Browsing by Subject "Niemann-Pick Disease, Type C"
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Item Evaluating the Mechanisms of 2-Hydroxypropyl-β-Cyclodextrin and Liver X Receptor Agonists as Potential Therapies for Niemann-Pick Type C Disease(2013-02-12) Taylor, Anna Marie 1986-; Mendelson, Carole R.; Kliewer, Steven A.; Goodman, Joel M.; Repa, Joyce J.Cholesterol is essential to life; therefore, the synthesis, entry, and efflux of cholesterol are tightly regulated. In some rare disease states, such as in Niemann-Pick Type C, cholesterol balance is lost leading to detrimental effects. In Niemann-Pick Type C, mutations in either of the cholesterol trafficking proteins, NPC1 or NPC2, lead to the entrapment of unesterified cholesterol within the lysosome. The accumulated cholesterol promotes increased inflammation and apoptosis throughout the body resulting in premature death, which typically occurs during adolescence in humans. Currently there are no therapies proven to halt the progression of Niemann-Pick Type C in patients; however, two separate compounds (an LXR agonist and a cyclodextrin) have been shown to increase lifespan in the Npc1-/- mouse model. Although both of these potential therapies are known to alter cholesterol dynamics in the cell, the molecular mechanism(s) through which they are able to correct the defect in Niemann-Pick Type C and ultimately to enhance survival have not been fully elucidated, which is the goal of this work. As Abcg1 is a LXR target gene and a potential mechanism through which cholesterol is trafficked from Npc1-/- cells after LXR agonist treatment, the Npc1-/-Abcg1-/- mouse was generated and evaluated. These mice die significantly earlier than Npc1-/- littermates and suggest that ABCG1 plays a vital role in reducing inflammation in Niemann-Pick Type C. In addition, comprehensive studies were done within 24 hours of cyclodextrin administration in Npc1-/- mice. These results show that cyclodextrin works in Npc1-/- mice by freeing the trapped cholesterol from the lysosome of each cell very rapidly and then releasing the cholesterol intracellularly for normal sterol processing. Finally, Npc1-/- mice were treated in combination with the LXR agonist and cyclodextrin to test if dual treatment had an additive effect on relieving Niemann-Pick Type C disease progression. The combination therapy had no further benefit over cyclodextrin alone, which implies that the two agents are acting by similar mechanism(s). Overall, this work further clarifies the molecular mechanism(s) of LXR agonists and cyclodextrins in Niemann-Pick Type C disease progression, which could lead to the development of more effective therapies for patients.Item Sterol Binding to Niemann-Pick Type C1 Disease Protein (NPC1) - Implications for its Function in Cholesterol Transport(2009-06-15) Abi-Mosleh, Lina Fouad; Brown, Michael S.; Goldstein, Joseph L.Through studies of inborn errors of metabolism, scientists have uncovered many pathways involved in the transport of cholesterol within cells. Despite intense scientific interest, the mechanism by which cholesterol is transported between membrane compartments in animal cells remains obscure. Studies on Niemann-Pick type C disease (NPC) defined the requirement of at least two proteins involved in the transport of lipoprotein-derived cholesterol from lysosomes. Both proteins are located in the lysosomes and mutations in either NPC1, a membrane bound protein, or NPC2, a soluble protein, causes the accumulation of large amounts of cholesterol throughout the body. We encountered NPC1 in the course of isolating a cholesterol-homeostasis membrane protein that binds oxygenated metabolites of cholesterol, oxysterols. We then showed that NPC1‘s sterol binding domain was localized to its N-terminal luminal soluble domain (NTD) and that this domain bound cholesterol as well. We first attempted to understand the functional significance of NPC1 binding to oxysterols. We were able to demonstrate that NPC1 is not required for the regulatory actions of oxysterols since the oxysterol-mediated inhibition of the proteolytic processing of the sterol regulatory element-binding protein (SREBP) and activation of acyl-CoA:cholesterol acyltransferase (ACAT) was not defective in NPC1 mutant fibroblasts. Many studies emphasized the importance of NPC1 in the transport of LDL-derived cholesterol out of the lysosomes. However, there is contradictory evidence for the role of NPC1 in transporting plasma membrane cholesterol and endogenously synthesized cholesterol to the compartment of the endoplasmic reticulum (ER) that contains ACAT. Experiments presented in this study characterize NPC1 dependent and independent transport of cholesterol in cells. We definitively show that NPC1 is only required for the transport of cholesterol from the LDL particle that has entered the cell through receptor-mediated endocytosis. After defining that NPC1‘s role is only in the transport of cholesterol out of the lysosome, we began to study the mechanism involved in this transport. Inside the lysosome reside the two proteins, NPC1 and NPC2, that when mutated cause the NPC disease. Both NPC1 and NPC2 bind cholesterol with ~ 100nM affinity, but they bind to opposite ends of the cholesterol molecule. Recently an in vitro assay using purified proteins was developed to measure transfer of [³H]cholesterol between the NPC1(NTD), NPC2 and phosphatidylcholine liposomes. NPC1(NTD) donates or accepts cholesterol from liposomes very slowly, whereas NPC2 acts quickly. NPC2 stimulates the bidirectional transfer of cholesterol between NPC1(NTD) and liposomes. This evidence suggested that NPC2 could stimulate NPC1‘s rates of dissociation or association of cholesterol. This was the first evidence suggesting a direct interaction between both proteins. Using mutational analysis, we identified two functional subdomains of NPC1(NTD) – one for sterol binding and the other for NPC2-mediated transfer. NPC1 with point mutations in either one of the subdomains cannot accept cholesterol from NPC2 and therefore cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. These studies explain the dual requirement of these proteins in the export of cholesterol from lysosomes, defining how mutations in either protein produce similar clinical phenotypes. Based on these biochemical and tissue culture studies, in addition to the availability of the crystal structure of both proteins bound to sterols, we propose a working model: after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds to NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer-limiting lysosomal membranes.