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• W1993207934 abstract "Ceramides, which are membrane sphingolipids and key mediators of cell-stress responses, are generated by a family of (dihydro) ceramide synthases (Lass1–6/CerS1–6). Here, we report that brain development features significant increases in sphingomyelin, sphingosine, and most ceramide species. In contrast, C16:0-ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C16:0-ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca2+-loading capacity, which could be rescued by addition of ceramide. Selective CerS6 complexing with the inner membrane component of the mitochondrial permeability transition pore was detected by immunoprecipitation. This suggests that CerS6-generated ceramide could prevent mitochondrial permeability transition pore opening, leading to increased Ca2+ accumulation in the mitochondrial matrix. We examined the effect of high CerS6 expression on cell survival in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of de novo ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect: the pro-apoptotic role of CerS6 was not stimulus-specific. Knockdown of CerS6 with siRNA improved cell survival in response to nerve growth factor-induced OL apoptosis. Also, blocking mitochondrial Ca2+ uptake or decreasing Ca2+-dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. Thus, our data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca2+ homeostasis and calpain, which appears to be important in OL apoptosis during brain development. Ceramides, which are membrane sphingolipids and key mediators of cell-stress responses, are generated by a family of (dihydro) ceramide synthases (Lass1–6/CerS1–6). Here, we report that brain development features significant increases in sphingomyelin, sphingosine, and most ceramide species. In contrast, C16:0-ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C16:0-ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca2+-loading capacity, which could be rescued by addition of ceramide. Selective CerS6 complexing with the inner membrane component of the mitochondrial permeability transition pore was detected by immunoprecipitation. This suggests that CerS6-generated ceramide could prevent mitochondrial permeability transition pore opening, leading to increased Ca2+ accumulation in the mitochondrial matrix. We examined the effect of high CerS6 expression on cell survival in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of de novo ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect: the pro-apoptotic role of CerS6 was not stimulus-specific. Knockdown of CerS6 with siRNA improved cell survival in response to nerve growth factor-induced OL apoptosis. Also, blocking mitochondrial Ca2+ uptake or decreasing Ca2+-dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. Thus, our data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca2+ homeostasis and calpain, which appears to be important in OL apoptosis during brain development. IntroductionSphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation, migration, and apoptosis (1Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2395) Google Scholar). Most sphingolipids are ubiquitous, but complex sphingolipids, including sphingomyelin (SM) 2The abbreviations used are: SM, sphingomyelin; CI, caspase inhibitor; CerS, ceramide synthase; CLC, Ca2+-loading capacity; CSR, control siRNA; CSA, cyclosporin A; FB1, fumonisin B1; Glc-ceramide, glucosyl-ceramide; Lac-ceramide, lactosyl-ceramide; MPTP, mitochondrial permeability transition pore; OL, oligodendrocyte; p75NTR, p75 neurotrophin receptor; SPH, sphingosine; S1P, sphingosine-1-phosphate; TrkA, receptor tyrosine kinase; TMPD, N,N,N′,N′-tetramethyl-p-phenelenediamine; ANT, adenine nucleotide translocator. and glycosphingolipids, are more abundant in the brain and in myelin formed by oligodendrocytes (OLs). The building block of many complex sphingolipids is ceramide, which has numerous cellular signaling functions (2Kolesnick R.N. Goñi F.M. Alonso A. J. Cell. Physiol. 2000; 184: 285-300Crossref PubMed Scopus (374) Google Scholar). Ceramides are a family of distinct molecular species characterized by various acyl chains as well as the desaturation and hydroxylation of those chains. Highly hydrophobic ceramides are generated by membrane-associated enzymes and exert their effects proximal to the ceramide generation site, or they require specific transporter proteins to reach their targets in other intracellular compartments (1Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2395) Google Scholar, 3Futerman A.H. Riezman H. Trends Cell Biol. 2005; 15: 312-318Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar).Ceramides are synthesized de novo at the cytosolic side of the endoplasmic reticulum (4Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 5Mandon E.C. Ehses I. Rother J. van Echten G. Sandhoff K. J. Biol. Chem. 1992; 267: 11144-11148Abstract Full Text PDF PubMed Google Scholar), serving as precursors for the biosynthesis of glycosphingolipids and SM in the Golgi (6Kolter T. Proia R.L. Sandhoff K. J. Biol. Chem. 2002; 277: 25859-25862Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 7Futerman A.H. Stieger B. Hubbard A.L. Pagano R.E. J. Biol. Chem. 1990; 265: 8650-8657Abstract Full Text PDF PubMed Google Scholar). Mitochondria are another important intracellular compartment of sphingolipid metabolism (8Novgorodov S.A. Gudz T.I. J. Cardiovasc. Pharmacol. 2009; 53: 198-208Crossref PubMed Scopus (70) Google Scholar), and several sphingolipid-metabolizing enzymes were found to be associated with mitochondria, including neutral ceramidase (9El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), novel neutral sphingomyelinase (10Wu B.X. Rajagopalan V. Roddy P.L. Clarke C.J. Hannun Y.A. J. Biol. Chem. 2010; 285: 17993-18002Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and (dihydro) ceramide synthase (EC 2.3.1.24), a key enzyme in de novo ceramide synthesis (11Bionda C. Portoukalian J. Schmitt D. Rodriguez-Lafrasse C. Ardail D. Biochem. J. 2004; 382: 527-533Crossref PubMed Scopus (200) Google Scholar, 12Yu J. Novgorodov S.A. Chudakova D. Zhu H. Bielawska A. Bielawski J. Obeid L.M. Kindy M.S. Gudz T.I. J. Biol. Chem. 2007; 282: 25940-25949Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Recently, mitochondrial ceramide engagement in apoptosis has been shown using loss-of-function mutants of ceramide synthase in the germ cell line of Caenorhabditis elegans (13Deng X. Yin X. Allan R. Lu D.D. Maurer C.W. Haimovitz-Friedman A. Fuks Z. Shaham S. Kolesnick R. Science. 2008; 322: 110-115Crossref PubMed Scopus (146) Google Scholar). Specifically, ionizing radiation-induced apoptosis of germ cells was blocked upon inactivation of ceramide synthase, and apoptosis was restored upon microinjection of long-chain ceramide. Radiation-induced increases in ceramide localized to the mitochondria were required for activation of CED-3 caspase and apoptosis.Each of the 6 mammalian ceramide synthase (CerS, originally known as Lass) genes appears to regulate synthesis of a specific subset of ceramides, and each has a unique substrate specificity for chain-length and/or saturation of fatty acid acyl-CoA. Overexpression of any CerS protein in mammalian cells resulted in increases in a specific subset of ceramide species. CerS1 has high specificity for C18:0-CoA generating C18:0-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar, 15Spassieva S. Seo J.G. Jiang J.C. Bielawski J. Alvarez-Vasquez F. Jazwinski S.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2006; 281: 33931-33938Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). CerS2, CerS4, and CerS3 appear to have broader specificity (16Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2006; 398: 531-538Crossref PubMed Scopus (137) Google Scholar, 17Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). CerS2 or CerS4 mainly synthesizes C20:0-, C22:0-, C24:1-, C24:0-, C26:1-, and C26:0-ceramide, but is unable to synthesize C16:0- or C18:0-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar, 17Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). CerS3 generates C18:0-, C20:0-, C22:0-, and C24:0-ceramide (16Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2006; 398: 531-538Crossref PubMed Scopus (137) Google Scholar). It has been shown that CerS5 generates C14:0-, C16:0-, C18:0-, and C18:1-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar, 18Lahiri S. Futerman A.H. J. Biol. Chem. 2005; 280: 33735-33738Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar); and CerS6 produces C14:0-, C16:0-, and C18:0-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar).Our studies described here were designed to ascertain the functional role of ceramide and CerS6 in mitochondria during postnatal animal brain development. Herein, we report that, contrary to most ceramide species, C16:0-ceramide was down-regulated, as was CerS6 expression, in mitochondria. The data imply that CerS6 could be a primary ceramide synthase, generating C16:0-ceramide in brain mitochondria. Functional analysis revealed a significant decrease in Ca2+-loading capacity in mitochondria from the adult rat brain compared with the postnatal day 10 (P10) brain, and this decrease occurred with lower CerS6 expression and decreased C16:0-ceramide. Exogenously added C16:0-ceramide completely restored the Ca2+-loading capacity of adult mitochondria to that of the young rat brain. Co-immunoprecipitation studies exposed selective CerS6 association with adenine nucleotide translocator (ANT), the mitochondrial permeability transition pore (MPTP) component in the inner mitochondrial membrane. This suggests that CerS6 could generate C16:0-ceramide in close proximity of MPTP and prevent pore opening that results in an increased mitochondrial Ca2+-buffering capacity. Gene knockdown experiments revealed a critical role for CerS6 in promoting OL apoptosis. Thus, knocking down CerS6 enhanced OL survival in response to glutamate- or nerve growth factor-induced apoptosis. Investigation of downstream targets of the CerS6-mediated signaling pathway revealed an important contribution of mitochondrial Ca2+ and calpain in promoting ceramide-dependent apoptosis in OLs. Specifically, OL exposure to inhibitors of mitochondrial Ca2+ uptake or calpain activity enhanced cell survival in response to glutamate and NGF. Knocking down CerS6 reduced calpain activation. These studies identify CerS6 as an important regulator of mitochondrial Ca2+ homeostasis and suggest a pro-apoptotic role in OLs during postnatal brain development.DISCUSSIONThe present studies are unique in establishing a novel pro-apoptotic signaling pathway mediated by CerS6 in OLs. We have shown that an apoptotic stimulus triggers activation of CerS6 and generation of ceramide, thereby disturbing mitochondrial Ca2+ homeostasis and calpain activation, which results in OL death. Furthermore, CerS6 is down-regulated during postnatal brain development and appears to generate C16:0-ceramide in brain mitochondria. This is the first demonstration of an essential role of CerS6 in the neural cell apoptosis.Apoptosis is important during brain development, eliminating excess cells and ensuring the establishment of a proper synaptic connection network. In contrast to OLs, neuronal apoptosis has been extensively studied, and two waves of neuronal cell death have been described. The first wave consists of a large number of dividing neurons being eliminated during a peak of neurogenesis at mid-embryogenesis due to competition for a limited supply of neurotrophic factors and intracellular processes (56Meier P. Finch A. Evan G. Nature. 2000; 407: 796-801Crossref PubMed Scopus (801) Google Scholar). The second wave consists of differentiated neurons dying while migrating toward their target location or while connecting to target cells during the early postnatal period (57de la Rosa E.J. de Pablo F. Trends Neurosci. 2000; 23: 454-458Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar).To provide electrical insulation and maximize their conduction velocity, the axonal tracts in the central nervous system are myelinated by OLs in early postnatal life. The myelin biogenesis is coordinated by neuronal signals that control OL proliferation, differentiation, and survival (36Barres B.A. Hart I.K. Coles H.S. Burne J.F. Voyvodic J.T. Richardson W.D. Raff M.C. Cell. 1992; 70: 31-46Abstract Full Text PDF PubMed Scopus (1160) Google Scholar, 58Barres B.A. Raff M.C. J. Cell Biol. 1999; 147: 1123-1128Crossref PubMed Scopus (261) Google Scholar). OLs are greatly overproduced and the cell number is adjusted to the number and length of axons requiring myelination (36Barres B.A. Hart I.K. Coles H.S. Burne J.F. Voyvodic J.T. Richardson W.D. Raff M.C. Cell. 1992; 70: 31-46Abstract Full Text PDF PubMed Scopus (1160) Google Scholar). Only the OLs that manage to ensheath the axon survive, whereas those that fail degenerate (36Barres B.A. Hart I.K. Coles H.S. Burne J.F. Voyvodic J.T. Richardson W.D. Raff M.C. Cell. 1992; 70: 31-46Abstract Full Text PDF PubMed Scopus (1160) Google Scholar). Given the significance of neuron-derived factors in regulation of OL survival, the characterization of a ceramide-mediated apoptotic pathway triggered by glutamate and nerve growth factor is a valuable contribution to our understanding of sphingolipid signaling in OL apoptosis during brain development.Our studies identify CerS6 as a novel determinant of the pro-apoptotic signaling cascade in OLs. Here, we show that OL exposure to high concentrations of glutamate activates de novo ceramide biosynthesis, leading to cell death. Blocking ceramide production with a specific inhibitor of de novo ceramide biosynthesis or an inhibitor of ceramide synthase enhanced OL survival (Fig. 7, D and C). Gene knockdown experiments suggested involvement of CerS6, but not CerS5, in glutamate-induced OL death (Fig. 8, B–D). Further investigations revealed that CerS6-mediated OL death involves apoptotic mechanisms through activation of functionally similar executioner caspases 3 and 7 (Fig. 8A). Based on the knock-out mice studies, caspases 3 and 7 have been implicated as key mediators of apoptotic events downstream of mitochondria (59Lakhani S.A. Masud A. Kuida K. Porter Jr., G.A. Booth C.J. Mehal W.Z. Inayat I. Flavell R.A. Science. 2006; 311: 847-851Crossref PubMed Scopus (899) Google Scholar). Caspase activation requires the release of pro-apoptotic proteins from mitochondria due to mitochondrial dysfunction and loss of integrity. Indeed, glutamate-induced OL apoptosis appears to involve a disturbance in mitochondrial Ca2+ homeostasis and activation of the Ca2+-dependent protease, calpain (Fig. 11, A and C). Knocking down CerS6 reduced calpain activation in response to glutamate, suggesting that calpain is a downstream target of CerS6.Furthermore, these studies show that a CerS6/ceramide-mediated pro-apoptotic signaling pathway is essential for p75NTR-induced OL apoptosis (Fig. 10). Whereas p75NTR-induced responses in OLs are certainly understudied, ceramide participation in p75NTR-initiated signaling activities, ranging from growth and differentiation to apoptosis, is well established in neurons (60Brann A.B. Scott R. Neuberger Y. Abulafia D. Boldin S. Fainzilber M. Futerman A.H. J. Neurosci. 1999; 19: 8199-8206Crossref PubMed Google Scholar, 61Blöchl A. Blöchl R. J. Neurochem. 2007; 102: 289-305Crossref PubMed Scopus (102) Google Scholar). Thus, stimulation of p75NTR activates neutral and/or acid sphingomyelinases in the vicinity of the receptor in the plasma membrane that results in SM hydrolysis and ceramide generation. It has been emphasized that transient ceramide production upon sphingomyelinase activation takes place within 1–5 min and mainly serves the membrane structure, thereby facilitating the clustering of the death receptors localized in lipid rafts and promoting apoptosis (62Grassmé H. Cremesti A. Kolesnick R. Gulbins E. Oncogene. 2003; 22: 5457-5470Crossref PubMed Scopus (234) Google Scholar, 63van Blitterswijk W.J. van der Luit A.H. Veldman R.J. Verheij M. Borst J. Biochem. J. 2003; 369: 199-211Crossref PubMed Scopus (380) Google Scholar). Our studies point to an important pro-apoptotic role of ceramide generated by CerS6 in mitochondria, and they agree with the concept that endogenous ceramide production should be considered in its topological context (1Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2395) Google Scholar, 63van Blitterswijk W.J. van der Luit A.H. Veldman R.J. Verheij M. Borst J. Biochem. J. 2003; 369: 199-211Crossref PubMed Scopus (380) Google Scholar).Mitochondria are being appreciated as vital intracellular compartments for ceramide metabolism. Mitochondria have been shown to contain a variety of sphingolipids, including SM and ceramide (64Ardail D. Popa I. Alcantara K. Pons A. Zanetta J.P. Louisot P. Thomas L. Portoukalian J. FEBS Lett. 2001; 488: 160-164Crossref PubMed Scopus (60) Google Scholar, 65Tserng K.Y. Griffin R. Anal. Biochem. 2003; 323: 84-93Crossref PubMed Scopus (61) Google Scholar). Although several enzyme activities involved in ceramide metabolism have been shown in mitochondria, the nature of ceramide biosynthesis enzymes in this organelle is still a matter of debate (17Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar).Ceramide synthase activity was first detected (66Morell P. Radin N.S. J. Biol. Chem. 1970; 245: 342-350Abstract Full Text PDF PubMed Google Scholar, 67Ullman M.D. Radin N.S. Arch. Biochem. Biophys. 1972; 152: 767-777Crossref PubMed Scopus (57) Google Scholar) and partially purified from a bovine brain mitochondria-enriched fraction (68Shimeno H. Soeda S. Yasukouchi M. Okamura N. Nagamatsu A. Biol. Pharm. Bull. 1995; 18: 1335-1339Crossref PubMed Scopus (29) Google Scholar). Mitochondrial enzymes had ∼2-fold higher specific ceramide synthase activity than the ceramide synthase from the ER. The mitochondrial enzyme had a pH optimum ∼7.5 and maximal catalytic efficiency with C16:0- or C18:0-acyl-CoA (68Shimeno H. Soeda S. Yasukouchi M. Okamura N. Nagamatsu A. Biol. Pharm. Bull. 1995; 18: 1335-1339Crossref PubMed Scopus (29) Google Scholar). Purification of ceramide synthase from bovine liver mitochondria yielded two major protein bands: 62 and 72 kDa (69Shimeno H. Soeda S. Sakamoto M. Kouchi T. Kowakame T. Kihara T. Lipids. 1998; 33: 601-605Crossref PubMed Scopus (85) Google Scholar). Detailed analysis of ceramide synthase activity in highly purified mitochondria by Bionda et al. (11Bionda C. Portoukalian J. Schmitt D. Rodriguez-Lafrasse C. Ardail D. Biochem. J. 2004; 382: 527-533Crossref PubMed Scopus (200) Google Scholar) essentially confirmed previous findings. Thus, ceramide synthase activity was shown in rat liver mitochondria and in the subcompartment of the ER that is closely associated with mitochondria. Further submitochondrial investigation of ceramide synthase activity revealed enzyme localization to both outer and inner mitochondrial membranes (11Bionda C. Portoukalian J. Schmitt D. Rodriguez-Lafrasse C. Ardail D. Biochem. J. 2004; 382: 527-533Crossref PubMed Scopus (200) Google Scholar).Our studies describing CerS1, CerS2, CerS4, and CerS6, in purified brain mitochondria support the idea that several ceramide synthesizing enzymes could be localized to the mitochondria and/or to ER fragments tethered to the outer mitochondrial membrane (25Futerman A.H. Biochim. Biophys. Acta. 2006; 1758: 1885-1892Crossref PubMed Scopus (55) Google Scholar, 70Csordás G. Hajnóczky G. Biochim. Biophys. Acta. 2009; 1787: 1352-1362Crossref PubMed Scopus (242) Google Scholar). The results of our studies suggest that CerS6 could be localized to the inner mitochondrial membrane proximal to the MPTP, whereas CerS1, CerS2, and CerS4 are likely to be found in the outer mitochondrial membrane. The additional source of ceramide in mitochondria is a reverse reaction of a neutral ceramidase, e.g. formation of ceramide as a result of condensation of palmitate and sphingosine (71El Bawab S. Birbes H. Roddy P. Szulc Z.M. Bielawska A. Hannun Y.A. J. Biol. Chem. 2001; 276: 16758-16766Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). On the basis of molecular cloning and confocal microscopy data, this activity was ascribed to mitochondria (9El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and it was demonstrated in purified mitochondria (11Bionda C. Portoukalian J. Schmitt D. Rodriguez-Lafrasse C. Ardail D. Biochem. J. 2004; 382: 527-533Crossref PubMed Scopus (200) Google Scholar). A recent report suggests that ceramide could be generated by novel mitochondrial neutral sphingomyelinase hydrolyzing SM (10Wu B.X. Rajagopalan V. Roddy P.L. Clarke C.J. Hannun Y.A. J. Biol. Chem. 2010; 285: 17993-18002Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Continued research efforts are required to better understand the mechanisms of mitochondrial ceramide generation and utilization along with its influence on mitochondrial functions.Our studies provide further support for the concept of distinct roles of ceramide species in cell metabolism. As expected, sphingolipids and most ceramide species were increased in mitochondria during postnatal brain growth and development (Figs. 1 and 2). In contrast, C16:0-ceramide was severely reduced concomitantly with the down-regulation of CerS6 expression (Figs. 3 and 4). Although overexpressed in mammalian cells, CerS6 could generate C14:0-, C16:0-, and C18:0-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar); in brain tissue, CerS6 appears to specifically regulate C16:0-ceramide content in brain mitochondria during organ development. Increasing evidence suggests that the fatty acid chain of ceramide is an important characteristic of the biological effect mediated by the individual ceramide species. Generation of C18:0-ceramide, and not C16:0-ceramide, has been shown to repress the human telomerase reverse transcriptase promoter in lung carcinoma cells (72Wooten-Blanks L.G. Song P. Senkal C.E. Ogretmen B. FASEB J. 2007; 21: 3386-3397Crossref PubMed Scopus (55) Google Scholar). Activation of acid sphingomyelinase in the salvage pathway brought about a selective accumulation of C16:0-ceramide (20Chudakova D.A. Zeidan Y.H. Wheeler B.W. Yu J. Novgorodov S.A. Kindy M.S. Hannun Y.A. Gudz T.I. J. Biol. Chem. 2008; 283: 28806-28816Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 73Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) due to the involvement of CerS5 (73Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Another study revealed a specific role for dihydro-C16:0-ceramide in the adaptive cardiac tissue response to hypoxia (74Noureddine L. Azzam R. Nemer G. Bielawski J. Nasser M. Bitar F. Dbaibo G.S. Prostaglandins Other Lipid Mediat. 2008; 86: 49-55Crossref PubMed Scopus (20) Google Scholar). Although certain ceramide species could have different effects on biophysical properties of the membrane lipid bilayer (75Sot J. Aranda F.J. Collado M.I. Goñi F.M. Alonso A. Biophys. J. 2005; 88: 3368-3380Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), it remains unclear how ceramides containing different fatty acids exert their effects upon cell physiology. These studies suggest that regulated expression of specific CerS in intracellular compartments in conjunction with availability of certain fatty acyl-CoA species could be an important mechanism for controlling the fatty acid composition of ceramides and their biological effects on cell metabolism.In the present study, we investigated the effect of differential CerS6 protein expression on mitochondrial functions in brain mitochondria from young (high CerS6 expression) or adult (low CerS6 expression) rat brains (Fig. 5). Assessment of respiratory chain enzyme activities revealed no changes in oxidative phosphorylation parameters between brain mitochondria from animals at different ages. Consistent with previous reports (76Wang X. Carlsson Y. Basso E. Zhu C. Rousset C.I. Rasola A. Johansson B.R. Blomgren K. Mallard C. Bernardi P. Forte M.A. Hagberg H. J. Neurosci. 2009; 29: 2588-2596Crossref PubMed Scopus (109) Google Scholar), mitochondria from young animal brains were characterized by higher CLC compared with brain mitochondria from adult rats (Fig. 5). Remarkably, long-chain or very long-chain ceramide addition to brain mitochondria from adult rats enhanced their ability to retain Ca2+ such that the mitochondria were similar to mitochondria from young animal brains. Ceramide-mediated blockade of MPTP opening seems to be the underlying mechanism of the increased CLC. Further studies reveal selective association of CerS6 with ANT the inner mitochondrial membrane component of MPTP thereby linking CerS6 to the regulation of MPTP activity (Fig. 6). These studies suggest a novel role for CerS6/ceramide in governing Ca2+ homeostasis in brain mitochondria.The results from our study implicate CerS6 as an upstream regulator of calpain activity in the cellular response to apoptotic stimuli in OLs (Fig. 11). Calpains are part of a broad family of intracellular cysteine proteases that are independent from caspases. Typically, calpains function as key regulators in cytoskeletal remodeling through their substrates, including the microtubule-associated proteins neurofilament, Tau, and actin (77Potter D.A. Tirnauer J.S. Janssen R. Croall D.E. Hughes C.N. Fiacco K.A. Mier J.W. Maki M. Herman I.M. J. Cell Biol. 1998; 141: 647-662Crossref PubMed Scopus (229) Google Scholar). Conversely, calpain activation was found to increase as intracellular Ca2+ increased during oxidative stress, leading to induction of apoptotic pathways (55Das A. Sribnick E.A. Wingrave J.M. Del Re A.M. Woodward J.J. Appel S.H. Banik N.L. Ray S.K. J. Neurosci. Res. 2005; 81: 551-562Crossref PubMed Scopus (76) Google Scholar, 78Ishihara I. Minami Y. Nishizaki T. Matsuoka T. Yamamura H. Neurosci. Lett. 2000; 279: 97-100Crossref PubMed Scopus (36) Google Scholar).Whereas calpains are mainly cytosolic proteins, numerous reports exist of mitochondrial calpain-like activity (51Kar P. Samanta K. Shaikh S. Chowdhury A. Chakraborti T. Chakraborti S. Arch. Biochem. Biophys. 2010; 495: 1-7Crossref PubMed Scopus (68) Google Scholar). Cytosolic contamination of mitochondrial preparations has been a concern, and a few studies report the presence of calpain in the inner membrane fraction (47Kar P. Chakraborti T. Samanta K. Chakraborti S. Arch. Biochem. Biophys. 2008; 470: 176-186Crossref PubMed Scopus (22) Google Scholar). Mitochondrial calpains are thought to facilitate apoptosis-inducing factor release from the intra-membrane space, inducing caspase-independent apoptosis, but direct experimental evidence has been elusive (79Joshi A. Bondada V. Geddes J.W. Exp. Neurol. 2009; 218: 221-227Crossref PubMed Scopus (34) Google Scholar). Relevant to this study is the ability of mitochondrial calpain to modulate the activity of the MPTP, and the subsequent release of proteins initiating the caspase-dependent apoptotic cascade. Overexpression of calpain 10 induced mitochondrial fragmentation and swelling, consistent with the MPTP opening at a high conductance state and this altered mitochondrial morphology was blocked by MPTP inhibitors in kidney cells (49Arrington D.D. Van Vleet T.R. Schnellmann R.G. Am. J. Physiol. Cell Physiol. 2006; 291: C1159-C1171Crossref PubMed Scopus (157) Google Scholar).Conceivably, an apoptotic stimulus triggers a cytosolic Ca2+ influx into the mitochondria in OLs and an activation of mitochondrial CerS6 that then elevates ceramide. Ceramide blocks the MPTP opening at a low conductance state, leading to increased Ca2+ in the mitochondrial matrix. Rising mitochondrial Ca2+ activates calpain 10, which could cleave protein components of the MPTP resulting in the MPTP opening at a high conductance state, swelling, and rupture of the outer mitochondrial membrane and release of cytochrome c to initiate caspase activation. Noteworthy, all 8 calpain 10 splice variants seem to possess a mitochondrial targeting sequence localized to the NH2-terminal 15 amino acids (49Arrington D.D. Van Vleet T.R. Schnellmann R.G. Am. J. Physiol. Cell Physiol. 2006; 291: C1159-C1171Crossref PubMed Scopus (157) Google Scholar). Mitochondrial calpain expression appears to be tissue-specific. Also, it has been shown that smooth muscle and rat liver mitochondria do not contain calpain 10, whereas kidney mitochondria express only calpain 10 and not calpains 1 or 2 (51Kar P. Samanta K. Shaikh S. Chowdhury A. Chakraborti T. Chakraborti S. Arch. Biochem. Biophys. 2010; 495: 1-7Crossref PubMed Scopus (68) Google Scholar). Identification of the calpain isoform activated by the CerS6/ceramide-dependent pro-apoptotic pathway in OL mitochondria would be helpful, and these studies are currently underway in our laboratory.In summary, this study provides experimental evidence that apoptotic stimuli trigger activation of CerS6 and accumulation of ceramide that results in an increased Ca2+ in mitochondrial matrix and activation of calpain in OLs, and the data shed more light on the compartmentalization of sphingolipid metabolism and function in brain. IntroductionSphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation, migration, and apoptosis (1Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2395) Google Scholar). Most sphingolipids are ubiquitous, but complex sphingolipids, including sphingomyelin (SM) 2The abbreviations used are: SM, sphingomyelin; CI, caspase inhibitor; CerS, ceramide synthase; CLC, Ca2+-loading capacity; CSR, control siRNA; CSA, cyclosporin A; FB1, fumonisin B1; Glc-ceramide, glucosyl-ceramide; Lac-ceramide, lactosyl-ceramide; MPTP, mitochondrial permeability transition pore; OL, oligodendrocyte; p75NTR, p75 neurotrophin receptor; SPH, sphingosine; S1P, sphingosine-1-phosphate; TrkA, receptor tyrosine kinase; TMPD, N,N,N′,N′-tetramethyl-p-phenelenediamine; ANT, adenine nucleotide translocator. and glycosphingolipids, are more abundant in the brain and in myelin formed by oligodendrocytes (OLs). The building block of many complex sphingolipids is ceramide, which has numerous cellular signaling functions (2Kolesnick R.N. Goñi F.M. Alonso A. J. Cell. Physiol. 2000; 184: 285-300Crossref PubMed Scopus (374) Google Scholar). Ceramides are a family of distinct molecular species characterized by various acyl chains as well as the desaturation and hydroxylation of those chains. Highly hydrophobic ceramides are generated by membrane-associated enzymes and exert their effects proximal to the ceramide generation site, or they require specific transporter proteins to reach their targets in other intracellular compartments (1Hannun Y.A. Obeid L.M. Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2395) Google Scholar, 3Futerman A.H. Riezman H. Trends Cell Biol. 2005; 15: 312-318Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar).Ceramides are synthesized de novo at the cytosolic side of the endoplasmic reticulum (4Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 5Mandon E.C. Ehses I. Rother J. van Echten G. Sandhoff K. J. Biol. Chem. 1992; 267: 11144-11148Abstract Full Text PDF PubMed Google Scholar), serving as precursors for the biosynthesis of glycosphingolipids and SM in the Golgi (6Kolter T. Proia R.L. Sandhoff K. J. Biol. Chem. 2002; 277: 25859-25862Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 7Futerman A.H. Stieger B. Hubbard A.L. Pagano R.E. J. Biol. Chem. 1990; 265: 8650-8657Abstract Full Text PDF PubMed Google Scholar). Mitochondria are another important intracellular compartment of sphingolipid metabolism (8Novgorodov S.A. Gudz T.I. J. Cardiovasc. Pharmacol. 2009; 53: 198-208Crossref PubMed Scopus (70) Google Scholar), and several sphingolipid-metabolizing enzymes were found to be associated with mitochondria, including neutral ceramidase (9El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), novel neutral sphingomyelinase (10Wu B.X. Rajagopalan V. Roddy P.L. Clarke C.J. Hannun Y.A. J. Biol. Chem. 2010; 285: 17993-18002Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and (dihydro) ceramide synthase (EC 2.3.1.24), a key enzyme in de novo ceramide synthesis (11Bionda C. Portoukalian J. Schmitt D. Rodriguez-Lafrasse C. Ardail D. Biochem. J. 2004; 382: 527-533Crossref PubMed Scopus (200) Google Scholar, 12Yu J. Novgorodov S.A. Chudakova D. Zhu H. Bielawska A. Bielawski J. Obeid L.M. Kindy M.S. Gudz T.I. J. Biol. Chem. 2007; 282: 25940-25949Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Recently, mitochondrial ceramide engagement in apoptosis has been shown using loss-of-function mutants of ceramide synthase in the germ cell line of Caenorhabditis elegans (13Deng X. Yin X. Allan R. Lu D.D. Maurer C.W. Haimovitz-Friedman A. Fuks Z. Shaham S. Kolesnick R. Science. 2008; 322: 110-115Crossref PubMed Scopus (146) Google Scholar). Specifically, ionizing radiation-induced apoptosis of germ cells was blocked upon inactivation of ceramide synthase, and apoptosis was restored upon microinjection of long-chain ceramide. Radiation-induced increases in ceramide localized to the mitochondria were required for activation of CED-3 caspase and apoptosis.Each of the 6 mammalian ceramide synthase (CerS, originally known as Lass) genes appears to regulate synthesis of a specific subset of ceramides, and each has a unique substrate specificity for chain-length and/or saturation of fatty acid acyl-CoA. Overexpression of any CerS protein in mammalian cells resulted in increases in a specific subset of ceramide species. CerS1 has high specificity for C18:0-CoA generating C18:0-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar, 15Spassieva S. Seo J.G. Jiang J.C. Bielawski J. Alvarez-Vasquez F. Jazwinski S.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2006; 281: 33931-33938Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). CerS2, CerS4, and CerS3 appear to have broader specificity (16Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2006; 398: 531-538Crossref PubMed Scopus (137) Google Scholar, 17Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). CerS2 or CerS4 mainly synthesizes C20:0-, C22:0-, C24:1-, C24:0-, C26:1-, and C26:0-ceramide, but is unable to synthesize C16:0- or C18:0-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar, 17Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). CerS3 generates C18:0-, C20:0-, C22:0-, and C24:0-ceramide (16Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2006; 398: 531-538Crossref PubMed Scopus (137) Google Scholar). It has been shown that CerS5 generates C14:0-, C16:0-, C18:0-, and C18:1-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar, 18Lahiri S. Futerman A.H. J. Biol. Chem. 2005; 280: 33735-33738Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar); and CerS6 produces C14:0-, C16:0-, and C18:0-ceramide (14Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (302) Google Scholar).Our studies described here were designed to ascertain the functional role of ceramide and CerS6 in mitochondria during postnatal animal brain development. Herein, we report that, contrary to most ceramide species, C16:0-ceramide was down-regulated, as was CerS6 expression, in mitochondria. The data imply that CerS6 could be a primary ceramide synthase, generating C16:0-ceramide in brain mitochondria. Functional analysis revealed a significant decrease in Ca2+-loading capacity in mitochondria from the adult rat brain compared with the postnatal day 10 (P10) brain, and this decrease occurred with lower CerS6 expression and decreased C16:0-ceramide. Exogenously added C16:0-ceramide completely restored the Ca2+-loading capacity of adult mitochondria to that of the young rat brain. Co-immunoprecipitation studies exposed selective CerS6 association with adenine nucleotide translocator (ANT), the mitochondrial permeability transition pore (MPTP) component in the inner mitochondrial membrane. This suggests that CerS6 could generate C16:0-ceramide in close proximity of MPTP and prevent pore opening that results in an increased mitochondrial Ca2+-buffering capacity. Gene knockdown experiments revealed a critical role for CerS6 in promoting OL apoptosis. Thus, knocking down CerS6 enhanced OL survival in response to glutamate- or nerve growth factor-induced apoptosis. Investigation of downstream targets of the CerS6-mediated signaling pathway revealed an important contribution of mitochondrial Ca2+ and calpain in promoting ceramide-dependent apoptosis in OLs. Specifically, OL exposure to inhibitors of mitochondrial Ca2+ uptake or calpain activity enhanced cell survival in response to glutamate and NGF. Knocking down CerS6 reduced calpain activation. These studies identify CerS6 as an important regulator of mitochondrial Ca2+ homeostasis and suggest a pro-apoptotic role in OLs during postnatal brain development." @default.
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• W1993207934 title "Developmentally Regulated Ceramide Synthase 6 Increases Mitochondrial Ca2+ Loading Capacity and Promotes Apoptosis" @default.
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