Cell models of lipid-rich α-synuclein aggregation validate known modifiers of α-synuclein biology and identify stearoyl-CoA desaturase

Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of alpha-synuclein (alpha S) but also contain vesicles and other membranous material. We previously reported the existence of native alpha S tetramers/multimers and described engineered mutations of the alpha S KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the alpha S 3K (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of alpha S biochemistry and then identify compounds via a medium-throughput automated screen. alpha S 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, L-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the alpha S inclusions: for example, their rapid clearance by certain known modulators of alpha S toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the alpha S inclusions, reduce alpha S 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of alpha S E46K. SCD inhibition restores the E46K alpha S multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type alpha S. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the alpha S phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.