A central question in the cell biology relevant to Parkinson's disease (PD) is the roles for normal and pathogenic forms of alpha-synuclein protein in neurons and neurodegeneration. Recent evidence showed that alpha-synuclein is phosphorylated in Lewy body, a hallmark of pathogenesis of PD, and phosphorylation of alpha-synuclein is critical in mediating neurotoxicity. However, the mechanisms or cellular pathways that lead to alpha-synuclein phosphorylation, cytotoxicity, and nigral degeneration in PD are not clear. Recent discovery of mutations of LRRK2 which cause familial PD presents an opportunity to identify such cellular mechanisms or pathways. The genetic studies indicated that the mutations of LRRK2 cause the most common autosomal dominant PD to date. Although the patients with LRRK2 mutations all exhibited typical PD, the postmortem studies revealed neuro-pathological heterogeneity as shown in neurodegeneration, formation of Lewy bodies, or neurofibrillary tangles. LRRK2 encodes a large complex protein (280kD) containing several conserved domains such as sequence for Ras GTPase and MAPKKK kinase. Mutations of LRRK2 (such as R1441G) that are associated with PD are located in those domains, and likely cause altered biochemical activities of LRRK2 with deregulation of related cellular pathways. This proposal aims to 1) detect LRRK2-mediated cellular functions or pathways and to test the hypothesis of the phosphorylation of alpha-synuclein by LRRK2 kinase activity; 2) and to investigate the pathogenic mechanisms of familial PD mutation R1441G of LRRK2. To accomplish these goals, we generated transgenic mice producing epitope-tagged LRRK2 wild type or mutant which allows us to isolate LRRK2-associated protein complexes under normal and pathological conditions. These mice provide unique tools to assess the phosphorylation of alpha-synuclein (or other substrates) by LRRK2 kinase activity and to study pathogenesis of PD in vivo. In addition, we developed a specific anti-LRRK2 antibody which allows us to detect the expression and localization of endogenous LRRK2. These important reagents will help us uncover the cellular functions or pathways implicated in neuropathogenesis of PD. It will also reveal roles of LRRK2 in regulating or interacting with other molecular pathways. This proposal will provide valuable information for PD therapeutic design directed towards enzymatic activity of LRRK2. ?
