Splice Isoform And Pharmacological Studies Reveal That Sterol Depletion Relocalizes Alpha-Synuclein And Enhances Its Toxicity

Synucleinopathies are neurodegenerative diseases associated with toxicity of the lipid-binding protein alpha-synuclein (alpha-syn). When expressed in yeast, alpha-syn associates with membranes at the endoplasmic reticulum and traffics with vesicles out to the plasma membrane. At higher levels it elicits a number of phenotypes, including blocking vesicle trafficking. The expression of alpha-syn splice isoforms varies with disease, but how these isoforms affect protein function is unknown. We investigated two of the most abundant isoforms, resulting in deletion of exon four (alpha-syn Delta 4) or exon six (alpha-syn Delta 6). alpha-syn Delta 4, missing part of the lipid-binding domain, had reduced toxicity and membrane binding. alpha-syn Delta 6, missing part of the protein-protein interaction domain, had reduced toxicity but no reduction in membrane binding. To compare the mechanism by which the splice isoforms exert toxicity, equally toxic strains were probed with genetic modifiers of alpha-syn-induced toxicity. Most modifiers equally altered the toxicity induced by the splice isoforms and full-length alpha-syn (alpha-synFL). However, the splice isoform strains responded differently to a sterol-binding protein, leading us to examine the effect of sterols on alpha-syn-induced toxicity. Upon inhibition of sterol synthesis, alpha-synFL and alpha-syn Delta 6, but not alpha-syn Delta 4, showed decreased plasma membrane association, increased vesicular association, and increased cellular toxicity. Thus, higher membrane sterol concentrations favor plasma membrane binding of alpha-synFL and alpha-syn Delta 6 and may be protective of synucleinopathy progression. Given the common use of cholesterol-reducing statins and these potential effects on membrane binding proteins, further investigation of how sterol concentration and alpha-syn splice isoforms affect vesicular trafficking in synucleinopathies is warranted.