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• W3003296071 abstract "Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses. Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses. Cholesterol is an essential component of mammalian cell membranes that plays a major role in tuning membrane fluidity, thickness, and permeability to regulate membrane function and support the needs of specific organelles. Different cellular membranes vary widely in cholesterol content, ranging from the cholesterol-rich plasma membrane (PM) and endosomes to the cholesterol-poor ER and mitochondria (1Wüstner D. Solanko K. How cholesterol interacts with proteins and lipids during its intracellular transport.Biochim. Biophys. Acta. 2015; 1848: 1908-1926Crossref PubMed Scopus (47) Google Scholar, 2Chen F.W. Li C. Ioannou Y.A. Cyclodextrin induces calcium-dependent lysosomal exocytosis.PLoS One. 2010; 5: e15054Crossref PubMed Scopus (78) Google Scholar, 3Das A. Brown M.S. Anderson D.D. Goldstein J.L. Radhakrishnan A. Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis.eLife. 2014; 3: e02882Crossref Scopus (151) Google Scholar). Due to its hydrophobicity, cholesterol does not transit between membranes through the aqueous phase. Rather, cholesterol transfer is facilitated by lipid-binding proteins or through membrane-fusion events (1Wüstner D. Solanko K. How cholesterol interacts with proteins and lipids during its intracellular transport.Biochim. Biophys. Acta. 2015; 1848: 1908-1926Crossref PubMed Scopus (47) Google Scholar). Although a number of proteins have been shown to function in cholesterol movement, the precise time-resolved itinerary of cholesterol trafficking between membranes and the mechanisms of regulation of this trafficking remain to be determined. Moreover, how cells maintain steep gradients of cholesterol concentration across different membranes in the face of rapid and dynamic cholesterol trafficking is not well understood. Mammalian cells acquire cholesterol through endogenous cholesterol synthesis at the ER or through the uptake of cholesterol and cholesteryl ester-laden lipoprotein particles into the endosomal/lysosomal system. Receptor-mediated endocytosis of LDL by the LDL receptor or acetylated LDL (acLDL) by the scavenger receptor A (SRA) are responsible for cholesterol delivery into the lysosomal compartment. Here, the concerted actions of lysosomal acid lipase (LAL) and the Niemann-Pick disease type C (NPC) proteins NPC1 and NPC2 are critical for the mobilization of LDL cargo. LAL cleaves cholesteryl esters and thereby liberates free cholesterol, which is bound by NPC2, a soluble lysosomal protein. NPC2 transfers cholesterol to NPC1, a transmembrane protein embedded in the limiting lysosomal membrane (4Deffieu M.S. Pfeffer S.R. Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding.Proc. Natl. Acad. Sci. USA. 2011; 108: 18932-18936Crossref PubMed Scopus (111) Google Scholar). In the presence of functional LAL, NPC1, and NPC2, cholesterol is efficiently trafficked to the PM and ER as well as other cellular membranes. At the PM, excess cholesterol is effluxed through ABCA1, ABCG1, and SRB1 to apoA1/HDL particles (5Phillips M.C. Molecular mechanisms of cellular cholesterol efflux.J. Biol. Chem. 2014; 289: 24020-24029Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). In the ER membrane, cholesterol serves as a critical regulator of sterol homeostasis through the SREBP transcription factors, and excess cholesterol is esterified by the ER-resident protein ACAT for storage in lipid droplets (6Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Protein sensors for membrane sterols.Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). Interruption of the intralysosomal cholesterol trafficking network (NPC1, NPC2, or LAL) results in abnormal cholesterol homeostasis and lysosomal dysfunction. Mutations in NPC1 or NPC2 cause NPC, a fatal neurodegenerative disorder. Both cholesterol trafficking and homeostatic regulation are disrupted in NPC1-deficient cells, in which the accumulation of free cholesterol in the lysosome is accompanied by an elevated expression of cholesterol uptake and synthesis genes and decreased cholesterol esterification (7Abi-Mosleh L. Infante R.E. Radhakrishnan A. Goldstein J.L. Brown M.S. Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 19316-19321Crossref PubMed Scopus (121) Google Scholar, 8Peake K.B. Vance J.E. Normalization of cholesterol homeostasis by 2-hydroxypropyl-beta-cyclodextrin in neurons and glia from Niemann-Pick C1 (NPC1)-deficient mice.J. Biol. Chem. 2012; 287: 9290-9298Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). There is currently no U.S. Food and Drug Administration-approved therapy for NPC, but 2-hydroxypropyl-β-cyclodextrin (CD) has shown great promise in animal models and in human clinical trials (9Liu B. Turley S.D. Burns D.K. Miller A.M. Repa J.J. Dietschy J.M. Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1−/− mouse.Proc. Natl. Acad. Sci. USA. 2009; 106: 2377-2382Crossref PubMed Scopus (282) Google Scholar). CD is a cyclic oligosaccharide frequently used as an excipient in drug formulations because of its ability to solubilize hydrophobic molecules. At concentrations >1 mM, CD can extract cholesterol from cultured cells (10Christian A.E. Haynes M.P. Phillips M.C. Rothblat G.H. Use of cyclodextrins for manipulating cellular cholesterol content.J. Lipid Res. 1997; 38: 2264-2272Abstract Full Text PDF PubMed Google Scholar). At lower concentrations, in the range of effective doses in vivo, CD enhances cholesterol trafficking from lysosomes without changing total cellular cholesterol, and neither increases in serum cholesterol nor cholesterol excretion are observed (11Taylor A.M. Liu B. Mari Y. Liu B. Repa J.J. Cyclodextrin mediates rapid changes in lipid balance in Npc1−/− mice without carrying cholesterol through the bloodstream.J. Lipid Res. 2012; 53: 2331-2342Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In fact, in cell and animal models, CD treatment reduces the expression of SREBP2 gene targets and stimulates cholesterol esterification, consistent with a model in which lysosomal cholesterol is redistributed to the ER membranes (7Abi-Mosleh L. Infante R.E. Radhakrishnan A. Goldstein J.L. Brown M.S. Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 19316-19321Crossref PubMed Scopus (121) Google Scholar, 8Peake K.B. Vance J.E. Normalization of cholesterol homeostasis by 2-hydroxypropyl-beta-cyclodextrin in neurons and glia from Niemann-Pick C1 (NPC1)-deficient mice.J. Biol. Chem. 2012; 287: 9290-9298Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). While the mechanism of CD action remains unknown, recent studies provide evidence that CD promotes lysosomal exocytosis (2Chen F.W. Li C. Ioannou Y.A. Cyclodextrin induces calcium-dependent lysosomal exocytosis.PLoS One. 2010; 5: e15054Crossref PubMed Scopus (78) Google Scholar, 12Demais V. Barthelemy A. Perraut M. Ungerer N. Keime C. Reibel S. Pfrieger F.W. Reversal of pathologic lipid accumulation in NPC1-deficient neurons by drug-promoted release of LAMP1-coated lamellar inclusions.J. Neurosci. 2016; 36: 8012-8025Crossref PubMed Scopus (11) Google Scholar, 13Vacca F. Vossio S. Mercier V. Moreau D. Johnson S. Scott C.C. Montoya J.P. Moniatte M. Gruenberg J. Cyclodextrin triggers MCOLN1-dependent endo-lysosome secretion in Niemann-Pick type C cells.J. Lipid Res. 2019; 60: 832-843Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). However, if release from the cells of cholesterol-laden exosomes is responsible for the beneficial effects of CD treatment, this would be predicted to lower cellular cholesterol, a change inconsistent with the observed suppression of SREBP2 gene targets or with enhanced reesterification by ACAT. To address these disparate findings, we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient cells. Our data support a model in which CD promotes the redistribution of lysosomal cholesterol to the PM, where it is exchanged with cholesterol carried by extracellular acceptors, and to the ER, where it directs cholesterol homeostatic responses. U2OS cells expressing the human scavenger receptor A (U2OS-SRA) and U2OS-SRA cells with shNPC1 knockdown (U2OS-SRAshNPC1) were a gift from the Maxfield Laboratory (14Pipalia N.H. Subramanian K. Mao S. Ralph H. Hutt D.M. Scott S.M. Balch W.E. Maxfield F.R. Histone deacetylase inhibitors correct the cholesterol storage defect in most Niemann-Pick C1 mutant cells.J. Lipid Res. 2017; 58: 695-708Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). U2OS-SRAshNPC1 cells were transduced with a lentiviral vector for the expression of TMEM192-HA-RFP as previously described (15Abu-Remaileh M. Wyant G.A. Kim C. Laqtom N.N. Abbasi M. Chan S.H. Freinkman E. Sabatini D.M. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.Science. 2017; 358: 807-813Crossref PubMed Scopus (172) Google Scholar). Cells were cultured in McCoy's medium containing 10% FBS, 1.2 g/l sodium bicarbonate, and 1 mg/ml G418. Lipoprotein-deficient medium (LPDM) contained lipoprotein-depleted FBS instead of regular serum. Media depleted of extracellular vesicles were prepared as previously described (16Théry C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol . 2006; 30: 3.22.1-3-3.22.29Crossref Google Scholar). Unless otherwise specified, experiments were performed using a standard protocol. On day 1, 2 × 105 cells were plated in 6-well dishes and grown overnight. On day 2, growth media were replaced with media containing 10 μM lalistat-1, an inhibitor of LAL (Tocris Bioscience), with 25 μg/ml acLDL or acLDL reconstituted with d7 cholesteryl ester (d7-acLDL), and cells were incubated for an additional 18 h. For reesterification experiments, 36 μg/ml d9 oleate complexed to BSA was included during this period. On day 3, cells were washed twice with PBS and then incubated in fresh growth media containing 500 μM CD (Janssen Pharmaceuticals) or vehicle (H2O) for 6 h. Reconstituted acLDL was prepared as previously described (17Krieger M. Reconstitution of the hydrophobic core of low-density lipoprotein.Methods Enzymol. 1986; 128: 608-613Crossref PubMed Scopus (39) Google Scholar). Lipoproteins were obtained from Alfa Aesar or Kalen Biomedical. Human apoA1 was obtained from Millipore Sigma. U2OS-SRAshNPC1 cells expressing TMEM192-HA-RFP were plated at 12 × 105 in 10 cm dishes loaded with d7-acLDL and treated with CD according to the standard protocol. Lysosomes were isolated as previously described (15Abu-Remaileh M. Wyant G.A. Kim C. Laqtom N.N. Abbasi M. Chan S.H. Freinkman E. Sabatini D.M. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.Science. 2017; 358: 807-813Crossref PubMed Scopus (172) Google Scholar) using PierceTM Anti-HA Magnetic Beads (Thermo Fisher Scientific) and eluted from the beads with a 5 min incubation in 50 mM NaOH. Eluates were neutralized immediately with 100 mM Tris (pH 6.8). Cholesterol and cholesteryl ester content were measured in cell lysates, postnuclear supernatants, or elution fractions by LC/MS/MS. For Western blot analysis, lysosomal proteins were eluted from beads in RIPA buffer [50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1X Protease Complete (Roche)], separated on 4% to 12% Bis-Tris gels, transferred to nitrocellulose membranes, and analyzed for protein markers of lysosomes (LAMP1, HEXA), ER (calreticulin), PM (Na/K ATPase), mitochondria (cytochrome oxidase XIV), and nuclei (histone H3). See supplemental Table S1 for antibody sources and dilutions. To extract lipids, a portion of the fraction, cell homogenate, or media was added to a 1:1:1:0.5 mixture of chloroform-methanol-water-5M NaCl in glass tubes along with internal standards for cholesterol and cholesteryl ester. Mixtures were vortexed and centrifuged at 1,000 g for 5 min, and the chloroform phase was transferred to a 1.2 ml glass tube, dried under N2, and resuspended in 1:1 methanol-chloroform. Twenty percent of this solution was transferred to a new tube for the derivatization of cholesterols with 0.05 M nicotinic acid, 0.05 M 4-(dimethylamino)pyridine, and 0.05 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for 1 h at 55°C. Lipids were analyzed by LC/MS/MS. Another portion of the fraction or homogenate was used for the determination of protein concentration using BCA assay (Pierce). Conditioned media were collected and centrifuged at 550 g to remove debris and frozen prior to extraction. Lysosomal fractions, extracellular vesicles, and lipoprotein fractions were thawed and then added directly to the extraction mixture. Cells were recovered in PBS by scraping and centrifuged at 2,500 g for 10 min at 4°C and then frozen at −20°C. Pellets were thawed and homogenized with a 25-gauge needle in PBS before extraction. To quantify PM cholesterol and cholesteryl ester, cells were washed three times with 1% BSA in Tris-buffered saline (140 mM NaCl, 3 mM KCl, 25 mM Tris base, pH 7.4), washed twice with PBS, and then fixed for 10 min in 1% glutaraldehyde. Cells were washed twice more with PBS and then incubated in McCoy's medium containing 2 units/ml cholesterol oxidase and 0.1 units/ml sphingomyelinase for 30 min at 37°C to convert PM cholesterol to cholestenone. Cells were washed again and then incubated in 9:1 methanol-chloroform supplemented with internal standards for cholestenone and cholesteryl ester for 30 min to extract cellular lipids. The lipid extract was transferred to 1.2 ml glass tubes, dried under nitrogen, and resuspended in 1:1 methanol:chloroform. Twenty percent of this solution was transferred to a new tube for the derivatization of cholestenones with 2:1 5 mg/ml O-benzylhydroxylamine hydrochloride/formic acid for 1 h at 55°C, followed by LC/MS/MS analyses. The protein concentration of cellular homogenates prepared in parallel was determined using BCA assay. For lipoprotein-dependent efflux assays, U2OS-SRAshNPC1 were treated in LPDM or LPDM supplemented with 25 μg/ml LDL or 25 μg/ml HDL and harvested as described above, except that cells were plated at 4 × 104 in 24-well dishes. For extracellular vesicle (EV) isolations, 12 × 105 cells were plated in each of 5 plates (10 cm) per condition, loaded with 10 μg/ml d7-acLDL in the presence of 10 μM lalistat, and incubated overnight to load the lysosomal compartment. Cells were washed twice with PBS and then incubated in fresh media (depleted of EVs) with or without 500 μM CD for 6 h. EVs were isolated from conditioned media by differential ultracentrifugation as previously described (16Théry C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol . 2006; 30: 3.22.1-3-3.22.29Crossref Google Scholar). Cholesterol content was quantified in media, EVs, and media following the depletion of EVs by LC/MS/MS. EV proteins were separated on 4% to 12% Bis-Tris precast gels, transferred to PVDF membranes, and probed for nuclear (lamin B1) and EV (CD63) markers. For transient knockdowns of SRB1, U2OS-SRAshNPC1 cells were incubated with 25 μg/ml d7-acLDL and 10 μM lalistat in 2.1 ml media that also contained 0.5 ml Opti-MEM/RNAiMax/siRNA. A set of cells was harvested to evaluate protein knockdown by Western blot and baseline d7 cholesteryl oleate content by LC/MS/MS. Cells were then incubated in complete media with or without 500 μM CD for 6 h, and media were collected for the analysis of d7 cholesterol content. For stable knockdown of ABCA1 or ABCG1, shRNA plasmids were obtained from Origene and used to produce lentivirus in HEK 293T cells according to the TransIT-Lenti protocol using TransIT-Lenti transfection reagent and Mission Lentiviral Packaging Mix. U2OS-SRAshNPC1 cells were transduced with viral supernatants (obtained 48–72 h after transfection) and expanded, and the top 10% GFP-expressing cells were isolated by flow cytometry. Protein knockdown was evaluated by Western blot. U2OS-SRAshNPC1 cells were plated and treated according to the standard protocol. Conditioned media were removed from cells and spun at 550 g to remove debris. Cleared media were transferred to a 1.4 ml polycarbonate tube, the density was adjusted to 1.21 g/ml with KBr, and 150 μl 0.9% NaCl was added to the top of the suspension. After centrifugation for 18 h at 259,000 g at 4°C, the top 200 μl were removed as fraction 1, the second 200 μl were removed as fraction 2, and the remaining volume was removed as fraction 3. Cholesterol content was measured for whole media and fractionated media by LC/MS/MS. Proteins were analyzed on NuPage 3% to 12% Bis-Tris gels using NativePage Running Buffer (100 V × 30 min, 150 V × 30 min, and 200 V until the dye front reached the bottom). Gels were fixed and stained using Sypro Ruby according to the manufacturer's protocol (Thermo Fisher Scientific). U2OS-SRAshNPC1 cells were plated at 4 × 104 cells per well in 24-well dishes. After overnight growth, cells were preincubated with 10 μM dynasore (DYN) hydrate (Millipore Sigma) or vehicle (DMSO) in complete media for 30 min. Media were then replaced with LPDM with or without DYN, with or without 25 μg/ml dil-lipoprotein (dil-LDL or dil-HDL; Kalen Biomedical), and with or without 500 μM CD. After 6 h, cells were washed and then lysed in RIPA buffer with protease complete for 30 min at 4°C. The lysate was spun at 16,000 g for 10 min to remove debris. Lysate fluorescence was quantified using a TECAN scanner (excitation/emission: 550/580 nm) in a black flat-bottom 96-well plate. Percentage uptake was calculated as the ratio of cell-associated fluorescence relative to the fluorescence in the media at baseline. U2OS-SRAshNPC1 cells were plated and treated as for fluorescent lipoprotein uptake except that lipoproteins were labeled with d7 cholesterol instead of dil. After CD treatment, cellular cholesterol was recovered as described above. Percentage exchange was calculated as the ratio of cell-associated d7 cholesterol relative to the d7 cholesterol in the media at baseline. To prepare lipoproteins with isotope-labeled cholesterol, d7 cholesterol was dried under nitrogen and then incubated with LDL or HDL at a ratio of 15 μg d7 cholesterol to 100 μg protein at a final protein concentration of 2 mg/ml. After overnight incubation at 4°C, lipoprotein mixtures were centrifuged at 10,000 g for 10 min at 4°C. Ninety percent of the supernatant was recovered and diluted to 25 μg/ml in LPDM for the administration to cells. LC/MS/MS analysis was conducted using a Shimadzu HPLC system coupled to a TSQ Quantum Ultra Plus mass spectrometer (Thermo Fisher Scientific) operating in the positive mode and using selected reaction monitoring. Chromatography for cholesterol and cholestenone was performed on an Eclipse XDB-C18 column (3.0 × 100 mm; Agilent) at 50°C with 1% formic acid in isopropanol-methanol (1:1) as the mobile phase, a flow rate of 0.4 ml/min, and a run time of 4.5 min. Chromatography for cholesteryl esters utilized a BetaSilTM C18 column (100 × 2.1 mm; Thermo Fisher Scientific) at 50°C, with 97% 10 mM ammonium acetate in isopropanol-methanol (1:1) and 3% 10 mM ammonium acetate in acetonitrile-H2O (3:7) as the mobile phase, a flow rate of 0.4 ml/min, and a run time of 11 min. Collision energies for cholesterol, cholestenone, and cholesteryl ester were 22, 30, and 14 V, respectively. Monitored transitions are reported in supplemental Table S2. Additional assay parameters are reported in supplemental Table S3. Data was analyzed using Xcalibur software. Calibration curves were constructed by plotting peak ratios of standard/internal standard versus analyte concentration. U2OS-SRA cells efficiently take up the cholesteryl ester probe reconstituted in acLDL to lysotracker positive compartments (18Feltes M. Moores S. Gale S.E. Krishnan K. Mydock-McGrane L. Covey D.F. Ory D.S. Schaffer J.E. Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking.J. Lipid Res. 2019; 60: 707-716Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). In the presence of lalistat, which prevents LAL-mediated ester hydrolysis (19Pipalia N.H. Huang A. Ralph H. Rujoi M. Maxfield F.R. Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells.J. Lipid Res. 2006; 47: 284-301Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Rosenbaum A.I. Cosner C.C. Mariani C.J. Maxfield F.R. Wiest O. Helquist P. Thiadiazole carbamates: potent inhibitors of lysosomal acid lipase and potential Niemann-Pick type C disease therapeutics.J. Med. Chem. 2010; 53: 5281-5289Crossref PubMed Scopus (67) Google Scholar), cargo is retained in the compartment; upon inhibitor washout, cargo is released. We adapted this approach to specifically monitor the NPC1-independent trafficking of lysosomal cholesterol after CD treatment. In NPC1-deficient mice, CD is rapidly cleared from the plasma in 3 h and from the whole body in 6 h (11Taylor A.M. Liu B. Mari Y. Liu B. Repa J.J. Cyclodextrin mediates rapid changes in lipid balance in Npc1−/− mice without carrying cholesterol through the bloodstream.J. Lipid Res. 2012; 53: 2331-2342Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and CD is estimated to reach concentrations of 0.1–1 mM in vivo (21Dai S. Dulcey A.E. Hu X. Wassif C.A. Porter F.D. Austin C.P. Ory D.S. Marugan J. Zheng W. Methyl-beta-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK.Autophagy. 2017; 13: 1435-1451Crossref PubMed Scopus (40) Google Scholar, 22Ory D.S. Ottinger E.A. Farhat N.Y. King K.A. Jiang X. Weissfeld L. Berry-Kravis E. Davidson C.D. Bianconi S. Keener L.A. et al.Intrathecal 2-hydroxypropyl-beta-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1–2 trial.Lancet. 2017; 390: 1758-1768Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). On the basis of these studies, we chose to analyze the effects of CD after 6 h of treatment with 0.5 mM CD. U2OS-SRAshNPC1 cells were incubated with d7-acLDL in the presence of lalistat (Fig. 1). After loading, d7-acLDL and lalistat were removed and replaced with media containing CD. Trafficking of deuterated cholesterol away from the lysosome or to the PM, ER, and culture media was monitored using LC/MS/MS-based biochemical trafficking assays. Although previous studies have used filipin staining to demonstrate that total lysosomal cholesterol is decreased by CD treatment (8Peake K.B. Vance J.E. Normalization of cholesterol homeostasis by 2-hydroxypropyl-beta-cyclodextrin in neurons and glia from Niemann-Pick C1 (NPC1)-deficient mice.J. Biol. Chem. 2012; 287: 9290-9298Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), these studies have not metabolically traced lipoprotein-derived cholesterol. We used U2OS-SRAshNPC1 cells expressing TMEM192-RFP-HA, a tagged lysosomal protein, to quantify changes in lysosomal cholesterol cargo derived from endocytosed lipoproteins. Lysosomes from U2OS-SRAshNPC1-TMEM192-RFP-HA cells contained LAMP1 and HEXA, membrane-bound and soluble lysosomal proteins, respectively (Fig. 2A). The isolated lysosomal fraction was depleted of contaminating membranes from the nucleus and PM, as indicated by histone H3 and Na/K ATPase markers, respectively. While substantially depleted, the ER marker calreticulin and mitochondria marker cytochrome oxidase XIV could still be detected in the isolated fractions. The lysosome-enriched fraction isolated from cells treated with CD had less d7 cholesterol relative to the vehicle-treated control (Fig. 2B). D7 cholesteryl oleate associated with the enriched fraction did not differ significantly between CD and vehicle-treated cells (Fig. 2C). Some LAMP1-containing membranes were not immunoisolated and were detected in the flow through. These LAMP+ membranes could represent lysosome-related organelles that do not contain TMEM197 or HEXA but may harbor cholesterol. To trace the fate of lipoprotein cholesterol cargo to other cellular compartments, we analyzed cholesterol movement in U2OS-SRAshNPC1 cells. Similar to our findings in the lysosomes, total cellular d7 cholesteryl oleate was unchanged by CD treatment (Fig. 2D), suggesting LAL activity was not affected. To measure PM cholesterol, cells were fixed and treated with cholesterol oxidase and sphingomyelinase to oxidize d7 cholesterol to d7 cholestenone. Under vehicle-treated conditions, d7 cholesterol trafficked to the PM (Fig. 2E). There was a trend for increased trafficking of lysosomal cholesterol to the PM following CD treatment, but this did not reach significance. To assess reesterification, d9 oleate was included during loading to provide substrate for ACAT-mediated reesterification of d7 cholesterol to d16 cholesteryl ester. CD administration resulted in a 2-fold increase in the formation of d16 cholesteryl esters from cholesterol cargo originating in the lysosome (Fig. 2F). We also analyzed d7 cholesterol content of conditioned media to assess efflux. Most strikingly, compared with the vehicle treatment, CD treatment increased the efflux of d7 cholesterol to the culture medium 28-fold (Fig. 2G). Overall, the magnitude of d7 cholesterol arriving at the ER was small compared with the d7 cholesterol arriving at the PM and into the media. To further characterize the mechanism of release of d7 cholesterol into the culture medium, we performed a time course. D7 cholesterol was detectable in the culture medium as early as 3 h after lalistat washout and CD treatment. Between 3 and 6 h, d7 cholesterol in the media increased ∼5-fold (Fig. 3A). The appearance of lysosomal cholesterol in the PM preceded its appearance in the media (Fig. 3B). Similar levels of PM d7 cholesterol were measured after ionomycin or DMSO vehicle treatment. In contrast to CD, ionomycin did not promote the release of the cholesterol into the media over 6 h. These data show that the distribution of d7 cholesterol from the lysosome to the PM is an order of magnitude greater and occurs more rapidly than distribution into the media. A recent report that CD induces lysosomal exocytosis in H" @default.
• W3003296071 created "2020-02-07" @default.
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• W3003296071 date "2020-03-01" @default.
• W3003296071 modified "2023-10-18" @default.
• W3003296071 title "Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment" @default.
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• W3003296071 doi "https://doi.org/10.1194/jlr.ra119000571" @default.
• W3003296071 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7053843" @default.
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