Transcriptional activation of lysosomal exocytosis promotes cellular clearance

Abstract

Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca²⁺-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca²⁺ levels through the activation of the lysosomal Ca²⁺ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular storage.

Graphical abstract

Figure 1

TFEB Overexpression Induces Lysosomal Exocytosis (A) Confocal microscopy images showing the exposure of LAMP1 on the PM in both nonpermeabilized NSC cells and MEFs transfected with either a bicystronic plasmid expressing TFEB-GFP or with an empty vector. LAMP1 was detected using an antibody against its luminal portion (LAMP1-1DB4). TFEB-transfected cells were localized by the expression of GFP; nontransfected cells are indicated by asterisks. (B) Quantitative analysis by flow cytometry of LAMP1 levels on the PM in both NSCs and MEFs that express either a bi-cystronic TFEB-GFP plasmid or GFP. Bars represent the fold increase of LAMP1 fluorescence on PM in TFEB-transfected versus GFP-transfected (control) cells. (C) TFEB overexpression increases the release of lysosomal enzymes in the culture medium of MEFs, NSCs, HeLa, and COS-7 cells. Activities of lysosomal enzymes acid phosphatase, β-galactosidase, and β-hexosaminidase were determined in the culture medium and in cells transfected with either an empty vector or with a TFEB-expression vector. The figure shows percentages of enzyme activities released compared with total activities. (D) Representative immunoblots showing LAMP1 levels in both total lysates and enriched PM extracts from MEFs transfected with either TFEB or with an empty vector. Results were normalized using an antibody against actin and the PM protein β1-integrin, respectively. The histogram shows the quantification of LAMP1 detected by the immunoblot. (E) Total and surface LAMP1 levels after TFEB overexpression were analyzed by flow cytometry analyses using the LAMP1-1DB4 and the LAMP1-L1418 antibodies that are able to detect PM- and intracellular-LAMP1, respectively. Data represent mean ± SEM; ^∗p < 0.05 (B, C, D, and E). Scale bar represents 10 μm (A).

Figure 2

TFEB Enhances Trafficking and PM Proximity of Lysosomes (A) TFEB enhances the movement of lysosomes to PM. Merged time-lapse frames of control and stable TFEB-overexpressing HeLa cells (HeLa-CF7) transfected with a LAMP1-GFP vector showing the trajectories of lysosomes. The mean velocity was calculated using IMAGEJ software (see Experimental Procedures for details). (B) Immuno-EM reveals increase in lysosomal exocytosis upon overexpression of TFEB. Control and stable TFEB-overexpressing HeLa cells (HeLa-CF7) were fixed directly or 60 min after addition of 0.5% TA to the medium (see Experimental Procedures). Then cells were labeled with antibodies against LAMP1 and prepared for immuno-EM (see Supplemental Experimental Procedures). In control HeLa cells, lysosomes decorated by LAMP1 are often detected at a substantial distance from the cell surface (arrows), while treatment with TA only slightly increases the frequency of lysosomes that are close to the PM (arrowheads). TFEB-overexpressing cells (CF7) contain numerous lysosomes located near the cell surface (arrowheads), although several LAMP1-positive structures could be detected toward the cell interior as well (arrow). TA further induced accumulation of lysosomes in subsurface area of TFEB-overexpressing cells with many lysosomes almost touching the PM (arrows). The distance between lysosomes and PM was estimated in thin sections as described in Experimental Procedures in control and TFEB-overexpressing cells incubated with or without TA (100 lysosomes were counted for each condition). The number of lysosomes, as a function of their distance from the cell surface, was counted over 200 nm intervals (100 lysosomes were counted for each experimental condition). Scale bars represent 10 μm (A) and 350 nm (B). Data represent mean ± SEM; ^∗p < 0.05 (A).

Figure 3

TFEB-Mediated Induction of Lysosomal Exocytosis Is Associated with Intracellular Ca²⁺ Release (A) Analysis of intracellular Ca²⁺ by confocal microscopy of HeLa cells transfected with a bi-cystronic TFEB-GFP construct. Data are displayed as the percentage of cells with Ca²⁺ response compared with the nontransfected cells. (B) TFEB overexpression elevates cytosolic Ca²⁺ levels. HeLa cells were infected with either control adenovirus (Ad. Null) or adenovirus expressing TFEB (Ad. TFEB-FLAG) and loaded with Fura-2AM. Cytosolic Ca²⁺ concentration was calculated as described in Supplemental Experimental Procedures. (C) TFEB-mediated lysosomal exocytosis is Ca²⁺-dependent. MEFs and HeLa cells transfected with either TFEB or an empty vector were treated with either the Ca²⁺ ionophore ionomycin (10 micromolar) or the membrane-permeable Ca²⁺ chelator BAPTA-AM (30 micromolar), or both. Mean values of cell surface LAMP1 fluorescence were quantified by flow cytometry. (D) Ca²⁺ depletion in HeLa-overexpressing TFEB (HeLa CF7) does not affect endolysosomes' distance to the PM. HeLa q < CF7 loaded with BSA gold, following the same protocol described in (B), were treated with the Ca²⁺ chelator BAPTA-AM (30 micromolar) for 30 min before the fixation. Morphometric analysis was performed to visualize the distance of endo-lysosomes to the PM. Scale bar represents 10 μm (A). Data represent mean ± SEM; ^∗p < 0.05 (A, B, and C).

Figure 4

TFEB Elevates Intracellular Ca²⁺ Levels through the Activation of MCOLN1 (A) Flow cytometric Ca²⁺ flux assay of stable TFEB-transfected HeLa (CF7) and control cells transfected with a vector containing a scramble shRNA or a specific shRNA against MCOLN1. Data represent the mean values of the Fluo3/FuraRed ± SD ratio. (B) Flow-cytometry Ca²⁺ flux assay in NSCs transfected with a control vector (CTRL), a vector containing a scramble shRNA plus TFEB plasmid, and a vector containing a specific shRNA against MCOLN1 plus TFEB plasmid. Ca²⁺ was determined in resting condition and after stimulation with ionomycin (10 micromolar). (C and D) Analyses of intracellular Ca²⁺ by confocal microscopy of (C) a stable HeLa clone expressing specific shRNA against MCOLN1 (HeLa ^shMCOLN1) and (D) human MLIV fibroblasts transfected with a bi-cystronic TFEB-GFP construct. Data are displayed as the percentage of cells with Ca²⁺ response compared with nontransfected cells. (E) Flow-cytometry analysis of LAMP1 on the PM of HeLa^shMCOLN1 cells transfected with TFEB. (F) Silencing of MCOLN1 reduces TFEB-mediated translocation of LAMP1 to the PM. Representative confocal microscopy images of nonpermeabilized wild-type MEFs transfected with either a bicystronic TFEB-GFP (green in the figure) or with a bi-cystronic TFEB-GFP vector. Cells were cotransfected with a vector containing a specific shRNA against MCOLN1 tagged with RFP (gray in the figure), and stained for luminal LAMP1 (LAMP1-1DB4; red in the figure). (G) Secretion of lysosomal β-galactosidase in HeLa^shMCOLN1 cells. The displayed values represent secretion efficiency of the indicated enzyme, which was calculated as the % of enzymatic activity in the medium with respect to the total activity (medium and cellular pellet). Data represent mean ± SEM; ^∗p < 0.05 (A–E and G). Scale bars represent 10 μm (C and F) and 25 μm (D).

Figure 5

TFEB Overexpression Reduces Storage in LSD Cells (A) NSCs isolated from the cerebral cortex of MSD and MPSIIIA mice were nucleofected with either a TFEB plasmid or with an empty plasmid and cultured on coverslips. Alcian blue was then used to stain the GAGs. (B) Wild-type, MSD, and MPSIIIA NSCs were nucleofected either with a TFEB plasmid or with an empty plasmid. After 16 hr, cells were pulsed with H³-glucosamine in differentiation medium and chased for the indicated time-points. Cell extracts obtained at different time points were quantified to determine the levels of labeled GAGs. (C) Glia-differentiated MSD and MPS-IIIA NSCs were nucleofected with either TFEB or with an empty vector, fixed on glutaraldehyde, and processed for standard electron microscopy. The number of vacuoles per cell was counted from 20 different cells and displayed as mean ± SEM. (D) TFEB promotes clearance of lipofuscin in fibroblasts from a patient with Batten disease. Cells were transfected with a vector carrying TFEB-Ruby (continuous red staining). After 24 hr, cells were examined by live imaging confocal analysis. Cells with increased TFEB (i.e., cells with red signal in the picture and outlined by dashed white lines in the middle panel) display highly reduced levels of lipofuscin (punctate green signal) and a normal cellular morphology compared with nontransfected cells (i.e., cells with intense green staining). (E) Human Pompe disease fibroblasts were transfected with either TFEB or with an empty vector, loaded with the fluorescent sugar 2-NBDG, and analyzed by epifluorescence microscopy. (F) Secretion efficiency of radioactive GAGs was measured in the culture medium of NSCs from MSD mice. Cells were nucleofected with either TFEB or an empty vector after pulse-chase incorporation of H³-glucosamine. Scale bars represent 100 μm (A), 10 μm (C, MSD cells), 500 nm (C, MPSIIIA cells), and 10 μm (D). Data represent mean ± SEM; ^∗p < 0.05 (B, C, and F).

Figure 6

TFEB Overexpression Reduces Storage and Tissue Pathology in a Mouse Model of MSD (A) Alcian blue staining of GAG content in skeletal muscle and liver from mice injected systemically with either an AAV2/9-CMV-GFP or with an AAV2/9-CMV-TFEB3xflag viral vector. (B) Quantitative analysis of GAG content in skeletal muscle and liver of MSD mice injected with either an AAV2/9-CMV-GFP or with an AAV2/9-CMV-TFEB3xflag viral vector. GAG content was displayed as mg of GAGs/mg of tissue extract. At least four mice per group were analyzed for each tissue examined (^∗p < 0.05). Data are mean ± SEM. (C) TFEB reduces inflammation in the liver of MSD mice. Macrophages and macrophage-related inflammatory cells were detected in liver sections from mice injected systemically with either an AAV2/9-CMV-GFP or with an AAV2/9-CMV-TFEB3xflag viral vector by immunofluorescence analysis using an antibody against CD68. (D) Reduction of TUNEL-positive cells (arrows) in 4-month-old MSD mice injected with an AAV2/9-CMV-TFEB3xflag viral vector compared with age-matched MSD noninjected mice. At least four mice per group were analyzed for each tissue examined). Data are mean ± SEM; ^∗p < 0.05 (B and D). Scale bars represent 100 μm (A, C, and D).