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.