Mechanistic dissection of P-body formation and abnormal mRNA degradation in alpha-synucleinopathy

项目摘要 目前还没有预防或减缓帕金森病（PD）和痴呆症病程的措施， 路易小体（DLB）。这些常见的破坏性"突触核蛋白病"的特征是， 与囊泡膜相关蛋白α-突触核蛋白（α-synuclein）聚集相关的神经变性 syn）。a-syn基因位点的显性点突变、倍增和常见风险变异导致或 增加PD和DLB的风险，明确地将这种蛋白质与疾病病因联系起来。这引起了 在靶向α-syn毒性的治疗中，但这种毒性仍然知之甚少。多年来，我们的团队和 其他人已经开发了几种不同的α-syn毒性的细胞模型来研究这种毒性的性质， 包括源自PD患者的神经元。我们已经绘制了a-syn的遗传和物理相互作用。他们 不出所料，我们注意到囊泡运输，但出乎意料的是，mRNA代谢出现了一个新的 与a-syn生物学有关的途径。在这里，我们提出a-syn直接与加工体（P-体）相连， 参与mRNA降解和储存的细胞质无膜细胞器。我们用了一种新的蛋白质- 干扰传感器，以缩小RNA结合蛋白质组中的a-syn特异性干扰，和 因此发现增加的P-体形成与α-syn介导的毒性相关。p体 在PD患者来源的神经元中，这种形成是保守的。这不是一般的应激反应。例如， 与在其他蛋白质错误折叠的背景下发生的典型"应激颗粒"反应高度不同， 事件此外，我们的初步数据强烈表明P体的形成与a-syn生物学直接相关 因为我们发现a-syn与P体的中心成分有物理相互作用，即去帽， 蛋白质和相关因子。现在，我们已经优化了一种方法，使用CLICK化学为基础的代谢 标记以定量测量mRNA降解速率。从技术上讲，我们已经准备好充分 研究α-syn聚集和错误定位导致mRNA代谢紊乱的机制。 我们现在提出在疾病相关的细胞中测定α-syn-P-体相互作用和mRNA降解， 模型，特别是在来源于携带a-synA53T突变和a-syn-野生型的患者的神经元中 三倍我们已经彻底表征了这些iPSC系，并产生了同基因突变校正的 对照最后，我们将研究PD/DLB患者的死后脑组织中是否存在升高的水平， P体的错误定位以确认疾病相关性。我们提供了一个路线图来测试我们的假设， 通过在细胞和体内模型中进行遗传分析来预测未来。我们相信P- 身体和a-syn将开始新的途径来理解a-syn毒性的病理后果， 以及PD和其他突触核蛋白病的潜在新治疗选择。

PROJECT SUMMARY There are currently no measures that prevent or slow the course of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). These common and devastating “synucleinopathies” are characterized by neurodegeneration associated with aggregation of the vesicle membrane-associated protein alpha-synuclein (a- syn). Dominant point mutations, multiplications, and common risk variants at the a-syn gene locus cause or confer increased risk for PD and DLB, definitively tying this protein to disease etiology. This has raised interest in therapies that target a-syn toxicity, but this toxicity remains poorly understood. Over the years, our group and others have developed several distinct cellular models of a-syn toxicity to address the nature of this toxicity, including PD patient-derived neurons. We have mapped genetic and physical interactions for a-syn. They predictably drew our attention to vesicle trafficking but, unexpectedly, mRNA metabolism emerged as a novel pathway tied to a-syn biology. Here, we propose that a-syn is directly linked to Processing-bodies (P-bodies), cytoplasmic membraneless organelles involved in mRNA degradation and storage. We used a novel protein- perturbation sensor to narrow down the a-syn specific perturbations in the RNA-binding proteome, and consequently discovered that increased P-body formation is associated with a-syn mediated toxicity. P-body formation is conserved in PD patient-derived neurons. This is not a generic stress response. For example, it is highly distinct from the typical “stress granule” response that occurs in the context of other protein misfolding events. Furthermore, our preliminary data strongly suggest P-body formation is directly tied to a-syn biology because we found that a-syn has physical interactions with central components of P-bodies, namely decapping proteins and associated factors. Now, we have optimized a method using CLICK chemistry-based metabolic labeling to quantitatively measure mRNA degradation rates. Technologically, we are thus poised to fully investigate the mechanism by which a-syn aggregation and mislocalization lead to perturbed mRNA metabolism. We now propose to assay a-syn-P-body interactions and mRNA degradation in a disease-relevant cellular model, specifically in neurons derived from patients harboring a-synA53T mutation and a-syn-wild type triplication. We have thoroughly characterized these iPSC lines and generated isogenic mutation-corrected controls. Finally, we will investigate postmortem brain tissues of PD/DLB patients for elevated levels or mislocalization of P-bodies to confirm disease-relevance. We provide a roadmap for testing our hypothesis in the future through genetic analysis in cellular and in vivo models. We believe that the novel link between P- bodies and a-syn will commence new avenues for understanding the pathologic consequences of a-syn toxicity, and potentially new therapeutic options for PD and other synucleinopathies.