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Niemann Pick type C (NPC) disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH) depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE), but not N-acetylcysteine (NAC), restored the mGSH pool in liver and brain of Npc1-/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1-/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1-/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1-/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1-/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options.

MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

Lysosomal storage disorders (LSD) are characterized by the accumulation of diverse lipid species in lysosomes. Niemann-Pick type A/B (NPA/B) and type C diseases Niemann-Pick type C (NPC) are progressive LSD caused by loss of function of distinct lysosomal-residing proteins, acid sphingomyelinase and NPC1, respectively. While the primary cause of these diseases differs, both share common biochemical features, including the accumulation of sphingolipids and cholesterol, predominantly in endolysosomes. Besides these alterations in lysosomal homeostasis and function due to accumulation of specific lipid species, the lysosomal functional defects can have far-reaching consequences, disrupting intracellular trafficking of sterols, lipids and calcium through membrane contact sites (MCS) of apposed compartments. Although MCS between endoplasmic reticulum and mitochondria have been well studied and characterized in different contexts, emerging evidence indicates that lysosomes also exhibit close proximity with mitochondria, which translates in their mutual functional regulation. Indeed, as best illustrated in NPC disease, alterations in the lysosomal-mitochondrial liaisons underlie the secondary accumulation of specific lipids, such as cholesterol in mitochondria, resulting in mitochondrial dysfunction and defective antioxidant defense, which contribute to disease progression. Thus, a better understanding of the lysosomal and mitochondrial interactions and trafficking may identify novel targets for the treatment of Niemann-Pick disease.