Mutations of human Mucolipin transient receptor potential 1 (TRPML1) cause type IV mucolipidosis (ML4), a devastating neurodegenerative disease in young children. ML4 patients exhibit motor defects, mental retardation, and retinal degeneration. Currently there is no treatment for ML4. By developing a patch-clamp method to directly measure the functions of TRPML proteins in the late endosome and lysosome (LEL), we found that TRPML1 mediates Ca2+ and Fe2+ efflux from endosomes and lysosomes, and that PI(3,5)P2, a low-abundance endolysosome-specific phosphoinositide, binds and specifically activates TRPMLs. Under physiological conditions, TRPMLs regulate membrane trafficking by transducing information about PI(3,5)P2 levels into changes in juxtaorganellar Ca2+, thereby triggering membrane fusion/fission events in the late endocytic pathways (endolysosomal trafficking). In addition, TRPML1 mediates release of Fe2+ from late endosomes and lysosomes, which is essential for cellular iron metabolism. Thus impaired ion (Ca2+ and Fe2+) homeostasis underlies lysosomal dysfunction and ML4 phenotypes. In ML4 cells, the problems are twofold. First, defective Ca2+-dependent membrane trafficking due to TRPML1-deficiency causes accumulation of lipids and other bio-materials in the lysosome. The second strike comes from the lysosomal iron overload (due to impairment of TRPML1's Fe2+ conductivity), which converts the accumulated materials into the non-degradable (resistant to lysosomal degradation) lipofuscin (also called aging pigment) in the lysosome. Lipofuscin accumulation dramatically compromises the functions of lysosomes. Thus TRPML1 appears to be an essential regulator of lysosome ion homeostasis. We specifically hypothesize that stimulating TRPML1's Fe2+/Ca2+ channel activity in the lysosome using synthetic agonists can alleviate neurodegeneration associated with ML4.
Our first aim i s to identify novel small molecule activators and inhibitors of TRPML1 by high throughput screening (HTS) using a Ca2+-imaging assay against the MLSCN (Molecular Libraries Screening Center Network) compound collection.
Our second aim i s to characterize and optimize the TRPML1 activators and inhibitors identified from HTS using Ca2+ imaging and electrophysiology assays.
Our third aim i s to validate the trafficking-rescue and storage-reducing functions of candidate compounds using cells lines derived from patients with ML4 and NPC diseases. Overall, the demonstration of defective lysosomal Fe2+/Ca2+ efflux as the cause of ML4 makes it highly significant to obtain pharmacological agents that can manipulate TRPML1 channel activity. The ultimate goal of our proposed research is to develop therapeutic strategies for ML4.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Small Research Grants (R03)
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Special Emphasis Panel (ZRG1-BST-F (50))
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Yao, Yong
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
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