TFEB overexpression or pharmacological activation of TFEB ameliorates cholesterol accumulation in U18666A-induced HeLa NPC1 model.

(A) Overexpression of TFEB/ TFEB S211A reduced cellular cholesterol levels in U18666A-induced HeLa NPC1 model. Filipin staining of HeLa transiently transfected by mCherry, mCherry-TFEB and mCherry-TFEB S211A plasmid for 48 h, followed by U18666A (2.5 μM) for 24 h. Overlay phase contrast images are shown together with the red (mCherry-TFEB/TFEB S211A) and white (filipin). In each image the red circles point to the successful transfected cells, green circles represent the untransfected cells, Scale bar, 20 μm. (B) Quantification of cholesterol accumulation from A. N=15 randomly selected cells from n=3 independent experiments. (C) SFN reduces lysosomal (LAMP1) cholesterol accumulation (Filipin) in HeLa NPC1 cells. HeLa cells were exposed to U18666A (2.5 μM) in the absence or the presence of SFN (15 μM) for 24 h. Each panel shows fluorescence images taken by confocal microscopes. The red signal is LAMP1-mCherry driven by stable transfection, and the green signal is Filipin. (D) Each panel shows the fluorescence intensity of a line scan (white line on the blown-up image) through the double labeled object indicated by the white arrow. Scale bar, 20 μm or 2 μm (for zoom-in images). (E) Quantification of cholesterol levels shown in C. N=15 randomly selected cells from n=3 experiments. (F) SFN (15 μM, 24 h) induced TFEB nuclear translocation in HeLa NPC1 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (G) Average ratios of nuclear vs. cytosolic TFEB immunoreactivity shown in F. N=20 randomly selected cells from n=3 experiments. (H) SFN induced mRNA expression of TFEB target genes in HeLa NPC1 model. HeLa cells were cotreated with U18666A (2.5 µM) and SFN (15 μM) for 24 h (n=3). (I) Western blot analysis of TFEB phosphorylation by SFN (15 μM, 24 h) in HeLa NPC1 cells. (J) Quantification of ratios of pS211-, pS142-, pS122- TFEB vs. total TFEB as shown in I. (n=3). For all the panels, average data are presented as mean ± s.e.m.; *** P < 0.001.

SFN promotes cholesterol clearance in various human NPC1 cell models.

(A) Western blot analysis of the KD efficiency of a specific NPC1-targeting siRNA in HeLa cells (n = 3 independent repeats). (B) SFN induced TFEB nuclear translocation in NPC1 KD HeLa cells. Detection of TFEB immunoreactivity in HeLa cells transiently transfected with siNPC1 for 48 h, followed by SFN (15 μM) treatment for 24 h. Scale bar, 20 μm. (C) Average ratios of nuclear vs. cytosolic TFEB immunoreactivity shown in B. N=20 randomly selected cells from 3 independent repeats. (D) In NPC1 KD HeLa cells, SFN (15 μM, 24 h) upregulated expression of TFEB target genes (n=3 independent repeats). (E) SFN promoted cholesterol clearance in NPC1 KD HeLa cells. HeLa cells was transiently transfected by siNPC1 for 48 h, followed by SFN (15 μM) treatment for 24 h. Scale bar, 20 μm. (F) Quantification of cholesterol accumulation in NPC1 KD HeLa cells shown in E. N=15 randomly selected cells from 3 independent repeats. (G) SFN (15 μM, 24 h)-induced TFEB nuclear translocation in human NPC1 fibroblasts. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (H) Quantification of nuclear vs. cytosolic TFEB ratio as shown in G. N=20 randomly selected cells from at least 3 independent experiments. (I) SFN promoted cholesterol clearance in human NPC1-patient fibroblasts. Human NPC1 fibroblasts were treated with SFN (15 µM, 24 h) and filipin staining was carried out. Scale bar, 20 µm. (J) Quantification of cholesterol accumulation as shown in I. N=30 randomly selected cells from 3 independent repeats. For all the panels, average data are presented as mean ± s.e.m.; *** P < 0.001.

TFEB is required for SFN-promoted cholesterol clearance.

(A) Western blot analysis of the efficiency of siTFEB KD and mCherry-TFEB OE in HeLa cells. (B) HeLa cells were transfected with siTFEB or mCherry-TFEB for 48 h, followed by cotreatment with U18666A (2.5 μM) and SFN (15 μM) for 24 h and cholesterol accumulation was analyzed by Filipin assay. Scale bar, 20 μm. (C) Quantification of cholesterol levels as shown in B. N=15 randomly selected cells from 3 independent repeats. (D) Western blot analysis the efficiency of TFEB KO in HeLa cells. (E) HeLa, HeLa TFEB KO and HeLa TFEB KO cells transient expressing mCherry-TFEB (TFEB OE, 48 h) were cotreatment with U18666A (2.5 μM) and SFN (15 μM) for 24 h and cholesterol levels were analyzed by filipin assay. Scale bar, 20 μm. (F) Quantification analysis of cholesterol accumulation as shown in E. N=15 randomly selected cells from at least 3 independent experiments. Average data are presented as mean ± s.e.m.; ***P < 0.001.

SFN promotes lysosomal exocytosis and biogenesis in NPC1 cell models.

(A) Confocal microscopy images showing the exposure of LAMP1 on the PM in nonpermeabilized HeLa NPC1 cells treated with SFN (15 μM) for 24 h using an antibody against LAMP1 luminal portion. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (B) Quantitative analysis of LAMP1 levels on the PM in HeLa NPC1 cells shown in A. Bars represent the fold increase of LAMP1 fluorescence on PM in SFN treated cells. N=15 randomly selected cells from 3 independent repeats. (C) SFN increased the release of free cholesterol into medium. HeLa cells were cotreated with U18666A (2.5 μM) and SFN (15 μM) for 24 h, and then examined for cholesterol. The levels of cholesterol in the medium or cell lysates were measured by cholesterol assay in a reaction mixture with (measuing total cholesterol content) or without (measuring free cholesterol content) cholesterol esterase enzyme (n=6 independent repeats). (D) SFN increased the release of lysosomal enzyme NAGases and ACP in HeLa NPC1 cells. HeLa cells were cotreated with U18666A (2.5 μM) and SFN for 24 h, and the activities of NAGases and ACP were analyzed in the medium and cell lysates (n=6 independent repeats). (E) LAMP1 staining in HeLa cells upon U18666A treatment (2.5 μM) in the presence and absence of SFN (15 μM). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (F) Quantification analysis of LAMP1 immunofluorescence shown in E. N=20 randomly selected cells from at least 3 independent experiments. (G) Effects of SFN on lysosome acidity. HeLa cells were treated with 2.5 μM U18666A (24 h) in the presence and absence of 15 μM SFN (12 h) and lysosomal pH was analyzed by LysoTracker Red DND-99 (50 nM). Scale bar, 20 μm. (H) Quantification of LysoTracker intensity shown in G. N=20 randomly selected cells from at least 3 independent experiments. (I) Effects of SFN on lysosomal acidity using a ratiometric pH dye. HeLa cells were treated with U18666A (2.5 μM) in the presence and absence of SFN (15 μM), lysosomal pH was determined using a ratiometric pH dye combination (pHrodo Green dextran and CF555 dextran). Scale bar, 20 μm or 2 μm (for zoom-in images). (J) Quantification analysis of lysosomal pH shown in I. Randomly selected cells from at least 3 independent experiments. For all the panels, data are presented as mean ± s.e.m.; **P < 0.01, ***P < 0.001.

SFN ameliorates cholesterol accumulation in Npc1-/- MEF cells.

(A) SFN (15 μM) treatment induced TFEB nuclear translocation in NPC1 MEF cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (B) Average ratios of nuclear vs. cytosolic TFEB immunoreactivity shown in A. N=20 from 3 independent repeats. (C) SFN (15 μM, 24-72 h) reduced cholesterol accumulation in NPC1 MEF cells by filipin assay. Scale bar, 20 μm. (D) Quantification analysis of cholesterol accumulation levels shown in C. N=15 randomly selected cells from at least 3 independent experiments. (E) Effects of SFN (15 μM, 12 h) on intensity of LAMP1 in NPC1 MEF cells. Scale bar, 20 μm. (F) Quantification of LAMP1 intensity shown in E. N=20 randomly selected cells from at least 3 independent experiments. (G) Effects of SFN (15 μM, 12 h) on lysosome acidity in MEF cells. Scale bar, 20 μm. (H) Quantification analysis of LysoTracker intensity shown in G. N=20 randomly selected cells from at least 3 independent experiments. For all the panels, data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001.

SFN rescues the loss of Purkinje cells and body weight in NPC in vivo model. mice.

(A) SFN promoted TFEB dephosphorylation in mice brain. 4-week-old BALB/cJ mice were intraperitoneally (i.p) injected with SFN (50 mg/kg) or vehicle for 12 h and brain tissues including cerebellum and hippocampus were collected and subjected to detect pS211- TFEB and total TFEB levels by Western blotting. (B) Quantification of the ratios of p-TFEB vs. total TFEB as shown in A. (C) Cerebella from vehicle and SFN treated NPC mice were analyzed at 8 weeks of age for calbindin by immunohistochemistry. SFN and vehicle were intraperitoneally injected daily in 4-week-old NPC mice for 4 weeks. Scale bar = 200 μm. (n=6 for each group). (D) Quantification of the number of Purkinje cells as indicated in the anterior lobules (II-V) as shown in C. (E) Body Weight was registered during the treatment. For all the panels, data are presented as mean ± s.e.m.; **P < 0.01, ***P < 0.001.

A working scheme to illustrate that small-molecule TFEB agonist promotes cholesterol clearance in NPC model via TFEB-upregulated lysosomal exocytosis and biogenesis.

Pharmacological or genetic activation/overexpression of TFEB dramatically ameliorates cholesterol accumulation in NPC1 cells. Small-molecule, BBB-permeable TFEB agonist-SFN induces TFEB nuclear translocation by dephosphorylation of TFEB at S142 and S211 residues, promoting lysosomal biogenesis and exocytosis, resulting in mitigating lysosomal cholesterol levels.