one corresponding with a more immediate response, associated with the identification of stress responsive proteins such as oxireductases and phosphatases; and the other corresponding to an adaptive and long term response associated with the strong representation of proteins involved in gene expression. Moreover, it was possible to confirmed that: i) DJ‐1 not only acts at different pathways but also controls the same mechanisms at different stages, such as in the case of the regulation of protein expression and of the intrinsic and extrinsic apoptotic pathways, and ii) to point out for the first time, the involvement of DJ‐1 with stress granules and with the response to DNA damage, which relevance for cell survival and implication in neurodegeneration (including in PD) is increasing in importance within the scientific community. In conclusion, this study resulted in the identification of several novel proteins involved in DJ‐1 network contributing to the elucidation of DJ‐1‐mediated neuronal protection against oxidative stress, and pointing out some new mechanisms. In addition, many of the proteins identified are well established in distinct cellular functions implicated in PD. Thus, these results may also contribute to a better understanding of PD and the distinct pathways involved in the establishment and progression of the disease.