Fig 1: Torin1 upregulates TFEB-mediated lysosomal biogenesis in GD iPSC neurons. (A) Representative fluorescence images for control (left) and GD2a (right) neurons expressing TFEB-GFP fusion protein (green). Neurons were either untreated (NT) or treated with 100 nM Torin1 or 200 nM rapamycin for 18 h. Arrows point to the overlap of TFEB-GFP signal with nuclear DAPI (blue). (B) Quantitation of TFEB-GFP nuclear translocation in control and GD2 neurons (data from GD2a and GD2b combined) that were either untreated or treated with Torin1 or rapamycin. Bar graph represents the percentage of neurons with nuclear TFEB-GFP normalized to the number of GFP-expressing neurons in the same field, in three independent experiments. (C) Fluorescence quantitation of the ratio of nuclear/ cytoplasmic TFEB-GFP signal in control and GD2 neurons (data from GD2a and GD2b combined) that were either untreated or treated with Torin1 or rapamycin. Data from >50 cells, assayed in at least four different fields per group in two independent experiments. (D) Representative z-stack fluorescence images of control and GD2a neurons expressing TFEB-GFP fusion protein labeled for LAMP1. Neurons were either untreated or treated with 100 nM Torin1 or 200 nM rapamycin for 18 h. (E) Fluorescence quantitation of the LAMP1 area in control and GD2 neurons (data from GD2a and GD2b combined) that were either untreated or treated with Torin1 or rapamycin. Data from >50 cells, assayed in at least four different fields in three independent experiments. Data are mean±s.e.m. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 (one-way ANOVA between indicated groups). Scale bars: 50 µm in A (magnification 20×); 50 µm in D (magnification 60×).
Fig 2: Increased p-TEFB(Ser142) level in GD iPSC NPCs. (A) Representative western blot showing p-TEFB(Ser142) levels in control and GD2a NPCs that were treated with the proteasome inhibitor Clasto-lactacystin ß-lactone for 18 h. Also shown is ß-actin loading control. (B) Bar graph shows western blot quantitation of fold p-TEFB(Ser142) level in GD2 NPCS (data from GD2a and GD2b combined) relative to control, n=3 per group. (C) Representative immunoprecipitation for control, GD2a, and GD2b NPCs using p-TFEB(Ser142) antibody and probed with anti-ubiquitin antibody. Cells were treated with the proteasome inhibitor for 18 h and either untreated or co-treated with Torin1 for 8 h. ß-Actin loading levels in the input lysates are also shown. (D) Representative western blot (left) showing total TEFB level in control and GD2a NPCs. Cells were treated with the proteasome inhibitor for 18 h and were either untreated or co-treated with Torin1 for 8 h. Bar graph (right) shows western blot quantitation of total TEFB in GD NPCs (GD2a and GD3) relative to untreated control, n=3 per group. (E) Schematic for a proposed mechanism of TFEB dysfunction in neuronopathic GD, in which glycosphingolipid accumulation leads to increased mTORC1 activity. mTORC1 hyperactivation results in increased TFEB phosphorylation, which targets TFEB for proteasomal degradation. mTOR inhibition by Torin1 stabilizes TFEB and allows its nuclear translocation, thus upregulating lysosomal functions. Data are mean±s.e.m. *P<0.05, ***P<0.0005 (Student's t-test).
Fig 3: Decreased TFEB levels in GBA1 mutant PD neurons. (A) Representative immunofluorescence images for WT control and GBA1 mutant PD neurons co-labeled with anti-Tuj1 (red) and anti-TFEB (green) antibodies. Also shown is nuclear DAPI (blue), (images with separate fluorescence channels are provided in Supplementary Figure S3A). Scale bar = 50 µm. Bar graph represents quantitation of TFEB fluorescence signal intensity. Compiled data from >200 cells per group assayed in at least 3 different fields per experiment in 2–5 independent experiments. Error bars = SEM. **p = 0.002 and ****p < 0.0001 between the indicated groups as assessed by One-way ANOVA. (B) Representative immunofluorescence images for WT control, GBA1 mutant, and gene-corrected neurons co-labeled with anti-MAP2 (red) and anti-TFEB (green) antibodies. Also shown is nuclear DAPI (blue). Scale bar = 25 µm. Bar graph represents quantitation of TFEB fluorescence signal intensity. Compiled data from >200 cells per group assayed in at least 3 different fields per experiment in 2–5 independent experiments. Error bars = SEM. *p = 0.01, **p = 0.001, ***p = 0.0002, and ****p < 0.0001 between the indicated groups as assessed by One-way ANOVA. (C) Western blot analysis for TFEB levels in WT control and GBA1 mutant PD DNCs. Also shown is ß-Actin loading control. Bar graph represents fold TFEB relative to control, Data represent average ± SEM, n = 3 per group. *p = 0.01 (WT vs. PD4), *p = 0.02 (WT vs. PD2), and *p = 0.04 (WT vs. PD1) as assessed by Student’s t-test. (D) Western blot analysis for TFEB levels in GBA1 mutant and gene-corrected PD DNCs. Also shown is ß-Actin loading control. Bar graph represents fold TFEB relative to the gene-corrected cells. Data represent average (PD2-GBAWT/N370S and PD3-GBAWT/N370S combined) ± SEM, n = 4 per group. *p = 0.02 as assessed by Student’s t-test. (E) Western blot analysis for total TFEB and pTFEB (Ser211) levels in control and GBA1 mutant PD DNCs. Also shown is ß-Actin loading control. Bar graph represents fold pTFEB (Ser211) relative to control. Data represent average ± SEM, n = 2–3 per group. *p = 0.01 as assessed by Student’s t-test. (F) Western blot analysis for pTFEB (Ser211) levels in WT control, GBA1 mutant, and gene-corrected PD DNCs. Also shown is ß-Actin loading control. Bar graph represents fold pTFEB (Ser211) relative to control. Data represent average ± SEM, n = 2–4 per group. *p = 0.03 (WT vs. mutant) and *p = 0.02 (mutant vs. gene-corrected) as assessed by One-way ANOVA. (G) Western blot analysis for pTFEB (Ser211) levels in 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 fold pTFEB (Ser211) relative to gene-corrected cells. Data represent average ± SEM, n = 2–3 per group. *p = 0.01 between the indicated groups as assessed by One-way ANOVA. (H) Western blot analysis for p-TEFB (Ser142) levels in 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 pTFEB (Ser142) relative to untreated control. Data represent average ± SEM, n = 3 per group. ***p = 0.0007 (WT vs. mutant), and ***p = 0.0003 (mutant vs. mutant with Torin) as assessed by One-way ANOVA.
Fig 4: Decreased nuclear TFEB levels in GBA1 mutant PD neurons. (A) Representative immunofluorescence images for WT control, GBA1 mutant, and gene-corrected PD neurons co-labeled with anti-MAP2 (red) and anti-TFEB (green) antibodies. Also shown is nuclear DAPI (blue). Arrows point to nuclear TFEB fluorescence signal, (images with separate fluorescence channels are provided in Supplementary Figure S4). Scale bar = 25um. (B) Representative immunofluorescence images for gene-corrected and GBA1 mutant neurons that was untreated or treated with 200 nM Torin1 for 18 h. Cells were co-labeled with anti-MAP2 (red) and anti-TFEB (green) antibodies. Also shown is nuclear DAPI (blue). Arrows point to nuclear TFEB signal. (C) Quantitation of nuclear TFEB fluorescence signal intensity in WT control, GBA1 mutant, and gene-corrected neurons. Data were collected from >50 cells per group, assayed in 3 different fields in a representative experiment. Data represent average ± SEM. ****p < 0.0001 between the indicated groups as assessed by One-way ANOVA. (D) Quantitation of nuclear TFEB fluorescence signal intensity in gene-corrected, GBA1 mutant, and Torin-treated GBA1 mutant neurons. Data were collected from >50 cells per group, assayed in 3 different fields in a representative experiment. Data represent average ± SEM. *p = 0.04, **p = 0.008, and ***p = 0.0001 between the indicated groups as assessed by One-way ANOVA.
Fig 5: Decreased TFEB activity in GBA1 mutant PD neurons. Measurement of TFEB activity in: (A) WT control and GBA1 mutant PD DNCs. *p = 0.04 and ***p = 0.0003 between the indicated groups as assessed by Student’s t-test. (B) GBA1 mutant PD iPSCs and the corresponding isogenic gene-corrected line. *p = 0.03, **p = 0.001, and ***p = 0.0002 between the indicated groups as assessed by One-way ANOVA. (C) WT control, GBA1 mutant, and gene-corrected PD DNCs. **p = 0.009 (WT vs. PD2), **p = 0.006 (PD2 vs. PD2 gene-corrected), **p = 0.005 (WT vs. PD4), and **p = 0.001 (PD4 vs. PD4 gene-corrected) as assessed by One-way ANOVA. Cells were either untreated or treated with 200 nM Torin1 for 18 h where indicated. Data represent average fold activity relative to control ± SEM, n = 3–4 per group. (D) qRT-PCR analysis showing expression of selected TFEB target genes in WT control and GBA1 mutant PD DNCs. Data represent fold relative to control ±SEM, n = 3–4 per group. ****p < 0.0001 (TFEB), *p = 0.01 (LAMP), *p = 0.04 (CTSB), ****p<0.0001 (CTSD), ***p = 0.0006 (HEXA), and **p = 0.001 (PSAP), as assessed by Student’s t-test. (E) qRT-PCR analysis showing expression of TFEB target genes in GBA1 mutant and gene-corrected PD DNCs. Data represent fold relative to gene-corrected cells ±SEM in 2–3 independent experiments. *p = 0.03 (MCOLN), *p = 0.02 (CTSD), **p = 0.001 (HEXA), **p = 0.009 (CTSB), ***p = 0.0001 (PSAP), ***p = 0.0004 (LAMP,GBA), and ****p < 0.0001 (TFEB, ATP6, GNS) as assessed by Student’s t-test. (F) qRT-PCR analysis showing fold expression of TFEB target genes in GBA1 mutant and gene-corrected PD NPCs. Data represent fold relative to gene-corrected cells ±SEM in 3–4 independent experiments. **p = 0.001 (ATP6,CTSA),**p = 0.007 (CTSD), **p = 0.009 (MCOLN), **p = 0.004 (LAMP,PSAP),**p = 0.001 (GBA),***p = 0.0003 (TFEB), and ****p < 0.0001 (HEXA, GNS) as assessed by Student’s t-test. (G) qRT-PCR analysis showing expression of TFEB target genes in GBA1 mutant NPCs that were untreated or treated for 18 h with 200 nM Torin1. Data represent fold relative to untreated cells ±SEM in 2–3 independent experiments. *p = 0.04, **p = 0.002 (CTSD), **p = 0.009 (TFEB), ***p = 0.0003 (CLCN7), and ***p = 0.0002 (ATP6, HEXA) as assessed by Student’s t-test.
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