MCOLN1 is a ROS sensor in lysosomes that regulates autophagy

Abstract

Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes 'host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.

Figure 1. Direct and specific activation of lysosomal TRPML1 channels by ROS.

(a) Representative time course of whole-endolysosome TRPML1-mediated currents (I_TRPML1, open circles, measured at −140 mV) activated by bath application of ChT (75 μM), followed by a 4-min washout and then ML-SI3 (50 μM) application. I_TRPML1 was recorded from an enlarged vacuole isolated from COS-1 cells overexpressing EGFP–TRPML1. The currents were elicited by repeated voltage ramps (−140 to +140 mV; 200 ms) with a 4-s inter-step interval. (b) Representative traces of basal (blue), ChT-activated (magenta) and ML-SI3-inhibited (black) I_TRPML1 at the three time points indicated in a. Only a portion of the voltage protocol is shown; holding potential was 0 mV. (c) Dose-dependence of ChT activation (n=4–8 patches for each data point). (d) Oxidant-specific activation of I_TRPML1. Active oxidants included ChT (150 μM), NaOCl (3 mM), N-chlorosuccinimide (NCS, 500 μM), thimerosal (TMS, 50 μM), H₂O₂ (10 mM) and t-butyl hydroperoxide (TBHP, 1 mM). Representative traces are in Supplementary Fig. 2. Other tested oxidants included cysteine-specific oxidants (DTNB and DTNP, both at 100 μM), an NO-donor (SNAP, 100 μM) and a reactive lipid (4-HNE, 300 μM). The effects of oxidants were normalized to that of ML-SA1 (20 μM). Numbers of patches tested for each oxidant are shown in brackets. (e) Representative traces of DTNP-insensitive (red), ChT-activated (magenta) I_TRPML1. (f) ChT induced single-channel openings in an inside-out patch isolated from TRPML1-4A-expressing cells. (g) The single-channel conductance of ChT- and ML-SA1-activated I_TRPML1. (h) ChT activated mTRPML1 specifically, but not mTRPML2, mTRPML3, zTRPML1.1 or mTPC2. Numbers of patches tested for each constructs are shown in brackets. (i) Insensitivity of zTRPML1.1 to ChT. (j) Insensitivity of whole-endolysosome TRPML1^Va currents to ChT. (k,l) ChT activated endogenous whole-endolysosome I_TRPML1 in WT but not TRPML1 KO mouse macrophages. Data are presented as mean±s.e.m. in c,d and h.

Figure 2. Endogenous mitochondrion-generated ROS activate lysosomal TRPML1 channels and Ca²⁺ release.

(a) Upper panels: application of CCCP increased the fluorescence intensity of CM-H2DCFDA (green) versus the DMSO-treated control (CTL) group. The increase was inhibited by co-application of NAC (5 mM). Lower panels: representative traces of basal whole-endolysosomal currents under each condition (CTL, CCCP, CCCP+NAC) in TRPML1-expressing COS-1 cells. Scale bar, 50 μm. (b) Summary of CCCP pretreatment effects on basal I_TRPML1 and I_zTRPML1.1 from at least five patches for each experimental condition. Data are presented as mean±s.e.m. *P<0.05, ANOVA. Numbers of patches for each experimental condition are shown in brackets. (c) Effects of CCCP pretreatment on endogenous I_TRPML1 in HEK293 cells (mean±s.e.m., n=4–5 patches for each treatment).*P<0.05, ANOVA. (d) Pretreatment of CCCP (10 μM) for 1 h increased ML-SA1-induced Ca²⁺ release measured by Fura-2 imaging in TRPML1-expressing HEK293 cells. (e) CCCP pretreatment reduced GPN-induced lysosomal Ca²⁺ release, which is presumed to reflect the lysosomal Ca²⁺ store. Mean values (±s.e.m.) are shown for >30 cells per coverslip. (f) Quantification of results shown in d,e from at least three independent experiments (mean±s.e.m.). *P<0.05, paired t-tests.

Figure 3. ROS-dependent autophagy induction requires Ca²⁺ and TRPML1.

(a) In HeLa cells stably expressing mRFP–GFP–LC3, CCCP treatment (5 μM for 3 h) increased the formation of autophagosomes, ‘visualized' as mRFP⁺ GFP⁺ puncta. Co-treatment with NAC, BAPTA-AM, or ML-SI3 abolished the increases. Scale bar, 10 μm. (b) Quantification of various treatment conditions on CCCP-induced autophagosome formation (mean±s.e.m., n≥30 randomly-selected cells for each treatment). *P<0.05, ANOVA. (c) NAC did not affect ML-SA5-induced autophagosome formation. Means are shown with s.e.m. (n≥40 randomly-selected cells for each treatment). *P<0.05, ANOVA. (d) Western blot analysis of LC3-I and -II (arrows) protein expression in CCCP (10 μM, 3 h) -treated WT and ML-IV human fibroblasts. Torin 1 (1 μM) was used as a positive control to induce autophagy, and bafilomycin A1 (Baf, 0.5 μM) was used to inhibit lysosomal degradation. (e) Quantitative analysis of LC3-II levels under various experimental conditions shown in d. Data are presented as mean±s.e.m. (from at least three independent experiments); *P<0.05, paired t-tests.

Figure 4. TRPML1 is required for autophagic clearance of damaged mitochondria and removal of excessive ROS.

(a) Effects of ML-SI3 (10 μM) co-administration on the accumulation of PARKIN-positive puncta (red) induced by CCCP treatment (10 μM for 3 h) followed by 1 h recovery (without CCCP) in PARKIN stable cells. Scale bar, 10 μm. (b) Quantitative analysis of ML-SI3 and ML-SI4 effects on the clearance of PARKIN puncta. (c) Effects of CCCP treatment on mitochondrial membrane potential monitored by JC-1 fluorescent dyes in WT and ML-IV fibroblasts. After CCCP (10 μM for 3 h) treatment, removal of CCCP for 1 h led to repolarization (re-energization) of mitochondrial membrane potential (green, J-monomer; de-energized; red, J-aggregates; energized) in WT but not ML-IV cells. Scale bar, 10 μm. (d) The ratio of red to green fluorescence of JC-1 was quantified for >30 randomly-selected cells. Data are presented as mean±s.e.m. *P<0.05, ANOVA. (e) Basal ROS levels in WT, ML-IV and NPC fibroblasts. (f) Effect of ML-SI4 (10 μM) on ROS levels measured by CM-H₂DCFDA (green) imaging in HeLa cells. Scale bar, 50 μm. (g) Quantification of results shown in f. Means are shown with s.e.m.; *P<0.05, ANOVA.

Figure 5. TRPML1 channel activity is required for ROS-induced TFEB-nuclear translocation.

(a) Differential effects of BAPTA-AM and NAC on CCCP- and Torin-1-induced TFEB-nuclear translocation in HEK293 cells stably expressing mCherry–TFEB. Cells were treated with CCCP (5 μM) and Torin 1 (1 μM) for 1 h to induce TFEB-nuclear translocation. Nuclei were counterstained with DAPI (pseudo-colored in green). Scale bar, 10 μm. (b) Ratio of nuclear versus cytosolic TFEB (>100 cells per experimental condition). (c) Western blot analysis of cytosolic versus nuclear pools of TFEB proteins with and without CCCP treatment, and in the presence and absence of NAC and BAPTA-AM. Tubulin and Lamin are proteins abundant in the cytosolic and nuclear fractions, respectively. (d) Averaged effects of NAC or BAPTA-AM on CCCP-induced TFEB-nuclear translocation, based on multiple repeated experiments as shown in c. (e) Differential effects of ML-SI3 on CCCP- and Torin-1-induced TFEB-nuclear translocation, shown with western blot analyses of TFEB. (f) The quantitative effects of ML-SI3 on CCCP-induced TFEB-nuclear translocation based on the multiple repeated experiments as shown in e. (g) CCCP (10 μM for 1 h) induced accumulation of TFEB, detected by anti-human TFEB antibody, in the nuclei of WT, but not ML-IV cells. In contrast, Torin-1 induced TFEB-nuclear translocation in both WT and ML-IV cells. Nuclei were labelled with DAPI (pseudo-colored in red). Scale bar, 10 μm. (h) Average ratios of nuclear versus cytosolic TFEB immuoreactivity (>100 randomly-selected cells per experiment). ML-SI4 (10 μM) inhibited TFEB-nuclear translocation induced by CCCP (10 μM) treatment for 1 h. (i) The effects of ML-SA5 (1 μM for 1 h) on TFEB-nuclear translocation in the presence and absence of ML-SI4 (10 μM) in WT and ML-IV cells. Scale bar, 10 μm. (j) Average ratios of nuclear versus cytosolic TFEB immuoreactivity (>50 randomly-selected cells per experiment). All quantification data are presented as mean±s.e.m.; *P<0.05, paired t-test for western blots and ANOVA for all other comparisons.

Figure 6. ROS sensitivity of TRPML1 is required for TFEB activation by mitochondrial ROS.

(a) Effect of ML-SI3 on Lamp1 expression 7 h after CCCP treatment (5 μM for a duration of 1 h). Note that there was a progressive increase in Lamp1 expression levels following CCCP withdrawal (see Supplementary Fig. 35). (b) Quantification of a from three independent experiments (mean±s.e.m.); *P<0.05, paired t-test. (c) Rescue of CCCP-induced TFEB-nuclear translocation in ML-IV fibroblasts by transfection of mTRPML1, but not zTRPML1.1 constructs. ML-SA5 induced TFEB-nuclear translocation in ML-IV cells transfected with either mTRPML1 or zTRPML1.1. Scale bar, 10 μm. (d) Quantification of experimental results as shown in c. Data are presented as mean±s.e.m.; *P<0.05, ANOVA. (e) A working model to illustrate the role of TRPML1 in ROS-induced TFEB activation and autophagy. An increase in mitochondrial ROS (for example, by CCCP-mediated mitochondrial depolarization) may activate TRPML1 channels on the perimeter membranes of lysosomes, inducing lysosomal Ca²⁺ release of that activates calcineurin. Subsequently, Ca²⁺-bound calcinurin dephosphorylates TFEB, which is otherwise kept in its phosphorylated form by the nutrient-sensitive lysosome-localized mTOR kinase. Nucleus-localized TFEB then activates the transcription of a unique set of genes related to autophagy induction, autophagosome biogenesis and lysosome biogenesis. Lysosomal Ca²⁺ release may also directly promote lysosome reformation/biogenesis. Subsequently, autophagy is promoted to facilitate clearance of damaged mitochondria and removal of excessive ROS.