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Title: TRPML1 : role in autophagy and potential target to treat lysosomal storage disorders
Author: Scotto Rosato, Anna
ISNI:       0000 0004 7234 0143
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2018
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The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signaling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release via mucolipin 1 (TRPML1) activates calcineurin, which binds and de-phosphorylates TFEB, thus promoting its nuclear translocation. Induction of autophagy and lysosomal biogenesis via TFEB required TRPML1-mediated calcineurin activation, linking lysosomal calcium signaling to both calcineurin regulation and autophagy induction. In addition to the role of TRPML1 on sustaining transcriptional autophagy program, through the activation of TFEB, we also found that TRPML1-activation induces the recruitment of PtdIns(3)P-binding proteins to the nascent autophagosome, whereas genetic or pharmacological inhibition of TRPML1 channel inhibits autophagy initiation. Importantly, alteration of this function has pathological consequences, and thus we found that autophagosome formation is impaired in human fibroblasts from patients affected of mucolipidosis IV (MLIV; a severe lysosomal storage disorder caused by mutations in TRPML1). By using specific compound inhibitors during starvation, we found that TRPML1-mediated induction of autophagosome biogenesis requires calmodulin, CaMKKβ, and the PtdIns(3)-generating enzyme VPS34. Therefore, we hypothezise that during starvation, TRPML1 activation releases lysosomal calcium that activates a calcium-depedent pathway involving CaMKKβ and the induction of two essential protein complexes involved in autophagy initiation such as ULK1 and PIK3C3 complexes. In parallel studies, we used high content (HC) screening approaches to identify small molecules able to ameliorate the MLIV phenotype. In one of them, we tested whether previously identified drugs (150 FDA compounds) inducing TFEB translocation and cellular clearance might be active in MLIV patient cells. This screening resulted in the identification of 3 drugs able to induce TFEB nuclear translocation confirming the importance of the TRPML1-mediated signalling to promote TFEB activity. In addition, only one of these hits were able to reduce the pathological accumulation of autophagic substrates such as p62 and NBR1 in MLIV human fibroblasts. In a second independent HC-screening, we developed a cell-based assay to identify FDA-drugs able to reduce cholesterol accumulation in MLIV cells. We identify 8 small molecules able to reduce cholesterol accumulation in MLIV human fibroblasts that will need further characterization to define their ability to ameliorate the phenotype of this devastating disease. In summary, we found two novel signaling pathways triggered by TRPML1-dependent lysosomal calcium release that regulate cellular homeostasis by both promoting autophagy initiation and sustaining transcriptional programs inducing autophagic and lysosomal genes during starvation. Finally, we use part of this knowledge to develop cell-based high content screening assays that identified 9 FDA-approved compounds able to ameliorate the autophagic impairment and reduce lipid storage in MLIV disease cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral