The additional lysine in fPD-linked αS E46K was proposed to stabilize the monomeric, membrane-associated helix of αS ( 16).Ĭurrent therapeutics for synucleinopathies are symptomatic, not disease-modifying. These helices are promoted by hydrophobic aa that interact with the fatty acyl chains of membrane lipids and by lysine residues that interact with negatively charged phospholipid head groups ( 15). αS binding to small vesicles is well-documented in vitro and mediated by the formation of transient αS amphipathic helices at highly curved membranes ( 12– 14). Transient lipid association has been implicated in αS multimer assembly ( 11). Inhibiting glycolipid anabolism via the compound miglustat restores metastable physiological αS multimerization ( 7), which can be trapped in situ by live-cell cross-linking and Western blot (WB) ( 8– 10). GCase functional deficiency leads to αS dyshomeostasis, including a lack of native multimerization. Lipid-related risk genes have emerged from genetic studies, most importantly glucocerebrosidase A ( GBA, GCase), a key enzyme in glycolipid catabolism proposed to form a “bidirectional pathogenic loop” with αS ( 6). Recent data suggest that Lewy-type inclusions are rich in lipids/membranes and thus not principally fibrillar as previously thought ( 5). Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. αS 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. Here, we use the αS “3K” (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. Microscopy of Lewy bodies in Parkinson’s disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material.
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