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

α-突触核蛋白病中 P 体形成和异常 mRNA 降解的机制解析

• 批准号: 9808391
• 负责人: Vikram Khurana
• 金额: $ 49.23万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808391
• 关键词: Address; Affect; Amyloid; Antibodies; Attention; Autopsy; Biological Assay; Biology; Biotin; Brain; Cell Culture Techniques; Cell Line; Cell model; Cells; Charge; Chemistry; Chronic; Co-Immunoprecipitations; Collaborations; Cultured Cells; Cytoplasm; Data; Disease; Disease model; Dissection; Epitopes; Equilibrium; Etiology; Eukaryotic Cell; Event; FYN gene; Future; G3BP1 gene; Genes; Genetic; Grant; Healthcare Systems; Human; Investigation; Label; Lead; Lewy Bodies; Lewy Body Dementia; Ligase; Light; Link; Mammalian Cell; Maps; Measures; Mediating; Messenger RNA; Metabolic; Metabolism; Methods; Modeling; Mus; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Parkinson Disease; Parkinson' s Dementia; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Physiologic pulse; Physiological; Physiology; Point Mutation; Prevention strategy; Proteins; Proteome; RNA Binding; RNA-Binding Proteins; Risk; Streptavidin; Structure; Substantia nigra structure; Techniques; Testing; Toxic effect; Untranslated RNA; Vesicle; Yeasts; alpha synuclein; base; biological adaptation to stress; brain tissue; case control; deep sequencing; design; dopaminergic neuron; experimental study; fly; frontal lobe; genetic analysis; in vivo; in vivo Model; induced pluripotent stem cell; interest; mRNA Decay; mRNA Transcript Degradation; mRNA decapping; mutant; neuropathology; new therapeutic target; novel; novel therapeutics; prevent; protein misfolding; protein protein interaction; response; risk variant; sensor; stress granule; synucleinopathy; targeted treatment; temporal measurement; trafficking; vesicle-associated membrane protein

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.

项目总结 目前还没有预防或减缓帕金森氏病(PD)和痴呆症的措施 路易氏小体(DLb)。这些常见且具有破坏性的“联体核病”的特点是 与囊泡膜相关蛋白α-突触核蛋白聚集相关的神经退行性变 SYN)。A-syn基因座上的显性点突变、倍增和常见的风险变异可引起 增加了帕金森病和DLB的风险，明确地将这种蛋白与疾病病因联系在一起。这引起了人们的兴趣 在针对a-syn毒性的治疗中，但这种毒性仍然知之甚少。多年来，我们集团和 其他人已经开发了几种不同的a-SYN毒性的细胞模型来解决这种毒性的性质， 包括帕金森病患者来源的神经元。我们已经绘制了a-syn的遗传和物理相互作用图。他们 不出所料地引起了我们对囊泡运输的注意，但出乎意料的是，信使核糖核酸代谢作为一种新的 一条与a-syn生物学有关的途径。这里，我们提出a-syn与加工小体(P小体)直接相连， 参与mRNA降解和储存的细胞质无膜细胞器。我们使用了一种新的蛋白质- 扰动传感器，以缩小RNA结合蛋白质组中a-syn特异性扰动的范围，以及 结果发现，P小体的形成增加与a-syn介导的毒性有关。P形体 帕金森病患者来源的神经元中的形成是保守的。这不是一般的应激反应。例如，它是 与典型的“应激颗粒”反应截然不同，这种反应发生在其他蛋白质错误折叠的背景下 事件。此外，我们的初步数据强烈表明，P-小体的形成与a-syn生物学直接相关 因为我们发现a-syn与P小体的中枢成分有物理上的相互作用，即去顶 蛋白质及相关因子。现在，我们已经优化了一种使用基于点击化学的代谢的方法 标记法定量测定信使核糖核酸降解速率。在技术上，我们因此做好了充分的准备 研究a-syn聚集和错位导致mRNA代谢紊乱的机制。 我们现在建议在与疾病相关的细胞中检测a-syn-p小体的相互作用和mRNA的降解。 模型，特别是来自携带a-synA53T突变和a-synn-野生型的患者的神经元 三倍增长。我们已经对这些IPSC株系进行了彻底的鉴定，并产生了已纠正的等基因突变 控制。最后，我们将调查PD/DLB患者的死后脑组织中是否存在水平升高或 错误定位P小体以确认与疾病的相关性。我们提供了一个路线图来检验我们的假设 通过在细胞和体内模型中的遗传分析来展望未来。我们相信P-P之间的新联系- Body和a-syn将开始新的途径来了解a-syn毒性的病理后果， 并有可能为帕金森氏症和其他联核病提供新的治疗选择。