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.

项目概要 目前尚无任何措施可以预防或减缓帕金森病 (PD) 和痴呆症的病程 路易体 (DLB)。这些常见且具有破坏性的“突触核蛋白病”的特点是 与囊泡膜相关蛋白 α-突触核蛋白 (a-synuclein) 聚集相关的神经变性 同步）。 a-syn 基因位点的显性点突变、倍增和常见风险变异会导致或 增加患 PD 和 DLB 的风险，明确地将这种蛋白质与疾病病因联系起来。这引起了人们的兴趣 在针对 a-syn 毒性的疗法中，但这种毒性仍然知之甚少。多年来，我们集团和 其他人开发了几种不同的 a-syn 毒性细胞模型来解决这种毒性的本质， 包括 PD 患者来源的神经元。我们绘制了 a-syn 的遗传和物理相互作用图。他们 不出所料，我们注意到囊泡运输，但出乎意料的是，mRNA 代谢作为一种新的代谢途径出现了。 与a-syn生物学相关的途径。在这里，我们建议 a-syn 直接链接到处理体（P-bodies）， 细胞质无膜细胞器参与 mRNA 降解和储存。我们使用了一种新型蛋白质—— 扰动传感器缩小 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-体形成的增加与a-syn介导的毒性有关。 P体 PD患者来源的神经元的形成是保守的。这不是一般的压力反应。例如，它是 与其他蛋白质错误折叠背景下发生的典型“应激颗粒”反应截然不同 事件。此外，我们的初步数据强烈表明 P-body 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，即脱帽 蛋白质和相关因子。现在，我们优化了一种使用基于 CLICK 化学的代谢方法 标记以定量测量 mRNA 降解率。从技术上讲，我们已做好充分准备 研究 a-syn 聚集和错误定位导致 mRNA 代谢紊乱的机制。 我们现在建议测定疾病相关细胞中的 a-syn-P-body 相互作用和 mRNA 降解 模型，特别是来自携带 a-synA53T 突变和 a-syn 野生型的患者的神经元 三倍。我们已经彻底表征了这些 iPSC 系并生成了同基因突变校正的 控制。最后，我们将调查 PD/DLB 患者死后脑组织的水平是否升高或 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检验我们的假设 通过细胞和体内模型的遗传分析来预测未来。我们相信 P- 之间的新颖联系 身体和a-syn将为了解a-syn毒性的病理后果开辟新途径， 以及 PD 和其他突触核蛋白病的潜在新治疗选择。