Lysosomal Calcium Channel TRPML1 in Physiology and Pathology

Abstract

Not only as the degradation center of the cell, the lysosome has recently been demonstrated to sense and respond to various cellular stresses. Failures of this lysosomal adaptation process to environmental and cellular cues are linked to many diseases. Transient receptor potential mucolipin 1 (TRPML1 or ML1) is the principle Ca2+ channel localized to lysosomes. Lysosomal Ca2+ release is known to regulate various aspects of lysosomal function, including membrane trafficking, fusion, fission, and lysosomal exocytosis. As an important signal in the cell, lysosomal Ca2+ release through ML1 has recently been suggested to act as a trigger for activation of transcription factor (TF)EB. Upon translocating into the nucleus, TFEB upregulates a set of autophagic and lysosomal genes, boosting lysosomal biogenesis and function. Besides, overexpression of TFEB has been shown to activate autophagy and lysosomal exocytosis to promote cellular clearance and ameliorate pathologies in a wide range of disease models. Therefore, ML1, lysosomal Ca2+ release, and TFEB together form a pathway to regulate lysosome function under both physiological and pathological conditions. In this dissertation work, I am trying to answer two questions: 1) What is the endogenous cue that activates the ML1-TFEB pathway and its physiological significance (Chapter II); 2) Can activation of ML1 in vivo, especially through small molecule agonists, be sufficient to activate TFEB and lysosomal exocytosis, thus ameliorating muscular dystrophy (Chapter III & IV). Reactive oxygen species (ROS) is an important signal regulating many cellular processes. We found that ML1 channels can be directly and specifically activated by oxidants and endogenous mitochondria-originated ROS, mediating lysosomal Ca2+ release and TFEB nuclear translocation. Our data also showed that ML1 activity is required for ROS-induced autophagosome formation and clearance of damaged mitochondria. Together, our data suggest that the ML1-TFEB mechanism is activated by physiological levels of ROS, initiating autophagy to clean damaged mitochondria and ROS. Duchenne muscular dystrophy (DMD) is an inherited muscle disease caused by impairment of sarcolemmal repair. Lysosomal exocytosis is an important mechanism that the cell utilizes to repair damaged plasma membrane. ML1 and lysosomal Ca2+ release are required for lysosomal exocytosis, and loss of ML1 function causes muscular dystrophy (MD) in both human and mouse. Here, by utilizing genetical and pharmacological tools, we found that activation of ML1 in vivo ameliorated all pathological hallmarks of MD mice, including acute necrosis, progressive fibrosis, central-nucleated fibers, reduced muscle force and compromised exercise ability. Sarcolemmal permeability, assayed by serum creatine kinase (CK) release and Evan’s blue dye, was also improved upon ML1 activation. Additionally, TFEB was activated by ML1 overexpression and agonists, correcting lysosomal insufficiency seen in MD mice. Finally, we discovered that ML1 agonists activate TFEB and lysosomal biogenesis in vitro, preventing human DMD myoblasts from cell death. Taken together, our data demonstrated that ML1 and lysosomal exocytosis can be a potential therapeutic target for treating DMD.