Fig 1: mTOR inhibition improves autophagic clearance in GBA1 mutant PD neurons. (A) Representative immunofluorescence images of WT control and GBA1 mutant NPCs labeled with an antibody to LAMP1. Also shown is nuclear DAPI. Scale bar = 25 µm. Bar graph represent mean LAMP1 fluorescence signal intensity. Data were collected from >30 cells per group, assayed in 3 different fields in a representative experiment. Data represent average ± SEM, *p = 0.04 (WT vs. PD1) and *p = 0.02 (WT vs. PD2) as assessed by One-way ANOVA. (B) Representative immunofluorescence images of WT control and GBA1 mutant neurons co-labeled with anti-LAMP1 and anti-TH. Last panel is the overlay of the two markers. Arrows point to LAMP1-labeled puncti in TH expressing neurons. Scale bar = 25 µm. Bar graph represent mean LAMP1 fluorescence signal intensity in TH positive neurons. Data were collected from >30 cells per group, assayed in 2–3 different fields in 3 independent experiments. Data represent average ± SEM, **p = 0.007 between the indicated groups as assessed by Student’s t-test. (C) Western blot analysis for LAMP1 levels in WT control, GBA1 mutant, and gene-corrected PD DNCs. Also shown is ß-Actin loading control. Bar graph represents fold LAMP1 relative to control, Data represent average ± SEM, n = 3–4 per group. *p = 0.01, ***p = 0.0002 and ****p < 0.0001 between the indicated groups as assessed by One-way ANOVA. (D) Western blot analysis for Galectin3 (Gal3) levels in WT control, GBA1 mutant, and gene-corrected PD DNCs. Also shown is ß-Actin loading control. Bar graph represents fold Gal3 relative to control. Data represent average ± SEM, n = 3–4 per group. *p = 0.01, **p = 0.001 (WT vs. PD2), and **p = 0.004 (PD2 vs. gene-corrected) as assessed by One-way ANOVA. (E) Western blot analysis for LC3II levels in WT control, GBA1 mutant, and gene-corrected PD DNCs. Also shown is ß-Actin loading control. Bar graph represents fold LC3II relative to control. Data represent average ± SEM, n = 3–4 per group. **p = 0.005 (WT vs. PD1), **p = 0.009 (WT vs. PD2), and **p = 0.002 (PD2 vs. gene-corrected) as assessed by One-way ANOVA. (F) Western blot analysis for NBR1 and P62 levels in WT control, GBA1 mutant, and gene-corrected PD DNCs. Also shown is ß-Actin loading control. Bar graph represents folds relative to control. Data represent average ± SEM, n = 3–6 per group. For NBR1, ****p<0.000 (WT vs. PD1) and *p=0.02 (WT vs. PD2). For P62, *p = 0.03 (PD2 vs. gene-corrected), **p = 0.009 and (WT vs. PD2), **p = 0.003 and (WT vs. PD1) as assessed by One-way ANOVA. (G) Western blot analysis for LC3II levels in WT control and GBA1 mutant PD DNCs. Also shown is ß-Actin loading control. Cells were either untreated or treated with 200 nM Torin1 for 18 h. Bar graph represents fold LC3II relative to untreated control. Data represent average ± SEM, n = 3–4 per group. **p = 0.002 between the indicated groups as assessed by One-way ANOVA. (H) Western blot analysis for P62 levels in WT control, GBA1 mutant, and gene-corrected PD DNCs. Also shown is ß-Actin loading control. Cells were either untreated or treated with 200 nM Torin1 for 18 h. Bar graph represents folds relative to untreated control. Data represent average ± SEM, n = 3–4 per group. **p = 0.008 (WT vs. PD1) and *p = 0.01 (WT vs. PD2) as assessed by One-way ANOVA. (I) Western blot analysis for autophagic flux in WT control, and GBA1 mutant PD DNCs. Cells was either untreated or treated with Torin1 or Torin1 plus Chloroquine (chlq) for18 h as indicated. Bar graph represents quantification of LC3 flux normalized to actin. Data represent average (from PD1-GBAWT/N370S and PD2-GBAWT/N370S combined) ± SEM, n = 3–6 per group. *p = 0.02 between the indicated groups as assessed by One-way ANOVA.