Functional proteomics of mutant LRRK2 induced Parkinson's Disease

点击翻译按钮获取中文摘要

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.