Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment

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 Google Scholar). cells were with a for the expression of as M. C. M. D.M. and regulation of acid from 2017; PubMed Scopus Google Scholar). were cultured in and 1 of of extracellular were as C. S. A. and of exosomes from cell and 2006; Google Scholar). were a cells were in and were with an of LAL with or with cholesteryl and cells were for an reesterification to was during this cells were with and in CD or for was as M. of the hydrophobic of PubMed Scopus Google Scholar). were from or was from cells expressing were at in with and with CD to the were as M. C. M. D.M. and regulation of acid from 2017; PubMed Scopus Google and from the with a in were with Cholesterol and cholesteryl were in cell or by lysosomal proteins were from in to to and for protein of lysosomes ER PM mitochondria and for and To extract a of the cell or was to a of in with for cholesterol and cholesteryl were and at for and the was to a and in of this was to a for the of with and for 1 at were by of the or was used for the of protein concentration were and at to and to extracellular and lipoprotein were and to the were in by and at for at and at were and with a in To PM cholesterol and cholesteryl cells were with in with and for in were with and in cholesterol and for at to PM cholesterol to were and in with for and cholesteryl for to extract cellular lipid extract was to and in of this was to a for the of with acid for 1 at by protein concentration of cellular in was were in or with LDL or and as that cells were at in extracellular cells were in of with in the presence of and to the lysosomal compartment. were with and in of with or without CD for were from by as C. S. A. and of exosomes from cell and 2006; Google Scholar). Cholesterol was in and the of by proteins were to to and for and of cells were with and in that A of cells was to protein knockdown by and cholesteryl by were in with or without CD for and were for the of cholesterol stable knockdown of or ABCG1, were from and used to in cells to the and cells were with and and the cells were by Protein knockdown was by cells were and to the were from cells and at to were to a the was to with and was to the of the for at at the were as the were as and the was as Cholesterol was for and by were to and the the were and to the cells were at cells well in cells were with or in for were with with or without with or without or and with or without cells were and in with for at was at for to was a in a uptake was as the of to the in the at cells were and as for lipoprotein uptake that were with cholesterol of CD treatment, cellular cholesterol was as exchange was as the of cholesterol to the cholesterol in the at To with cholesterol, cholesterol was and with LDL or at a of cholesterol to protein at a protein concentration of at lipoprotein were at for at of the was and to in for the to cells. was a to a in the and for cholesterol and was an at with acid in as the a of and a of for cholesteryl esters a at with in and in as the a of and a of for cholesterol, and cholesteryl were and are in are in was were by of U2OS-SRA cells efficiently the cholesteryl in to M. S. K. L. J.E. and of for the of intracellular cholesterol Lipid Res. 2019; 60: Full Text Full Text PDF PubMed Scopus Google Scholar). In the presence of which N.H. A. Ralph H. M. Maxfield F.R. for that cholesterol accumulation in Niemann-Pick C cells.J. Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, C.C. Maxfield F.R. inhibitors of lysosomal acid lipase and Niemann-Pick type C disease Chem. 2010; 53: PubMed Scopus Google cargo is in the cargo is We this to the trafficking of lysosomal cholesterol CD In NPC1-deficient CD is from the plasma in and from the in (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 and CD is to concentrations of in S. A.E. J. in Niemann-Pick cells through of 2017; PubMed Scopus Google Scholar, L. S. 2-hydroxypropyl-beta-cyclodextrin disease in Niemann-Pick type a 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). the of these we to the effects of CD of treatment with cells were with in the presence of and were and with of cholesterol from the lysosome or to the PM, ER, and was trafficking Although studies have used to demonstrate that total lysosomal cholesterol is decreased by CD treatment 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 these studies have not cholesterol. We used cells expressing a lysosomal to changes in lysosomal cholesterol cargo from from cells and and soluble lysosomal lysosomal was of membranes from the and PM, as by and While the ER and mitochondria be in the from cells with CD cholesterol to the cholesteryl associated with the not between CD and cells membranes were not and were in the These membranes that do not or but cholesterol. 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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. 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F. of and and a of PubMed Scopus Google Scholar). In with lipoprotein 1 and from of cells with CD or that with human LDL or human lower a CD treatment, the lipoprotein of the cholesterol found in the cells were with CD in cholesterol was no in the of with or LDL cholesterol concentrations of or not support cholesterol that accumulation in the requires Although the of lysosomal cholesterol in the PM was not different between and cells in cholesterol in the PM were in the of cholesterol, as cholesteryl was enhanced in the absence of was to the increased delivery of cholesterol to the ER for esterification increased esterification, as the of was not different between and these support a model in which CD enhances the trafficking of lysosomal cholesterol to the PM, where it is released into the if are or trafficked to the ER, where it is the release of lysosomal cholesterol from the CD treatment in not alter total cellular cholesterol we cells with to cholesterol release was by the of lipoprotein cholesterol from the during CD of protein of the was and CD was by the consistent with a mechanism of uptake of was the of these we that in the presence of CD the release of cholesterol from the PM to in the as an exchange with lipoprotein cholesterol. To this we cells with CD in carrying cholesterol. CD treatment increased cholesterol in the presence of LDL or of the cellular uptake of with not the CD-dependent transfer of or cholesterol CD increased the transfer of cholesterol from LDL to cells the transfer of cholesterol from to the cells was data support a model in which CD the exchange of with lipoprotein in the with LDL the mechanism by which CD cholesterol from the lysosomal in the of NPC1 is not well understood. To the lysosomal cholesterol we lysosomes with cholesteryl esters in to CD treatment and the itinerary of the cholesterol We observed increased esterification and release of cholesterol into the cholesterol released into the was associated with lipoprotein but the release was of and not with and In the absence of a release not and cholesterol in the In the presence of total cellular cholesterol was through the movement of cholesterol between and the cell these data support a model by which CD enhances the trafficking of lysosomal cholesterol to the PM, where it is for exchange with extracellular or for esterification There is evidence that CD its effects cholesterol homeostasis by lysosomal exocytosis, a by the of lysosomal with the PM and release of a lysosomal into the in NPC1-deficient cells with CD (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). a lysosomal that has been in lysosomal exocytosis, and trafficking L. J. S. M. M. is a in lysosomes that 2016; PubMed Scopus Google Scholar, K. role of in trafficking and 2015; 58: PubMed Scopus Google Scholar, J.M. M. J. D. Brown exocytosis is in type 2006; PubMed Scopus Google is for of lysosomal cholesterol accumulation in cells 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). of lysosomes to the PM in a provide a mechanism by which CD increases the trafficking of lysosomal cholesterol to the However, these studies not the movement of or cholesterol of the cells. Our data do not support a model in which lysosomal cholesterol is released from the cell in exosomes. Although exosomes are in total cholesterol, cholesterol that from the lysosome not be in is that cells do not of to cell However, the of lysosomal exocytosis with not the release of lysosomal cholesterol into the the other in the presence of we observed cholesterol movement from the PM to extracellular that was by the delivery of lipoprotein cholesterol to the cells. These actions are consistent with a of the cholesterol of CD J. J. J. L. and of different cyclodextrins in Niemann-Pick C 2016; 3: PubMed Scopus Google Scholar). While that lysosomal cholesterol accumulates in the CD treatment, the mechanism is of exchange and not efflux, and it is with to cholesterol trafficking to the exchange mechanism is consistent with in that CD treatment does not total cellular cholesterol or cellular cholesterol (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). these data that CD cholesterol exchange but does not support to CD as a functional and reesterification of lysosomal cholesterol at the ER is the of restoration of cholesterol homeostatic responses. was in cells, lysosomal cholesterol to the PM trafficking to the ER R.E. Radhakrishnan A. transport of a of plasma membrane cholesterol to reticulum total cellular 2017; PubMed Scopus Google Scholar). Our that the treatment with CD in which suitable extracellular cholesterol acceptors, to PM accumulation of lysosomal cholesterol a lysosome to PM itinerary for the movement of cholesterol in NPC1-deficient cells. in the esterification of cholesterol in with the of cholesterol the PM for esterification cholesteryl the absence of changes in esterification cholesteryl this we the movement of lysosomal cholesterol cargo CD studies have to lysosomal cholesterol or to total cholesterol is with to the trafficking of cholesterol, and the is because it cholesterol esterification the esterification of from the Moreover, these are by the of cholesterol from the stable for specific of the lysosomal with cargo and the release of cholesterol through the of a LAL of by and treatment with cholesterol we the trafficking of the of cholesterol that accumulates in lysosomes in These provide with which to the itinerary of cholesterol and to mechanisms that its acetylated LDL 2-hydroxypropyl-β-cyclodextrin acetylated LDL with cholesteryl extracellular lysosomal acid lipase Niemann-Pick disease type C plasma membrane scavenger receptor A U2OS cells expressing the human scavenger receptor A U2OS-SRA cells with shNPC1 knockdown

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