MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein
2009; Elsevier BV; Volume: 51; Issue: 5 Linguagem: Inglês
10.1194/jlr.m002345
ISSN1539-7262
AutoresMark Charman, Barry E. Kennedy, Nolan Osborne, Barbara Karten,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoNiemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder, caused in most cases by mutations in the late endosomal protein NPC1. A hallmark of NPC disease is endosomal cholesterol accumulation and an impaired cholesterol homeostatic response, which might affect cholesterol transport to mitochondria and, thus, mitochondrial and cellular function. This study aimed to characterize mitochondrial cholesterol homeostasis in NPC disease. Using wild-type and NPC1-deficient Chinese hamster ovary cells, stably transfected with a CYP11A1 complex to assess mitochondrial cholesterol import by pregnenolone production, we show that cholesterol transport to the mitochondrial inner membrane is not affected by loss of NPC1. However, mitochondrial cholesterol content was higher in NPC1-deficient than in wild-type cells. Cholesterol transport to the mitochondrial inner membrane increased markedly upon exposure of cholesterol-deprived cells to lipoproteins, indicating transport of endosomal cholesterol to mitochondria. Reduction of endosomal metastatic lymph node protein 64 (MLN64) by RNA interference decreased cholesterol transport to the mitochondrial inner membrane and reduced mitochondrial cholesterol levels in NPC1-deficient cells, suggesting that MLN64 transported cholesterol to mitochondria even in the absence of NPC1. In summary, this study describes a transport pathway for endosomal cholesterol to mitochondria that requires MLN64, but not NPC1, and that may be responsible for increased mitochondrial cholesterol in NPC disease. Niemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder, caused in most cases by mutations in the late endosomal protein NPC1. A hallmark of NPC disease is endosomal cholesterol accumulation and an impaired cholesterol homeostatic response, which might affect cholesterol transport to mitochondria and, thus, mitochondrial and cellular function. This study aimed to characterize mitochondrial cholesterol homeostasis in NPC disease. Using wild-type and NPC1-deficient Chinese hamster ovary cells, stably transfected with a CYP11A1 complex to assess mitochondrial cholesterol import by pregnenolone production, we show that cholesterol transport to the mitochondrial inner membrane is not affected by loss of NPC1. However, mitochondrial cholesterol content was higher in NPC1-deficient than in wild-type cells. Cholesterol transport to the mitochondrial inner membrane increased markedly upon exposure of cholesterol-deprived cells to lipoproteins, indicating transport of endosomal cholesterol to mitochondria. Reduction of endosomal metastatic lymph node protein 64 (MLN64) by RNA interference decreased cholesterol transport to the mitochondrial inner membrane and reduced mitochondrial cholesterol levels in NPC1-deficient cells, suggesting that MLN64 transported cholesterol to mitochondria even in the absence of NPC1. In summary, this study describes a transport pathway for endosomal cholesterol to mitochondria that requires MLN64, but not NPC1, and that may be responsible for increased mitochondrial cholesterol in NPC disease. STARTing to understand MLN64 function in cholesterol transportJournal of Lipid ResearchVol. 51Issue 8PreviewIn most cell types, intracellular transport mechanisms that mediate cholesterol movement into the mitochondrial outer and inner membranes are not well understood. By contrast in steroidogenic tissues, the steroidogenic acute regulatory (StAR) protein in conjunction with the benzodiazepine receptor have been defined as key mediators of cholesterol flux toward the mitochondrial inner membrane (1, 2). Furthermore, StAR-mediated cholesterol transfer is critical for steroid synthesis, and deficiencies in this protein are responsible for the rare but severe disorder of human steroidogenesis known as congenital lipoid adrenal hyperplasia (1, 2). Full-Text PDF Open Access Niemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder caused by mutations in NPC1 or NPC2 (1Carstea 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.Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Crossref PubMed Scopus (1238) Google Scholar, 2Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Crossref PubMed Scopus (714) Google Scholar). NPC1 is a late endosomal transmembrane protein with a sterol-sensing domain homologous to the cholesterol sensor sterol regulatory element binding protein cleavage activating protein (3Davies J.P. Ioannou Y.A. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein.J. Biol. Chem. 2000; 275: 24367-24374Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 4Millard E.E. Gale S.E. Dudley N. Zhang J. Schaffer J.E. Ory D.S. The sterol-sensing domain of the Niemann-Pick C1 (NPC1) protein regulates trafficking of low density lipoprotein cholesterol.J. Biol. Chem. 2005; 280: 28581-28590Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar); NPC2 is a small lumenal protein in late endosomes/lysosomes (5Friedland N. Liou H.L. Lobel P. Stock A.M. Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease.Proc. Natl. Acad. Sci. USA. 2003; 100: 2512-2517Crossref PubMed Scopus (260) Google Scholar, 6Ko D.C. Binkley J. Sidow A. Scott M.P. The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels.Proc. Natl. Acad. Sci. USA. 2003; 100: 2518-2525Crossref PubMed Scopus (167) Google Scholar). Both NPC1 and NPC2 can bind cholesterol (5Friedland N. Liou H.L. Lobel P. Stock A.M. Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease.Proc. Natl. Acad. Sci. USA. 2003; 100: 2512-2517Crossref PubMed Scopus (260) Google Scholar, 6Ko D.C. Binkley J. Sidow A. Scott M.P. The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels.Proc. Natl. Acad. Sci. USA. 2003; 100: 2518-2525Crossref PubMed Scopus (167) Google Scholar, 7Infante R.E. Wang M.L. Radhakrishnan A. Kwon H.J. Brown M.S. Goldstein J.L. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes.Proc. Natl. Acad. Sci. USA. 2008; 105: 15287-15292Crossref PubMed Scopus (345) Google Scholar, 8Ohgami N. Ko D.C. Thomas M. Scott M.P. Chang C.C. Chang T.Y. Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain.Proc. Natl. Acad. Sci. USA. 2004; 101: 12473-12478Crossref PubMed Scopus (171) Google Scholar); NPC1 also has affinity for oxysterols (9Infante R.E. Abi-Mosleh L. Radhakrishnan A. Dale J.D. Brown M.S. Goldstein J.L. Purified NPC1 protein. I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein.J. Biol. Chem. 2008; 283: 1052-1063Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). All NPC1-deficient cells accumulate cholesterol in late endosomal multivesicular bodies and have an impaired homeostatic response of the sterol regulatory element binding protein pathway to exogenous cholesterol (10Liscum L. Niemann-Pick type C mutations cause lipid traffic jam.Traffic. 2000; 1: 218-225Crossref PubMed Scopus (134) Google Scholar). Based on these observations, it was proposed that NPC1 acts as a cholesterol sensor or transporter and is required for cholesterol egress from endosomes to plasma membrane and to the regulatory pool in the endoplasmic reticulum (7Infante R.E. Wang M.L. Radhakrishnan A. Kwon H.J. Brown M.S. Goldstein J.L. NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes.Proc. Natl. Acad. Sci. USA. 2008; 105: 15287-15292Crossref PubMed Scopus (345) Google Scholar, 10Liscum L. Niemann-Pick type C mutations cause lipid traffic jam.Traffic. 2000; 1: 218-225Crossref PubMed Scopus (134) Google Scholar). NPC1-deficient cells also sequester a variety of other lipids in their endosomes (11Walkley S.U. Suzuki K. Consequences of NPC1 and NPC2 loss of function in mammalian neurons.Biochim. Biophys. Acta. 2004; 1685: 48-62Crossref PubMed Scopus (223) Google Scholar, 12Lloyd-Evans E. Morgan A.J. He X. Smith D.A. Elliot-Smith E. Sillence D.J. Churchill G.C. Schuchman E.H. Galione A. Platt F.M. Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium.Nat. Med. 2008; 14: 1247-1255Crossref PubMed Scopus (650) Google Scholar), and the possibility remains that cholesterol accumulation is not the primary defect. The exact function of NPC1 and how its loss leads to the observed neuropathology remain unknown. Regardless of the primary storage material in NPC1-deficient endosomes, the sequestration of cholesterol has a widespread impact on cellular cholesterol distribution. Recently, it has been proposed that changes in mitochondrial cholesterol contribute to NPC pathology (13Schneiter R. Intracellular sterol transport in eukaryotes, a connection to mitochondrial function?.Biochimie. 2007; 89: 255-259Crossref PubMed Scopus (18) Google Scholar). Cholesterol is required as a precursor for steroid and oxysterol synthesis at the mitochondrial inner membrane and as a component of mitochondrial membranes. Increased mitochondrial cholesterol can lead to mitochondrial dysfunction, including reduced fluidity of mitochondrial membranes (14Colell A. Garcia-Ruiz C. Lluis J.M. Coll O. Mari M. Fernandez-Checa J.C. Cholesterol impairs the adenine nucleotide translocator-mediated mitochondrial permeability transition through altered membrane fluidity.J. Biol. Chem. 2003; 278: 33928-33935Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 15Mari M. Caballero F. Colell A. Morales A. Caballeria J. Fernandez A. Enrich C. Fernandez-Checa J.C. Garcia-Ruiz C. Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.Cell Metab. 2006; 4: 185-198Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), reduced ATP generation (16Yu W. Gong J.S. Ko M. Garver W.S. Yanagisawa K. Michikawa M. Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function.J. Biol. Chem. 2005; 280: 11731-11739Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), and decreased mitochondrial glutathione import (15Mari M. Caballero F. Colell A. Morales A. Caballeria J. Fernandez A. Enrich C. Fernandez-Checa J.C. Garcia-Ruiz C. Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.Cell Metab. 2006; 4: 185-198Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). In line with the theory that cholesterol entrapment in NPC1-deficient endosomes limits its availability to the rest of the cell, it was found that steroid and oxysterol levels were decreased in NPC1-deficient murine brain and fibroblasts (17Griffin L.D. Gong W. Verot L. Mellon S.H. Niemann-Pick type C disease involves disrupted neurosteroidogenesis and responds to allopregnanolone.Nat. Med. 2004; 10: 704-711Crossref PubMed Scopus (326) Google Scholar, 18Mellon S. Gong W. Griffin L.D. Niemann pick type C disease as a model for defects in neurosteroidogenesis.Endocr. Res. 2004; 30: 727-735Crossref PubMed Scopus (27) Google Scholar, 19Chen G. Li H.M. Chen Y.R. Gu X.S. Duan S. Decreased estradiol release from astrocytes contributes to the neurodegeneration in a mouse model of Niemann-Pick disease type C.Glia. 2007; 55: 1509-1518Crossref PubMed Scopus (60) Google Scholar, 20Fluegel M.L. Parker T.J. Pallanck L.J. Mutations of a Drosophila NPC1 gene confer sterol and ecdysone metabolic defects.Genetics. 2006; 172: 185-196Crossref PubMed Scopus (73) Google Scholar, 21Roff C.F. Strauss 3rd, J.F. Goldin E. Jaffe H. Patterson M.C. Agritellis G.C. Hibbs A.M. Garfield M. Brady R.O. Pentchev P.G. The murine Niemann-Pick type C lesion affects testosterone production.Endocrinology. 1993; 133: 2913-2923Crossref PubMed Scopus (45) Google Scholar, 22Zhang J.R. Coleman T. Langmade S.J. Scherrer D.E. Lane L. Lanier M.H. Feng C. Sands M.S. Schaffer J.E. Semenkovich C.F. et al.Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking.J. Clin. Invest. 2008; 118: 2281-2290PubMed Google Scholar). In seeming contradiction, several groups have reported increased cholesterol in mitochondria isolated from brain or liver of NPC1-deficient mice (15Mari M. Caballero F. Colell A. Morales A. Caballeria J. Fernandez A. Enrich C. Fernandez-Checa J.C. Garcia-Ruiz C. Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.Cell Metab. 2006; 4: 185-198Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 16Yu W. Gong J.S. Ko M. Garver W.S. Yanagisawa K. Michikawa M. Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function.J. Biol. Chem. 2005; 280: 11731-11739Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 23Fernandez A. Llacuna L. Fernandez-Checa J.C. Colell A. Mitochondrial cholesterol loading exacerbates amyloid beta peptide-induced inflammation and neurotoxicity.J. Neurosci. 2009; 29: 6394-6405Crossref PubMed Scopus (130) Google Scholar). The mechanisms by which cholesterol is transported to the mitochondrial outer and inner membranes under basal conditions are not well defined. In steroidogenic cells, cholesterol transport to the mitochondrial inner membrane, which is rate limiting for steroid synthesis, is mediated by the steroidogenic acute regulatory (StAR) protein in conjunction with the translocator protein (formerly known as peripheral benzodiazepine receptor) (24Miller W.L. Mechanism of StAR's regulation of mitochondrial cholesterol import.Mol. Cell. Endocrinol. 2007; 265–266: 46-50Crossref PubMed Scopus (45) Google Scholar, 25Papadopoulos V. Liu J. Culty M. Is there a mitochondrial signaling complex facilitating cholesterol import?.Mol. Cell. Endocrinol. 2007; 265–266: 59-64Crossref PubMed Scopus (109) Google Scholar). However, StAR-mediated transport is low under basal, nonstimulated conditions and in nonsteroidogenic cells, which do not express significant amounts of StAR. Since even under these conditions, cholesterol is needed for the upkeep of mitochondrial membranes and in some cases for oxysterol synthesis, other mechanisms of mitochondrial cholesterol import must exist. Recently, it was proposed that plasma membrane cholesterol transported via cytosolic transport proteins served as a source for mitochondrial oxysterol production (26Lange Y. Steck T.L. Ye J. Lanier M.H. Molugu V. Ory D.S. Regulation of fibroblast mitochondrial 27-hydroxycholesterol production by active plasma membrane cholesterol.J Lipid Res. 2009; 50: 1881-1888Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), but the mechanism was not defined in detail. Other potential mediators of mitochondrial cholesterol import include proteins that contain a lipid-binding domain homologous to the C terminus of StAR [START proteins (27Alpy F. Tomasetto C. Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.J. Cell Sci. 2005; 118: 2791-2801Crossref PubMed Scopus (297) Google Scholar)]. Among these, endosomal metastatic lymph node protein 64 (MLN64) is of particular interest, since its START domain binds cholesterol and it has been shown to transport cholesterol to mitochondria when expressed as a soluble protein lacking the transmembrane N terminus of MLN64 (28Alpy F. Stoeckel M.E. Dierich A. Escola J.M. Wendling C. Chenard M.P. Vanier M.T. Gruenberg J. Tomasetto C. Rio M.C. The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein.J. Biol. Chem. 2001; 276: 4261-4269Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 29Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss 3rd, J.F. MLN64 mediates mobilization of lysosomal cholesterol to steroidogenic mitochondria.J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 30Tuckey R.C. Bose H.S. Czerwionka I. Miller W.L. Molten globule structure and steroidogenic activity of N-218 MLN64 in human placental mitochondria.Endocrinology. 2004; 145: 1700-1707Crossref PubMed Scopus (51) Google Scholar). In view of the strong link between mitochondrial cholesterol and mitochondrial function and the central role of mitochondria in neurodegenerative disease (31Beal M.F. Mitochondria take center stage in aging and neurodegeneration.Ann. Neurol. 2005; 58: 495-505Crossref PubMed Scopus (788) Google Scholar), this study was designed to elucidate mechanisms of basal mitochondrial cholesterol import in wild-type and NPC1-deficient cells. We show that mitochondrial cholesterol import does not require functional NPC1 and that MLN64 can mediate cholesterol transport from endosomes to mitochondria under basal conditions, its contribution likely depending on the relative availability of cholesterol from different pools. Furthermore, our data indicate that in NPC1-deficient cells, transfer of cholesterol from outer to inner mitochondrial membrane may become rate limiting, leading to cholesterol buildup in mitochondrial outer membranes. Cell culture media, FBS, and supplements were obtained from Invitrogen. Geneticin was from Wisent Inc. Trilostane and 22R-hydroxycholesterol (22-OH-Chol) were purchased from Steraloids. The rabbit anti-pregnenolone antibody was purchased from MP Biologicals. Charcoal-coated dextran, filipin complex III, and mouse antitubulin antibodies were purchased from Sigma. Rabbit anti-human NPC1 antibodies that fully cross-react with NPC1 from mouse and hamster were obtained from Novus Biologicals. MLN64 antibodies were from Affinity Bioreagents or Abcam, goat anti-actin and rabbit anti-mouse Tom20 antibodies were from Santa Cruz Biotechnologies, and mouse anti-protein disulphide isomerase was from Assay Designs. Rabbit antibodies directed against the voltage-dependent anion channel (VDAC) or against lysosome-associated membrane protein (LAMP1), which cross-react with hamster VDAC or LAMP1, and rabbit anti-ferredoxin reductase antibodies were obtained from Abcam. Mitotracker Red CMXRos and Dynabeads M500 Subcellular were purchased from Invitrogen. Short interfering RNA (siRNA) sequences and transfection agent (dharmaFECT 4) were obtained from Dharmacon. [7-3H(N)]pregnenolone (1 mCi/ml and 11.5 Ci/mmol) was obtained from Perkin-Elmer Life Sciences. All other chemicals were from Sigma or Fisher Scientific. Human LDL was isolated from EDTA-plasma of apparently healthy, normolipidemic volunteers by KBr density gradient ultracentrifugation (32Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.Methods Enzymol. 1994; 233: 469-489Crossref PubMed Scopus (288) Google Scholar). Informed consent was obtained from all human volunteers, and the protocol was approved by the Human Research Ethics Board, Dalhousie University. Lipoprotein-deficient serum (LPDS) was prepared by density ultracentrifugation from FBS (32Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.Methods Enzymol. 1994; 233: 469-489Crossref PubMed Scopus (288) Google Scholar). All protein assays were performed with the BCA kit (Pierce). Wild-type Chinese hamster ovary (CHO) and NPC1-deficient 4-4-19 cell lines were a generous gift from L. Liscum (Tufts University, Boston, MA) and have been described and characterized previously (33Dahl N.K. Reed K.L. Daunais M.A. Faust J.R. Liscum L. Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.J. Biol. Chem. 1992; 267: 4889-4896Abstract Full Text PDF PubMed Google Scholar, 34Wojtanik K.M. Liscum L. The transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in NPC1 cells.J. Biol. Chem. 2003; 278: 14850-14856Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The 4-4-19 cell line expresses a nonfunctional NPC1 protein with a point mutation (Gly660Arg) in the sterol-sensing domain (L. Liscum, personal communication). To simplify the description of these cell lines together with the murine model carrying a null mutation in NPC1, we use the term "NPC1-deficiency" to include NPC1 dysfunction and loss of NPC1 protein. The expression vector F2-pcDNA3 encodes a fusion protein (F2) of human CYP11A1 (P450 side chain cleavage complex), ferredoxin reductase, and ferredoxin 1 (35Harikrishna J.A. Black S.M. Szklarz G.D. Miller W.L. Construction and function of fusion enzymes of the human cytochrome P450scc system.DNA Cell Biol. 1993; 12: 371-379Crossref PubMed Scopus (136) Google Scholar, 36Huang M.C. Miller W.L. Creation and activity of COS-1 cells stably expressing the F2 fusion of the human cholesterol side-chain cleavage enzyme system.Endocrinology. 2001; 142: 2569-2576Crossref PubMed Scopus (11) Google Scholar) and was a kind gift from W. L. Miller (University of California, San Francisco, CA). The open reading frame was excised from the pcDNA3 backbone using the restriction enzymes EcoRI and KpnI and inserted into the pcDNA3.1(+) backbone (Invitrogen). To create cell lines stably expressing F2-pcDNA3.1, CHO and 4-4-19 cells were transfected with the F2-pcDNA3.1 vector using electroporation (Microporator; Montreal Biotech). Monoclonal colonies were selected for their survival in 500 µg/ml geneticin following serial dilution. Cells were cultured in Ham's F12 medium containing 5% FBS, antibiotics, and 300 µg/ml geneticin to keep the cells under selection pressure. Hereafter, cells stably expressing the F2-fusion protein will be designated with the prefix "F2." Expression of F2-fusion complex was measured by RT-PCR. Total RNA was extracted from F2 cells using Trizol and reverse transcribed using Superscript II (Invitrogen), and cDNA was amplified using primers directed against cyclophilin and F2 [cyclophilin forward, 5′-TCTTCTTGCTGGTCTTGCCATTCC-3′; reverse, 5′-TCCAAAGACAGCAGAAAACTTTCG-3′; F2 forward (P450scc), 5′-AGTGGCCATCTATGCTCTGG-3′; reverse (ferredoxin reductase), 5′-ATGTCCGTTCTCTCCAGGTG-3′]. PCR products and DNA size standards (Fermentas) were separated on a 1.5% agarose gel and visualized with GelRed (Biotium). Cells were harvested into PBS and homogenized in 1 ml ice-cold mitochondria isolation (MI) buffer (5 mM HEPES, 250 mM mannitol, 1 mM EGTA, 70 mM sucrose, pH 7.4, and protease inhibitors) with 30 strokes in a Dounce homogenizer. Cell homogenates were centrifuged at 800 g, 4°C for 5 min to remove nuclei and unbroken cells, followed by centrifugation of the supernatant at 12,000 g, 4°C for 15 min to yield crude mitochondria. Samples were used immediately or frozen at −20°C for no longer than 1 week. CYP11A1 activity was determined essentially as described (37Papadopoulos V. Guarneri P. Kreuger K.E. Guidotti A. Costa E. Pregnenolone biosynthesis in C6–2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.Proc. Natl. Acad. Sci. USA. 1992; 89: 5113-5117Crossref PubMed Scopus (219) Google Scholar). Cell homogenates, crude mitochondria, or the 12,000 g supernatant were incubated for 1 h at 37°C in reaction buffer (250 mM sucrose, 20 mM KCl, 15 mM Tris-EDTA-HCl, 10 mM KH2PO4, and 5 mM MgCl2, pH 7.2) with 2 mM NADPH, 2 mM malate/pyruvate, 5 μM 22-OH-Chol, 0.3% Tween, and an NADPH regenerating system (BD Biosciences). Trilostane (10 μM) was added during all incubations for pregnenolone measurement to inhibit conversion of pregnenolone to downstream steroids (38Potts G.O. Creange J.E. Hardomg H.R. Schane H.P. Trilostane, an orally active inhibitor of steroid biosynthesis.Steroids. 1978; 32: 257-267Crossref PubMed Scopus (227) Google Scholar). Reaction mixtures were assayed for pregnenolone by radioimmunoassay (RIA) according to the protocol provided by MP Biologicals. Cells were plated at a density of 200 cells/mm2 and grown for 48 h in growth medium. Cells were then washed twice in phenol red-free, serum-free Ham's F12/DMEM (1:1, v/v) (import medium) and incubated for 6 or 24 h in import medium containing 10 µM trilostane. In experiments to determine the maximum rate of pregnenolone formation, 5 µM 22-OH-Chol was added to the import medium. Import medium was collected for measurement of pregnenolone by RIA. Each assay included a standard curve of known amounts of pregnenolone and a positive control of import medium with trilostane spiked with 300 pg pregnenolone to test recovery. A negative control of import medium (containing trilostane) that was not incubated with cells was routinely included but did not give readings above background. Where indicated, cells were incubated for 48 h in Ham's F12 medium containing 5% LPDS with or without 50 µg/ml LDL and then washed and incubated for 24 h in import medium with 10 µM trilostane with or without 50 µg/ml LDL. Import medium and cells were collected and analyzed as above. CHO cells grown on coverslips were transiently transfected with F2-pcDNA3.1 vector by electroporation. Two days after transfection, cells were incubated with 50 nM Mitotracker Red CMXRos (Invitrogen) according to the manufacturer's protocol and then washed and fixed with 4% paraformaldehyde. Cells were permeabilized with 0.1% Triton X-100 for 5 min and blocked with 1% BSA in PBS followed by sequential incubation with antiferredoxin reductase antibody (1:100) and Cy2-conjugated affinity-purified donkey anti-rabbit IgG (Jackson Immunoresearch). Images were acquired on a Nikon TE2000 epifluorescence microscope equipped with a CCD camera (Orca-AG; Hamamatsu) at filter settings of 474/23 nm and 585/29 nm (excitation), dual-band dichroic, and 572/42 nm and 645/49 nm (emission), using a 60× oil immersion objective. Using these conditions, Mitotracker staining alone did not yield measurable fluorescence in the green channel. Cells grown on glass coverslips were fixed and stained with 50 µg/ml filipin as described (39Karten B. Vance D.E. Campenot R.B. Vance J.E. Cholesterol accumulates in cell bodies, but is decreased in distal axons, of Niemann-Pick C1-deficient neurons.J. Neurochem. 2002; 83: 1154-1163Crossref PubMed Scopus (131) Google Scholar). Images were acquired on a Nikon TE2000 epifluorescence microscope with CCD camera at filter settings of 387/11 nm (excitation) and 447/60 nm (emission) using a 20× objective. Cells were incubated for 48 h in Ham's F12 medium containing 5% LPDS, with or without 50 µg/ml LDL, washed twice in import medium, and incubated for 24 h in import medium containing 1 µCi/ml [14C]acetate, with or without 50 µg/ml LDL. Cellular lipids were extracted and separated by TLC with the solvent phase cyclohexane/ethyl acetate (3:2, v/v) (39Karten B. Vance D.E. Campenot R.B. Vance J.E. Cholesterol accumulates in cell bodies, but is decreased in distal axons, of Niemann-Pick C1-deficient neurons.J. Neurochem. 2002; 83: 1154-1163Crossref PubMed Scopus (131) Google Scholar). Radioactivity was determined in the band corresponding to unesterified cholesterol by liquid scintillation counting. Transfection with siRNA was performed according to the manufacturer's protocol using DharmaFECT 4 (Dharmacon). siRNA (Dharmacon) was added to the cells at a final concentration of 50 nM for siNT or siMLN64, or 25 nM each for transfection with a mixture of three siRNAs against NPC1, using DharmaFECT 4 at a final concentration of 1:250 (v/v). The cells were grown with the siRNA for 24 h, and then medium was replaced with growth medium for 24 h, before import medium with trilostane was added for 24 h. Pregnenolone formation was measured by RIA. siRNA sequences were as follows: hamster NPC1: NPC1-1, sense 5′-GGAAAGAGUUCAUGAAAUUUU-3′ and antisense 5′-AAUUUCAUGAACUCUUUCCUU-3′; NPC1-2, sense 5′-GGGAAAGAGUUCAUGAAAUUU-3′ and antisense 5′-AUUUCAUGAACUCUUUCCCUU-3′; NPC1-3, sense 5′-CCGAGUAAGCCGAGCAGAAUU-3′ and antisense 5′-UUCUGCUCGGCUUACUCGGUU-3′; MLN64, siGENOME duplex (1Carstea 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.Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Crossref PubMed Scopus (1238) Google Scholar), D-048833-01; siGENOME duplex (3Davies J.P. Ioannou Y.A. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein.J. Biol. Chem. 2000; 275: 24367-24374Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), D-048833-03; nontargeting siRNA: siCONTROL nontargeting siRNA #1, D-001210-01-05. The MLN64 siGENOME sequences were predesigned against mouse MLN64, and sequences 1 and 3 were effective against hamster MLN64. The degree of protein depletion was tested by immunoblotting. Cells were harvested into PBS, collected by centrifugation, resuspended in 5 ml ice-cold MI buffer, and ruptured by nitro
Referência(s)