Analysis of a suppressor of the Niemann-Pick C phenotype

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DESCRIPTION (provided by applicant): Niemann-Pick C (NPC) is caused by mutations in one of two genetic loci, NPC1 and NPC2. Our focus is on NPC1 because mutations in this locus are responsible for 95% of the clinical cases. The most striking consequence of NPC1 dysfunction is aberrant cholesterol movement, which results in lysosomal storage of cholesterol and glycosphingolipids. The mechanism by which NPC1 facilitates lipid transport from endocytic compartments to other cellular membranes is unknown. Currently, there is no definitive therapy for NPC. Elucidation of cellular factors that suppress the NPC phenotype may reveal new therapeutic targets. We have isolated a somatic cell model of NPC disease with an unusual phenotype. Chinese hamster ovary (CHO) mutants 4-4-D (disease) and 4-4-S (suppressed) belong to the same complementation group as NPC fibroblasts and contain the identical base insertion in the NPC1 gene, which results in a frameshift and termination. Mutant 4-4-D shows the classical NPC disease phenotype of lysosomal cholesterol storage; however, mutant 4-4-S shows no cholesterol storage by filipin fluorescence microscopy. The 4-4-S phenotype is likely due to expression of a gene that suppresses the mutant phenotype. Surprisingly, mutant 4-4-S still shows defective low-density lipoprotein stimulation of acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER), which is characteristic of NPC. Our hypothesis is that the 4-4-S suppressor is a protein that mobilizes cholesterol out of endosomes, but fails to deliver the cholesterol to the ER. To test this hypothesis, we will perform the following Aims. Specific Aim #1: To identify the gene product(s) that suppresses the phenotype of mutant 4-4. Specific Aim #2: To determine the fate of LDL-cholesterol in mutant 4-4-S. Specific Aim #3: To examine intracellular trafficking of glycosphingolipids in 4-4-D and 4-4-S cells. Elucidation of cellular factors responsible for cholesterol clearance from NPC lysosomes will reveal potential therapeutic targets for this devastating neurodegenerative disease.