Characterization of an endosome to mitochondria cholesterol import pathway mediated by MLN64

Niemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder caused by mutations in one of two late endosomal proteins, NPC1 and NPC2. The main biochemical hallmarks of NPC disease are endosomal cholesterol accumulation and an impaired cholesterol homeostatic response. Defective regulation of cholesterol homeostasis contributes to the pathogenesis of a wide variety of disorders. The disruption in cholesterol trafficking might affect cholesterol transport to mitochondria, consequently affecting mitochondrial and overall cellular function. Using wild-type and NPC1-deficient Chinese hamster ovary cells, stably transfected with a CYP11A1 complex to evaluate mitochondrial cholesterol import by the production of pregnenolone, we show that cholesterol transport to the inner mitochondrial membrane was not affected by loss of functional NPC1. We demonstrate that cholesterol from multiple cellular sources is imported into mitochondria. Reduction of NPC2, MLN64, and VDAC1 by RNA interference decreased cholesterol transport to the inner mitochondrial membrane. A novel assay using semi-intact cells demonstrated direct transfer of cholesterol from endosomes to mitochondria, providing important insight into the mechanisms of mitochondrial cholesterol import. This work demonstrates a novel pathway for transport of endosomal cholesterol to mitochondria that requires NPC2 and MLN64, but not NPC1.