Psychosine, the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy, alters membrane architecture
2013; Elsevier BV; Volume: 54; Issue: 12 Linguagem: Inglês
10.1194/jlr.m039610
ISSN1539-7262
AutoresJacqueline A. Hawkins-Salsbury, Archana R. Parameswar, Xuntian Jiang, Paul H. Schlesinger, Ernesto R. Bongarzone, Daniel S. Ory, Alexei V. Demchenko, Mark S. Sands,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoGloboid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2–5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions. Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2–5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions. Globoid cell leukodystrophy (GLD) is a rapidly progressing pediatric neurodegenerative disease caused by missing or dysfunctional lysosomal enzyme galactosylceramidase (GALC). Hallmarks of thi s disease include macrophage (globoid cell) infiltration into the brain parenchyma, loss and dysfunction of myelin and oligodendrocytes, and axonal damage (1Wenger D. Suzuki K. Suzuki Y. Galactosylceramide Lipidosis: Globoid Cell Leukodystrophy (Krabbe Disease)..in: Scriver C. Beaudet A. Sly W. Valle D. Childs B. Kinzler K. Vogelstein B. InThe Metabolic & Molecular Basis of Disease. McGraw-Hill Medical Publishing Division, New York2001: 2669-3694Google Scholar, 2Castelvetri L.C. Givogri M.I. Zhu H. Smith B. Lopez-Rosas A. Qiu X. van Breemen R. Bongarzone E.R. Axonopathy is a compounding factor in the pathogenesis of Krabbe disease.Acta Neuropathol. 2011; 122: 35-48Crossref PubMed Scopus (75) Google Scholar). The GALC enzyme is responsible for cleaving the glycosydic linkage of galactosylceramide and galatosylsphingosine (psychosine or psy) (3Wenger D.A. Sattler M. Hiatt W. Globoid cell leukodystrophy: deficiency of lactosyl ceramide beta-galactosidase.Proc. Natl. Acad. Sci. USA. 1974; 71: 854-857Crossref PubMed Scopus (69) Google Scholar). Although galactosylceramide can be degraded by other enzymes, psy cannot and subsequently accumulates to high levels in the brains of GALC-deficient individuals (4Svennerholm L. Vanier M.T. Mansson J.E. Krabbe disease: a galactosylsphingosine (psychosine) lipidosis.J. Lipid Res. 1980; 21: 53-64Abstract Full Text PDF PubMed Google Scholar). Psychosine is a highly cytotoxic lipid, capable of inducing cell death in a wide variety of cell types including, most relevantly to GLD, oligodendrocytes (5Nagara H. Ogawa H. Sato Y. Kobayashi T. Suzuki K. The twitcher mouse: degeneration of oligodendrocytes in vitro.Brain Res. 1986; 391: 79-84Crossref PubMed Scopus (13) Google Scholar). Psychosine accumulation was postulated to be the pathogenic mechanism leading to GLD nearly 40 years ago (4Svennerholm L. Vanier M.T. Mansson J.E. Krabbe disease: a galactosylsphingosine (psychosine) lipidosis.J. Lipid Res. 1980; 21: 53-64Abstract Full Text PDF PubMed Google Scholar, 6Miyatake T. Suzuki K. Additional deficiency of psychosine galactosidase in globoid cell leukodystrophy: an implication to enzyme replacement therapy.Birth Defects Orig. Artic. Ser. 1973; 9: 136-140PubMed Google Scholar, 7Vanier M. Svennerholm L. Chemical pathology of Krabbe disease: the occurrence of psychosine and other neutral sphingoglycolipids.Adv. Exp. Med. Biol. 1976; 68: 115-126Crossref PubMed Scopus (45) Google Scholar). However, the mechanism of psychosine toxicity has remained elusive (8Suzuki K. Twenty five years of the "psychosine hypothesis": a personal perspective of its history and present status.Neurochem. Res. 1998; 23: 251-259Crossref PubMed Scopus (243) Google Scholar). Psychosine induces pleiotropic effects, including dysfunctions in several cellular pathways and compartments with no clear mechanistic connection between them (9Yamada H. Suzuki K. Responses to cyclic AMP is impaired in the twitcher Schwann cells in vitro.Brain Res. 1999; 816: 390-395Crossref PubMed Scopus (5) Google Scholar, 10Haq E. Giri S. Singh I. Singh A.K. Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line.J. Neurochem. 2003; 86: 1428-1440Crossref PubMed Scopus (94) Google Scholar, 11Khan M. Haq E. Giri S. Singh I. Singh A.K. Peroxisomal participation in psychosine-mediated toxicity: implications for Krabbe's disease.J. Neurosci. Res. 2005; 80: 845-854Crossref PubMed Scopus (53) Google Scholar, 12Contreras M.A. Haq E. Uto T. Singh I. Singh A.K. Psychosine-induced alterations in peroxisomes of twitcher mouse liver.Arch. Biochem. Biophys. 2008; 477: 211-218Crossref PubMed Scopus (14) Google Scholar, 13Giri S. Khan M. Nath N. Singh I. Singh A.K. The role of AMPK in psychosine mediated effects on oligodendrocytes and astrocytes: implication for Krabbe disease.J. Neurochem. 2008; 105: 1820-1833Crossref PubMed Scopus (62) Google Scholar). No unified cause for these dysfunctions has been proposed. Given the wide-ranging nature of psy's effects, it seems unlikely that psy toxicity is mediated through a single protein-binding partner, such as a receptor. It is similarly unlikely that psy specifically binds many proteins independently. As an amphipathic molecule, psy would be expected to partition largely into cellular membranes. Indeed, White et al. (14White A.B. Givogri M.I. Lopez-Rosas A. Cao H. van Breemen R. Thinakaran G. Bongarzone E.R. Psychosine accumulates in membrane microdomains in the brain of krabbe patients, disrupting the raft architecture.J. Neurosci. 2009; 29: 6068-6077Crossref PubMed Scopus (129) Google Scholar) demonstrated that psy preferentially partitions into detergent-resistant membrane microdomains rich in cholesterol. Cholesterol has a chemical affinity for sphingolipids such as psy (15Leventis R. Silvius J.R. Use of cyclodextrins to monitor transbilayer movement and differential lipid affinities of cholesterol.Biophys. J. 2001; 81: 2257-2267Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar); therefore, this association is not surprising. It is possible that psy disrupts the biophysical properties of membranes it inserts into, thereby causing widespread downstream effects. Membrane perturbations have been implicated in other diseases, including Alzheimer's (16Yang X. Askarova S. Lee J.C. Membrane biophysics and mechanics in Alzheimer's disease.Mol. Neurobiol. 2010; 41: 138-148Crossref PubMed Scopus (47) Google Scholar), cancer (17Hendrich A.B. Michalak K. Lipids as a target for drugs modulating multidrug resistance of cancer cells.Curr. Drug Targets. 2003; 4: 23-30Crossref PubMed Scopus (156) Google Scholar), and diabetes (18Waczuliková I. Cagalinec M. Ulicna O. Slezak P. Ziegelhoffer A. Biophysical investigation on left ventricular myocytes in rats with experimentally induced diabetes.Physiol. Res. 2010; 59: S9-S17Crossref PubMed Google Scholar), providing evidence that this mechanism is possible and perhaps even common. Enantiomers are powerful tools with which to investigate biological processes (19Covey D.F. ent-Steroids: novel tools for studies of signaling pathways.Steroids. 2009; 74: 577-585Crossref PubMed Scopus (57) Google Scholar). Enantioselectivity has recently been used to distinguish between the protein-mediated and membrane-mediated effects of cholesterol and other steroids (20Langmade S.J. Gale S.E. Frolov A. Mohri I. Suzuki K. Mellon S.H. Walkley S.U. Covey D.F. Schaffer J.E. Ory D.S. Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann-Pick C disease.Proc. Natl. Acad. Sci. USA. 2006; 103: 13807-13812Crossref PubMed Scopus (132) Google Scholar, 21Gale S.E. Westover E.J. Dudley N. Krishnan K. Merlin S. Scherrer D.E. Han X. Zhai X. Brockman H.L. Brown R.E. et al.Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions.J. Biol. Chem. 2009; 284: 1755-1764Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 22Bielska A.A. Schlesinger P. Covey D.F. Ory D.S. Oxysterols as non-genomic regulators of cholesterol homeostasis.Trends Endocrinol. Metab. 2012; 23: 99-106Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Similarly, enantioselectivity can be used to study psy modes of action. Enantiomers (psy and ent-psy) have identical physicochemical properties but are mirror images of each other. Typically, only one enantiomer of lipids, proteins, sugars, nucleic acids, and cofactors are found within cells. Proteins are chiral molecules with a defined three-dimensional structure and generally only recognize one enantiomer of their binding partners (psy or ent-psy) (19Covey D.F. ent-Steroids: novel tools for studies of signaling pathways.Steroids. 2009; 74: 577-585Crossref PubMed Scopus (57) Google Scholar). Accordingly, psy interactions with proteins are expected to be largely enantioselecitve. Conversely, the lipid membrane, even though it is composed of chiral molecules, is fluid and does not maintain well-defined "binding sites" for other lipids or sterols. Thus, lipid-lipid and lipid-sterol membrane interactions of psy are nonenantioselective. Additionally, any membrane property altered by psy (i.e., polarity, fluidity, and packing capacity) will be altered equivalently by ent-psy because of its identical chemical and physical properties. This point is important because if psy and ent-psy had different physicochemical properties then they might alter membrane properties differently even if their membrane interactions were not enantioselective. Because of this fundamental difference between membranes and proteins, protein-mediated actions are usually enantiosensitive, whereas membrane-mediated actions are not. Therefore, enantiomers of natural molecules can be used as probes to distinguish between enantio-specific (protein-mediated) and nonenantio-specific (lipid membrane-based) functions (19Covey D.F. ent-Steroids: novel tools for studies of signaling pathways.Steroids. 2009; 74: 577-585Crossref PubMed Scopus (57) Google Scholar). We previously synthesized the unnatural enantiomer of psy (ent-psy) (23Parameswar A.R. Hawkins J.A. Mydock L.K. Sands M.S. Demchenko A.V. Consise synthesis of the unnatural sphingosine and psychosine enantiomer.Eur. J. Org. Chem. 2010; : 3269-3274Crossref Scopus (12) Google Scholar). In this study, the underlying causes of psy toxicity are investigated using ent-psy as a probe to distinguish between protein- and membrane-based mechanisms. Psychosine was synthesized as previously described (23Parameswar A.R. Hawkins J.A. Mydock L.K. Sands M.S. Demchenko A.V. Consise synthesis of the unnatural sphingosine and psychosine enantiomer.Eur. J. Org. Chem. 2010; : 3269-3274Crossref Scopus (12) Google Scholar) Psychosine was obtained from Matreya (Pleasant Gap, PA). Both lipids were dissolved in DMSO to 20 mM before use unless otherwise noted. Cells from the human glial (oligodendrocytic) cell line MO3.13 (Cedarlane Labratories, Burlington, NC) were maintained in DMEM with 10% FBS. Before lipid treatment, cells were plated to a density of 2 × 104 cells/cm2 on uncoated plastic dishes. After 24 h, the media was replaced with serum-free media. After an additional 24 h, media containing lipids or vehicle was added. Cells were exposed to the lipids for 24 h before measuring cell survival. Cell viability was measured using the commercially available MTT assay (ATCC, Manassas, VA). This procedure is based on the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a blue product by mitochondrial dehydrogenase in viable cells. Absorbance was measured at 570 nm and compared with the reference wavelength of 650 nm to determine cell viability. Cell survival after lipid treatment was normalized to the vehicle (DMSO) control. The proportion of cells undergoing apoptosis mediated by exposure to psy or its ent-psy was measured using Annexin V according to the manufacturer's instructions. Briefly, oligodendrocyte cells, M03.13, were plated at ∼80% confluence on day 1. The cells were serum-starved for 24 h, and then the appropriate concentration (10, 20, and 40 μM) of psychosine or the enantiomer was added for an additional 24 h in the absence of fetal calf serum. The cells were harvested, washed once in PBS, and resuspended at ∼1 × 106 cells/ml in Annexin V binding buffer (10 mM, HEPES [7.4], 140 mM NaCl, 2.5 mM CaCl2). Allophycocyanin-conjugated Annexin V (cat# A35110; Life Technologies, Eugene, OR) and 7AAD (viability) were added to the cell suspensions and incubated 15 min at room temperature. The Annexin V-positive cells were detected by fluorescence activated cell sorting using a Beckman Coulter Gallios instrument (Brea, CA) and analyzed using FloJo software (Ashland, OR). MO3.13 cells were pulsed with 10 μM psy, ent-psy or an equivalent volume of vehicle for 30 min, after which they were washed and returned to normal media at 37°C. At 0, 15, 30, and 60 min postpulse, cells were washed, harvested, spun down, and flash frozen. Once thawed, cells were quickly homogenized in 0.04 M citric acid by passing five times through a 25 gauge needle . Fifty microliters of each sample was added to 20 μl of N,N-dimethylpsychosine (250 ng/ml) internal standard and 200 μl MeOH. Samples were vortexed and centrifuged, and the supernatant was collected. This extraction was repeated on the remaining pellet and the supernatants pooled. Psy or ent-psy concentrations were obtained using a column-switching LC-MS/MS method. Detection was achieved using an AB SCIEX 4000QTRAP tandem mass spectrometer (Applied Biosystems/MDS Sciex Inc., Ontario, Canada) using ESI in the positive ion mode along with multiple reaction monitoring. Analyst software (version 1.5.1; Applied Biosystems/MDS Sciex Inc.) was used for the data analysis. The calibration curves (analyte peak area/internal standard peak area for y-axis and analyte concentration for x-axis) of psychosine were obtained using the least square linear regression fit (y = ax + b) and a weighting factor of 1/x2. The coefficient of determination (r 2) was set as >0.98 for acceptance criteria of calibration curves. HeLa cells were plated at a density of 2 × 104 cells/cm2 on poly-L-lysine (Sigma, St. Louis MO) coated glass coverslips in serum-free standard media (DMEM-F12). After 24 h of growth, the cells were exposed to lipids or vehicle for an additional 24 h. After this time, stimulated cells were treated with 20 ng/ml platelet-derived growth factor for 15 min at 37°C. After stimulation, the cells were fixed with 4% paraformaldehyde. Plasma membranes were stained with fluorescently labeled cholera toxin B (CTXB, Sigma) before incubation with anti-phosphoPKC primary antibody (Cell Signaling, Danvers, MA). After incubation with secondary antibodies (Invitrogen, Grand Island, NY), the coverslips were mounted and imaged with a Zeiss confocal microscope in the Alafi Neuroimaging Core (Washington University in St. Louis, Hope Center). Images from random fields were acquired. Membrane translocation was quantified by an independent observer blinded to treatment who scored each cell as "membrane associated" or "cytoplasmic." More than 30 cells per treatment were scored in this manner. Carboxyfluorescein (CF)-loaded DOPC (Sigma) liposomes were generated essentially as previously described (24Szoka Jr, F. Papahadjopoulos D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation.Proc. Natl. Acad. Sci. USA. 1978; 75: 4194-4198Crossref PubMed Scopus (2358) Google Scholar). Dried DOPC was dissolved in 0.5 ml ethyl ether, and an equal volume of 20 mM CF (Molecular Probes, Eugene, OR) in elution buffer (100 mM KCl, 10 mM HEPES [pH 7.0], 1 mM EDTA) was added. This mixture was sonicated three times for 20 s each to generate a thick emulsion before evaporation of the ether under reduced pressure. The evaporated solution was passed five times through a 22 guage needle and a mini-extruder (Avanti, Alabaster, AL) containing a 200 nM membrane to limit liposome size (Nuclepore, Pleasanton, CA). All CF not contained within a liposome was removed by passing the solution over a Sephadex G-25-80 column. Liposome concentration was normalized against the maximum fluorescence achieved after the vesicles were lysed with 20% Triton-X-100. Liposomes were diluted in elution buffer to a concentration appropriate for the assay (generally 1:500 to 1:2,000) to a final volume of 1 ml. Baseline fluorescence was measured for 10 min. Lipids were then added, and the change in fluorescence was measured for an additional 60 min. At this time, 10 μl of 20% Triton-X-100 was added to lyse all liposomes so that the maximal fluorescence could be obtained. Data are presented as the percent maximal fluorescence after the baseline is subtracted. Detergent-resistant membranes (DRMs) were isolated and analyzed essentially as previously reported (14White A.B. Givogri M.I. Lopez-Rosas A. Cao H. van Breemen R. Thinakaran G. Bongarzone E.R. Psychosine accumulates in membrane microdomains in the brain of krabbe patients, disrupting the raft architecture.J. Neurosci. 2009; 29: 6068-6077Crossref PubMed Scopus (129) Google Scholar). Briefly, HeLa cells were treated with lipid at a concentration of 10 μM for 6 h. The cells were scraped from the plates, pelleted, then resuspended in 2 ml cold lysis buffer (25 mM Tris-HCL [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.5% Lubrol, protease inhibitor cocktail, 1 mM PMSF, 1 mM okadaic acid, and 2 mM sodium orthovanadate). This suspension was passed through a 25 gauge needle five times to lyse the cells. An equal volume of 90% sucrose was added to the cellular lysate to give a final concentration of 45% sucrose. Four milliliters of 35% sucrose then 5% sucrose was loaded on top of the homogenates using an auto densi-flow density gradient fractionator (Labonco, Kansas City, MO). Tubes were ultracentrifuged using a SW-41 rotor (Beckman-Coulter, Brea, CA) at 39,000 rpm overnight at 4°C. Fractions were collected in 1ml volumes from the top of the fractions down, with raft markers found in fractions 4 and 5. Psy or ent-psy was measured using LC-MS. To prepare the samples, each fraction was extracted with chloroform/methanol/water then analyzed as described (25Galbiati F. Basso V. Cantuti L. Givogri M.I. Lopez-Rosas A. Perez N. Vasu C. Cao H. van Breemen R. Mondino A. et al.Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease.J. Neurosci. 2007; 27: 13730-13738Crossref PubMed Scopus (50) Google Scholar). Positive ion electrospray precursor ion scanning was performed using a triple quadrapole mass spectrometer (API 4000, Applied Biosystems) equipped with a Shimadzu HPLC system and Leap autosampler (26Whitfield P.D. Sharp P.C. Taylor R. Meikle P. Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry.J. Lipid Res. 2001; 42: 2092-2095Abstract Full Text Full Text PDF PubMed Google Scholar). Ceramide was quantified in these samples using an AB SCIEX 4000QTRAP tandem mass spectrometer (Applied Biosystems/MDS Sciex Inc.). Western blots of membrane fractions were carried out essentially as described previously (14White A.B. Givogri M.I. Lopez-Rosas A. Cao H. van Breemen R. Thinakaran G. Bongarzone E.R. Psychosine accumulates in membrane microdomains in the brain of krabbe patients, disrupting the raft architecture.J. Neurosci. 2009; 29: 6068-6077Crossref PubMed Scopus (129) Google Scholar). Pooled aliquots from DRMs (fractions 4 and 5) were solubilized in 0.25% SDS. Volume of sample loaded was normalized, and samples were resolved in a precast 7.5% polyacrylamide gel (Biorad, Hercules, CA) using the MiniProtean electrophoresis system (Biorad). Proteins were then transferred to a PVDF membrane (Biorad), blocked with 5% nonfat milk, and probed with primary antibodies for phospho-PKC (Cell Signaling) or total PKC (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with peroxidase-labeled secondary antibodies, the blots were visualized with chemiluminescent ECL substrate (Pierce, Rockford, IL). All treatment groups (DMSO, psy, and ent-psy) were exposed to the same film for the same amount of time. Signal was quantified using ImageJ software. CHO cells were grown on uncoated plastic dishes under standard conditions in DMEM-F12 media containing 5% FBS. Twenty-four hours before testing, cells were loaded with 1 μCi/ml 3H-Cholesterol (PerkinElmer, Waltham, MA). At this time, cells were also treated with 20 μM lipids or vehicle control. After 24 h, the media was washed off, and cells were trypsinized and pelleted. The cell pellet was washed three times with HEPES/DMEM (50 mM HEPES in DMEM) to remove any remaining 3H-cholesterol and resuspended in HEPES/DMEM at 4 × 106 cells/ml. Six hundred microliters of this cell suspension was added to 3 ml acceptor medium (50 ml 2-hydroxypropyl-β-cyclodextrin in HEPES/DMEM 50% cholesterol saturated) and incubated at 37°C. Aliquots (200 μl) were removed after 0, 0.5, 1, 2, 4, 6, 10, and 20 min. These aliquots were filtered to remove the cells, and the flow-through containing CD-extracted 3H-cholesterol was collected. The amount of 3H-cholesterol in the samples was measured by scintillation counting and normalized to the time zero measurement. Control samples were treated with lyso-phosphatidylserine (25 μM; decreases cholesterol extractability), 25-hydroxycholesterol (1 μM; increases cholesterol extractability), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1 μM; has no effect on cholesterol extractability). Graphpad Prism software (GraphPad Software, Inc., La Jolla, CA) was used to generate all graphs and perform all statistical analyses. All data are the average of at least three experiments and are presented as the mean ± SEM unless otherwise noted. Student's t-tests, one-way ANOVA, or repeated measures ANOVA were used where appropriate. P values <0.05 were considered significant. We first confirmed that ent-psy (Fig. 1A) could not interact with the only known psy-binding protein, GALC, as predicted. We measured the degradation of psy and ent-psy over time in MO3.13 cells (which have functional GALC). Whereas psy was rapidly degraded to a steady-state level after 1 h, levels of ent-psy remained high over this time course (Fig. 1B). Therefore, GALC does not recognize and degrade ent-psy. These data also clearly demonstrate that exogenously added lipid does enter cells and lysosomes because it could not otherwise be available to the lysosomal enzyme GALC for degradation. The oligodendrocytic cell line MO3.13 has been used frequently for psy-toxicity studies. After a 24-hr serum-starvation, these cells were treated with psy, ent-psy, or vehicle (DMSO) at a lethal (20 μM) or sublethal (5 μM) dose. These concentrations were empirically determined. Psy at 20 μM has been shown to be toxic, and a dose response curve revealed that 5 μM is not. After 24 h, cell viability was assessed (Fig. 2A). As expected, neither psy nor ent-psy demonstrated toxicity at 5 μM. Both lipids were highly cytotoxic at 20 μM, showing only 20–30% cell viability after 24 h. At concentrations higher than 20 μM, 0% cell viability was observed. A small but significant increase in toxicity of ent-psy relative to psy was observed. Psychosine causes cell death at least in part via apoptosis (10Haq E. Giri S. Singh I. Singh A.K. Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line.J. Neurochem. 2003; 86: 1428-1440Crossref PubMed Scopus (94) Google Scholar, 27Jatana M. Giri S. Singh A.K. Apoptotic positive cells in Krabbe brain and induction of apoptosis in rat C6 glial cells by psychosine.Neurosci. Lett. 2002; 330: 183-187Crossref PubMed Scopus (68) Google Scholar, 28Zaka M. Wenger D.A. Psychosine-induced apoptosis in a mouse oligodendrocyte progenitor cell line is mediated by caspase activation.Neurosci. Lett. 2004; 358: 205-209Crossref PubMed Scopus (70) Google Scholar). It was theoretically possible that psy and ent-psy could cause cell death through different mechanisms. To investigate this, we treated cells with psy or ent-psy and measured the percentage of cells staining positively for the apoptosis marker Annexin V. Treatment with either lipid resulted in a dose-dependent increase in Annexin V-positive cells, and both lipids induced apoptosis equivalently (Fig. 2B). Therefore, not only is psy toxicity not enantioselective, but both enantiomers likely induce death through the same mechanism(s). Protein kinase C is an important signal transduction protein involved in regulating numerous cellular functions. Psychosine is a known inhibitor of PKC. White et al. (14White A.B. Givogri M.I. Lopez-Rosas A. Cao H. van Breemen R. Thinakaran G. Bongarzone E.R. Psychosine accumulates in membrane microdomains in the brain of krabbe patients, disrupting the raft architecture.J. Neurosci. 2009; 29: 6068-6077Crossref PubMed Scopus (129) Google Scholar) demonstrated that phosphorylated PKC (p-PKC, active) does not translocate to the plasma membrane of cells treated with psy under PKC stimulating conditions. However, it was unclear whether this is due to direct binding of psy to either of PKC's lipid-binding domains. Phospholipid binding is required for PKC translocation to the membrane and function. Although it is unlikely that psy would bind in a pocket optimized for phospholipids, it is possible that psy may be able to aberrantly enter the lipid-binding site of PKC. It is equally possible that psy may act by altering the plasma membrane in such a way as to exclude PKC association without binding to PKC. To test this, we treated cells with psy or ent-psy to determine whether psy bound to PKC directly because ent-psy should not be capable of such an interaction. The lipid-binding domain of PKC is stereospecific and would be predicted not to recognize the enantiomer of a lipid binding partner (29Dekker L.V. Protein Kinase C. 2nd ed. Landes Bioscience/Eurekah.com United States, Georgetown, TX2004Google Scholar). We observed that psy and ent-psy inhibit the translocation of PKC to the membrane surface under stimulating conditions (Fig. 3). Previously, activated PKC (p-PKC) has been shown to be diminished at the DRMs in the brains of Twi mice compared with normal animals (14White A.B. Givogri M.I. Lopez-Rosas A. Cao H. van Breemen R. Thinakaran G. Bongarzone E.R. Psychosine accumulates in membrane microdomains in the brain of krabbe patients, disrupting the raft architecture.J. Neurosci. 2009; 29: 6068-6077Crossref PubMed Scopus (129) Google Scholar). Significantly less p-PKC is found associated with the DRM fraction of psy or ent-psy treated cells, so this inhibition of association, even in an unstimulated cell, is nonenantioselective. These data suggest that psy inhibition of PKC is most likely due to an alteration of the lipid environment rather than a direct binding of psy to PKC itself. To further investigate what effects psy may have on membranes, we used a liposome-based system. Disruptive lipids cause the liposomes to swell, whereas more inert lipids have no effect (30Pajewski R. Djedovic N. Harder E. Ferdani R. Schlesinger P.H. Gokel G.W. Pore formation in and enlargement of phospholipid liposomes by synthetic models of ceramides and sphingomyelin.Bioorg. Med. Chem. 2005; 13: 29-37Crossref PubMed Scopus (14) Google Scholar). Lipid vesicles composed of DOPC and filled with CF at a self-quenching concentration were exposed to psy at concentrations of 5, 20, and 50 μM. These concentrations of lipid are physiologically relevant because they represent nontoxic, moderat
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