Fig 1: Exogenous expression of GBA1 can rescue the lysosomal integrity(A) GD-I fibroblasts (–), and GD-I fibroblasts stably expressing EGFP (mock) and C-terminally HA-tagged GBA1 were treated with LLOMe (0.5 mM), fixed, and stained for Gal-3. Bar, 40 μm. (B) The number of Gal-3-positive puncta was counted. In total, >100 cells per group were analyzed. The presented data represents a typical result from two independent experiments. The graph displays the average number of Gal-3-positive puncta ± SEM from all counted cells. Variance assessment was conducted through a one-way ANOVA, followed by Tukey’s post-hoc analysis for comparisons. **p<0.01, ns, not significant. (C) GD-I fibroblasts stably expressing EGFP (mock) and C-terminally HA-tagged GBA1 were lysed and GBA activity was measured. GBA activity was normalized to the protein concentration of each cell lysate. The presented data a representative of two independent experiments and the graph represents the mean ± SD of triplicate samples. Variance analysis was performed through a one-way ANOVA, followed by Tukey’s post-hoc analysis for comparisons. ****p<0.0001. ns, not significant. (D) GD-I fibroblasts stably expressing EGFP (mock) and C-terminally HA-tagged GBA1 were fixed, permeabilized, and incubated with anti-HA and anti-PDI (marker for the endoplasmic reticulum), anti-Lamp-2, or anti-CD63 antibodies followed by Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-mouse secondary antibodies. Bar, 20 μm. The boxed regions are enlarged and shown on the right side (Bar, 2 μm). (E) Expression levels of HA-tagged GBA1 were analyzed by immunoblotting with anti-HA and anti-β-actin (as an internal control) antibodies.
Fig 2: Gba1 mutant mice develop a progressive motor decline and an insecure gait.(A) Graph quantifies balancing on a 4–40 rpm accelerating rotarod (avg. of 6 trials on 3 consecutive days) at 12 months. (B) Automated gait scans of mouse paw pattern on a horizontal treadmill (Cleversys) at 12 months. Schematic of stepping pattern derived from gait scans display insecure (bradykinetic) gait in Gba1 mutant mice. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001. One-way ANOVA, Tukey’s post hoc test.
Fig 3: 5b SCD inhibition improves the motor performance, lowers the fatty acid desaturation index, and restores striatal DAergic fiber densities and DA level.(A) SCD-inhibitor 5b treatment study of symptomatic (12 months old) L444P and E326K Gba1 versus Ctl mice. All mice were treated either with 15 mg/kg 5b or Plb. Some additional L444P Gba1 mice were treated with 7.5 mg/kg 5b (low dose [LD]; see Supplemental Figure 3). (B) Graph quantifies balancing skill learning on a 4–40 rpm accelerating rotarod. (C) Fatty acid saturation indices in brain cortex of 5b versus Plb treated mice validating efficacy reducing the specific MUFAs (C16:1, C18:1 versus C16:0, C18:0) ratio by 5b treatment (see also Supplemental Figure 1, D and E). Heatmaps show the calculated desaturation index for GBA L444P and E326K Plb and 5b. Note: Planned pairwise comparisons showed a relative increase of FADI C16:1/C16:0 in Plb L444P and E326K versus Ctl. Quantifying FADI C16 and FADI C18 in Gba1 (E326K+L444P) showed a significant decrease in 5b versus Plb. (D) Representative images of TH+ nerve terminals and fibers of Ctl, L444P, and E326K Gba1 mice. Scale bar: 600 μm. (E) Relative TH optical density (total of 12 sections; n = 3–4 mice each cohort) was analyzed in the dorsal striatum. (F) HPLC assay of striatal dopamine measured by HPLC. Data are shown as mean ± SEM. Two-way ANOVA with Bonferroni (C) or Tukey’s (B, E, and F) post hoc tests. Two-tailed, unpaired 2-tailed t test comparing Gba1 (E326K+L444P) Plb versus 5b. *P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001
Fig 4: Monounsaturated fatty acids (C16:1 and C18:1) are increased in GBA1 L444P mutant iPSC neurons and SCD inhibition increases αS T:M ratio of patient-derived GBA1 L444P and E326K mutant iPSC neurons.GBA L444P mutant and isogenic corrected neurons were differentiated (DIV 20) and harvested for FA analysis by gas chromatography. n = 6. (A–C) Total cellular C16:0, C16:1n9, and C16:1n7 of GBA1 L444P mutant neurons and isogenic corrected control neurons were measured by gas chromatography. Data are reported relative to the isogenic corrected line. GraphPad Prism 10, unpaired 2-tailed t test. (D–F) Total cellular C18:0, C18:1n9, C18:1n7 of GBA1 L444P mutant neurons and isogenic corrected control neurons were measured by gas chromatography. Data is reported relative to the isogenic corrected line. Statistical analysis: Graphpad Prism 10, unpaired 2-tailed t test. (G) Heatmap shows the calculated desaturation index for C16:1n9+C16:1n7/C16:0 for GBA L444P mutant neurons versus isogenic corrected control neurons. (H) Heatmap shows the calculated desaturation index for C18:1n9+C18:1n7/C18:0 for GBA L444P mutant neurons versus isogenic corrected control neurons. (G and H) n = 6. (I and L) L444P and E326K neurons were treated with 1 μM 5b or DMSO. Cells were crosslinked using DSG. Cell lysates were immunoblotted to detect and quantify αS14, αS60, and DJ-1 (crosslinking control). (J) L444P: Quantification of αS60:αS14 (T:M) ratio. Two-way ANOVA: statistically significant effects of condition (F1,46 = 18.75, P = 0.0011) and treatment (F1,46 = 21.69, P = 0.0005), Tukey’s’s HSD test, Corr without 5b (Corr–5b) versus L444P–5b, P = 0.0060, L444P–5b versus L444P with 5b (L444P+5b), P = 0.0028; N = 2, n = 12–19). (M) E326K: Quantification of αS60:αS14 (T:M) ratio. (K and N) No statistical differences in DJ-1 (P > 0.05). Data are shown as mean ± SD. Unpaired 2-tailed t test, P = 0.0036; N = 1, n = 8–9. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 5: 5b treatment reduces PK-resistant and vesicle/lipid-rich αS aggregates and normalizes lysosomal clustering and biogenesis.(A and B) Representative images of PK-resistant pS129+ aggregates in the cortex of Plb- and 5b-treated Gba1 mice and quantification (n = 2–3 sections, n = 3–4 mice per cohort). Note only background staining was seen in Ctl mice independent of treatment. (C and D) Confocal microscopy of cortical and midbrain (S. Nigra) region labeled with Plin2 (red) and quantification of puncta sizes in cortex and S. Nigra. (E and F) Midbrain sections triple labeled with pS129 (red), LAMP1 (green), and DAPI (blue) and quantification of LAMP1+pS129 clusters. (G and H) Adjacent sections were additionally stained for lysosomal biogenesis marker TFEB and tyrosine hydroxylase and graphs quantify the relative proportion of dopaminergic neurons displaying nuclear TFEB immunolabeling. Data are shown as mean ± SEM. Two-way ANOVA, Tukey’s post hoc test (B, D, F, and H, left panels). Two-tailed, unpaired 2-tailed t test comparing in Gba1 (E326K+L444P) Plb versus 5b (B, D, F, and H, right panels). *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001. Scale bars: 25 μm.
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