Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment

Abstract

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Keywords: Parkinson’s disease; alpha-synuclein; diglyceride; inclusions; lipid droplets; oleic acid; stearoyl-CoA-desaturase; synucleinopathy; tetramer; triglyceride; unsaturated fatty acid.

Fig 1–. αS expression alters lipid metabolism in yeast. LDs protect against αS toxicity in yeast

A) Lipid profiles of vector and human αS expression in yeast 12 h post induction. Lipid species (116) indicated by color and in the order of the key. B) Primary pathway for LD formation. DG and TG metabolic pathways are highly conserved between mammals (enzymes-red) and yeast (enzymes-blue). ACC1, cytosolic acetyl-CoA carboxylase; ATGL, adipose triglyceride lipase; DGAT1, 2: diacylglycerol acyltransferases; DGK, diglyceride kinase (multiple isoforms in mammals); OA, oleic acid; HSL, hormone-sensitive lipase; LCAT, lecithin:cholesterol acyltransferase; Seipin, integral membrane protein; SCD1, stearoyl-coA-desaturase; LPIN, lipid phosphatases; ER, endoplasmic reticulum; LD, lipid droplet. Whether lipolysis-derived DG enters the ER is currently unknown. C) αS expression increases LD formation in yeast. Green: BODIPY (LDs). Bar chart: integrated density (ImageJ) fold difference of uninduced vs induced (12h post induction). n = 22/condition. p<0.0005, t-test. D) LDs (TG) protect against αS toxicity. Differences in αS toxicity in 4 different strain backgrounds: (i) wt; (ii) dga1Δ lro1Δ; (iii) are1Δ are2Δ; (iv) LDΔ = dga1Δ lro1Δ are1Δ are2Δ. Samples induced at 5 nM estradiol. Table S2: statistical analysis of all yeast growth curves.

Fig 2–. DG accumulation induced by αS expression is toxic and causes a trafficking defect

A) Neutral lipid profiles of human αS expression in wt and dga1Δ lro1Δ yeast 6 h post induction. B) αS-induced ER accumulation of CPY is exacerbated in dga1Δ lro1Δ vs wt. CPY Immunoblot (ImageJ quantified). n=3. p=0.01, t-test. C) Deletion of lipases TGL3 and TGL4 rescues αS toxicity. D) Neutral lipid profiles of human αS expression in wt and tgl3Δ tgl4Δ yeast 12 h post induction. E) αS-induced ER accumulation of CPY is reduced in the tgl3Δ tgl4Δ strain (ImageJ quantified). n=3.p=0.007,t-test. F) Choline addition rescues αS toxicity. G) αS-induced ER accumulation of CPY is alleviated upon 0.5 mM choline addition (ImageJ quantified). n=3. p=0.002, t-test.

Fig 3-. OA exacerbates αS toxicity

A) OA is increased upon αS expression. Intracellular FA analysis of αS-expressing wt yeast 12 h post induction. ** p<0.005; ****p<0.0001 (one way Anova). B) The αS-associated OA phenotype is more pronounced in dga1Δ lro1Δ vs. wt. Intracellular FA analysis of αS-expressing wt yeast 6 h post induction. ****p<0.0001 (one way Anova).C) The αS-associated OA phenotype is reduced in a tgl3Δ tgl4Δ strain vs. wt. Intracellular FA analysis of αS-expressing wt yeast 12 h post induction. ****p<0.0001 (one way Anova). D) Treatment with exogenous OA enhances αS toxicity. E) Dampening of OLE1 expression suppresses αS toxicity. F) Dampening OLE1 expression mitigates the αS-induced neutral lipid profile phenotype 12 h post induction. G) αS-induced ER accumulation of CPY is ameliorated in an OLE1 damp strain. (ImageJ quantified). n=3.p=0.0018, t-test.

Fig 4-. αS expression alters lipid metabolism in rat and C. elegans synucleinopathy models

A) Lipid profiles of human αS expression in rat cortical neurons. Lipid species (516) are indicated by color and in the order of the key on the right of the map. B) αS expression increases LD formation in rat cortical neurons. Microscopy-Green: BODIPY (LDs); Red: αS. Blue: Hoechst (nucleus). Neurons were imaged at 14d (ImageJ quantified). Bar chart: integrated density signal fold difference for MOI1 vs MOI5. n=16 cells. p<0.0001,t-test. C) LDs protect against αS toxicity in rat cortical neurons. Neuronal survival was measured following expression of αS in control rat cortical neurons and in neurons with knockdown of DGAT1 and DGAT2 (D1+D2). Fig S3E, RTPCR knockdown data. % Viability (Resazurin to Resorufin conversion). n=6. ****p<0.0001 *p=0.02 (one way Anova). D) Reduction in LPIN expression suppresses αS toxicity in rat cortical neurons. Neuronal survival was measured following expression of αS in control rat cortical neurons and in neurons with knockdown of LPIN1, LPIN2, LPIN3. Fig S3F, RTPCR knockdown data. %Viability (Resazurin to Resorufin conversion). n=6 ****p<0.0001 **p=0.007 (one way Anova). E) OA content is increased upon human αs expression in rat cortical neurons. Intracellular FA analysis was performed in control and human αS expressing rat cortical neurons. *p=0.02, t-test. F) Reduction in SCD1 rescues αS toxicity. Neuronal survival was measured following expression of human αS in control rat cortical neurons and in neurons with SCD1 knockdown. Fig S4E, RT-PCR knockdown data. % Viability (Resazurin to Resorufin conversion). n=6. ****p<0.0001 (one way Anova). G) Inhibition of SCD1 rescues αS toxicity (% ATP). Survival of neurons was measured following treatment with SCD1 inhibitor in control and human αS-expressing rat cortical neurons. ****p<0.0001 ***p≤0.0005 (one way Anova). H) SCD1 inhibition rescues DGAT1+DGAT2+αS-associated toxicity in rat cortical neurons. DGAT1 and DGAT2 (D1+D2) were knocked down in control vs human αS-expressing rat cortical neurons + DMSO or SCD inhibitor. %Viability (Resazurin to Resorufin conversion). n=6. ***p<0.0005 (one way Anova). I) SCD knockdown in a C. elegans model of dopaminergic neuron degeneration rescued an αS-induced dopaminergic neuron degeneration phenotype. Open arrowheads-CEP dendrites (white), ADE dendrites (black);closed arrowheads-CEP cell bodies (white), ADE cell bodies (black); *p<0.05.

Fig 5–. αS-associated lipid metabolism phenotypes in PD-relevant human neuron models

A)Lipid profiles of αS overexpression in human iPSCs-derived neurons. Lipid species indicated by color and in the order of the key. B)αS expression increases LD formation in human iPSC-derived neurons. Microscopy-Green:BODIPY(LDs); Blue:Hoechst (nucleus);Red:αS (ImageJ quantified). Bar chart:fold difference in integrated density signal MOI1 vs MOI5. n=7 cells.p<0.0005,t-test. C)OA is increased upon αS overexpression in human iPS neurons. Intracellular FA analysis of αS overexpressing human iPSC-derived neurons.**p0.008, t-test. D)Treatment with exogenous OA enhances αS toxicity in human iPS neurons. Treatment of αS-expressing human iPS neurons expressing vector or αS with 1μM OA, ST (stearic), POA (palmitoleic), PA (palmitic). % Viability (Resazurin to Resorufin conversion). n=6. ***p<0.0005, **p<0.01 (one way Anova). E) Neutral lipid profiles of patient triplication and isogenic corrected neurons identify increased DG in the triplication line. Lipid species indicated by color and in the order of the key. Lipid profiling performed 23 d after differentiation to neurons. F) Neutral lipid profiles of human neurons, wt vs. E46K αS. Lipid species indicated by color and in the order of the key. Lipid profiling performed 36 d after differentiation to neurons. WB confirmed an equal amount of wt and E46K αS.

Fig 6-. Expression of fPD αS E46K alters brain FA composition in mice displaying motor deficits

12 mo male WT and E46K αS mouse cortices were analyzed: A) UFAs; B) DG; C) TG levels; n=3; N=2 independent expts. Evaluation of motor behavior: D) quantification of time to descend a pole; n=6; E) endurance in wire hanging; n=5–6. F) WB shows equal αS expression. * p<0.02 **p<0.01,t-test.

Fig 7–. OA impacts αS inclusion formation, tetramer:monomer ratio and pS129

A) Exogenous OA increases αS inclusions in αS3K-expressing neuroblastoma cells. *p<0.05 ****p<0.0001 (one way Anova). B) SCD1 knockdown decreases αS inclusions in αS3K-expressing neuroblastoma cells. C: control (scrambled DsiRNA). ***p<0.0005; ****p<0.0001. C) SCD inhibition decreases αS inclusions. *p<0.05 ****p<0.0001 (one way Anova). D) SCD inhibition decreases αS inclusions (microscopy) in αS3K-expressing neuroblastoma cells. E&F) SCD1 inhibition increases 60kDa αS:14kDa αS in αS3K-expressing neuroblastoma cells. *non-specific band (Perrin et al., 2003).*p<0.05 **p<0.01. G) SCD inhibition decreases pS129 αS: total αS. n=6. *p<0.05 ***p<0.005,t-test. H) SCD1 inhibition increases 60kDa αS:14kDa αS and 80kDa αS: 14kDa αS in fPD E46K-expressing neuroblastoma cells. N=2, n=6. I) SCD1 inhibition decreases pS129 αS: total αS in E46K-expressing cells. N=2, n=10. **p<0.01, t-test.