Article Figures & Data
Figures
- Figure 1.
RNA-Seq identifies SCD as a αSyn 3K neurotoxicity regulated gene in rat cortical neuron cultures. A, Western blotting showing equivalent expression of αSyn WT, αSyn 3K, or αSyn 3K S129 phosphorylation mutant (S129A) in rat cortical neuron cultures on respective AAV transduction. B, Viability assessed by ATP levels (CellTiter-Glo) in rat cortical neuron cultures under respective AAV9 transduction, two-way ANOVA with Dunnett’s multiple test correction. Dose-dependent and time-dependent toxicity analysis in mouse and rat cortical neuron cultures are shown in Extended Data Figure 1-1. C, Principal component analysis of RNA-Seq data from rat cortical neurons with AAV9-EV or AAV9-αSyn 3K (6, 12, and 19 d after AAV9 transduction). D, Relative transcripts per million of SCD1 transcripts in AAV9-EV and AAV9-αSyn 3K samples. E, Relative transcripts per million of endogenous rat SNCA (αSyn) transcripts in AAV9-EV and AAV9-αSyn 3K samples. F, qPCR confirmation of SCD1 transcript suppression from AAV9-αSyn 3K in rat cortical neuron cultures 12 d after AAV9 transduction, two tailed t test. Data displayed as boxplots or as line charts showing error bars with SD; *p < 0.05, **p < 0.01, ***p < 0.001. Extended Data Figure 1-2 displays additional information on statistical tests applied in present study.
- Figure 2.
Inhibition of SCD in αSyn 3K-GFP neuroblastoma model reduces accumulations and cytotoxicity. A, Dose-dependent increase in αSyn 3K-GFP accumulations from 48-h OLA treatment (1–100 μm). B, 48-h induction of αSyn 3K-GFP leads to caspase 3/7 activation and 100 μm OLA treatment further increases it, whereas SCD1 siRNA reduces it (Extended Data Fig. 2-1A) C, 5 nm siRNA knock-down of SCD1 or SCD5 for 48 h decreases the number of cells with accumulations. Please see Extended Data Figure 2-1B–E for further siRNA knock-down characterization. D, mRNA levels of SCD1 and SCD5 in our neuroblastoma model showing SCD5 has ∼80% less expression than SCD1 and that accumulations can increase SCD1 levels. E, Relative fraction of cells with accumulations under increasing concentration of CAY for 48 h with and without 100 μm OLA. F, Relative fraction of cells with accumulations under increasing concentration of MF for 48 h with and without 100 μm OLA. Both prophylactic and therapeutic treatment paradigms (Extended Data Fig. 2-1F) reduce accumulations. G, Relative αSyn 3K-GFP accumulation-induced caspase activity with increasing concentration of CAY or MF with and without 100 μm OLA for 48 h (* denotes comparison between non-OLA and OLA treatments, # denotes respective inhibitor concentration vs DMSO control). Note that OLA does not reestablish the αSyn 3K accumulation-induced caspase activity under SCD inhibition ≥1 μm. H, Representative high-content images of αSyn 3K-GFP neuroblastoma model with induced αSyn 3K accumulations showing OLA-induced increase, SCD inhibitor-induced decrease, and OLA rescue of accumulations from SCD inhibition. Additional αSyn 3K accumulation characterization including comparison to LBs is shown in Extended Data Figure 2-2. One-way or two-way ANOVA run with Dunnett’s multiple test correction, data displayed as boxplots or as line charts showing error bars with SD, *p < 0.05, **p < 0.01, ***p < 0.001. All plots n ≥ 3 independent experiments.
- Figure 3.
High SCD inhibitor concentrations suppress αSyn 3K-GFP mRNA and protein in neuroblastoma model. A, Representative Licor western blot depicting αSyn 3K-GFP, βactin, and SCD1 protein levels from samples treated with SCD inhibitors (0.01 to 10 μM), αSyn 3K-GFP induction and SCD inhibitor treatment was 48 hours before protein isolation. B, Quantification of αSyn 3K protein levels as in A, CAY10566 and MF-438 data was equivalent and merged for this plot. C, mRNA levels of αSyn 3K-GFP at equivalent time points and treatments as in C. Note the drop in αSyn 3K protein and mRNA under SCD inhibition ≥ 1 μM. D, Quantification of SCD1 protein levels under increasing concentrations of SCD inhibitor, note the dose dependent increase. Please see Extended Data Figure 3-1 for further SCD mRNA and protein characterization under inhibitor treatment. One-way ANOVA run with Dunnett’s multiple test correction, all data displayed as boxplots, *** p < 0.001. All plots n ≥ 3 independent experiments.
- Figure 4.
Early primary neuron cultures are sensitive to SCD inhibitor indued toxicity, while late cultures are resistant. A, B, CellTiter-Glo analysis on primary rat cortical neuron cultures after 12-d treatment ± SCD inhibitors (0.01 or 0.1 μm) ± OLA (100 μm) starting at DIV7 (A, early) or DIV18 (B, late). C, Representative high-content images of both early and late cultures as in A, B with OLA, CAY, or both CAY and OLA. Note the SCD inhibitor induced toxicity in early cultures that is rescued by OLA, while there is no toxicity in the late cultures. Two-way ANOVA run with Dunnett’s multiple test correction, all data displayed as boxplots, ***p < 0.001. All plots n ≥ 3 independent experiments.
- Figure 5.
Late primary neuron cultures are protected from αSyn 3K toxicity by SCD inhibition (CAY). A, Schematic of treatment schedule in early and late cultures. Primary rat cortical neuron cultures were treated for 12-d ± SCD inhibitors (0.01 or 0.1 μm) ± OLA (100 μm) + AAV9-EV or AAV9-αSyn 3K starting at DIV7 (early) or DIV18 (late). Neurons and astrocytes were counted by staining with MAP2 and GFAP markers, respectively. B, Neuron counts from early primary cultures. C, Neuron counts from late primary cultures. D, Astrocyte counts from early cultures. E, Astrocyte counts from late cultures. F, Representative high-content images of late cultures as in B, D. Note that late cultures are rescued from αSyn 3K toxicity by SCD inhibition, which can be reversed by OLA treatment. Extended Data Figure 5-1 shows results with MF. Two-way ANOVA run with Dunnett’s multiple test correction, all data displayed boxplots, *p < 0.05, **p < 0.01, ***p < 0.001. All plots n ≥ 3 independent experiments.
- Figure 6.
Late human iPSC neurons are protected from αSyn 3K toxicity by SCD inhibition (CAY) and are less sensitive to SCD inhibition toxicity. A, Schematic of treatment schedule in early and late cultures. B, CellTiter-Glo analysis on human iPSC neuron cultures after 12-d treatment ± CAY (0.01, 0.1, 1, or 10 μm) ± OLA (10 μm) starting at DIV7 (early) or DIV21 (late). C, D, Same as B but only with 0.1 μm CAY and with AAV9-EV or AAV9-αSyn 3K for 12-d in late (C) or early (D) cultures. Note the rescue of viability in late iPSC neuron cultures by 0.1 μm CAY (a concentration non-toxic to late neurons but toxic to early neurons). Extended Data Figure 6-1 shows results with MF, and Extended Data Figure 6-2 shows SCD single-cell expression in human substantia nigra. Two-way ANOVA run with Dunnett’s multiple test correction, all data displayed as boxplots, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. All plots n ≥ 3 independent experiments.
Tables
Rank Gene name log2FC Combined FDR p value 1 Lpcat1 0.84 2.64E-86 2 Rnf145 0.61 5.14E-35 3 Rgs4 0.44 2.02E-17 4 Scd1 −0.67 5.96E-13 5 Fam102b 0.84 8.20E-12 6 Acsbg1 −0.25 1.34E-10 7 Snn 0.45 5.31E-10 8 Psat1 −0.29 1.34E-09 9 Rgcc −0.4 1.07E-08 10 Pcdh8 0.26 2.01E-08 A meta-analysis was performed to identify the top differentially regulated genes at 6, 12, and 19 d after AAV9 transduction time points. These are the top 10 genes from this meta-analysis (see Extended Data Table 1-1 for the full results, and Extended Data Table 1-2 for pathway analysis); p values are corrected for false discovery (for details, see Materials and Methods).
Extended Data Figure 1-1
αSyn WT versus 3K toxicity in rat and mouse cortical neuron cultures. Time (A) and dose (B; MOI of 62.5K, 125K, or 250K)-dependent toxicity measured by CellTiter-Glo in primary rat cortical neuron cultures treated with AAV9-EV or AAV9-αSyn 3K. C, D, Same as A, B except carried out in mouse cortical neuron cultures. Time-dependent plots A, C were done with AAV9 MOI of 250K. Dose-dependent plots B, D were assessed 19 d after AAV9 transduction. Note that AAV9-αSyn 3K transduction is always more toxic than WT or EV control. E, Relative transcripts per million of SCD2 transcripts in AAV9-EV and AAV9-αSyn 3K samples. Data displayed as line charts showing error bars with SD. Download Figure 1-1, TIF file.
Extended Data Figure 1-2
Statistical table. Download Figure 1-2, XLS file.
Extended Data Table 1-1
Meta-analysis RNAseq full data set. Download Table 1-1, XLS file.
Extended Data Table 1-2
Reactome Pathway analysis. Download Table 1-2, XLS file.
Extended Data Figure 2-1
siRNA knock-down of SCD characterization in αSyn 3K-GFP neuroblastoma model. A, Relative caspase activation under 5 nm siRNA knock-down of SCD1 in 3K neuroblastoma model. Note the rescue of αSyn 3K-GFP accumulation induced toxicity and caspase activation with SCD1 knock-down. B, Relative expression of SCD1 under 5 nm siRNA knock-down of SCD1 or SCD5. C, Same as B except SCD5 expression. Note that knock-down of SCD1 increases expression of SCD5. D, Representative Licor Western blotting depicting αSyn 3K-GFP, βactin, and SCD1 protein levels from samples treated as in A–C. siRNA treatment was performed 6 h before induction of αSyn 3K, all assessments were done 48 h after induction. E, Quantification of αSyn 3K-GFP protein levels as depicted in D, note that siRNA knock-down does not alter 3K protein levels. F, Accumulations were induced for 2 d before 1 d of SCD inhibitor (CAY or MF) treatment, n = 3 technical replicates. One-way or two-way ANOVA run with Dunnett’s multiple test correction, all data displayed as boxplots, *p < 0.05, **p < 0.01, ***p < 0.001. All plots n ≥ 3 independent experiments unless indicated otherwise. Download Figure 2-1, TIF file.
Extended Data Figure 2-2
Triton X-100 solubility of αSyn 3K-GFP accumulations. Sequential extraction with 750 mm NaCl (HS), 1% Triton TX-100 (TX soluble), and 1% SDS (TX insoluble) buffer in equal volume of (A) amygdala from control donor and donor with PD and (B) M17 cells overexpressing αSyn 3K GFP under Dox-inducible promoter. Western blotting for αSyn (MJFR1 at 1:1000 for A and 1:50,000 for B) and pSer129 αSyn (EP1536Y at 1:1000 for A and 1:25,000 for B). C, Dot blot analysis probing total extracts of M17 cells overexpressing αSyn 3K GFP (+/–Dox), control and PD extracts with total αSyn (Syn-1 1:1000) and oligomeric/fibrillar αSyn antibody (MJFR 14-6-4-2 1:10,000). D, Representative images of samples from E. E, Relative accumulation levels in 4% PFA fixed neuroblastoma cells with and without 1% Triton X-100 permeabilization, n = 18 technical replicates, Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001. Download Figure 2-2, TIF file.
Extended Data Figure 3-1
SCD1 mRNA compensatory upregulation in neurons and neuroblastoma cells under SCD inhibition. A, Representative Licor Western blotting depicting endogenous SNCA (αSyn), βactin, and SCD1 protein levels from neuroblastoma cells, SCD inhibitor treatment was 48 h before protein isolation. B, Relative SCD1 mRNA levels in neuroblastoma αSyn 3K-GFP model under 0.01 or 0.1 μm SCD inhibition ± 100 μm OLA (48-h treatments). C, Relative SCD1 mRNA levels in primary rat cortical neuron cultures under 0.01 or 0.1 μm SCD inhibition ± 100 μm OLA (12-d treatments starting in DIV7 early cultures). Note that SCD1 mRNA increases under SCD inhibitor in both primary neurons and neuroblastoma cells and that this increase is reversed by OLA. Two-way ANOVA run with Dunnett’s multiple test correction, all data displayed as boxplots, *p < 0.05, **p < 0.01, ***p < 0.001. Download Figure 3-1, TIF file.
Extended Data Figure 5-1
Late primary neuron cultures are protected from αSyn 3K toxicity by SCD inhibition (MF). A, Neuron counts from early primary rat cortical neuron cultures treated with MF (0.01 or 0.1 μm) ± OLA (100 μm) + AAV9-EV or AAV9-αSyn 3K for 12 d. B, Same as A except treatments starting in late cultures. C, Astrocyte counts from early cultures. D, Astrocyte counts from late cultures. Experimental paradigm for A–D is equivalent to Figure 5, with the addition of AAVs and high-content image counting with MAP2 and GFAP markers for neurons and astrocytes, respectively. E, Representative high-content images of late cultures as in B, D. Note that late cultures are rescued from αSyn 3K toxicity by SCD inhibition, which can be reversed by OLA treatment. Two-way ANOVA run with Dunnett’s multiple test correction, all data displayed as boxplots, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All plots n ≥ 3 independent experiments. Download Figure 5-1, TIF file.
Extended Data Figure 6-1
Late human iPSC neurons are protected from αSyn 3K toxicity by SCD inhibition (MF) and are less sensitive to SCD inhibitor toxicity. A, CellTiter-Glo analysis on human iPSC neuron cultures after 12-d treatment ± MF (0.01, 0.1, 1, or 10 μm) ± OLA (10 μm) starting at DIV7 (early) or DIV21 (established). B, C, Same as A but only with 0.1 μm MF and with AAV9-EV or AAV9-αSyn 3K for 12 d in early (B) or late (C) cultures. Note the rescue of viability in late iPSC neuron cultures by 0.1 μm MF (a concentration non-toxic to establish neurons but toxic to early neurons). Two-way ANOVA run with Dunnett’s multiple test correction, all data displayed as boxplot, **p < 0.01, ***p < 0.001. All plots n ≥ 3 independent experiments. Download Figure 6-1, TIF file.
Extended Data Figure 6-2
SCD1 and SCD5 cell type-specific expression in human substantia nigra. A, Relative expression of SCD1 and SCD5 from human substantia nigra single-cell RNA-Seq (Agarwal et al., 2020). Note that the expression of the SCD5 isoform is higher than SCD1 in all but the oligodendrocyte population. Astro, astrocytes; Endo, endothelial cells; MG, microglia; Neuron, neuron; OPC, oligodendrocyte precursor cells; Oligo, oligodendrocyte. Download Figure 6-2, TIF file.
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