EXPLORING THE ROLE OF MITOCHONDRIAL CHOLESTEROL IN NIEMANN-PICK TYPE C DISEASE

Abstract

Niemann-Pick type C (NPC) disease is an autosomal recessive neurodegenerative disorder caused by mutations in Npc1 or Npc2 gene. Npc1 and Npc2 encode a late-endosomal transmembrane and lumenal cholesterol-binding domain protein, respectively. Together NPC1 and NPC2 mediate the transport of endosomal cholesterol to the plasma membrane and endoplasmic reticulum. Loss of function mutations in NPC1 or NPC2 leads to cholesterol accumulation in late endosomes, neuronal dysfunction, neurodegeneration, and premature death. The link between cholesterol trafficking and NPC pathogenesis is unclear. I performed metabolomics analyses of different brain regions from wildtype and Npc1-/- mice at pre-symptomatic, early-symptomatic and late-stage disease by 1H-NMR spectroscopy. Metabolic profiling revealed increased lactate and decreased acetyl-CoA levels in Npc1-/- cerebellum and cerebral cortex at all ages. Protein and gene expression analyses indicated a deficiency in the oxidative decarboxylation of pyruvate to acetyl-CoA, an upregulation of glycolytic gene expression, and alterations in glutamate oxidation at the early-symptomatic stage. I also observed a pre-symptomatic increase in several indicators of oxidative stress and antioxidant response systems in all Npc1-/- brain regions. Furthermore, mitochondrial cholesterol levels were increased in Npc1-/- murine brain and NPC1-deficient Chinese hamster ovary (CHO) cells. Using CHO cells that express CYP11A1 fusion protein that converts cholesterol to pregnenolone at the mitochondrial inner membrane I determined that the endosomal cholesterol-binding protein, metastatic lymph node protein 64 (MLN64) and NPC2 but not NPC1 were required for transport of endosomal cholesterol to mitochondria. To investigate the effects of elevated mitochondrial cholesterol levels on energy metabolism I manipulated mitochondrial cholesterol by genetic alterations of NPC1, MLN64, and NPC2. I found that the depletion of NPC1 increased lactate secretion, decreased glutamine-dependent mitochondrial respiration, and decreased ATP transport across mitochondrial membranes in CHO cells. These metabolic alterations did not occur when transport of endosomal cholesterol to mitochondria was blocked. Additionally, the elevated mitochondrial cholesterol levels in NPC1-depleted cells and in NPC2-depleted cells expressing mutant NPC2 that allows endosomal cholesterol trafficking to mitochondria were associated with increased expression of the antioxidant response factor Nfe2l2. These results suggest that mitochondrial cholesterol accumulation causes oxidative stress and mitochondrial dysfunction and contributes to NPC disease pathogenesis.