Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The orthologue of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, PHI-33, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppress cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

Mucolipidosis type IV (MLIV) is a rare, autosomal recessive, neurodegenerative, lysosomal storage disorder. MLIV is caused by mutations in a gene (MCOLN1) encoding a TRP channel family member known as Mucolipin 1 or TRPML1. TRPML1 is a lysosomal transmembrane protein that appears to be required for normal lysosomal pH regulation, recycling of molecules and membrane reorganisation including lysosomal biogenesis, fusion and exocytosis. The exact function of the channel is unknown but it is permeable to multiple ions including Ca²⁺, Na⁺ and K⁺, possibly also Fe²⁺ and Zn²⁺. How normal TRPML1 function regulates lysosomal processes is not clearly understood. Mutations in the MCOLN1 gene can lead to complete loss of TRPML1 function, partial loss of function or mislocalisation, all of which lead to lysosomal dysfunction, lysosomal lipid storage and ultimately neurodegeneration. The disease processes that lead to neurodegeneration are poorly understood and at present no therapy exists for MLIV. We have discovered that TRPML1 results in regulating lysosomal Ca²⁺ homeostasis that is the opposite of the Ca²⁺ dysregulation associated with Niemann-Pick type C disease (NPC). Our findings indicate that disrupted function of TRPML1 leads to enhanced Ca²⁺ release via the NAADP receptor, recently shown to be the lysosomal two-pore channel TPC2. This indicates that TRPML1 is not the NAADP receptor as suggested by others, indeed NAADP mediated Ca²⁺ release is enhanced with multiple NAADP induced lysosomal Ca²⁺ release events occurring in TRPML1 null cells compared to single releases in normal cells. This phenotype appears to be responsible for the cellular dysfunction associated with MLIV disease cells, enhanced lysosomal fusion, defective endocytosis and potentially even altered lysosomal pH. Several of these phenotypes are normalised by the NAADP receptor specific antagonist Ned-19. These findings illustrate that the NAADP receptor is central to MLIV disease pathology and may be a novel candidate for disease therapy.