THE ROLE OF NPC2 IN CHOLESTEROL TRANSPORT AND THE ENDOCYTIC SYSTEM IN NEUROBLASTOMA CELLS

Abstract

Niemann-Pick type C (NPC) disease is an autosomal recessive, neurodegenerative disorder caused by mutations in either Niemann-Pick Type C1 (NPC1) or NPC2. NPC disease is a lysosomal lipid-storage disease characterized by accumulation of unesterified cholesterol in late endosomes/lysosomes (LE/L). NPC2, located to the LE/L lumen, binds cholesterol and hands it off to NPC1 in the LE/L perimeter membrane to be presented to the plasma membrane and the ER. Mature neurons do not synthesize cholesterol efficiently and rely on uptake for their cholesterol needs, thereby highlighting the importance of this cholesterol transport pathway. In human fibroblasts, depletion of either NPC1 or NPC2 leads to sequestration of cholesterol in LE/L. Here, I show that only NPC1, but not NPC2 depletion led to LE/L cholesterol accumulation in a human neuroblastoma cell line (SH-SY5Y), contrary to results in fibroblasts using the same RNA interference system. NPC2-depleted cells still showed impaired transport of endocytosed cholesterol to the ER, similarly to NPC1-depleted cells. Cellular cholesterol levels were lower in NPC2-depleted cells compared to control or NPC1-depleted cells. Cholesterol secretion was markedly reduced in NPC1- and NPC2-depleted SH-SY5Y cells, with NPC2-deficient cells showing a more severe reduction. This reduction was accompanied by a decreased capacity for lysosomal exocytosis in both NPC1- and NPC2-depleted compared to control SH-SY5Y cells. The most striking abnormalities were seen in the endocytic system of NPC2-depleted SH-SY5Y cells, where early and late endosomes were reduced in number but similar in size compared to control cells. In contrast, NPC1-depleted SH-SY5Y cells maintained the number of vesicles but showed a significant increase in their size. This effect of NPC2 depletion on the endocytic system was rescued by simultaneous expression of wildtype NPC2 but also of NPC2 variants that are unable to bind NPC1, are sterol binding-deficient, or glycosylation- and cellular retention-deficient. These results suggest a role of NPC2 in addition to cholesterol transport out of LE/L, which is not dependent on the ability of NPC2 to bind cholesterol or interact with NPC1, potentially regulating endosome homeostasis. These findings highlight the importance of NPC2 outside the dogmatic role as merely the cholesterol transport partner of NPC1.