Altered vitamin E status in Niemann-Pick type C disease
2011; Elsevier BV; Volume: 52; Issue: 7 Linguagem: Inglês
10.1194/jlr.m015560
ISSN1539-7262
AutoresLynn Ulatowski, Robert S. Parker, Cristin Davidson, Nicole M. Yanjanin, Thomas J. Kelley, Deborah A. Corey, Jeffrey Atkinson, Forbes D. Porter, Hiroyuki Arai, Steven U. Walkley, Danny Manor,
Tópico(s)Glycogen Storage Diseases and Myoclonus
ResumoVitamin E (α-tocopherol) is the major lipid-soluble antioxidant in many species. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by mutations in the NPC1 or NPC2 gene, which regulates lipid transport through the endocytic pathway. NPC disease is characterized by massive intracellular accumulation of unesterified cholesterol and other lipids in lysosomal vesicles. We examined the roles that NPC1/2 proteins play in the intracellular trafficking of tocopherol. Reduction of NPC1 or NPC2 expression or function in cultured cells caused a marked lysosomal accumulation of vitamin E in cultured cells. In vivo, tocopherol significantly accumulated in murine Npc1-null and Npc2-null livers, Npc2-null cerebella, and Npc1-null cerebral cortices. Plasma tocopherol levels were within the normal range in Npc1-null and Npc2-null mice, and in plasma samples from human NPC patients. The binding affinity of tocopherol to the purified sterol-binding domain of NPC1 and to purified NPC2 was significantly weaker than that of cholesterol (measurements kindly performed by R. Infante, University of Texas Southwestern Medical Center, Dallas, TX). Taken together, our observations indicate that functionality of NPC1/2 proteins is necessary for proper bioavailability of vitamin E and that the NPC pathology might involve tissue-specific perturbations of vitamin E status. Vitamin E (α-tocopherol) is the major lipid-soluble antioxidant in many species. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by mutations in the NPC1 or NPC2 gene, which regulates lipid transport through the endocytic pathway. NPC disease is characterized by massive intracellular accumulation of unesterified cholesterol and other lipids in lysosomal vesicles. We examined the roles that NPC1/2 proteins play in the intracellular trafficking of tocopherol. Reduction of NPC1 or NPC2 expression or function in cultured cells caused a marked lysosomal accumulation of vitamin E in cultured cells. In vivo, tocopherol significantly accumulated in murine Npc1-null and Npc2-null livers, Npc2-null cerebella, and Npc1-null cerebral cortices. Plasma tocopherol levels were within the normal range in Npc1-null and Npc2-null mice, and in plasma samples from human NPC patients. The binding affinity of tocopherol to the purified sterol-binding domain of NPC1 and to purified NPC2 was significantly weaker than that of cholesterol (measurements kindly performed by R. Infante, University of Texas Southwestern Medical Center, Dallas, TX). Taken together, our observations indicate that functionality of NPC1/2 proteins is necessary for proper bioavailability of vitamin E and that the NPC pathology might involve tissue-specific perturbations of vitamin E status. Niemann-Pick type C (NPC) disease is a heritable lysosomal storage disorder, in which the intracellular transport of lipids is perturbed (1Brady R.O. Carstea E.D. Pentchev P.G. The Niemann-Pick Diseases Group.in: Rosenberg R.N. Pruisner S.B. DiMauro S. Barchi R.L. The Molecular And Genetic Basis of Neurological Disease. Butterworth-Heinmann, Boston1997: 387-403Google Scholar). The cellular phenotype of NPC disease is massive accumulation of cholesterol and other lipids in membranous organelles derived from late endosomes and lysosomes (2Pentchev P.G. Comly M.E. Kruth H.S. Vanier M.T. Wenger D.A. Patel S. Brady R.O. A defect in cholesterol esterification in Niemann-Pick disease (type C) patients.Proc. Natl. Acad. Sci. USA. 1985; 82: 8247-8251Crossref PubMed Scopus (320) Google Scholar, 3Blanchette-Mackie E.J. Dwyer N.K. Amende L.M. Kruth H.S. Butler J.D. Sokol J. Comly M.E. Vanier M.T. August J.T. 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In light of these observations, we hypothesized that proper intracellular trafficking of vitamin E (and in turn, adequate antioxidant protection) depends on timely egress from the lysosome and, therefore, on the functionality of NPC1/2. We describe here our findings regarding α-tocopherol status in cells that express defective alleles or reduced expression of NPC1/2, in various tissues from mice in which expression of NPC1/2 is disrupted and in plasma from human NPC patients.MATERIALS AND METHODSCell cultureHuman fibroblasts harboring the p.P237S and p.I1061T missense mutations in the NPC1 gene were obtained from Coriell Cell Repository (GM03123; Camden, NJ) and grown in Eagle's minimum essential medium with Earle's salts, 2 mM L-glutamine and 15% fetal bovine serum at 37°C and 5% CO2 (38Manson M.E. Corey D.A. White N.M. Kelley T.J. cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells.Am. J. Physiol. Lung Cell. Mol. Physiol. 2008; 295: L809-L819Crossref PubMed Scopus (35) Google Scholar). Control human fibroblasts (CRL-2076) were obtained from American Type Culture Collection (Manassas, VA). Immortalized human hepatocytes (IHH) (39Basu A. Meyer K. Ray R.B. Ray R. Hepatitis C virus core protein is necessary for the maintenance of immortalized human hepatocytes.Virology. 2002; 298: 53-62Crossref PubMed Scopus (50) Google Scholar, 40Ray R.B. Meyer K. Ray R. Hepatitis C virus core protein promotes immortalization of primary human hepatocytes.Virology. 2000; 271: 197-204Crossref PubMed Scopus (146) Google Scholar) were a generous gift from R. Ray (Saint Louis University, St. Louis, MO) and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% calf serum. Lentiviral short hairpin RNA (shRNA) constructs targeted against human NPC1, human NPC2, and a control shRNA in the pLKO vector (Open Biosystems, Huntsville, AL) were transfected into HEK293T cells using Lipofectamine-Plus (Invitrogen, Carlsbad, CA). Culture media were harvested 24 and 48 h posttransfection, pooled, and pelleted by centrifugation at 100,000 g for 1.5 h. The pellet was resuspended in PBS and used for polybrene-mediated (4 μg/ml) transduction of IHH cells using standard protocols. Stable knockdown clones were selected in media supplemented with puromycin (10 μg/ml; Sigma Chemical Co., St. Louis, MO) 48 h after transduction. Knockdown efficiency was evaluated by immunoblotting using antibodies raised against NPC1 (Abcam, Cambridge, MA) or NPC2 (generous gift of Peter Lobel, Rutgers University, Rutgers, NJ). For evaluating the endogenous expression levels of the α-tocopherol transfer protein (TTP), samples were immunoblotted using the A8E5 anti-TTP antibody (H. Arai).Disease modelsThe Npc1−/− mice (BABLc/NPCnih) were originally described in Ref. 12Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. et al.Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.Science. 1997; 277: 232-235Crossref PubMed Scopus (692) Google Scholar, and the Npc2−/− mice were described in Ref. 41Sleat D.E. Wiseman J.A. El-Banna M. Price S.M. Verot L. Shen M.M. Tint G.S. Vanier M.T. Walkley S.U. Lobel P. Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport.Proc. Natl. Acad. Sci. USA. 2004; 101: 5886-5891Crossref PubMed Scopus (270) Google Scholar. Human serum samples were collected from NPC1 patients and healthy age-appropriate unaffected subjects under a clinical protocol (06-CH-0186) approved by the NICHD Institutional Review Board of the National Institute of Child Health and Human Development. Both consent and assent, if appropriate, were obtained. Serum samples were de-identified and maintained at −80 degrees centigrade.Fluorescence microscopyCells were plated on poly-L-lysine coated glass coverslips in 24-well tissue culture plates. NBD-cholesterol (Invitrogen) and NBD-tocopherol (42Morley S. Cross V. Cecchini M. Nava P. Atkinson J. Manor D. Utility of a fluorescent vitamin E analogue as a probe for tocopherol transfer protein activity.Biochemistry. 2006; 45: 1075-1081Crossref PubMed Scopus (30) Google Scholar, 43Nava P. Cecchini M. Chirico S. Gordon H. Morley S. Manor D. Atkinson J. Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein.Bioorg. Med. Chem. 2006; 14: 3721-3736Crossref PubMed Scopus (37) Google Scholar) were complexed to serum lipoprotein as described earlier (35Qian J. Morley S. Wilson K. Nava P. Atkinson J. Manor D. Intracellular trafficking of vitamin E in hepatocytes: Role of tocopherol transfer protein.J Lipid Res. 2005; 46: 2072-2082Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 44Asmis R. Physical partitioning is the main mechanism of alpha-tocopherol and cholesterol transfer between lipoproteins and P388D1 macrophage-like cells.Eur. J. Biochem. 1997; 250: 600-607Crossref PubMed Scopus (11) Google Scholar) and added to the culture media to a final concentration of 20 μM and incubated for 17 h at 37°C. The fluorescent lipid was "chased" by incubation in normal media for 3 h more. Cells were fixed for 20 min in 3.7% paraformaldehyde and mounted in SlowFade Gold antifade reagent (Invitrogen) prior to imaging on a confocal or inverted fluorescence microscope (Zeiss LSM 510 and Leica DMI 4000B, respectively). For quantitation of accumulated fluorophores, ten microscopic images were captured under identical conditions, each containing 30-60 cells. Fluorescence intensities were quantitated using Image J software (http://rsbweb.nih.gov/ij/index.html). The RGB images were converted to an 8-bit images; a common threshold set for all images. For colocalization studies, LysoTracker Red DND-99 (75 nM, Invitrogen) was added 30 min prior to fixing. For visualization of free cholesterol, fixed and permeabilized cells were incubated with 25 μg/ml filipin (Streptomyces filipinensis; Sigma Chemical) for 1 h at room temperature in the dark, prior to washing in PBS and visualization.Analytical determinationsTotal cholesterol.Cells were harvested, resuspended in PBS, and lysed by repeated passing through a 22-gauge needle. Total cholesterol was measured using the Amplex Red Cholesterol Assay kit (Invitrogen) according to manufacturer's protocol. Fluorescence was excited at 530 nm and emission was collected at 590 nm on a Tecan GENios Pro plate reader (Tecan, Durham, NC). Total cholesterol was normalized to total protein, as determined by the Bio-Rad protein assay kit.Tocopherols and free cholesterol.Serum and appropriate tissues from Npc1−/− and Npc2−/− mice and their wild-type littermates were freshly excised and flash-frozen as described previously (45Davidson C.D. Ali N.F. Micsenyi M.C. Stephney G. Renault S. Dobrenis K. Ory D.S. Vanier M.T. Walkley S.U. Chronic cyclodextrin treatment of murine Niemann-Pick C disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression.PLoS ONE. 2009; 4: e6951Crossref PubMed Scopus (345) Google Scholar). Lipids were extracted, silylated, and analyzed by GC-MS on a Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-Packard 5872 mass selective detector operated in selected ion mode as previously described (46Sontag T.J. Parker R.S. Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherol-omega-hydroxylase.J. Lipid Res. 2007; 48: 1090-1098Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Deuterated α-tocopherol added prior to extraction served as an internal standard. Monitored masses of trimethylsilyl ethers (TMS) were 511.6 (d9-α-tocopherol-TMS), 502.6 (d0-α-tocopherol-TMS), 488.6 (d0-γ-tocopherol-TMS), and 458.7 (cholesterol-TMS). A previously determined detector response correction factor was applied in quantitation of cholesterol. Tocopherol and unesterified cholesterol concentrations were normalized to tissue wet weight.Binding of tocopherol to purified NPC1 and NPC2The affinity of α-tocopherol to the purified NPC1/2 proteins was measured by Rodney Infante and Joseph Goldstein at the University of Texas Southwestern Medical Center (Dallas, TX) using a published assay based on competition with radio-labeled cholesterol (17Kwon H.J. Abi-Mosleh L. Wang M.L. Deisenhofer J. Goldstein J.L. Brown M.S. Infante R.E. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.Cell. 2009; 137: 1213-1224Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). Briefly, 4 pmol purified sterol binding domain (NTD) of NPC1, or 8 pmol purified full-length NPC2 were incubated overnight with 130 nmol [3H]cholesterol at 4°C. The proteins were then incubated with 6 μM unlabeled competitor (cholesterol, epicholesterol, 25-hydroxycholesterol, or dl-α tocopherol), and protein-bound radioactivity was measured after affinity chromatography with nickel-agarose and scintillation counting.Statistical analysesStatistical significance of data was determined using unpaired Student's t-test. P values < 0.05 were taken as the threshold of significance. Data were analyzed and graphed using the IgorPro software package (Wavemetrics, Inc., Portland, OR).RESULTSα-tocopherol accumulates in NPC-affected fibroblastsThe NPC1 and NPC2 proteins are residents of the lysosome that are required for proper transit of cholesterol through the endocytic pathway (15Storch J. Xu Z. Niemann-Pick C2 (NPC2) and intracellular cholesterol trafficking.Biochim. Biophys. Acta. 2009; 1791: 671-678Crossref PubMed Scopus (141) Google Scholar, 47Liscum L. Sturley S.L. Intracellular trafficking of Niemann-Pick C proteins 1 and 2: obligate components of subcellular lipid transport.Biochim. Biophys. Acta. 2004; 1685: 22-27Crossref PubMed Scopus (96) Google Scholar). Given that sphingomyelin, glycosphingolipids, and phospholipids also accumulate in NPC-affected lysosomes (2Pentchev P.G. Comly M.E. Kruth H.S. Vanier M.T. Wenger D.A. Patel S. Brady R.O. A defect in cholesterol esterification in Niemann-Pick disease (type C) patients.Proc. Natl. Acad. Sci. 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Using fluorescence microscopy, we visualized the accumulation of NBD-tocopherol in cultured fibroblasts isolated from an NPC-affected patient (harboring the c.709C>T and c.3182T>C substitutions in the NPC1 gene) and control fibroblasts. As shown in Fig. 1, control fibroblasts retained very little NBD-tocopherol. However, NPC-fibroblasts accumulated much higher (ca. 3-fold) levels of the fluorescent vitamin, appearing in a punctate, perinuclear distribution pattern. These observations indicate that egress of α-tocopherol from the endocytic compartment requires a functional NPC1 protein.Generation and characterization of NPC1 and NPC2 knockdown hepatocyte cell linesAlthough genetic defects in NPC1 and NPC2 lead to severe accumulation of cholesterol in the liver (55Beltroy E.P. Richardson J.A. Horton J.D. Turley S.D. Dietschy J.M. Cholesterol accumulation and liver cell death in mice with Niemann-Pick type C disease.Hepatology. 2005; 42: 886-893Crossref PubMed Scopus (102) Google Scholar), no hepatocyte cell culture model is presently available to study the disease. We therefore generated lentiviruses that encode s
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