NPC1 and NPC2 Regulate Cellular Cholesterol Homeostasis through Generation of Low Density Lipoprotein Cholesterol-derived Oxysterols

Abstract

Mutations in the Niemann-Pick disease genes cause lysosomal cholesterol accumulation and impaired low density lipoprotein (LDL) cholesterol esterification. These findings have been attributed to a block in cholesterol movement from lysosomes to the site of the sterol regulatory machinery. In this study we show that Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) mutants have increased cellular cholesterol, yet they are unable to suppress LDL receptor activity and cholesterol biosynthesis. Cholesterol overload in both NPC1 and NPC2 mutants results from the failure of LDL cholesterol tobothsuppresssterolregulatoryelement-bindingprotein-dependent gene expression and promote liver X receptor-mediated responses. However, the severity of the defect in regulation of sterol homeostasis does not correlate with endoplasmic reticulum cholesterol levels, but rather with the degree to which NPC mutant fibroblasts fail to appropriately generate 25-hydroxycholesterol and 27-hydroxycholesterol in response to LDL cholesterol. Moreover, we demonstrate that treatment with oxysterols reduces cholesterol in NPC mutants and is able to correct the NPC1I1061T phenotype, the most prevalent NPC1 disease genotype. Our findings support a role for NPC1 and NPC2 in the regulation of sterol homeostasis through generation of LDL cholesterol-derived oxysterols and have important implications for the treatment of NPC disease. Mutations in the Niemann-Pick disease genes cause lysosomal cholesterol accumulation and impaired low density lipoprotein (LDL) cholesterol esterification. These findings have been attributed to a block in cholesterol movement from lysosomes to the site of the sterol regulatory machinery. In this study we show that Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) mutants have increased cellular cholesterol, yet they are unable to suppress LDL receptor activity and cholesterol biosynthesis. Cholesterol overload in both NPC1 and NPC2 mutants results from the failure of LDL cholesterol tobothsuppresssterolregulatoryelement-bindingprotein-dependent gene expression and promote liver X receptor-mediated responses. However, the severity of the defect in regulation of sterol homeostasis does not correlate with endoplasmic reticulum cholesterol levels, but rather with the degree to which NPC mutant fibroblasts fail to appropriately generate 25-hydroxycholesterol and 27-hydroxycholesterol in response to LDL cholesterol. Moreover, we demonstrate that treatment with oxysterols reduces cholesterol in NPC mutants and is able to correct the NPC1I1061T phenotype, the most prevalent NPC1 disease genotype. Our findings support a role for NPC1 and NPC2 in the regulation of sterol homeostasis through generation of LDL cholesterol-derived oxysterols and have important implications for the treatment of NPC disease. Niemann-Pick type C (NPC) 1The abbreviations used are: NPC, Niemann-Pick type C; ACAT, acyl-CoA:cholesterol acyltransferase; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; GC/MS, gas chromatography/mass spectrometry; 24-HC, 24(S)-hydroxycholesterol; 25-HC, 25-hydroxycholesterol; 27-HC, 27-hydroxycholesterol; LDL, low density lipoprotein; LDLr, LDL receptor activity; LXR, liver X receptor; LPDS, lipoprotein-deficient serum; NPC1, Niemann-Pick type C1; NPC2, Niemann-Pick type C2; SREBP, sterol regulatory element-binding protein; SCAP, SREBP cleavage-activating protein; WT, wild type. disease is a fatal, autosomal recessive lipid storage disorder characterized by cholesterol accumulation in the liver, spleen, and central nervous system (1Ory D.S. Biochim. Biophys. Acta. 2000; 1529: 331-339Crossref PubMed Scopus (124) Google Scholar). Mutations in two independent genes result in the clinical and biochemical NPC phenotype (2Vanier M.T. Duthel S. Rodriguez-Lafrasse C. Pentchev P. Carstea E.D. Am. J. Hum. Genet. 1996; 58: 118-125PubMed Google Scholar, 3Steinberg S.J. Ward C.P. Fensom A.H. J. Med. Genet. 1994; 31: 317-320Crossref PubMed Scopus (98) Google Scholar). The NPC1 disease gene encodes a late endosomal protein that has 13 membrane-spanning domains, five of which share sequence homology with the sterol-sensing domains of hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein (SREBP), cleavage-activating protein (SCAP), and Patched (4Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. et al.Science. 1997; 277: 228-231Crossref PubMed Scopus (1229) Google Scholar, 5Davies J.P. Ioannou Y.A. J. Biol. Chem. 2000; 275: 24367-24374Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The minor disease locus, the NPC2 gene, encodes a 132-amino acid soluble lysosomal protein that has been shown to specifically bind cholesterol with a 1:1 stoichiometry and submicromolar affinity (6Okamura N. Kiuchi S. Tamba M. Kashima T. Hiramoto S. Baba T. Dacheux F. Dacheux J.-L. Sugita Y. Jin Y.-Z. Biochim. Biophys. Acta. 1999; 1438: 377-387Crossref PubMed Scopus (167) Google Scholar, 7Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Science. 2000; 290: 2298-2301Crossref PubMed Scopus (710) Google Scholar, 8Ko D. Binkley J. Sidow A. Scott M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2518-2525Crossref PubMed Scopus (166) Google Scholar, 9Friedland N. Liou H.-L. Lobel P. Stock A.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2512-2517Crossref PubMed Scopus (260) Google Scholar). The NPC disease genes are key participants in the intracellular trafficking of cholesterol. Cells with mutations in NPC1 and NPC2 accumulate unesterified cholesterol in an aberrant late endosomal/lysosomal organelle and have markedly impaired rates of esterification of LDL cholesterol (2Vanier M.T. Duthel S. Rodriguez-Lafrasse C. Pentchev P. Carstea E.D. Am. J. Hum. Genet. 1996; 58: 118-125PubMed Google Scholar, 10Steinberg S.J. Mondal D. Fensom A.H. J. Inher. Metab. Dis. 1996; 19: 769-774Crossref PubMed Scopus (29) Google Scholar). Fibroblasts from NPC1 patients also exhibit a defect in mobilization of endosomal cholesterol to the plasma membrane (11Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 12Millard E.E. Srivastava K. Traub L. Schaffer J.E. Ory D.S. J. Biol. Chem. 2000; 275: 38445-38451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 13Wojtanik K.M. Liscum L. J. Biol. Chem. 2003; 278: 14850-14856Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and delayed down-regulation of the LDL receptor and de novo cholesterol biosynthesis (14Pentchev P.G. Comly M.E. Kruth H.S. Vanier M.T. Wenger D.A. Patel S. Brady R.O. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8247-8251Crossref PubMed Scopus (327) Google Scholar, 15Liscum L. Faust J.R. J. Biol. Chem. 1987; 262: 17002-17008Abstract Full Text PDF PubMed Google Scholar). Similarly, in the NPC1–/– mice, which phenocopy human NPC disease, cholesterol synthesis in the whole animal is elevated, and there is increased accumulation of unesterified cholesterol (16Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Am. J. Physiol. 1999; 276: E336-E344PubMed Google Scholar). These sterol regulatory defects have been attributed to impaired delivery of LDL-derived free cholesterol to the endoplasmic reticulum (ER). Decreased ER cholesterol promotes SCAP-mediated trafficking of SREBP to the Golgi, enabling SREBP proteolysis and up-regulation of genes involved in cholesterol synthesis and uptake (17Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). The goal of the present study is to characterize the relationship between the NPC proteins and the sterol regulatory machinery. Thus, we examined the consequence of NPC1and NPC2 loss of function on the key cellular pathways that govern cholesterol homeostasis. We find that cholesterol overload in NPC1 and NPC2 mutants results from the failure of LDL cholesterol to both suppress SREBP-dependent gene expression and promote liver X receptor (LXR)-mediated responses. We show that the sterol regulatory defects in the NPC mutants correlate with the failure to generate oxysterols in response to LDL cholesterol loading, rather than with ER cholesterol levels. Our findings indicate that the NPC proteins regulate sterol homeostasis through production of LDL cholesterol-derived oxysterols and have implications for the treatment of NPC disease. Materials—Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, glutamine, and penicillin/streptomycin were obtained from Invitrogen. Lipoprotein-deficient fetal calf serum (LPDS) was obtained from Cocalico Laboratories. Fetal bovine serum (FBS), filipin complex and human LDL were obtained from Sigma. DiI-LDL was obtained from Molecular Probes. Oleic acid, triolein, and cholesteryl oleate were obtained from Nu-Check Prep. [9,10-3H]oleic acid (5 Ci/mmol), [cholesteryl-1,2,6,7-3H]linoleate (84 Ci/mmol), [3H]acetic acid (5.1 Ci/mmol), and [oleate-1-14C]cholesteryl oleate (59.5 mCi/mmol) were obtained from PerkinElmer Life Sciences. 27-Hydroxycholesterol was obtained from Research Plus, Inc. Cholesterol, 24(S)-hydroxycholesterol (24-HC), and 25-hydroxycholesterol (25-HC) were obtained from Steraloids, Inc. Plasmids—The LDLp-588luc construct containing the human LDL receptor (LDLr) promoter linked to a luciferase reporter was a gift of M. Issandou (18Grand-Perret T. Bouillot A. Commans S. Walker M. Issandou M. Nat. Med. 2001; 7: 1332-1338Crossref PubMed Scopus (68) Google Scholar). The TK-LXRE3-luc construct containing three copies of the LXR response element upstream of a luciferase reporter was a gift of D. Mangelsdorf (19Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf PubMed Scopus Google Scholar). The luciferase was from fibroblasts were obtained from The NPC1 mutant human with the NPC1I1061T and were by P. Pentchev of K. Am. J. Hum. Genet. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The NPC2 mutant human and were by A. Fensom of and M. Vanier (2Vanier M.T. Duthel S. Rodriguez-Lafrasse C. Pentchev P. Carstea E.D. Am. J. Hum. Genet. 1996; 58: 118-125PubMed Google Scholar, 3Steinberg S.J. Ward C.P. Fensom A.H. J. Med. Genet. 1994; 31: 317-320Crossref PubMed Scopus (98) Google Scholar). fibroblasts were in medium containing DMEM, Cholesterol esterification were E.E. Srivastava K. Traub L. Schaffer J.E. Ory D.S. J. Biol. Chem. 2000; 275: 38445-38451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). of cholesterol were medium containing of LDL, with for and was of cholesteryl of triolein, of and were The were by from acid and with oleate was by lipid were with and protein the of cellular cholesterol was by an the Cholesterol A. Srivastava K. D. Traub Schaffer J.E. Ory D.S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). LDL were for in DMEM, DMEM, LPDS, with DiI-LDL for and the of was by a were for and with LDL with LDL with LDL and with LDL both and Cells were with DiI-LDL for and examined by of de Cholesterol of de novo cholesterol was E.E. Srivastava K. Traub L. Schaffer J.E. Ory D.S. J. Biol. Chem. 2000; 275: 38445-38451Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). of SREBP-dependent gene fibroblasts were with of LDLp-588luc and of the of were in the human were lipoprotein-deficient were with LDL for by of and of luciferase and activity of luciferase activity to activity for of gene fibroblasts were with of TK-LXRE3-luc and of were lipoprotein-deficient were with LDL for by of and of luciferase and In Cholesterol were for in DMEM, FBS, for in DMEM, by with DMEM, in the and of LDL The in esterification was by and Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Cells were and in were in and on for The were with a with an to and to and bovine serum were by of and for with and of a of cholesterol, of cholesteryl of were oleate was by and were the fibroblasts were in DMEM, for and for DMEM, containing were from the and S. U. PubMed Scopus Google Scholar, Y. S.D. C.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). was not in the we used of an were to by treatment with for were on a gas to a The used for the study was a P. J. was The of was for and increased to The was increased from to and for The was in the and the and were and The and were The of and was with and for and were from production was the of oxysterols in the and the were in DMEM, for in the and of Cells were with in for with and with filipin in for was by on a and a of were on of two and to were of in NPC the function of the NPC1 and NPC2 we examined the consequence of NPC mutations on regulation of cellular cholesterol homeostasis. We the rates of cholesterol esterification in NPC1 and NPC2 In we examined NPC1 fibroblasts NPC1I1061T mutations and NPC2 with mutations K. Am. J. Hum. Genet. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. Sleat D.E. Fensom A.H. K. M. Lobel P. Vanier M.T. Am. J. Hum. Genet. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of cholesterol esterification in NPC is to than of fibroblasts both NPC1 and NPC2 mutants exhibit activity in response to by the of LDL cholesterol esterification is not to activity in These results are with for NPC1 and NPC2 function in trafficking of LDL cholesterol of the lysosomal We examined the of the NPC mutations on accumulation of cholesterol. we NPC1I1061T and to In NPC1I1061T and mutant we find that cellular cholesterol is and fibroblasts the increased cellular cholesterol in the NPC mutants was to regulation of LDL cholesterol uptake de novo cholesterol we the activity in the fibroblasts was an that the uptake of a LDL, by J. Full Text PDF PubMed Google Scholar, A. R. J. Full Text Full Text PDF PubMed Google Scholar). this uptake of DiI-LDL was in the of of LDL, that the DiI-LDL uptake is a of activity not the cholesterol overload in NPC activity in the NPC mutants to suppress in response to LDL In with is increased and in the NPC1I1061T and results were obtained an receptor not These findings the of LDL cholesterol to suppress de novo cholesterol synthesis in NPC1I1061T and mutants and In response to lipoprotein the mutant activity and de novo cholesterol that are for cholesterol the the NPC1I1061T mutant activity and de novo cholesterol that in the NPC1I1061T lysosomal cholesterol is for regulation of cholesterol responses. NPC to SREBP-dependent has been that the ER cholesterol to the cholesterol that cellular cholesterol through proteolysis of D. 1997; PubMed Scopus Google Scholar). the activity and de novo cholesterol synthesis in NPC mutants are to a failure to suppress SREBP we examined the of NPC mutations on SREBP-dependent gene an sterol regulatory reporter construct an of the of SREBP (18Grand-Perret T. Bouillot A. Commans S. Walker M. Issandou M. Nat. Med. 2001; 7: 1332-1338Crossref PubMed Scopus (68) Google Scholar). and we NPC mutants with and defects in sterol regulatory activity to were fibroblasts by a that in not with the NPC1I1061T mutant is to of SREBP-dependent activity low of LDL but is of LDL In the mutant of SREBP-dependent gene expression by LDL cholesterol the of examined to appropriately suppress gene is not in NPC cholesterol trafficking in in NPC with ER Cholesterol that failure to suppress SREBP in NPC results from failure to cholesterol to the ER, ER cholesterol correlate with the severity of the biochemical We cholesterol in the ER an in esterification Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In cholesterol in the is of lipoprotein and is treatment with LDL cholesterol 25-HC, with the in the ER regulatory In NPC1I1061T and mutant the of the ER cholesterol is to that of show activity and de novo cholesterol synthesis in the Similarly, in NPC ER cholesterol is with that of LDL the ER cholesterol are in both the NPC1I1061T and mutants with with impaired trafficking of LDL cholesterol. However, the failure to ER cholesterol in response to LDL is in the NPC1I1061T than the mutant the defect in regulation of sterol is in the Thus, the biochemical defects between the NPC mutants not correlate with in ER cholesterol levels. in NPC between ER cholesterol and that regulatory than free ER cholesterol important for of the NPC demonstrate that cholesterol are for which regulate cellular cholesterol by of genes that promote and of free cholesterol A. Evans R.M. Mangelsdorf Science. 2001; PubMed Scopus Google Scholar). with LXR has been shown to expression of sterol and a of genes that function in the cholesterol S. L. M.A. M. J. J. J.L. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Mangelsdorf Nat. Med. Scopus Google Scholar). the of NPC mutations on gene we examined in NPC mutant fibroblasts the expression of a reporter construct by an LXR response element (19Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf PubMed Scopus Google Scholar). In fibroblasts LXR activity with of LDL cholesterol In NPC on the of LXR activity in the NPC1I1061T mutant of LDL and of LXR activity is in the Thus, the of gene regulation with the severity of the biochemical phenotype in NPC of LDL in NPC the failure to suppress SREBP-dependent gene expression and the impaired activity in the NPC mutants a defect in the generation of sterol LDL-derived cholesterol in the trafficking of to site of production is in NPC we the of and in medium and lipid from LDL and NPC1I1061T and mutants We specifically examined the production of cholesterol they are the oxysterols by fibroblasts M. J. Biol. Chem. Full Text PDF PubMed Scopus Google they are of hydroxymethylglutaryl-CoA J. Biol. Chem. Full Text PDF PubMed Google Scholar, Science. PubMed Scopus Google Scholar, H. J. Full Text PDF PubMed Google and they are for LXR Y. S.D. C.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. J.R. 1996; PubMed Scopus Google Scholar). In NPC1I1061T and production of is by and with fibroblasts production of is by and in the NPC1I1061T and and 27-HC, in to and are not is that the oxysterols by were the Scopus Google Scholar). to in we in which free cholesterol was to the and the of lipid and of the and was by We were unable to of the in of the not that were not by Thus, the defects in generation correlate with the in esterification and the in cholesterol in NPC in NPC of the defect in generation in NPC we examined with oxysterols is able to regulation of cholesterol homeostasis in We activity in NPC1I1061T and mutants in response to LDL in the and of 25-HC, 27-HC, both In fibroblasts to LDL appropriately and is in the of In activity in NPC1I1061T fibroblasts with but in the of activity is to a with that of fibroblasts are than NPC1I1061T fibroblasts to LDL activity increased activity in is in the of with in with activity in both mutant The in production of with the of oxysterols to suppress that NPC1 and NPC2 in generation of oxysterols in response to failure to generate oxysterols to central to the of the defect in sterol homeostasis in NPC with Cholesterol in NPC the of oxysterols is able to activity in the NPC we treatment with and the cellular cholesterol and the NPC We cellular cholesterol and filipin in NPC1I1061T and fibroblasts that were in medium with of cholesterol accumulation is in the NPC1I1061T fibroblasts to in fibroblasts with and reduces cholesterol in the mutant by and cholesterol that of with and the in and of NPC1I1061T mutant of the mutant with 25-HC, but not 27-HC, reduces filipin but to a than is with the NPC1I1061T mutant and is unable to correct the filipin These results show that oxysterols are of cholesterol the sterol trafficking defect in the NPC1I1061T with oxysterols filipin in NPC1I1061T and mutant fibroblasts were for in DMEM, with DMEM, was In the present study we that NPC1 and NPC2 function in the regulation of cellular cholesterol homeostasis through generation of LDL cholesterol-derived Our results and the findings of Liscum et L. Faust J.R. J. Biol. Chem. 1987; 262: 17002-17008Abstract Full Text PDF PubMed Google Scholar). we demonstrate that in response to LDL cholesterol NPC1 and NPC2, loss of function mutants fail to appropriately suppress cholesterol synthesis and uptake to of SREBP-dependent gene we show that gene expression is in NPC we the ER cholesterol in the and demonstrate that ER cholesterol fail to for in of cholesterol esterification and the biochemical defects in the NPC we find that LDL production of and is in both NPC1 and NPC2 Moreover, in the NPC mutants the degree of with the severity of the sterol regulatory we show that treatment with oxysterols reduces cholesterol overload in both NPC1 and NPC2 and is able to correct the NPC1I1061T phenotype, the most prevalent NPC1 disease K. Am. J. Hum. Genet. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is not that a block in the of LDL cholesterol in NPC mutants to cellular of findings the that in production is linked to the of NPC disease. have shown that both NPC1 and NPC2 mutants accumulate free cholesterol in lysosomes and exhibit We show that cholesterol overload in both NPC1 and NPC2 mutants results from the of LDL cholesterol to both suppress SREBP-dependent gene expression and promote responses. on the mutant has a phenotype than the a between NPC1 and NPC2 in to from NPC1 and NPC2 present the NPC2 are NPC1 and NPC2 to the generation of LDL cholesterol-derived We that the NPC proteins LDL cholesterol to intracellular of Our findings that in fibroblasts NPC1 and NPC2 function in of free cholesterol to both the sterol and the cholesterol Biochim. Biophys. Acta. 2000; 1529: PubMed Scopus Google Scholar). Thus, in NPC disease, the failure to generate of and of SREBP-dependent gene expression and of of LDL uptake and de novo cholesterol with impaired cholesterol results in a accumulation of free cholesterol. of NPC1 mutants to and has been shown to cellular cholesterol Y. J. M. T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). We show that treatment with oxysterols the NPC1I1061T phenotype and a for Our findings that oxysterols of the NPC phenotype by cholesterol synthesis and we a role for oxysterols in mobilization of free cholesterol from the NPC The by which oxysterols suppress sterol synthesis and LDL cholesterol uptake is not has been that 25-HC, promote of free cholesterol from the plasma membrane to the ER Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). free cholesterol in the ER regulatory SCAP-mediated trafficking of to the Golgi, SREBP proteolysis (17Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). The that free cholesterol, but not 25-HC, the of in support to this the by which this to the regulation of sterol homeostasis is not yet L. M.S. Goldstein J.L. Full Text Full Text PDF PubMed Scopus Google Scholar). In the present study we an in esterification to the of cholesterol in the Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). have shown that the of the by this with ER membrane cholesterol in Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. M. T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Our that the ER cholesterol in NPC mutants is is in with an study Y. J. M. T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). does the ER cholesterol in NPC mutants fail to cellular cholesterol and have that the of the ER cholesterol is by the of the plasma membrane Y. J. M. T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). in NPC mutants the plasma membrane cholesterol is the of cholesterol between the and membrane is impaired Y. J. M. T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). is that production of LDL cholesterol-derived which is in NPC the of the plasma membrane by the of cholesterol to the oxysterols on the sterol regulatory through a These to in are to of sterol synthesis both in and in J. Biol. Chem. Full Text PDF PubMed Google Scholar, Science. PubMed Scopus Google Scholar, H. J. Full Text PDF PubMed Google Scholar) and are of of pathways (19Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf PubMed Scopus Google Scholar, Y. S.D. C.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). have sterol regulatory in fibroblasts from patients with a disorder by in sterol and characterized by of M. J. Biol. Chem. Full Text PDF PubMed Scopus Google Scholar). The role of a of whole sterol is in of a in whole cholesterol the in sterol synthesis was not that than production of to the regulation of sterol synthesis in Dietschy J.M. Turley S.D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). NPC on the fail to appropriately generate both and 27-HC, of a defect in production that the cholesterol overload with in NPC1–/– show increased cholesterol synthesis in (16Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Am. J. Physiol. 1999; 276: E336-E344PubMed Google Scholar). not regulate lipid and cholesterol but in the synthesis and of oxysterols a of cholesterol to sterol to the liver to lipid homeostasis Biochim. Biophys. Acta. 2000; 1529: PubMed Scopus Google Scholar, J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). In the cholesterol is 24-HC, which the a to of cholesterol homeostasis in this D. A. U. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). In NPC disease, failure to generate oxysterols not of cellular cholesterol, but the of cholesterol synthesis and lipoprotein to accumulation of free cholesterol. in the which of both NPC1 and J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, A. R. J. 2000; PubMed Scopus Google rates of to cellular homeostasis. a a for the loss of this in NPC disease C. H. S. Am. J. Google Scholar, J. H. S. J. PubMed Scopus Google Scholar, Y. S. Pentchev P.G. K. PubMed Scopus Google Scholar). Our that treatment with oxysterols the cellular NPC1I1061T phenotype the to the central nervous system used to NPC disease. We A. Fensom and M. Vanier for NPC mutant We are for P. Pentchev for of this