Elucidating the biological differences between distinct fibrillar and non-fibrillar alpha-synuclein inclusions in human stem-cell models

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Project Summary Aggregation of specific proteins within neurons and glia comprise the hallmark pathologies of neurodegenerative diseases (ND) like Alzheimer’s (AD) and Parkinson’s disease (PD). In AD, the aggregating protein is b-amyloid (Ab), and in PD it is a-synuclein (a-syn). Intriguingly, these two pathologies often coexist and the significance of this is unknown. Many cellular defects are detected in the presence of a-syn mutations or overexpression, prominently including defective vesicle trafficking. Outstanding questions remain—how does a-syn impart toxicity, and how does it exert effects on a wide span of cellular pathways? How does stage of the pathology, including the formation of distinct subtypes of a-syn inclusions, alter the vulnerability of the cell? What is the connection between a-syn and the AD-linked protein Ab that is often seen to coexist in the brains of patients with synucleinopathies? We previously conducted a genome-wide CRISPR/Cas9 screen for genetic modifiers of a-syn toxicity in a human cellular model that captures advanced membrane-rich a-syn aggregates highly reminiscent of human postmortem brain pathology in PD. We also conducted a targeted exome sequencing screen in synucleinopathy patients, focusing on known modulators of both a-syn and Ab cytotoxicity. Interestingly, top hits from both of these approaches converged on genes related to actin cytoskeleton regulation. The convergence of hits from these two screens led us to hypothesize that a key aspect of a-syn toxicity relates to altered actin cytoskeletal stabilization and that this may be a key point of convergence of both a-syn and Ab cellular toxicity. Indeed, the actin cytoskeleton orchestrates organization of cellular organelles and substructures and others have postulated its dysregulation may play a role in many of the a-syn- and Ab-mediated cellular defects. Our genetic investigations in human cells and patients now pinpoint a specific set of genes that might mediate this crosstalk between AD and PD pathologies. In this proposal, we aim to (1) examine whether the actin cytoskeleton contributes to toxicity in synucleinopathy iPSC-derived neuron models, (2) conduct a pooled secondary screen in iPSC-derived neuron models and validate genetic modifiers of a-syn toxicity, and (3) examine whether a-syn-mediated actin cytoskeleton defects are modulated by Ab to understand the convergence between a-syn and Ab toxicity. Understanding the molecular and genetic underpinnings of PD, and any possible connection with AD, will ultimately contribute to a better grasp of the disease and help uncover novel therapeutic targets.