To mediate the degradation of macromolecules delivered by endocytosis and autophagy, lysosomes must undergo extensive trafficking, including migration along microtubule tracks towards cargo-carrying endosomes and autophagosomes and subsequent membrane fusion and fission processes. Defects in lysosomal trafficking lead to lysosome storage diseases (LSDs). The long term goal of the proposed research is to understand how various cellular cues regulate lysosomal trafficking in autophagy and endocytosis. During the last funding period, we demonstrated that Ca2+ release from the lysosomal lumen plays a key signaling role in regulating multiple steps of lysosomal trafficking. By directly patch-clamping lysosomal membranes and by fluorescently imaging Ca2+ release from lysosomes, we showed that TRPML1 (transient receptor potential mucolipin 1, or ML1) is the principle Ca2+ release channel in the lysosome. By performing high-throughput screening we have identified several highly potent and selective synthetic agonists and antagonists of ML1. Whereas human mutations of TRPML1 cause a LSD characterized by disordered lysosomal trafficking, pharmacological activation of ML1 using synthetic agonists is sufficient to induce lysosomal biogenesis and trafficking. Furthermore, use of synthetic antagonists shows that ML1 is required for lysosomal exocytosis during phagocytosis and membrane repair. Our preliminary studies have identified two distinct classes of endogenous molecules that act as agonists for ML1. The central goal of this competitive renewal project is to test the hypothesis that cellular cues regulate lysosome trafficking and autophagy, by directly activating ML1 to elicit lysosomal Ca2+ release. We will use lysosome Ca2+ imaging, lysosome electrophysiology, mouse knockouts, transgenic mice, and CRISPR/Cas9 in vivo mutagenesis to test the hypothesis that boosting the activity of ML1 will facilitate lysosome biogenesis and autophagy in cells in culture, and alleviate lysosome storage and neurodegeneration in mouse models of several LSDs.
Aim 1 is to determine the role of ML1 in lysosome biogenesis and autophagy.
Aim 2 is to investigate the roles of ML1 in Ca2+-dependent lysosomal motility. Finally, we will study the in vivo effects of increasing ML1's activity on neurodegeneration associated with LSD. Our ultimate goal of this proposed research is to lay the groundwork necessary to develop new therapeutic strategies for lysosome-related pediatric and adult neurodegenerative diseases.
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