Niemann-Pick Type C (NPC) is fatal lipid storage disease caused by mutations in NPC1 (95%) and NPC2 (5%) genes. NPC is characterized by cholesterol and sphingolipid accumulation, complex progressive neurodegeneration, and a lifespan of less than 20 years. NPC1/2 function in late endosomes/lysosomes (LE/L), with soluble NPC2 binding unesterified cholesterol and transferring it to integral membrane NPC1 that facilitates its movement out of the LE/L. Recently it was discovered that HDAC inhibition can epigenetically increase expression of the low activity NPC1mut protein to levels that can correct the cholesterol defect. However, HDAC inhibitors now being tried for treatment NPC do not get into the CNS and do not treat the neurological sequelea of NPC. My published results, however, suggest that FTY720/Fingolimod, used for treatment of multiple sclerosis, is also a HDAC inhibitor that increases expression of NPC1/2 and corrects cholesterol and sphingolipid storage defects in NPC1mut fibroblasts. Furthermore, my preliminary data in mice show that FTY720 does accumulate in the cerebellum, increases NPC1/2 expression in brain as well as liver, and reduces cholesterol levels. Hence, in my first Aim, I will extend this approach to an in vivo mouse model of NPC that more accurately mimics the human disorder, monitoring brain and liver NPC1/2 expression, cholesterol and sphingolipid homeostasis, and disease progression. While accumulation of sphingolipids have been implicated in development of NPC-associated neurological defects, the mechanisms remain unknown. Recent published work and my preliminary data support the premise that a deficiency of sphingosine kinase 1 (SphK1), which converts the sphingolipid metabolite sphingosine to the bioactive mediator sphingosine-1- phosphate, may be important in pathogenesis of NPC. Consequently, my Aim 2a focuses on the role of SphK1 in sphingolipid accumulation on a cellular level through a detailed examination of its expression, localization, and regulation in NPC mutant fibroblasts. Furthermore, by employing a novel SphK1 activator we recently discovered, and CRISPR CAS9 mediated modulation of SphK1 expression and activity, I will examine whether increasing sphingolipid catabolism suppresses the NPC cellular phenotype. Finally, most therapeutic approaches focus on reducing cholesterol accumulation, but our preliminary data suggest that cholesterol and sphingolipid metabolic pathways are interdependent, decreased SphK1 activity leads to accumulation of both sphingolipids and cholesterol. Therefore, I will examine the benefits of targeting both cholesterol and sphingolipid accumulation through a novel ?dual lipid? reduction strategy in a mouse model of NPC. I expect that my results will validate the use of FTY720/Fingolimod to effectively restore normal NPC1/2 activity levels, answer longstanding questions about the mechanism of sphingolipid accumulation in NPC, and pave the way for a new therapeutic strategy targeting both pathogenic classes of lipids in NPC disease.
Unfortunately for children with the fatal lipid storage disease Niemann-Pick Type C (NPC), there are currently no effective treatments. Caused by mutations in genes (NPC1 and NPC2) encoding proteins responsible for the transport of cholesterol out of the endolysosomal system, NPC results in the accumulation of both cholesterol and sphingolipids. Our goal is to elucidate the mechanisms of pathogenic lipid accumulation in NPC, the roles that defects of the sphingolipid metabolizing enzyme, sphingosine kinase 1, play in this process, and to develop an innovative ?dual lipid? reduction strategy using FTY720/Fingolimod to epigenetically increase levels of low activity mutant NPC proteins, and a novel sphingosine kinase 1 activator that we discovered.