Querying the Interactome of Alpha-Synuclein During Pathological Processes

Mayo Clinic Jacksonville, Jacksonville FL

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Alpha-Synuclein (aSyn) is an intracellular neuronal protein which is implicated in a variety of neurodegenerative diseases including Lewy body (LB) dementias (consisting of Dementia with Lewy Bodies and Parkinson’s disease dementia), Parkinson’s disease, and Multiple Systems Atrophy. Endogenous healthy aSyn is involved in synaptic function; in pathogenic states, aSyn is believed to misfold, accumulate into oligomers and fibrils, and ultimately become part of large insoluble aggregates such as Lewy bodies and Lewy neurites. It is still unknown why aSyn aggregates begin to form. It is also unclear whether pathogenic aSyn exists in primarily monomeric or multimeric form, with the field currently believing that oligomeric aSyn may be toxic. Thus, there is great need to better understand cellular mechanisms involved in aSyn aggregation in disease pathology. The proposed study focuses on understanding the protein interactors of alpha-Synuclein during early aggregate formation and propagation. Protein-protein interactions and related mechanisms identified in this study have potential to facilitate biomarker development for early LB dementia diagnosis and/or promote new candidates for successful disease-modifying therapies. I will employ an in vitro proximity-dependent biotin identification (BioID) system to characterize the aggregate interactome of aSyn. This model is an unbiased antibody-free method for tagging proteins of interest; a mutant R118G BirA* biotin ligase enzyme will be fused to aSyn. Upon exogenous addition of biotin to in vitro assays, proteins within a 10nm radius will be biotinylated. Proteomic analysis of enriched biotinylated peptides will identify potential interactors. Aim 1 will characterize intracellular changes in the aSyn interactome in seeded and non-seeded states. Aim 2 will uncover proteins involved in aSyn secretion and propagation in seeded and non-seeded states. Validation of potential interactors will be performed using in vitro models such as split VenusYFP, split luciferase, siRNA knockdown, and co- immunoprecipitation assays. Analysis of post-mortem LB dementia patient tissue will investigate the presence of a protein target in late-stage LBs – suggesting true involvement of a protein target in pathological processes. Novelty of the proposed project comes from (1) the application of split BirA* fragments fused to aSyn to identify interactors of multimeric aSyn and (2) the use of LB dementia patient-derived pathogenic seeds to increase physiological relevance of this study. The proposed experiments will be carried out at Mayo Clinic, a research institution with state-of-the-art lab facilities and expert scientists leading advancements in neurodegeneration. With these resources, the long-term objective of this project is to reduce the burden of synucleinopathies for patients and their caregivers.