Protein Aggregation and Inflammasome Signaling in Manganese Neurotoxicity.

摘要 金属暴露日益被认为是慢性阻塞性肺疾病的潜在环境因素 神经退行性疾病包括帕金森氏病(PD)和阿尔茨海默病(AD)。慢性 锰(Mn)暴露与人类帕金森氏症样神经疾病有关。蛋白 聚集性及其类病毒繁殖被认为是黄斑狼疮的主要病理生理机制。 许多神经退行性疾病统称为蛋白质病。然而，金属在其中的作用 蛋白质聚集和驱动退变过程的神经毒理学机制并不是很好 明白了。α-突触核蛋白(α-syn)蛋白聚集与帕金森病有关，该蛋白的特征是 多种二价金属结合基序，已被认为在αSYN的成纤维和 神经毒性。由于锰与αSYN中的金属结合部位有亲和力，我们研究了锰对 神经元模型中的α突触。有趣的是，我们发现α-syn对锰诱导的神经毒性有保护作用 锰暴露的早期阶段，但长期的暴露促进了α-Syn的聚集。在与 我们还观察到，聚集的蛋白质通过胞外释放在细胞间传播的新概念 在锰暴露过程中，含有αsyn的外切体被促进释放到细胞外环境中。因此， 我们令人兴奋的发现是锰诱导的αSYN聚集并与αSYN Cargo一起释放外切体，这促使我们 进一步表征神经细胞退变过程的细胞和分子机制 神经毒性。我们的建议将检验这一新的假设，即暴露于锰促进αSYN错误折叠和 损害细胞内αSYN的运输，从而增加含有以下物质的外切体的形成和释放 NLRP3Syn蛋白聚集体，随后通过α炎症小体触发小胶质细胞激活 PKCδ依赖的途径。依赖蛋白激酶Cδ的NLRP3炎症体的持续激活 途径参与了锰的神经毒性作用。该提案的具体目标是：i)描述细胞 锰对心肌细胞内体转运、逆转录功能障碍和外切体释放的损伤机制 锰神经毒性的细胞培养和动物模型，II)测定NLRP2/3炎症性神经炎 锰诱导的小胶质细胞和星形胶质细胞内信号转导的α-Syn聚集体及其特征 蛋白激酶Cδ在锰神经毒性中NLRP2/3炎症体激活中的促炎调节作用 Iii)研究PKCδ在介导外体αSyn聚集体诱导的促炎反应中的作用 锰神经毒性动物模型的建立及α-Syn蛋白聚集性的证实 人脑组织。我们综合的细胞和分子方法来解开 锰的外体αSyn聚集体及其对神经炎性信号的功能影响 金属神经毒性将为环境相关的神经退行性变提供新的机制见解 精神错乱。

Abstract Metal exposure has increasingly been recognized as a potential environmental contributor to chronic neurodegnerative 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 extracelluar 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 misfolding 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.