c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder

Abstract

The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB. Here, we show that under pharmacological and genetic c-Abl inhibition, TFEB translocates into the nucleus promoting the expression of its target genes independently of its well-known regulator, mammalian target of rapamycin complex 1. Active c-Abl induces TFEB phosphorylation on tyrosine and the inhibition of this kinase promotes lysosomal biogenesis, autophagy, and exocytosis. c-Abl inhibition in Niemann-Pick type C (NPC) models, a neurodegenerative disease characterized by cholesterol accumulation in lysosomes, promotes a cholesterol-lowering effect in a TFEB-dependent manner. Thus, c-Abl is a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models.

Keywords: Biological Sciences; Cell Biology; Molecular Biology.

Graphical abstract

Figure 1

c-Abl Inhibition Increases TFEB Nuclear Translocation and Activity HeLa TFEB-GFP cells were treated with DMSO (control), Torin1 0.3 μM (positive control) and c-Abl inhibitors at different concentrations for 3 h. Then, the cells were fixed and stained with DAPI. (A) Representative images of the TFEB-GFP translocation assay obtained by confocal automated microscopy and. Scale bars, 10μM. (B) graph of the ratio value resulting from the average intensity of nuclear TFEB-GFP fluorescence divided by the average cytosolic intensity of TFEB-GFP fluorescence. Black bars represent Torin1 treatment (positive control). Differences are statistically significant compared to control conditions (DMSO). For each condition, 450–800 cells were analyzed (7 images per sample); n = 4 biological independent samples. (C) Western blot and quantification of TFEB-GFP normalized with histone 3 (H3), in a nuclear/cytoplasmic fractionation of HeLa TFEB-GFP cells treated with imatinib 10μM for 3 h. n = 3 independent experiments. (D) Representative Western blot of endogenous TFEB in a nuclear/cytoplasmic fractionation assay of control human fibroblast treated with imatinib 10μM for 24 hr n = 3 independent experiments. (E) Representative images of endogenous TFEB in HT22 cells treated with imatinib 10 μM for 24 hr n = 3 independent experiments. Scale bars, 10 μM. (F) HeLa TFEB-GFP cells were treated with a scramble siRNA and c-Abl siRNA for 72 hr. Representative images of the TFEB-GFP translocation assay and quantification. For each condition 3,000–5,000 cells were analyzed (16 images per sample) n = 4 biological independent samples, Scale bars, 10 μM. (G) The graph represents q-PCR analysis of c-Abl mRNA levels in HeLa cells treated with the scramble siRNA and siRNA against c-Abl for 24 hr n = 3 independent experiments. (H) The Western blot confirms the reduction of c-Abl protein levels in HeLa cells treated with siRNA against c-Abl for 24 hr n = 3 independent experiments. (I) The graph shows q-PCR analysis of mRNA levels of different TFEB target genes in HeLa cells treated with the scramble siRNA and a siRNA against c-Abl. n = 3 independent experiments. Statistical analysis with one-way ANOVA followed by Tukey's post-test and Student's t-tests when comparing two experimental groups. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001. Data represent mean ± SEM.

Figure 2

c-Abl Inhibition Increases Lysotracker Positive Organelles and Lysosomal Protein Levels (A) Representative Western blot of endogenous TFEB in HeLa cells (control), HeLa TFEB-KO cells (TFEB-KO), and HeLa TFEB-GFP cells (TFEB-GFP). GAPDH was used as loading control. (B) Representative Western blot and quantification of HeLa cells treated with imatinib 10μM for 24 hr using a Lamp1 antibody. n = 3 independent experiments. (C) Quantitative flow cytometry analysis of lysotracker in HeLa cells treated with imatinib 10μM for 24 hr n = 10,000 cells per conditions. (D) Quantitative flow cytometry analysis of lysotracker in the human wild type fibroblasts treated with imatinib 10μM for 24 hr n = 10,000 cells per conditions. (E) Representative immunofluorescence images of lysosomes using Lamp1 antibody in human fibroblast treated with imatinib 10μM for 24 hr, or transfected with a scramble siRNA or a siRNA against c-Abl for 48 hr n = 3 independent experiments. Scale bars, 50 μM. (F) Representative immunofluorescence images of lysosomes attached to the plasma membrane using the antibody Lamp1-DB4 in HeLa cells treated with imatinib 10μM for 24 hr n = 3 independent experiments. Scale bars, 10 μM. (G) H4 cells were treated with DMSO (control), Torin1 0.3 μM (positive control) and c-Abl inhibitor imatinib 10 μM for 3 h. The cells were then fixed and stained with DAPI. Representative confocal microscopy images. Scale bars, 10 μM. (H) Graph of the autophago-lysosome number (spots negative for GFP and positive for RFP). For each condition 450–800 cells were analyzed (19 images per sample). n = 3 biological independent samples. Statistical analysis with one-way ANOVA followed by Tukey's post test and Student's t-tests when comparing two experimental groups. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001. Data represent mean ± SEM.

Figure 3

c-Abl Regulates TFEB Independent of mTORC1 Activity (A) Western blots of endogenous TFEB in HeLa cells treated with imatinib 10μM for 3 h. Torin1 0.3μM and starvation media (STV) for 3 h were used as a positive control. (B) Representative Western blot and quantification of TFEB phosphorylated on S142 normalized against GAPDH in HeLa TFEB-GFP cells treated with imatinib 10μM for 3 h and siRNA c-Abl for 48 hr. STV media for 3 h was used as positive control. n = 3 independent experiments. (C) Representative Western blot and quantification using the 14-3-3 antibody that binds to phosphorylated TFEB on S211. For immunoprecipitated GFP from HeLa TFEB-GFP, cells treated with imatinib 10 μM and Torin1 0.3 μM for 3 h. n = 3 independent experiments. (D) Representative Western blot and quantification of phospho p70-S6K normalized against GAPDH in HeLa cells treated with imatinib and nilotinib 10μM for 3 h. Torin1 0.3μM and STV media treatment for 3 h were used as positive controls. n = 3 independent experiments. Scale bars, 10 μM. (E) Representative confocal microscopy images and quantification of TSC2 KO cells transfected with the TFEB-GFP plasmid. Cells were treated with imatinib 10μM for 3 h and with Torin1 0.3μM for 3 h as a positive control. n = 40 cells per conditions. Scale bars, 10 μM. (F) Representative images and quantification of percentage of nuclear TFEB-GFP in HeLa TFEB-GFP cells synchronized with STV media for 1 h. Then, cells were treated with imatinib 10μM for 1 h and Torin1 0.3 μM for 1 h as a positive control and re-fed with normal media plus imatinib and Torin1 for 2 h. n = 3 independent experiments. Scale bars, 10 μM. Statistical analysis with one-way ANOVA followed by Tukey's post-test. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001. Data represent mean ± SEM.

Figure 4

Active c-Abl Phosphorylates TFEB on Tyrosine (A) Schematic diagram showing that c-Abl-ERT2 under tamoxifen treatment phosphorylates in tyrosine (P-Tyr) its target proteins. (B) Representative Western blot of HeLa TFEB-GFP cells transfected with c-Abl-ERT2 and c-Abl-ERT KD plasmids and treated with Tamoxifen for 8 h. GFP was immunoprecipitated using beads-anti-GFP and then used a anti-phospho-tyrosine antibody. n = 3 independent experiments. (C) Autoradiography of an in vitro phosphorylation assay. TFEB-Flag IP was incubated with human recombinant c-Abl active and ATP-γ-³²P for 0 h, 0.5 hr, 1 h, and 2 h. ATP-γ-³²P incubation for 2 h without recombinant c-Abl active is showed as control (ct). (D) Immunoprecipitated (IP) CRKII was incubated with human recombinant c-Abl active and ATP-γ-³²P for 2 h. (E) Tyrosine 75 (Y75) and Y173 are highly conserved across different species and are included in the c-Abl phosphorylation motif YX_1-5P, which included the tyrosine (Y) and after one to five different amino acids, it recognize a proline (P). (F) Representative confocal microscopy images and quantification of subcellular localization of TFEB-GFP mutants and control plasmids. n = 3 independent experiments. Scale bars, 10 μM. (G) Western blot of HeLa cells transfected with a TFEB-GFP wild type plasmid or with plasmids carrying the Y75 or Y173 mutations. Western blot membranes were incubated with a specific antibody against S211, S138, and S142.

Figure 5

c-Abl Inhibitors Reduce Cholesterol Accumulation in a TFEB-Dependent Manner (A) Representative confocal microscopy images showing cholesterol accumulation. HeLa cells were treated with U18666A 0.5μg/mL and/or imatinib 10μM for 24 hr. Then, GST-PFO (red) immunofluorescence and DAPI (blue) staining were performed. Scale bars, 10 μM. (B) Quantification of cholesterol accumulation from (A). A high-content GST-PFO assay using confocal automated microscopy was performed. n = 3 independent experiments. (C) Representative images and quantification of cholesterol accumulation by filipin staining in HT22 cells and Hepa 1–6 cells treated with U18666A 0.5μg/mL and/or imatinib 10μM for 24 hr n = 3 independent experiments. Scale bars, 10 μM. (D) Representative images and quantification of cholesterol accumulation by filipin staining on hippocampal neurons cultures of 7 DIV from Wild-type (WT) mice and c-Abl KO mice (c-Abl KO) treated with U18666A 0.5μg/mL and/or imatinib 10μM for 24 hr n = 40 neurons per conditions. Scale bars, 10 μM. (E) HeLa cells; HeLa TFEB-GFP cells and HeLa TFEB-KO were treated with U18666A 0.5 μg/mL for 24 hr and cholesterol accumulation was analyzed by the high-content GST-PFO assay. For each condition 450–800 cells were analyzed (7 images per sample) n = 4 biological independent samples. (F) Representative images of cholesterol by GST-PFO and quantification of TFEB-GFP translocation assay in HeLa TFEB-GFP cells treated with U18666A 0.5μg/mL and/or imatinib 10μM for 24 hr. For each condition, 450–800 cells were analyzed (7 images per sample) n = 4 biological independent samples. Scale bars, 10 μM. (G) Representative images of cholesterol accumulation using GST-PFO of HeLa and HeLa TFEB-KO cells treated with U18666A 0.5μg/mL and/or imatinib 10 μM for 24 hr n = 3 independent experiments. Scale bars, 10 μM. (H) Quantification of cholesterol accumulation in (G) n = 3 independent experiments. Statistical analysis with one-way ANOVA followed by Tukey's post-test. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001. Data represent mean ± SEM.

Figure 6

c-Abl Inhibition Reduces Cholesterol Accumulation in NPC1 Human V1165M Fibroblasts (A) Representative Western blot of phospho-c-Abl (p-c-Abl) and phosphor-CRKII (p-CRKII) in wild-type and NPC1 human fibroblasts. GAPDH was used as loading control. n = 3 independent experiments. Scale bars, 10 μM. (B) Representative images and quantification of cholesterol accumulation detected through filipin staining in NPC1 human fibroblast treated with imatinib 10μM and nilotinib 10μM for 24 hr n = 80 cells per conditions. (C) Representative Western blot of endogenous TFEB in a nuclear/cytoplasmic fractionation assay of V1165M fibroblasts of patients with NPC1 treated with imatinib 10μM for 24 hr n = 3 independent experiments. (D) Quantitative analysis of lysotracker in V1165M fibroblasts of patients with NPC1 treated with imatinib and nilotinib with 10μM for 24 hr using flow cytometry. n = 10,000 cells per conditions. (E) q-PCR analysis of different mRNA TFEB target genes in V1165M fibroblasts of patients with NPC1 treated with imatinib and nilotinib 10 μM for 24 hr n = 3 independent experiments. (F) Representative immunofluorescence images of Lamp1 in V1165M fibroblasts of patients with NPC1 treated with imatinib 10μM for 24 hr n = 3 independent experiments. Scale bars, 50 μM. (G) Representative images of cholesterol accumulation in NPC1 human fibroblasts treated with imatinib 10 μM or nilotinib 10 μM for 24 hr. Scale bars, 50 μM. (H) Quantification of cholesterol accumulation by high-content GST-PFO assay in NPC1 human fibroblasts treated with imatinib or nilotinib at different concentrations for 24 hr. For each condition 450–800 cells were analyzed (7 images per sample). n = 4 biological independent samples. (I) Flow cytometry graphs of GST-PFO (cholesterol) in NPC1 human fibroblasts treated with imatinib or nilotinib 10μM for 24 hr (J) Quantitative analysis by flow cytometry of GST-PFO (cholesterol) in (I) n = 10,000 cells per conditions. Statistical analysis with one-way ANOVA followed by Tukey's post-test and Student's t-tests when comparing two experimental groups. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001. Data represent mean ± SEM.

Figure 7

c-Abl Deficiency Reduces Cholesterol Accumulation in NPC In Vivo Models (A) Representative immunofluorescence showing cholesterol by GST-PFO staining (red) in cerebellum sections from 8-month-old wild-type (WT) and NPC mice treated with vehicle or imatinib 12.5 mg/Kg, every day for 2 weeks by intraperitoneal injection. Arrows show purkinje neurons. Scale bars, 50 μM. (B) Quantification of cholesterol accumulation per Purkinje cell area in (A) (n = 2) (5 images per animal). (C–E) WT and NPC mice were treated for 4 weeks with vehicle and GNF-2 (5 mg/kg in 40% water, 30% polyethylene glycol 300 and 30% propylene glycol) starting at p28. Motor coordination was assessed weekly by beam test (C), weight was registered during the treatment (D) and (E) Cerebella from vehicle and GNF-2 treated female NPC mice were analyzed at 8 weeks of age for calbindin by immunohistochemistry. Quantification of Purkinje cell area/Purkinje cell layer (PCL) area is shown (n = 9). Scale bars, 100 μM. (F) WT and NPC mice were treated with GNF-2 (5 mg/Kg) for three weeks starting at p28. Upper panels show filipin staining of cerebellum sections and lower panels show filipin staining of liver sections. n = 3 independent experiments. Scale bars, 50 μM. (G) Representative immunofluorescence showing cholesterol by GST-PFO (red) and endogenous TFEB (green) in cerebellum sections from 8-month-old wild-type (WT) and c-Abl KO mice treated with vehicle or U18666A 10 mg/Kg, for 2 days by intraperitoneal injections. Arrows show purkinje neurons. Scale bars, 50 μM. Statistical analysis with one-way ANOVA followed by Tukey's post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data represent mean ± SEM.