mTOR hyperactivity mediates lysosomal dysfunction in Gaucher's disease iPSC-neuronal cells

Abstract

Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder. Neuronopathic manifestations in GD include neurodegeneration, which can be severe and rapidly progressive. GBA1 mutations are also the most frequent genetic risk factors for Parkinson's disease. Dysfunction of the autophagy-lysosomal pathway represents a key pathogenic event in GBA1-associated neurodegeneration. Using an induced pluripotent stem cell (iPSC) model of GD, we previously demonstrated that lysosomal alterations in GD neurons are linked to dysfunction of the transcription factor EB (TFEB). TFEB controls the coordinated expression of autophagy and lysosomal genes and is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). To further investigate the mechanism of autophagy-lysosomal pathway dysfunction in neuronopathic GD, we examined mTORC1 kinase activity in GD iPSC neuronal progenitors and differentiated neurons. We found that mTORC1 is hyperactive in GD cells as evidenced by increased phosphorylation of its downstream protein substrates. We also found that pharmacological inhibition of glucosylceramide synthase enzyme reversed mTORC1 hyperactivation, suggesting that increased mTORC1 activity is mediated by the abnormal accumulation of glycosphingolipids in the mutant cells. Treatment with the mTOR inhibitor Torin1 upregulated lysosomal biogenesis and enhanced autophagic clearance in GD neurons, confirming that lysosomal dysfunction is mediated by mTOR hyperactivation. Further analysis demonstrated that increased TFEB phosphorylation by mTORC1 results in decreased TFEB stability in GD cells. Our study uncovers a new mechanism contributing to autophagy-lysosomal pathway dysfunction in GD, and identifies the mTOR complex as a potential therapeutic target for treatment of GBA1-associated neurodegeneration.

Keywords: GBA1 mutations; Lysosomal storage disorder; TFEB; iPSC; mTORC1.

Fig. 1.

Increased mTOR activity in GD iPSC NPCs. (A) Representative immunofluorescence images of control and GD2a NPCs labeled for mTOR (green) or DAPI (blue). Bar graph shows mean mTOR fluorescence signal intensity in control and GD NPCs (GD2a, GD2b and GD3 combined). Data were collected from >100 cells per group, assayed in two different fields per experiment, in two independent experiments. P>0.05 between control and GD NPCs (Student's t-test). (B) Representative immunofluorescence images of control and GD2a NPCs labeled for phospho-mTOR-Ser2448 (p-mTOR) (green) and DAPI (blue). Cells were either untreated (NT) or treated with 100 nM Torin1 for 6 h. Bar graph shows mean p-mTOR fluorescence signal intensity in control and GD NPCs (GD2a and GD2b combined). Data were collected from >30 cells per group, assayed in three different fields in a representative experiment. (C) Representative western blot showing p-mTOR protein levels in control and GD NPCs derived from two GD iPSC lines (GD3 and GD2a). Cells were either untreated or treated with 100 nM Torin1 for 6 h. Also shown is β-actin loading control. Bar graph shows fold p-mTOR in treated relative to untreated control (GD2a, GD2b and GD3 NPCs combined), n=3-5 per group. (D) Representative immunofluorescence images of control and GD2a NPCs labeled for phospho-RPS6 Ser235/236 (p-RPS6). Cells were either untreated, treated with 200 nM rapamycin for 6 h or with 10 nM insulin for 30 min as indicated. (E) Representative immunofluorescence images of control and GD2b NPCs labeled for phospho-4EBP1 Thr37/46 (p-4EBP1). Cells were either untreated, treated with 200 nM rapamycin or 100 nM Torin for 6 h. Bar graph shows mean p-RPS6 and p-4EBP1 fluorescence signal intensity in control and GD NPCs (GD2a, GD2b and GD3 combined). Data were collected from >100 cells per group, assayed in two or three different fields in a representative experiment. (F) Representative western blot showing p-RPS6 protein levels in control and GD2a NPCs with or without Torin1 or insulin treatment. Also shown is β-actin loading control. Bar graph shows fold p-RPS6 (data from GD2a, GD2b and GD3 combined) relative to untreated control, n=3 per group. (G) Representative western blot showing p-4EBP1 levels in control and GD2a NPCs with or without rapamycin treatment. Also shown is β-actin loading control. Bar graph shows fold p-4EBP1 in GD NPCs (data from GD2a and GD2b combined) relative to untreated control, n=3 per group. 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: 100 µm in A,D,E (magnification 20×); 50 µm in B (magnification 60×).

Fig. 2.

Lipid substrate accumulation mediates mTOR hyperactivity in GD iPSC NPCs. (A) Representative immunofluorescence images of control and GD2a NPCs co-labeled for p-RPS6 (green) and DAPI (blue). GD NPCs were either untreated or treated with 5 µM of the substrate reduction compound (SRC) GZ-161 for 72 h. (B) Quantitation of p-RPS6 fluorescence signal intensity in control and GD2a NPCs with and without GZ-161 treatment. Data from >100 cells per group, assayed in at least four different fields per experiment, in three experiments. Bar graph represents fold change in the mean p-RPS6 fluorescence intensity relative to control. (C) Representative western blot showing p-mTOR (left) and p-RPS6 (right) levels in control, GD2a and GD2a NPCs treated with the SRC. Also shown is β-actin loading control. Bar graphs represent fold p-mTOR and p-RPS6 in GD NPCs (data from GD2a and GD2b combined) relative to control, n=3-5 per group. Data are mean±s.e.m.*P<0.05 and **P<0.005 (one-way ANOVA between indicated groups). Scale bar: 50 µm (magnification 20×).

Fig. 3.

Increased mTORC1 activity in GD iPSC neurons. (A) Representative immunofluorescence images of control and GD3 neurons labeled for p-mTOR (red) and DAPI (blue). Bar graph (right) shows p-mTOR mean fluorescence signal intensity in control and GD neurons (GD2a and GD3 combined). Data were collected from >25 cells per group, assayed in two different fields, in a representative experiment. P<0.05 (Student's t-test). (B) Representative western blot showing p-mTOR (top) and mTOR (bottom) protein levels in control and GD2b neurons untreated (NT) or treated with 100 nM Torin1 for 6 h. Also shown is β-actin loading control. Bar graph represents fold p-mTOR in GD2 neurons (data from GD2a and GD2b combined) relative to untreated control, n=3-5 per group. (C) Representative immunofluorescence images of control and GD2b neurons labeled for p-RPS6 and Tuj1. The overlay of both markers is shown in the last panel. Bar graph (below) represents p-RPS6 mean fluorescence signal intensity in control and GD2 neurons (data from GD2a and GD2b combined). Data were collected from >100 cells per group, assayed in two to four different fields, in two experiments. P<0.005 (Student's t-test). (D) Representative western blot showing p-RPS6 (top) and RPS6 (bottom) levels in control and GD2a neurons with or without 100 nM Torin1 treatment for 6 h. Also shown is β-actin loading control. Bar graph represents fold p-RPS6 in GD2 neurons (data from GD2a and GD2b combined) relative to untreated control, n=3 per group. (E) Representative western blot showing p-4EBP1 (top) and 4EBP1 (bottom) protein levels in control and GD2a neurons with or without 100 nM Torin1 treatment for 6 h. Also shown is β-actin loading control. Bar graph represents fold p-4EBP1in GD2 neurons (data from GD2a and GD2b combined) relative to untreated control, n=3-4 per group. Data are mean±s.e.m. *P<0.05, **P<0.005, ****P<0.00005 (one-way ANOVA between indicated groups). Scale bars: 50 µm in A (magnification 60×); 100 µm in C (magnification 20×).

Fig. 4.

Torin1 upregulates lysosomal marker expression in GD iPSC neurons. (A) qRT-PCR analysis showing fold expression of lysosomal genes in control and GD2 neurons (data from GD2a and GD2b combined). GD neurons were untreated (NT) or treated with either 100 nMTorin1 or 200 nM rapamycin for 18 h. The lysosomal genes examined were cathepsin B (CATB), cathepsin D (CATD), GNS, LAMP1, HEXA and GBA, n=3-6 per group. (B) Representative western blot showing LAMP1 protein levels in control and GD2a neurons that were either untreated (NT) or treated with Torin1 or rapamycin. Also shown is β-actin loading control. Bar graph represents fold LAMP1 in control and GD2 neurons (data from GD2a and GD2b combined) relative to untreated control, n=3-4 per group. Data are mean±s.e.m. *P<0.05, **P<0.005, ***P<0.0005 (one-way ANOVA between indicated groups).

Fig. 5.

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. 6.

Torin1 improves autophagic clearance in GD iPSC neurons. (A) Representative fluorescence images for control and GD2b neurons, expressing GFP-LC3 fusion protein. Neurons were either untreated (NT) or treated with 100 nM Torin1 for 18 h. Insets are enlargement of a small area in each panel. Arrows point to GFP-LC3-labeled puncta. (B) Fluorescence quantitation of GFP-LC3 puncta in GD2 neurons (data from GD2a and GD2b combined) with and without Torin1 treatment. The bar graphs show average GFP-LC3 puncta number (left) and average GFP-LC3 puncta fluorescence intensity (right). (C) Representative fluorescence images for control and GD2a neurons, expressing GFP-LC3 fusion protein (green) and LAMP1 (red). Neurons were either untreated or treated with 100 nM Torin1 for 18 h. The merged image of GFP-LC3 puncti and LAMP1-labeled lysosomes is also shown. Insets to the right are enlargement of the boxed areas to show the fluorescence signal colocalization. (D) Quantitation of the percentage colocalization of GFP-LC3 puncti and LAMP1 fluorescence signal in control and GD2 neurons (data from GD2a and GD2b combined) with and without Torin1 treatment for 18 h. Data from >100 cells per group assayed in at least four different fields in two independent experiments. (E) Representative western blot showing p62 and NBR1 protein levels in control and GD2b neurons that were either untreated or treated with 100 µm Torin1 for 18 h. Also shown is β-actin loading control. Bar graphs show fold p62 (left) and NBR1 (right) in GD neurons (Data from GD2a, GD2b and GD3 combined) relative to the untreated control, n=3-4 per group. 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 40×); 25 µm in A (inset); 25 µm in C (magnification 20×); 10 µm in C (inset).

Fig. 7.

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 β-lactone for 18 h. Also shown is β-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. β-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).