Cholesterol, which is released by lysosomal hydrolysis of LDL-cholesteryl esters, is transported from lysosomes to fulfill structural and metabolic roles throughout the cell. The mechanisms and pathways of directed cellular movement of cholesterol are unknown. We are studying intracellular cholesterol transport in: Niemann-Pick diseases, types C and D, which are autosomally recessive lipid storage diseases, and CHO cells treated with the pharmacological agent, U18666A. (i) Niemann-Pick disease, type C (NPC) fibroblasts express a defect in intracellular cholesterol transport that is specific for LDL-derived cholesterol. LDL-cholesterol accumulates in NPC lysosomes, which results in impaired movement of LDL-cholesterol to other cell membranes. We propose to develop an in vitro assay of cholesterol transport from isolated normal and NPC lysosomes in order to identify and purify cellular components required for intracellular cholesterol movement, and define biochemically the molecular defect in NPC disease. (ii) Niemann-Pick disease, type D (NPD) is clinically related to NPC; however, there is biochemical evidence that NPD cells express a unique mutation. We propose to determine if LDL-cholesterol transport is defective in Niemann-Pick, type D fibroblasts. We will assess if LDL-mediated regulation of cellular cholesterol metabolism and intracellular cholesterol transport are impaired. (iii) Chinese hamster ovary cells treated with the pharmacological agent, U18666A, express impaired LDL-cholesterol transport from lysosomes to other cell membranes. We propose to characterize U18666A-mediated inhibition of intracellular cholesterol transport in CHO cells. Somatic cell genetic and biochemical analyses of control, U18666A resistant mutants, and revertants will be used to identify the cell component affected by U18666A treatment of CHO cells. Our long term goal is to define the basic mechanism of intracellular cholesterol transport and identify and purify required cell components.
