Niemann-Pick type C (NPC) disease is a cholesterol-glycosphingolipid (GSL) lysosomal storage disorder caused most commonly by defects in NPC1, a transmembrane protein believed critical in retroendocytic trafficking of substrates from lysosomes. Most affected children appear normal at birth, develop progressive neurological disease in their early years and die in their second decade. We have pioneered the development of two compounds for this disorder. The first, N-butyldeoxynojirimycin (NB-DNJ) or miglustat is a documented inhibitor of GSL synthesis, whereas the second, hydroxypropyl -cyclodextrin (HPBCD), is an FDA-approved excipient used for drug solublization. Both compounds are efficacious in delaying onset of neurological disease and prolonging life (by 25% and 100%, respectively) in the mouse model of NPC1 disease. Yet neither drug is understood in terms of the precise mechanism responsible for its effectiveness. For miglustat, evidence for sustained reductions in ganglioside storage following oral administration to Npc1 mice is lacking. Similarly, for HPBCD, while both cholesterol and GSL storage are substantially reduced following treatment in Npc1 mice, the mechanism underlying this benefit is completely unknown, and indeed controversy continues even over its ability to cross the blood brain barrier. This proposal will carry out a series of complementary in vivo and in vitro studies employing current and novel reagents and animal models, and quantitative high-resolution imaging, biochemical and genetic evaluations, each directed at treatment mechanisms for NPC disease. Our first two aims are to precisely define HPBCD's mechanism of action in reducing cholesterol/GSL storage in neurons and to critically re-examine and assess miglustat's ability to reduce GSL synthesis as a basis for its beneficial impact on neuron survival. Our third aim uses an unbiased gene analysis approach to explore the full range of metabolic pathways impacted by each drug. Capitalizing on lessons learned in these aims, new combinatorial treatment strategies will be tested in the fourth aim as a means to substantially improve therapy for children with NPC disease. PUBLIC HEALTH RELEVANCE: Lysosomal storage disorders are a group of about 60 rare, fatal genetic diseases caused by defects in a wide range of proteins associated with the endosomal-lysosomal system. Niemann-Pick type C (NPC) disease is a cholesterol-glycosphingolipid (GSL) storage disorder caused most commonly by defects in NPC1, a transmembrane protein believed critical in retroendocytic trafficking of substrates from lysosomes. Affected children typically appear normal at birth but exhibit progressive neurological decline beginning at 4-6 years of age with death often occurring in the second decade of life. Therapeutic options for NPC disease are very limited, with enzyme replacement, cell-mediated, and gene therapies providing little hope of benefit since the NPC1 protein is not soluble and secreted by cells. Such limitations have driven development of drugs that can limit the build-up of offending substrates in brain and other organs - known as substrate reduction therapy (SRT). We have pioneered the study of two such agents, N- butyldeoxynojirimycin (miglustat) and 2-hydroxypropyl -cyclodextrin (HPBCD), both of which have shown efficacy for NPC disease in animal models. The purpose of this grant is to determine the mechanisms by which these two agents delay clinical disease and increase longevity in the murine model of NPC disease. This goal has now become all the more timely as a clinical trial involving HPBCD for treatment of children with NPC disease is being proposed to the FDA, to begin in 2012. Many of the individuals enrolling in this trial will also be under treatment with miglustat. Understanding the mechanisms of action and possible interactions (e.g., synergy) of HPBCD and miglustat are of paramount importance. Importantly, given similarities between NPC and other lysosomal diseases, as well as more common neurodegenerative conditions like Alzheimer's, successful treatments emerging here may provide benefit well beyond a single rare disease.