Acid sphingomyelinase promotes mitochondrial dysfunction due to glutamate-induced regulated necrosis

Inhibiting the glutamate/cystine antiporter system xc−, a key antioxidant defense machinery in the CNS, could trigger a novel form of regulated necrotic cell death, ferroptosis. The underlying mechanisms of system xc−-dependent cell demise were elucidated using primary oligodendrocytes (OLs) treated with glutamate to block system xc− function. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. A sphingolipid profile showed elevations of ceramide species and sphingosine that were preventable by inhibiting of an acid sphingomyelinase (ASM) activity. OL survival was enhanced by both downregulating ASM expression and blocking ASM activity. Glutamate-induced ASM activation seems to involve posttranscriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced reactive oxygen species production, augmented lipid peroxidation, and opening of the mitochondrial permeability transition pore that were attenuated by hindering ASM. Of note, knocking down sirtuin 3, a deacetylase governing the mitochondrial antioxidant system, reduced OL survival. The data highlight the importance of the mitochondrial compartment in regulated necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which is essential for pathobiology in stroke and traumatic brain injury. Inhibiting the glutamate/cystine antiporter system xc−, a key antioxidant defense machinery in the CNS, could trigger a novel form of regulated necrotic cell death, ferroptosis. The underlying mechanisms of system xc−-dependent cell demise were elucidated using primary oligodendrocytes (OLs) treated with glutamate to block system xc− function. Pharmacological analysis revealed ferroptosis as a major contributing factor to glutamate-initiated OL death. A sphingolipid profile showed elevations of ceramide species and sphingosine that were preventable by inhibiting of an acid sphingomyelinase (ASM) activity. OL survival was enhanced by both downregulating ASM expression and blocking ASM activity. Glutamate-induced ASM activation seems to involve posttranscriptional mechanisms and was associated with a decreased GSH level. Further investigation of the mechanisms of OL response to glutamate revealed enhanced reactive oxygen species production, augmented lipid peroxidation, and opening of the mitochondrial permeability transition pore that were attenuated by hindering ASM. Of note, knocking down sirtuin 3, a deacetylase governing the mitochondrial antioxidant system, reduced OL survival. The data highlight the importance of the mitochondrial compartment in regulated necrotic cell death and accentuate the novel role of ASM in disturbing mitochondrial functions during OL response to glutamate toxicity, which is essential for pathobiology in stroke and traumatic brain injury. For many years, apoptosis was used as a synonym of programmed cell death, whereas the term necrosis was reserved for nonregulated cell death, which was not amenable to therapeutic manipulation. The recent discovery of regulated necrotic cell death mechanisms presents exciting possibilities for gaining control over cell survival in disease (1.Conrad M. Angeli J.P. Vandenabeele P. Stockwell B.R. Regulated necrosis: disease relevance and therapeutic opportunities.Nat. Rev. Drug Discov. 2016; 15: 348-366Crossref PubMed Scopus (396) Google Scholar). The uncovering of necroptosis and ferroptosis as the alternative forms of programmed cell death has resulted in a few studies implicating regulated necrotic cell death as an important contributing factor in tumor suppression, neurodegeneration, and ischemia/reperfusion (IR) tissue injury (2.Degterev A. Huang Z. Boyce M. Li Y. Jagtap P. Mizushima N. Cuny G.D. Mitchison T.J. Moskowitz M.A. Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.Nat. Chem. Biol. 2005; 1: 112-119Crossref PubMed Scopus (2023) Google Scholar, 3.Friedmann Angeli J.P. Schneider M. Proneth B. Tyurina Y.Y. Tyurin V.A. Hammond V.J. Herbach N. Aichler M. Walch A. Eggenhofer E. et al.Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.Nat. Cell Biol. 2014; 16: 1180-1191Crossref PubMed Scopus (1507) Google Scholar). In the brain, cessation of blood flow followed by reoxygenation, IR, induces a complex cascade of events involving an energy failure and an alteration of ionic homeostasis that results in excessive release of neurotransmitters, in particular, glutamate, into the extracellular space (4.Benveniste H. Drejer J. Schousboe A. Diemer N. Elevation of extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis.J. Neurochem. 1984; 43: 1369-1374Crossref PubMed Scopus (2475) Google Scholar). Microdialysis studies in both humans and rodents have also demonstrated a rise in extracellular glutamate following traumatic brain injury (TBI) (5.Guerriero R.M. Giza C.C. Rotenberg A. Glutamate and GABA imbalance following traumatic brain injury.Curr. Neurol. Neurosci. Rep. 2015; 15: 27Crossref PubMed Scopus (254) Google Scholar). Glutamate can harm oligodendrocytes (OLs), unique myelin-forming cells in the CNS, and neurons by excitotoxicity, caused via sustained activation of ionotropic glutamate receptors and/or by blocking the cystine/glutamate antiporter, system xc−, leading to oxidative stress (6.Matute C. Domercq M. Sanchez-Gomez M.V. Glutamate-mediated glial injury: mechanisms and clinical importance.Glia. 2006; 53: 212-224Crossref PubMed Scopus (256) Google Scholar, 7.Rosin C. Bates T.E. Skaper S.D. Excitatory amino acid induced oligodendrocyte cell death in vitro: receptor-dependent and -independent mechanisms.J. Neurochem. 2004; 90: 1173-1185Crossref PubMed Scopus (70) Google Scholar, 8.Murphy T.H. Miyamoto M. Sastre A. Schnaar R.L. Coyle J.T. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress.Neuron. 1989; 2: 1547-1558Abstract Full Text PDF PubMed Scopus (850) Google Scholar). System xc− is an amino acid antiporter that imports cystine, the oxidized form of cysteine, into cells with 1:1 countertransport of glutamate. It is composed of a regulatory protein, SLC3A2 (4F2hc), linked by a disulfide bridge to the 12-pass transmembrane protein, SLC7A11 (xCT), which is credited for the transport activity of the dimer (9.Bridges R.J. Natale N.R. Patel S.A. System xc(-) cystine/glutamate antiporter: an update on molecular pharmacology and roles within the CNS.Br. J. Pharmacol. 2012; 165: 20-34Crossref PubMed Scopus (311) Google Scholar). The transport is driven by a transmembrane glutamate gradient and it can be inhibited by high extracellular glutamate, which is the only physiological inhibitor identified so far (10.Albrecht P. Lewerenz J. Dittmer S. Noack R. Maher P. Methner A. Mechanisms of oxidative glutamate toxicity: the glutamate/cystine antiporter system xc- as a neuroprotective drug target.CNS Neurol. Disord. Drug Targets. 2010; 9: 373-382Crossref PubMed Scopus (138) Google Scholar, 11.Lewerenz J. Hewett S.J. Huang Y. Lambros M. Gout P.W. Kalivas P.W. Massie A. Smolders I. Methner A. Pergande M. et al.The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities.Antioxid. Redox Signal. 2013; 18: 522-555Crossref PubMed Scopus (523) Google Scholar). Ferroptosis is believed to be distinct from other types of regulated cell death, such as apoptosis, necroptosis, and autophagic cell death at morphological, biochemical, and genetic levels (12.Xie Y. Hou W. Song X. Yu Y. Huang J. Sun X. Kang R. Tang D. Ferroptosis: process and function.Cell Death Differ. 2016; 23: 369-379Crossref PubMed Scopus (1533) Google Scholar, 13.Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). Ferroptosis has been described as a form of regulated necrotic cell death characterized by excessive reactive oxygen species (ROS) generation and iron-dependent accumulation of lipid peroxidation products (13.Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). Ferroptosis can be induced through inhibition of system xc− or by blocking glutathione peroxidase 4 function (3.Friedmann Angeli J.P. Schneider M. Proneth B. Tyurina Y.Y. Tyurin V.A. Hammond V.J. Herbach N. Aichler M. Walch A. Eggenhofer E. et al.Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.Nat. Cell Biol. 2014; 16: 1180-1191Crossref PubMed Scopus (1507) Google Scholar, 13.Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). System xc− negatively regulates lipid peroxidation by providing cysteine, a substrate for biosynthesis of GSH that is required for activity of glutathione peroxidase 4, which reduces the accumulation of phospholipid peroxides and protects cells (1.Conrad M. Angeli J.P. Vandenabeele P. Stockwell B.R. Regulated necrosis: disease relevance and therapeutic opportunities.Nat. Rev. Drug Discov. 2016; 15: 348-366Crossref PubMed Scopus (396) Google Scholar, 3.Friedmann Angeli J.P. Schneider M. Proneth B. Tyurina Y.Y. Tyurin V.A. Hammond V.J. Herbach N. Aichler M. Walch A. Eggenhofer E. et al.Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.Nat. Cell Biol. 2014; 16: 1180-1191Crossref PubMed Scopus (1507) Google Scholar). Blocking the system xc−-dependent pathway has been considered as a promising cancer therapy (1.Conrad M. Angeli J.P. Vandenabeele P. Stockwell B.R. Regulated necrosis: disease relevance and therapeutic opportunities.Nat. Rev. Drug Discov. 2016; 15: 348-366Crossref PubMed Scopus (396) Google Scholar). Yet, the mechanisms of system xc−-mediated cell death in the neural cells and its relevance to neurological disease remain unclear. We hypothesized that high extracellular glutamate, a key factor in cerebral IR and TBI, could suppress the activity of the system xc−, leading to excessive ROS formation and OL death via ferroptosis. Experimental evidence implicates sphingolipids, which are elevated in cerebral mitochondria after IR and TBI, as a causal factor of mitochondrial dysfunction and elevated ROS (14.Yu J. Novgorodov S.A. Chudakova D. Zhu H. Bielawska A. Bielawski J. Obeid L.M. Kindy M.S. Gudz T.I. JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction.J. Biol. Chem. 2007; 282: 25940-25949Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 15.Novgorodov S.A. Gudz T.I. Ceramide and mitochondria in ischemic brain injury.Int. J. Biochem. Mol. Biol. 2011; 2: 347-361PubMed Google Scholar, 16.Novgorodov S.A. Riley C.L. Yu J. Borg K.T. Hannun Y.A. Proia R.L. Kindy M.S. Gudz T.I. Essential roles of neutral ceramidase and sphingosine in mitochondrial dysfunction due to traumatic brain injury.J. Biol. Chem. 2014; 289: 13142-13154Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 17.Novgorodov S.A. Riley C.L. Keffler J.A. Yu J. Kindy M.S. Macklin W.B. Lombard D.B. Gudz T.I. SIRT3 deacetylates ceramide synthases: implications for mitochondrial dysfunction and brain injury.J. Biol. Chem. 2016; 291: 1957-1973Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 18.Zigdon H. Kogot-Levin A. Park J.W. Goldschmidt R. Kelly S. Merrill Jr., A.H. Scherz A. Pewzner-Jung Y. Saada A. Futerman A.H. Ablation of ceramide synthase 2 causes chronic oxidative stress due to disruption of the mitochondrial respiratory chain.J. Biol. Chem. 2013; 288: 4947-4956Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). We have previously reported the augmented sphingosine levels in brain tissue and mitochondria in an animal model of TBI (16.Novgorodov S.A. Riley C.L. Yu J. Borg K.T. Hannun Y.A. Proia R.L. Kindy M.S. Gudz T.I. Essential roles of neutral ceramidase and sphingosine in mitochondrial dysfunction due to traumatic brain injury.J. Biol. Chem. 2014; 289: 13142-13154Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Several studies showed that sphingolipid ceramide increases in the brain after experimental stroke, and reduction of ceramide generation was neuroprotective, leading to smaller infarct sizes (19.Yu Z.F. Nikolova-Karakashian M. Zhou D. Cheng G. Schuchman E.H. Mattson M.P. Pivotal role for acidic sphingomyelinase in cerebral ischemia-induced ceramide and cytokine production, and neuronal apoptosis.J. Mol. Neurosci. 2000; 15: 85-97Crossref PubMed Scopus (171) Google Scholar, 20.Herr I. Martin-Villalba A. Kurz E. Roncaioli P. Schenkel J. Cifone M.G. Debatin K.M. FK506 prevents stroke-induced generation of ceramide and apoptosis signaling.Brain Res. 1999; 826: 210-219Crossref PubMed Scopus (92) Google Scholar, 21.Ohtani R. Tomimoto H. Kondo T. Wakita H. Akiguchi I. Shibasaki H. Okazaki T. Upregulation of ceramide and its regulating mechanism in a rat model of chronic cerebral ischemia.Brain Res. 2004; 1023: 31-40Crossref PubMed Scopus (59) Google Scholar). Ceramides, a family of distinct molecular species characterized by various acyl chains, are synthesized de novo in the endoplasmic reticulum (ER) or generated through the recycling pathway from SM hydrolysis by acid sphingomyelinase (ASM) (15.Novgorodov S.A. Gudz T.I. Ceramide and mitochondria in ischemic brain injury.Int. J. Biochem. Mol. Biol. 2011; 2: 347-361PubMed Google Scholar). ASM is a phosphodiesterase that converts SM, a structural component of membranes, into ceramide and phosphocholine (22.Henry B. Ziobro R. Becker K.A. Kolesnick R. Gulbins E. Acid sphingomyelinase.Handb. Exp. Pharmacol. 2013; 215: 77-88Crossref PubMed Scopus (61) Google Scholar). Hereditary mutations of ASM result in a toxic accumulation of SM in lysosomes and are the cause of Niemann-Pick disease. More recently, it was discovered that inhibition of ASM activity mediates the effects of antidepressant drugs (23.Gulbins E. Palmada M. Reichel M. Luth A. Bohmer C. Amato D. Muller C.P. Tischbirek C.H. Groemer T.W. Tabatabai G. et al.Acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs.Nat. Med. 2013; 19: 934-938Crossref PubMed Scopus (247) Google Scholar) and diminishes symptoms associated with Alzheimer's disease (24.Lee J.K. Jin H.K. Park M.H. Kim B.R. Lee P.H. Nakauchi H. Carter J.E. He X. Schuchman E.H. Bae J.S. Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer's disease.J. Exp. Med. 2014; 211: 1551-1570Crossref PubMed Scopus (106) Google Scholar). In this study, we provide evidence for a critical role of ASM in mitochondrial dysfunction leading to system xc−-dependent regulated necrotic OL death. Importantly, we show that ASM inhibitors or Smpd1 gene ablation preserves mitochondrial function, reduces ROS generation and oxidative lipid damage, and augments OL survival in response to glutamate. These studies suggest a novel mechanism of ASM involvement in regulated necrosis that could be an important contributing factor to brain injury in stroke and TBI. Female timed-pregnant Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were acclimated for 1 week prior to experimentation. ASM-deficient mice were provided by the animal core facility at the Medical University of South Carolina (MUSC), Charleston SC (25.Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (411) Google Scholar). Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of MUSC and followed the National Institutes of Health guidelines for experimental animal use. DMEM/F12 and FBS used for cell culture were from GIBCO (Thermo Fisher, Waltham, MA). The complete protease inhibitor cocktail and PhosphoStop phosphatase inhibitor cocktail were from Roche Applied Science (Indianapolis, IN). Reclast (zoledronic acid), Z-VAD-fmk, and epoxyquinone G109 were from Enzo Biochem (Farmingdale, NY). Fumonisin B1 (FB1), myriocin, and GKT137831 were purchased from Cayman Chemical (Ann Arbor, MI). The 2′,7′-dichlorodihydrofluorescein (H2-DCF) diacetate, 4-acetamido-TEMPO, and MitoSox Red were from Thermo Fisher, C10-biphosphonate (C10-BPA) was from Avanti Polar Lipids (Alabaster, AL). Necrostatin-1 (methyl-thiohydantoin-tryptophan) and necrostatin-1s (7-Cl-O-necrostatin-1) were from BioVision (Milpitas, CA). LOXBlock-1 was from ChemBridge Corporation (San Diego, CA). GSK-872 and 4-amino-N-(3-chloro-4-fluorophenyl)-Nʹ-hydroxy-1,2,5-oxadiazole-3-carboximidamide indolamine 2,3-deoxygenase (IDO) inhibitor were from EMD Millipore (Billerica, MA). NIM811 was generously provided by Novartis (Cambridge, MA). LCL-521 was provided by the Lipidomics Core facility at MUSC. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit monoclonal anti-receptor-interacting protein kinase (RIPK)1, anti-RIPK3, anti-caspase-8, anti-LC3, anti-beclin-1 antibodies, and rabbit polyclonal anti-p62 antibodies were supplied by Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti-β-actin antibody was from Sigma-Aldrich. Mouse monoclonal anti-RIPK1 antibody was purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal anti-ASM antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-mixed-lineage kinase domain-like (MLKL) antibody was from Thermo Fisher. Secondary horseradish peroxidase-conjugated antibodies were supplied by Jackson ImmunoResearch (West Grove, PA). Dissociated cortices of rat and mouse 2-day-old pups were cultured on poly-l-lysine (PLL)-coated flasks as described (26.Chudakova D.A. Zeidan Y.H. Wheeler B.W. Yu J. Novgorodov S.A. Kindy M.S. Hannun Y.A. Gudz T.I. Integrin-associated Lyn kinase promotes cell survival by suppressing acid sphingomyelinase activity.J. Biol. Chem. 2008; 283: 28806-28816Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Briefly, the cerebra of pups were dissected and minced to generate a single-cell suspension. Cells were plated into 75 cm2 flasks and grown in DMEM/F12 medium with 10% FBS at 37°C and 5% CO2. By day 10, mixed glial cultures were obtained, consisting of OLs and microglia growing on an astrocyte monolayer. OLs were purified from mixed glial cell cultures using a shake-off procedure. OLs were collected by centrifugation at 1,000 g for 5 min. OLs were used immediately for transfections or further culturing. Cell culture dishes and plates were precoated with PLL (50 μg/ml). Cells were plated in cell culture dishes and 96-well (4 × 105 cells/well) plates in DMEM/F12 medium with 10% FBS and N2 supplement and allowed 24 h for attachment. Cells were treated with glutamate and/or inhibitors in DMEM medium without cystine for a defined time. All cultures contained less than 2% of GFAP+ astrocytes and nondetectable CD11+ microglia. To downregulate ASM (Smpd1) and sirtuin 3 (SIRT3), ON-TARGET plus SMARTpool silencing RNAs were obtained from GE Healthcare/Dharmacon (Rockford, IL). The set consists of four siRNAs targeting different regions of the gene to minimize the off-target effects. The following target sequences were used: Smpd1, 5′-GAACAUAGCGCCACUAAAU-3′, 5′-GCAACAGUCUCGACAAGAU-3′, 5′-GCAUAUAAUUGGGCACAUU-3′, 5′-CGCCUCAUCUCUCUCAAUA-3′ or Sirt3, 5′-GCUCAUGGGUCCUUUGUAU-3′, 5′-GGAUGGGACAGGACGGAUAA-3′, 5′-CAGCAA­GGUUCUUACUACA-3′, 5′-CAGCUUGUCUGAAUCGGUA-3′. OLs were transfected with siRNA using a Nucleofector electroporation system (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's instructions with efficiencies of >70% as described (26.Chudakova D.A. Zeidan Y.H. Wheeler B.W. Yu J. Novgorodov S.A. Kindy M.S. Hannun Y.A. Gudz T.I. Integrin-associated Lyn kinase promotes cell survival by suppressing acid sphingomyelinase activity.J. Biol. Chem. 2008; 283: 28806-28816Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Cells (6 × 106) were mixed with 100 μl of Nucleofector reagent and 20 nM (1 μl) siRNA in the cuvette of the Amaxa electroporation device. ON-TARGET plus nontargeting pool siRNA (GE Healthcare/Dharmacon) was used as a control. Cell death was measured using a lactate dehydrogenase (LDH)-based CytoTox-ONE™ homogeneous membrane integrity assay (Promega, Madison, WI) according to the manufacturer's recommendations. The fluorescence of the sample was measured at 590 nm emission with 560 nm excitation in a microplate reader (FLUOstar Optima; BMG LABTECH Inc., Durham, NC). The activities of executioner caspase 3/7 were determined using an Apo-One® homogeneous kit (Promega) according to the manufacturer's instructions. Cleavage of nonfluorescent substrate, Z-DEVD-Rodamine-110, by caspase 3/7 resulted in fluorescent Rodamine-110. The fluorescence of the sample was measured at 530 nm emission and 490 nm excitation in the microplate reader, Synergy H1 (BioTek, Winooski, VT). Mitochondria were isolated from OLs using a hypotonic swelling procedure (27.Panov A.V. Lund S. Greenamyre J.T. Ca2+-induced permeability transition in human lymphoblastoid cell mitochondria from normal and Huntington's disease individuals.Mol. Cell. Biochem. 2005; 269: 143-152Crossref PubMed Scopus (88) Google Scholar). OLs (3 × 105 cells/well) were plated on 96-well Seahorse XF-96 plates (Seahorse Biosciences, Billerica, MA) coated with PLL and maintained in DMEM/F12 medium for 24 h in humidified 5% CO2/95% air at 37°C. OLs were treated with glutamate in cystine-free medium for 6 h, then the medium was replaced with DPBS (PBS containing Ca2+ and Mg2+) supplemented with 10 mM glucose, respiration [oxygen consumption rate (OCR)] was measured in a Seahorse Bioscience XF-96 extracellular flux analyzer, and respiratory rates were calculated using Seahorse XF-96 software and the Direct ACOS fast algorithm with continuous averaging, as described (28.Gerencser A.A. Neilson A. Choi S.W. Edman U. Yadava N. Oh R.J. Ferrick D.A. Nicholls D.G. Brand M.D. Quantitative microplate-based respirometry with correction for oxygen diffusion.Anal. Chem. 2009; 81: 6868-6878Crossref PubMed Scopus (248) Google Scholar). Cells in modified HBSS were loaded (30 min at 37°C) with 200 nM TMRM or 5 μM MitoSox Red. After loading and washing, subsequent incubations were performed with 50 nM TMRM or 1 μM MitoSox Red to maintain equilibrium distribution of the fluorophore (29.Maldonado E.N. DeHart D.N. Patnaik J. Klatt S.C. Gooz M.B. Lemasters J.J. ATP/ADP turnover and import of glycolytic ATP into mitochondria in cancer cells is independent of the adenine nucleotide translocator.J. Biol. Chem. 2016; 291: 19642-19650Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Cells incubated in HBSS in humidified 5% CO2/air at 37°C were imaged with a Zeiss LSM 510 NLO inverted laser scanning confocal/multiphoton microscope (Thornwood, NJ) using a 63 × 1.4 N.A. plan apochromat oil immersion lens. Fluorescence of TMRM was detected at 560 nm (excitation 543 nm) or fluorescence of MitoSox Red was detected at 580 nm (excitation 510 nm) through a filter and a 1 airy unit-diameter pinhole. After the treatment with glutamate and test compounds, cells were washed three times with DPBS and incubated in the presence of 2 μM H2-DCF diacetate in DPBS supplemented with 10 mM glucose (DPBS buffer) at 37°C. Cellular ROS formation was assessed by oxidation of H2-DCF. Fluorescence of oxidized H2-DCF was measured at 520 nm emission and 490 nm excitation in the microplate reader, Synergy H1 (BioTek). Mitochondrial ROS formation was measured using MitoSox Red fluorescent dye. Cells were incubated with 5 μM MitoSox Red (Molecular Probes) in DPBS buffer for 30 min at 37°C to allow the dye to reach mitochondria. Cells were washed twice with DPBS buffer to remove excess dye. Fluorescence was measured at 580 nm emission and 510 nm excitation in the Synergy H1 microplate reader. ASM activity was measured using an ASM activity assay kit (Echelon Biosciences, Salt Lake City, UT) according to the manufacturer's instructions. Neutral sphingomyelinase (NSM) activity was determined as described for the ASM assay, except that the reaction mixture contained 100 mM Tris (pH 7.4) instead of 100 mM sodium acetate (pH 5.0) and was supplemented with 10 mM Mg2+, which is required for NSM activity (26.Chudakova D.A. Zeidan Y.H. Wheeler B.W. Yu J. Novgorodov S.A. Kindy M.S. Hannun Y.A. Gudz T.I. Integrin-associated Lyn kinase promotes cell survival by suppressing acid sphingomyelinase activity.J. Biol. Chem. 2008; 283: 28806-28816Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). GSH content was determined using a glutathione fluorometric assay kit (BioVision) according to the manufacturer's instructions. After the treatment with glutamate and test compounds for 18 h, cells were incubated in the presence of 1 μM of BODIPY 581/591 C11, a fluorescent reporter for lipid peroxidation, for 1 h. Upon oxidation, the reagent shifts the fluorescence excitation/emission peak from 581/591 nm to 488/510 nm. The ratio of fluorescence intensities at 590 nm to 510 nm gives the read-out for lipid peroxidation. Lipid peroxidation products were measured using a malondialdehyde (MDA) assay kit (Sigma-Aldrich) according to the manufacturer's instructions. Immunoprecipitations were performed as we previously described (17.Novgorodov S.A. Riley C.L. Keffler J.A. Yu J. Kindy M.S. Macklin W.B. Lombard D.B. Gudz T.I. SIRT3 deacetylates ceramide synthases: implications for mitochondrial dysfunction and brain injury.J. Biol. Chem. 2016; 291: 1957-1973Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 30.Novgorodov A.S. El-Alwani M. Bielawski J. Obeid L.M. Gudz T.I. Activation of sphingosine-1-phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration.FASEB J. 2007; 21: 1503-1514Crossref PubMed Scopus (154) Google Scholar, 31.Novgorodov S.A. Chudakova D.A. Wheeler B.W. Bielawski J. Kindy M.S. Obeid L.M. Gudz T.I. Developmentally regulated ceramide synthase 6 increases mitochondrial Ca2+ loading capacity and promotes apoptosis.J. Biol. Chem. 2011; 286: 4644-4658Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). For immunoprecipitation, cell lysates (500 μg) were precleared in buffer A [0.15 M NaCl, 0.5 mM EDTA, 0.5% Igepal CA-630, protease and phosphatase inhibitor cocktail, 0.05 M Tris (pH 7.5), and 0.2% BSA] by incubation with appropriate species-specific IgG-conjugated magnetic beads (Dynabeads, Dynal; Thermo Fisher) for 1 h. Antibodies were then added. After incubation at 4°C overnight with gentle mixing, antibody-antigen complexes were captured with Dynabeads and washed two times with buffer A (without BSA), and then washed twice with TBS (pH 7.5). The immunoprecipitates were eluted by boiling in SDS-sample buffer. As a control, the same immunoprecipitation procedure was performed except for the primary antibody application. Proteins were analyzed by Western blot (17.Novgorodov S.A. Riley C.L. Keffler J.A. Yu J. Kindy M.S. Macklin W.B. Lombard D.B. Gudz T.I. SIRT3 deacetylates ceramide synthases: implications for mitochondrial dysfunction and brain injury.J. Biol. Chem. 2016; 291: 1957-1973Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 30.Novgorodov A.S. El-Alwani M. Bielawski J. Obeid L.M. Gudz T.I. Activation of sphingosine-1-phosphate receptor S1P5 inhibits oligodendrocyte progenitor migration.FASEB J. 2007; 21: 1503-1514Crossref PubMed Scopus (154) Google Scholar). Proteins were separated by 4–15% SDS-PAGE, blotted to PVDF membrane, blocked with 5% nonfat dry milk (Bio-Rad, Hercules, CA) or 5% BSA in TBS-T buffer [10 mM Tris, 150 mM NaCl, and 0.2% Tween-20 (pH 8.0)] and subsequently probed with the appropriate primary antibody. Immunoreactive bands were visualized using a SuperSignal West Dura substrate (Thermo Fisher). Sphingolipid content was determined by MS/MS (14.Yu J. Novgorodov S.A. Chudakova D. Zhu H. Bielawska A. Bielawski J. Obeid L.M. Kindy M.S. Gudz T.I. JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction.J. Biol. Chem. 2007; 282: 25940-25949Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 17.Novgorodov S.A. Riley C.L. Keffler J.A. Yu J. Kindy M.S. Macklin W.B. Lombard D.B. Gudz T.I. SIRT3 deacetylates ceramide synthases: implications for mitochondrial dysfunction and brain injury.J. Biol. Chem. 2016; 291: 1957-1973Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Briefly, to extract lipids, 0.5 mg of mitochondria or cell lysate protein was added to 2 ml of the ethyl acetate/isopropanol/water (60:30:10%, v/v/v) solvent system. The lipid extracts were fortified with internal standards, dried under a stream of nitrogen gas, and reconstituted in 100 μl of methanol for ESI-MS/MS analysis, which was performed on a Thermo Fisher TSQ Quantum triple quadrupole mass spectrometer, operating in a multiple reaction-monitoring positive-ionization mode. The samples were injected onto the HP1100/TSQ Quantum liquid