Protein Aggregation and Inflammasome Signaling in Manganese Neurotoxicity

 描述（由申请人提供）：金属暴露越来越被认为是慢性神经退行性疾病（包括帕金森病（PD）和阿尔茨海默病（AD））的潜在环境因素。慢性锰（Mn）暴露与人类帕金森氏症样神经系统疾病有关。蛋白质聚集及其朊病毒样增殖现在被认为是许多神经退行性疾病（统称为蛋白质病）的中心病理生理机制。然而，金属在蛋白质聚集中的作用和驱动退行性过程的神经毒理学机制尚未得到很好的理解。α-突触核蛋白（α-Syn）蛋白聚集与PD有关，该蛋白具有多个二价金属结合基序，这些基序被认为在α-Syn的纤维化和神经毒性中发挥作用。由于Mn对αSyn中的金属结合位点具有亲和力，因此我们在神经元模型中研究了Mn对αSyn的影响。有趣的是，我们发现αSyn在Mn暴露的早期阶段保护Mn诱导的神经毒性，但延长Mn暴露促进αSyn聚集。与聚集蛋白通过外泌体释放在细胞间传播的新概念一致，我们还观察到在Mn暴露期间含有αSyn的外泌体向细胞外环境中的释放增强。因此，Mn诱导的αSyn聚集和具有αSyn货物的外泌体释放的令人兴奋的发现促使我们进一步表征Mn神经毒性中神经退行性过程的细胞和分子机制。我们的提案将检验新的假设，即Mn暴露促进αSyn错误填充并损害细胞内αSyn运输，从而增加含有αSyn蛋白聚集体的外泌体的形成和释放，其随后以PKCδ依赖性方式通过NLRP 3炎性体途径触发小胶质细胞活化。PKCδ依赖性NLRP 3炎性体通路的持续激活有助于Mn神经毒性。该提案的具体目标是：（i）表征Mn神经毒性的细胞培养物和动物模型中Mn诱导的内体运输、逆转录酶功能障碍和外泌体释放受损的细胞机制，（ii）确定由Mn诱导的外泌体αSyn聚集体触发的小胶质细胞和星形胶质细胞中的NLRP 2/3炎性体神经炎症信号传导，并表征NLRP 2/3中PKCδ的促炎调节功能。3 Mn神经毒性中的炎性小体激活，以及（iii）在Mn神经毒性动物模型中检查PKCδ在介导外泌体αSyn聚集体诱导的促炎反应中的作用，并证实Mn暴露的人脑组织中αSyn蛋白聚集体的存在。我们的综合细胞和分子方法来解开外泌体αSyn聚集体的形成和释放及其对锰金属神经毒性中神经炎症信号传导的功能性后果，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.