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

Abstract

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. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS 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 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. 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. 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. 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. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.

Rapid Alpha-Synuclein Toxicity in a Neural Cell Model and Its Rescue by a Stearoyl-CoA Desaturase Inhibitor

“…This indicates that fibrillar LB formation may be a later event in pathogenesis and more subtle changes in αS biology such as excess binding to (vesicle) membranes may be sufficient to cause marked damage to neurons. The inclusion phenotype of αS 3K, which can easily be tracked in live cells by yellow fluorescent protein (YFP)-tagging, has recently been used for phenotypic screening of compounds [12]. Inhibitors of stearoyl-CoA desaturase (SCD) were identified as suppressors of αS 3K inclusion formation, in line with independent reports on the benefits of SCD inhibition on αS-related phenotypes in a variety of yeast, cellular, and animal models [13,14].…”

“…In this system, we tested published modifiers of αS biology: trifluoperazine (TFP) [16], nortriptyline (NOR) [17], and a variety of SCD inhibitors [12][13][14]. As a negative control, we employed tafamidis, a chemical stabilizer of assembly of the unrelated protein transthyretin [18].…”

Soluble endogenous oligomeric α-synuclein species in neurodegenerative diseases: Expression, spreading, and cross-talk

“…Given the complexity of synucleinopathies, multi-therapy that targets multiple proteins at their oligomeric states as well as altered biological pathways might turn out to be superior to monotherapies. Lastly, immunotherapies may be combined with modifiers of intracellular ␣Syn dynamics [232,233] or lipid pathways that affect ␣Syn biology [234][235][236].…”

Interaction of Alpha-Synuclein With Lipids: Mitochondrial Cardiolipin as a Critical Player in the Pathogenesis of Parkinson’s Disease

“…These data point to increased PUFA levels in whole cortical tissues and reduced levels in lipid rafts. The relationship between MUFAs and α-Syn is underlined by recent studies showing that the inhibition of stearoyl-CoA desaturase (SCD), a key enzyme in MUFA biosynthesis, is able to reduce α-Syn cytotoxicity by increasing physiological α-Syn multimerization and solubility and decreasing α-Syn phosphorylation on Ser129-P ( Vincent et al, 2018 ; Fanning et al, 2019 ; Imberdis et al, 2019 ). Levels of MUFAs, specifically oleic acid levels, were found to be increased in α-Syn overexpression models, and excess oleic acid led to the formation of α-Syn inclusions ( Fanning et al, 2019 ).…”