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 kinase¹⁵. 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/biogenesis⁹. Subsequently, autophagy is promoted to facilitate clearance of damaged mitochondria and removal of excessive ROS.