Functional proteomics of mutant LRRK2 induced Parkinson's Disease

LRRK 2突变是PD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体，路易体主要由α-突触核蛋白（aSYN）组成。为了更好地理解突变型LRRK 2和PD病理之间的机制联系，我们之前已经开发了一种PD发病机制的体外模型，该模型使用携带不同LRRK 2突变的患者来源的诱导多能干细胞iPSC，然后特异性诱导突变型LRRK 2等位基因的基因校正。对这些细胞系的初步研究表明，含有突变型LRRK 2的中脑多巴胺能（mDA）神经元的iPSC衍生培养物具有增加的aSYN和TAU水平。在这里，我们建议通过使用我们先前证明的基因靶向技术，从具有特定遗传修饰的各种遗传背景以及同基因对照中生成iPSC文库来扩展这些结果。将使用先前已经建立和验证的方案分析这些iPSC系中的每一个的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这些变体中的每一种对磷酸化蛋白质组的影响，并确定由PD相关突变特异性诱导的LRRK 2磷酸化靶点。我们将使用上述测定与siRNA和过表达的组合来测试所提出的LRRK 2的靶标对于PD相关表型的表达是否是必需的和/或足够的。

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.