Protein Aggregation and Inflammasome Signaling in Manganese Neurotoxicity

 描述(申请人提供)：金属暴露日益被认为是包括帕金森氏病(PD)和阿尔茨海默病(AD)在内的慢性神经退行性疾病的潜在环境因素。慢性锰暴露与人类的帕金森氏症神经疾病有关。蛋白质聚集及其蛋白样增殖现在被认为是许多神经退行性疾病的中枢病理生理机制，统称为蛋白质病。然而，金属在蛋白质聚集中的作用以及驱动退化过程的神经毒理学机制还不是很清楚。α-突触核蛋白(αSyn)蛋白聚集与帕金森病有关，该蛋白具有多个二价金属结合基序，已被认为在αSynn的纤维化和神经毒性中发挥作用。由于锰与α突触蛋白中的金属结合部位有亲和力，我们在神经元模型中检测了锰对α突触蛋白的影响。有趣的是，我们发现，在锰暴露的早期阶段，α同步蛋白对锰诱导的神经毒性具有保护作用，但长期的锰暴露促进了α同步蛋白的聚集。与聚集蛋白通过胞外释放在细胞间传播的新概念一致，我们还观察到在锰暴露期间，含有αSYN的外切体在细胞外环境中的释放增强。因此，我们令人兴奋的发现，在锰诱导的α-Syn聚集和释放外切体与α-Syn货物，促使我们进一步表征细胞和分子机制的神经变性过程中的锰神经毒性。我们的建议将检验这一新的假设，即锰暴露促进αSyn错配并损害细胞内αSyn的运输，从而增加含有αSyn蛋白聚合体的外体的形成和释放，从而以δ依赖的方式通过NLRP3炎症体途径激活小胶质细胞。依赖于δ的PKC-NLRP3炎症体通路的持续激活参与了锰的神经毒性。该建议的具体目标是：(I)在细胞培养和锰神经毒性动物模型中，鉴定锰诱导的内体转运、逆转录功能障碍和外切体释放受损的细胞机制；(Ii)检测由锰诱导的外体αSyn聚集体触发的小胶质细胞和星形胶质细胞中的NLRP2/3炎性小体神经炎信号，并鉴定PKCδ在锰神经毒性中NLRP2/3炎症体激活中的促炎调节作用；以及(Iii)在锰神经毒性动物模型中，研究PKCδ在介导外体αSyn聚集体诱导的前炎症反应中的作用，并证实αSyn蛋白在锰暴露人脑组织中的存在。我们的细胞和分子集成方法解开了外体αSyn聚集体的形成和释放以及它们在金属锰神经毒性中对神经炎性信号的功能影响，这将为环境相关的神经退行性疾病提供新的机制见解。

 DESCRIPTION (provided by applicant): Metal exposure has increasingly been recognized as a potential environmental contributor to chronic neurodegenerative diseases including Parkinson's disease (PD) and Alzheimer's disease (AD). Chronic manganese (Mn) exposure has been implicated in Parkinson's-like neurological conditions in humans. Protein aggregation and its prion-like propagation are now considered the central pathophysiological mechanisms of many neurodegenerative diseases collectively known as proteinopathies. However, the role of metals in protein aggregation and the neurotoxicological mechanisms that drive degenerative processes are not well understood. α-Synuclein (αSyn) protein aggregation has been implicated in PD, and this protein features multiple divalent metal-binding motifs that have been suggested to play a role in αSyn's fibrillization and neurotoxicity. Since Mn shows affinity to the metal binding sites in αSyn, we have examined the effect of Mn on αSyn in neuronal models. Interestingly, we found that αSyn protected against Mn-induced neurotoxicity during early stages of Mn exposure, but prolonged Mn exposure promoted αSyn aggregation. In agreement with the emerging concept that aggregated proteins propagate cell-to-cell via exosomal release, we also observed enhanced release of exosomes containing αSyn into the extracellular environment during Mn exposure. Thus, our exciting finding of Mn-induced αSyn aggregation and release of exosomes with αSyn cargo prompts us to further characterize the cellular and molecular mechanisms of neurodegenerative processes in Mn neurotoxicity. Our proposal will test the novel hypothesis that Mn exposure promotes αSyn misfiling and impairs intracellular αSyn trafficking, thereby increasing the formation and release of exosomes containing αSyn protein aggregates, which subsequently trigger microglial activation through the NLRP3 inflammasome pathway in a PKCδ-dependent manner. Sustained activation of the PKCδ-dependent NLRP3 inflammasome pathway contributes to Mn neurotoxicity. Specific objectives of the proposal are: (i) to characterize the cellular mechanism of Mn-induced impairment in endosomal trafficking, retromer dysfunction and exosome release in cell culture and animal models of Mn neurotoxicity, (ii) to determine NLRP2/3 inflammasome neuroinflammatory signaling in microglia and astrocytes triggered by Mn-induced exosomal αSyn aggregates and to characterize the proinflammatory regulatory function of PKCδ in NLRP2/3 inflammasome activation in Mn neurotoxicity, and (iii), to examine the role of PKCδ in mediating the exosomal αSyn aggregate-induced proinflammatory response in animal models of Mn neurotoxicity and to confirm the presence of αSyn protein aggregation in Mn-exposed human brain tissues. Our integrated cellular and molecular approach to unraveling the formation and release of exosomal αSyn aggregates and their functional consequences on neuroinflammatory signaling in manganese metal neurotoxicity will provide novel mechanistic insights into environmentally-linked neurodegenerative disorders.