Functional proteomics of mutant LRRK2 induced Parkinson's Disease

突变体 LRRK2 诱导帕金森病的功能蛋白质组学

• 批准号: 234087398
• 负责人: Professor Dr. Thomas Gasser
• 金额: --
• 依托单位: Zentrum für Regenerative Therapien Dresden - CRTD
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/234087398?language=en
• 关键词: Functional; proteomics; mutant; LRRK2; induced

LRRK2 mutations are the most common genetic cause of PD. Patients carrying this mutation almost always harbor Lewy bodies, which are primarily composed of alpha-synuclein (aSYN). To better understand the mechanistic link between mutant LRRK2 and PD pathology, we have previously developed an in vitro model of PD pathogenesis using patient-derived induced pluripotent stem cells iPSCs harboring different LRRK2 mutations, and then specifically induced gene-correction of the mutant LRRK2 allele. Preliminary work with these lines demonstrates that iPSC-derived cultures of midbrain dopaminergic (mDA) neurons harboring mutant LRRK2 have increased levels of aSYN and TAU. Here, we propose to expand upon these results by generating a library of iPSCs from a variety of genetic backgrounds with specific genetic modifications as well as isogenic controls using our previously demonstrated gene-targeting technology. Each of these iPSC lines will be analyzed for PD-relevant phenotypes (aSYN and TAU processing, vesicle dynamics, and cytoskeletal aberrations) using protocols that have been previously established and validated. Further, we propose to use state-of-the-art proteomics technologies to assess the effects of each of these variants upon the phospho-proteome and identify LRRK2 phosphorylation targets specifically induced by PD-associated mutations. We will use the above-described assays in combination with siRNA and overexpression to test if the proposed targets of LRRK2 are necessary and/or sufficient for the expression of PD-related phenotypes.

LRRK2突变是帕金森病最常见的遗传原因。携带这种突变的患者几乎总是携带路易小体，其主要由α -突触核蛋白（aSYN）组成。为了更好地了解突变体LRRK2与PD病理之间的机制联系，我们先前利用含有不同LRRK2突变的患者来源的诱导多能干细胞iPSCs建立了PD发病机制的体外模型，然后特异性诱导突变体LRRK2等位基因的基因纠正。这些细胞系的初步研究表明，ipsc衍生的含有突变体LRRK2的中脑多巴胺能（mDA）神经元培养物增加了aSYN和TAU的水平。在这里，我们建议通过使用我们先前演示的基因靶向技术，通过具有特定遗传修饰和等基因控制的各种遗传背景生成iPSCs库，从而扩展这些结果。每个iPSC系将使用先前建立和验证的方案分析pd相关表型（aSYN和TAU加工，囊泡动力学和细胞骨架畸变）。此外，我们建议使用最先进的蛋白质组学技术来评估每种变体对磷酸化蛋白质组的影响，并确定pd相关突变特异性诱导的LRRK2磷酸化靶点。我们将使用上述分析结合siRNA和过表达来测试LRRK2的目标对于pd相关表型的表达是否必要和/或充分。