Title: Basal Levels of eIF2α Phosphorylation Determine Cellular Antioxidant Status by Regulating ATF4 and xCT Expression
Abstract: eIF2α is part of a multimeric complex that regulates cap-dependent translation. Phosphorylation of eIF2α (phospho-eIF2α) is induced by various forms of cell stress, resulting in changes to the proteome of the cell with two diametrically opposed consequences, adaptation to stress or initiation of programmed cell death. In contrast to the robust eIF2α phosphorylation seen in response to acute insults, less is known about the functional role of basal levels of eIF2α phosphorylation. Here we show that mouse embryonic fibroblasts expressing a nonphosphorylatable eIF2α have enhanced sensitivity to diverse toxic insults, including amyloid β-(1–42) peptide (Aβ), a key factor in the pathogenesis of Alzheimer disease. This correlates with impaired glutathione metabolism because of down-regulation of the light chain, xCT, of the cystine/glutamate antiporter system X-c. The mechanistic link between the absence of phospho-eIF2α and xCT expression is nuclear factor ATF4. Consistent with these findings, long term activation of the phospho-eIF2α/ATF4/xCT signaling module by the specific eIF2α phosphatase inhibitor, salubrinal, induces resistance against oxidative glutamate toxicity in the hippocampal cell line HT22 and primary cortical neurons. Furthermore, in PC12 cells selected for resistance against Aβ, increased activity of the phospho-eIF2α/ATF4/xCT module contributes to the resistant phenotype. In wild-type PC12 cells, activation of this module by salubrinal ameliorates the response to Aβ. Furthermore, in human brains, ATF4 and phospho-eIF2α levels are tightly correlated and up-regulated in Alzheimer disease, most probably representing an adaptive response against disease-related cellular stress rather than a correlate of neurodegeneration. eIF2α is part of a multimeric complex that regulates cap-dependent translation. Phosphorylation of eIF2α (phospho-eIF2α) is induced by various forms of cell stress, resulting in changes to the proteome of the cell with two diametrically opposed consequences, adaptation to stress or initiation of programmed cell death. In contrast to the robust eIF2α phosphorylation seen in response to acute insults, less is known about the functional role of basal levels of eIF2α phosphorylation. Here we show that mouse embryonic fibroblasts expressing a nonphosphorylatable eIF2α have enhanced sensitivity to diverse toxic insults, including amyloid β-(1–42) peptide (Aβ), a key factor in the pathogenesis of Alzheimer disease. This correlates with impaired glutathione metabolism because of down-regulation of the light chain, xCT, of the cystine/glutamate antiporter system X-c. The mechanistic link between the absence of phospho-eIF2α and xCT expression is nuclear factor ATF4. Consistent with these findings, long term activation of the phospho-eIF2α/ATF4/xCT signaling module by the specific eIF2α phosphatase inhibitor, salubrinal, induces resistance against oxidative glutamate toxicity in the hippocampal cell line HT22 and primary cortical neurons. Furthermore, in PC12 cells selected for resistance against Aβ, increased activity of the phospho-eIF2α/ATF4/xCT module contributes to the resistant phenotype. In wild-type PC12 cells, activation of this module by salubrinal ameliorates the response to Aβ. Furthermore, in human brains, ATF4 and phospho-eIF2α levels are tightly correlated and up-regulated in Alzheimer disease, most probably representing an adaptive response against disease-related cellular stress rather than a correlate of neurodegeneration. eIF2α is part of the multimeric eIF2 complex that is involved in the initiation of cap-dependent protein translation (for reviews see Refs. 1Wek R.C. Jiang H.-Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (995) Google Scholar, 2Proud C.G. Semin. Cell Dev. Biol. 2005; 16: 3-12Crossref PubMed Scopus (299) Google Scholar). The eIF2 complex brings the 40 S ribosomal subunit together with the initiating tRNAMet when eIF2 is bound to GTP. Upon hydrolysis of GTP to GDP, the complex is no longer active, and protein synthesis is not initiated. GDP/GTP exchange requires the activity of the guanine nucleotide exchange factor eIF2B. However, when eIF2α is phosphorylated on Ser-51 (phospho-eIF2α), the affinity of eIF2α for eIF2B increases, and thus it can sequester eIF2B, thereby inhibiting GDP/GTP exchange. As cells have considerably higher amounts of eIF2α compared with eIF2B, even modest increases in phospho-eIF2α can modulate eIF2B reactivation. Although these changes slow down cap-dependent initiation, they favor cap-independent translation. Proteins up-regulated by this mechanism include transcription factors such as activating transcription factor-4 (ATF4). Therefore, eIF2α phosphorylation orchestrates significant changes in the proteome of the cell. There are four known eIF2α kinases as follows: protein kinase R, heme-regulated eIF2α kinase, protein kinase R-like kinase (PERK), and GCN2 (general control nonderepressible-2) (for reviews see Refs. 1Wek R.C. Jiang H.-Y. Anthony T.G. Biochem. Soc. Trans. 2006; 34: 7-11Crossref PubMed Scopus (995) Google Scholar, 2Proud C.G. Semin. Cell Dev. Biol. 2005; 16: 3-12Crossref PubMed Scopus (299) Google Scholar, 3DeGracia D.J. Kumar R. Owen C.R. Krause G.S. White B.C. J. Cereb. Blood Flow Metab. 2002; 22: 127-141Crossref PubMed Scopus (195) Google Scholar), all of which are activated by distinct forms of stress. In addition, two different phosphatase complexes have been described that can mediate eIF2α dephosphorylation (4Novoa I. Zeng H. Harding H.P. Ron D. J. Cell Biol. 2001; 153: 1011-1021Crossref PubMed Scopus (1001) Google Scholar). In general, the changes to the proteome induced by changes in eIF2α phosphorylation lead to adaptation of the cell to stress with two possible, diametrically opposed consequences, survival or initiation of programmed cell death. The outcome seems to be determined by the duration of the insults, the interplay of different branches of the stress response, and their time courses (5Lin J.H. Li H. Yasumura D. Cohen H.R. Zhang C. Panning B. Shokat K.M. Lavail M.M. Walter P. Science. 2007; 318: 944-949Crossref PubMed Scopus (1033) Google Scholar). In addition to a response to cellular stress, basal levels of phospho-eIF2α are present in vitro (6Scheuner D. Van der Mierde D. Song B. Flamez D. Creemers J.W. Tsukamoto K. Ribick M. Schuit F.C. Kaufman R.J. Nat. Med. 2005; 11: 757-764Crossref PubMed Scopus (305) Google Scholar) and in vivo (7Gietzen D.W. Ross C.M. Hao S. Sharp J.W. J. Nutr. 2004; 134: 717-723Crossref PubMed Scopus (47) Google Scholar, 8Hussain S.G. Ramaiah K.V.A. Biochem. Biophys. Res. Commun. 2007; 355: 365-370Crossref PubMed Scopus (113) Google Scholar), and eIF2α phosphorylation was shown to be involved in biochemical processes as diverse as cell cycle regulation (9Grallert B. Boye E. Cell Cycle. 2007; 6: 2768-2772Crossref PubMed Scopus (27) Google Scholar), glucose homeostasis (10Scheuner D. Song B. McEwan E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar), and synaptic plasticity (11Costa-Mattioli M. Gobert D. Stern E. Gamache K. Colina R. Cuello C. Sossin W. Kaufman R.J. Pelletier J. Rosenblum K. Krnjevic K. Lacaille J.C. Nader K. Sonenberg N. Cell. 2007; 129: 195-206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). There is good evidence that eIF2α phosphorylation can modulate the resistance of nerve cells to oxidative stress. For example, in early work from our laboratory (12Tan S. Somia N. Maher P. Schubert P. J. Cell Biol. 2001; 152: 997-1006Crossref PubMed Scopus (61) Google Scholar), infection of the HT22 nerve cell line with a construct expressing the S51D mutant of eIF2α, which acts as a constitutively phosphorylated form of the protein, was shown to bring about an increase in the resistance of the cells to oxidative stress, which correlated with an ability to maintain a high GSH concentration in the presence of oxidative stress. Further studies from the David Ron laboratory using a different approach to generate constitutively phosphorylated eIF2α in the HT22 cells confirmed these results (13Lu P.D. Jousse C. Marciniak S.J. Zhang Y. Novoa I. Scheuner D. Kaufman R.J. Ron D. Harding H.P. EMBO J. 2004; 23: 169-179Crossref PubMed Scopus (301) Google Scholar). GSH and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of stress (14Maher P. Ageing Res. Rev. 2005; 4: 288-314Crossref PubMed Scopus (302) Google Scholar). In addition, the GSSG/GSH redox pair forms the major redox couple in cells and as such plays a critical role in regulating redox-dependent cellular functions. GSH is a tripeptide containing the amino acids cysteine, glutamate, and glycine. Because glutamate and glycine occur at relatively high intracellular concentrations, cysteine is limiting for GSH synthesis in many types of cells. In the extracellular environment, cysteine is readily oxidized to form cystine, so for most cell types cystine transport mechanisms are essential to provide them with the cysteine needed for GSH synthesis. Cystine uptake in many types of cells is mediated by the Na+-independent cystine/glutamate antiporter, system X-c. System X-c is a member of the disulfide-linked heterodimeric amino acid transporter family and consists of a light chain (xCT) that confers substrate specificity and a heavy chain (4F2hc) that is shared among a number of different amino acid transporters (15Sato H. Tamba M. Ishii T. Bannai S. J. Biol. Chem. 1999; 274: 11455-11458Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). It transports cystine into cells in a 1/1 exchange with glutamate and is thus inhibited by high concentrations of extracellular glutamate (16Bannai S. J. Biol. Chem. 1986; 261: 2256-2263Abstract Full Text PDF PubMed Google Scholar). The importance of system X-c for the maintenance of GSH levels in cells is demonstrated by the loss of GSH and subsequent cell death seen in neural cells following exposure to millimolar concentrations of extracellular glutamate, a pathway termed oxidative glutamate toxicity or oxytosis (17Tan S. Schubert D. Maher P. Curr. Top. Med. Chem. 2001; 1: 497-506Crossref PubMed Scopus (351) Google Scholar). System X-c is expressed in the brain (18Baker D.A. McFarland K. Lake R.W. Shen H. Tang X.-C. Toda S. Kalivas P.W. Nat. Neurosci. 2003; 6: 743-749Crossref PubMed Scopus (588) Google Scholar, 19Burdo J. Dargusch R. Schubert D. J. Histochem. Cytochem. 2006; 54: 549-557Crossref PubMed Scopus (124) Google Scholar, 20Shih A.Y. Erb H. Sun X. Toda S. Kalivas P. Murphy T.H. J. Neurosci. 2006; 26: 10514-10523Crossref PubMed Scopus (238) Google Scholar, 21La Bella V. Valentino F. Piccoli T. Piccoli F. Neurochem. Res. 2007; 32: 1081-1090Crossref PubMed Scopus (36) Google Scholar), and mice lacking system X-c function show signs of redox imbalance (22Sato H. Shiiya A. Kimata M. Maebara K. Tamba M. Sakakura Y. Makino N. Sugiyama F. Yagami K. Moriguchi T. Takahashi S. Bannai S. J. Biol. Chem. 2005; 280: 37423-37429Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) and brain atrophy (20Shih A.Y. Erb H. Sun X. Toda S. Kalivas P. Murphy T.H. J. Neurosci. 2006; 26: 10514-10523Crossref PubMed Scopus (238) Google Scholar). In this study we investigate further the role of eIF2α phosphorylation in regulating GSH metabolism and the response of cells to oxidative stress. Using multiple, distinct cell lines, we show a tight linkage between eIF2α phosphorylation, ATF4, and system X-c expression. We further show that the phospho-eIF2α/ATF4 module is activated in the brains of patients with Alzheimer disease (AD). 3The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β; UTR, untranslated region; ANOVA, analysis of variance; MEF, mouse embryonic fibroblast; DMEM, Dulbecco's modified Eagle's medium; siRNA, short interfering RNA; BCNU, 1,3-bis(2-chloroethyl)-N-nitrosourea; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HBSS, Hanks' balanced salt solution; HCA, homocysteic acid; TBS, Tris-buffered saline; AARE, amino acid-response element; tBHQ, tert-butylhydroquinone.3The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β; UTR, untranslated region; ANOVA, analysis of variance; MEF, mouse embryonic fibroblast; DMEM, Dulbecco's modified Eagle's medium; siRNA, short interfering RNA; BCNU, 1,3-bis(2-chloroethyl)-N-nitrosourea; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HBSS, Hanks' balanced salt solution; HCA, homocysteic acid; TBS, Tris-buffered saline; AARE, amino acid-response element; tBHQ, tert-butylhydroquinone. Together, these results suggest that the increase in eIF2α phosphorylation in AD brains that has repeatedly been interpreted as a sign of neuronal degeneration (23Chang R.C. Wong A.K. Ng H.K. Hugon J. Neuroreport. 2002; 13: 2429-2432Crossref PubMed Scopus (233) Google Scholar, 24Kim H.S. Choi Y.J. Shin K.Y. Joo Y. Lee Y.K. Jung S.Y. Suh Y.H. Kim J.H. J. Neurosci. Res. 2007; 85: 1528-1537Crossref PubMed Scopus (32) Google Scholar, 25Unterberger U. Hoftberger R. Gelpi E. Flicker H. Budka H. Voigtlander T. J. Neuropathol. Exp. Neurol. 2006; 65: 348-357Crossref PubMed Scopus (188) Google Scholar, 26Page G.A. Rioux B. Ingrand S. Lafay-Chebassier C. Pain S. Perault Pochat M.C. Bouras C. Bayer T. Hugon J. Neuroscience. 2006; 139: 1343-1354Crossref PubMed Scopus (84) Google Scholar) should rather be interpreted as an adaptive response to Aβ that promotes neuronal survival. Cell Culture and Viability Assays—Mouse embryonic fibroblasts derived from embryos genetically engineered to homozygously express the nonphosphorylatable S51A mutation (A/A MEFs) and wild-type controls (S/S MEFs) (10Scheuner D. Song B. McEwan E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar) were a kind gift from Randal J. Kaufman and Donalyn Scheuner (Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI). MEFs were propagated in high glucose DMEM (Invitrogen) with 10% fetal calf serum (Hyclone, Logan, UT), additionally supplemented with nonessential and essential amino acids (Invitrogen). For passaging, confluent cells were detached with trypsin/EDTA (Invitrogen). Cells were re-plated after trypsinization when confluent for no more than 10 passages. Aβ-resistant PC12 clone r7 was described previously (27Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-822Abstract Full Text PDF PubMed Scopus (2031) Google Scholar, 28Sagara Y. Dargusch R. Klier F.G. Schubert D. Behl C. J. Neurosci. 1996; 16: 497-505Crossref PubMed Google Scholar, 29Soucek T. Cumming R. Dargusch R. Maher P. Schubert D. Neuron. 2003; 39: 43-56Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 30Cumming R.C. Dargusch R. Fischer W.H. Schubert D. J. Biol. Chem. 2007; 282: 30523-30534Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Wild-type and Aβ-resistant PC12 cells were grown in high glucose DMEM with 10% horse serum (Hyclone) and 5% fetal calf serum. Cells were split once a week after dislodgement by repeated pipetting. HT22 cells were grown on tissue culture dishes in high glucose DMEM supplemented with 10% fetal calf serum as described (31Davis J.B. Maher P. Brain Res. 1994; 652: 169-173Crossref PubMed Scopus (295) Google Scholar). For viability assays, 1 × 104 MEFs, 2.5 × 103 HT22, or 2 × 103 PC12 cells were plated in 96-well plates, in some cases with 30 μm salubrinal (Axxora, San Diego) dissolved in DMSO or DMSO alone. After 24 h of culture, the medium was exchanged with fresh medium and the indicated concentrations of compounds. Sodium glutamate, homocysteic acid, SIN-1, 1,3-bis(2-chloroethyl)-N-nitrosourea (BCNU), ethacrynic acid, and tert-butylhydroperoxide were from Sigma. Amyloid β-(1–42) peptide (Aβ-(1–42)) was obtained from Bachem (Torrance, CA). MEFs and HT22 cells were treated for 24 h and PC12 cells for 48 h. Viability or toxicity of Aβ was measured by the MTT assay as described previously (32Lewerenz J. Letz J. Methner A. J. Neurochem. 2003; 87: 522-531Crossref PubMed Scopus (46) Google Scholar). Mouse cortical neurons were prepared as described previously (33Schubert D. Piasecki D. J. Neurosci. 2001; 21: 7455-7462Crossref PubMed Google Scholar) and seeded at a density of 1 × 105 cells per well onto poly-l-lysine- and laminin-coated 96-well plates using high glucose DMEM supplemented with 10% fetal calf serum with 30 μm salubrinal dissolved in DMSO or DMSO alone. After 24 h, glutamate was added at the indicated concentrations. Survival was measured by the MTT assay after another 24 h. Transfection and Luciferase Reporter Assays—For transfection, 5 × 104 S/S and 2 × 106 A/A MEFs were plated in 60-mm dishes and grown for 48 h. Because the two cell lines proliferate at different rates, these conditions yield similar cell densities at the end of the experiment. Transfection was performed with 2 μg of p-SV-β-galactosidase control vector (Promega, Madison, WI), 2 μg of xCT promoter luciferase constructs (generous gifts from Dr. Hideyo Sato, Yamagata University, Tsuruoka, Japan), and 5 μl of Lipofectamine 2000 (Invitrogen) in 2.5 ml of fresh DMEM with 10% fetal calf serum for 6 h. For some experiments, the transfection additionally included 40 pmol of ATF4 siRNA (catalog number sc-35113) or control siRNA (catalog number sc-37007), both from Santa Cruz Biotechnology (Santa Cruz, CA). For overexpression of ATF4, some experiments additionally included 2 μg of empty pRK7 vector (a generous gift form Dr. Robert C. Cumming, University of Western Ontario, London, Ontario, Canada) or a pRK7 vector expressing ATF4. This construct was obtained by excising the ATF4 open reading frame from a pcDNA3-hCD2 plasmid containing ATF4 (a generous gift from Dr. David Ron, Skirball Institute, New York University School of Medicine, New York, NY) and re-ligating into pRK7. To measure the translational activity of the ATF4 5′UTR, luciferase reporter promoter constructs were replaced by a vector containing the ATF4 5′-untranslated region (5′UTR) and AUG fused to luciferase by the TK promoter in a pGL3 backbone (a generous gift from David Ron). 24 h after transfection, the cells were lysed in 1× reporter lysis buffer (Promega), and enzyme activities were measured by luminescence using the Beta Glo and Luciferase Assay Systems (Promega) on a luminometer from Molecular Devices (Madison, WI). The luciferase/β-galactosidase ratio for each condition and cell line was normalized to the ratio obtained by transfection of pGL3 and pSV-β-Gal for the same condition and cell line. HT22 cells were plated at a density of 4.4 × 105 cells/100-mm dish and grown for 24 h. The medium was exchanged with 5 ml of fresh growth medium, and the cells were transfected with 4 μg of pRK7 or pRK7-ATF4 vector using 5 μl of Lipofectamine 2000 for 6 h. 24 h later, the cells were re-plated for the different assays performed in parallel as follows: 8.8 × 105 in 100-mm dishes for protein preparation; 1.65 × 105 in 60-mm dishes for GSH measurement; 3 × 104/well in 24-well plates for [35S]cystine uptake; and 2.5 × 103/well in 96-well plates for oxidative glutamate toxicity, respectively. Cells were harvested or glutamate was added 24 h after re-plating. Enzymatic Measurement of Total GSH—For measurement of GSH, 5 × 105 A/A and S/S MEFs, 1.65 × 105 HT22 cells or 4.5 × 105 PC12 cells were plated in 60-mm dishes. MEFs and HT22 cells with or without salubrinal were grown for 24 h and PC12 cells for 48 h. Cells were scraped into ice-cold phosphate-buffered saline, and 10% sulfosalicylic acid was added to a final concentration of 3.3%. GSH was assayed as described (34Maher P. Lewerenz J. Lozano C. Torres J.L. J. Neurochem. 2008; 107: 690-700Crossref PubMed Scopus (37) Google Scholar) and normalized to protein recovered from the acid-precipitated pellet by treatment with 0.2 n NaOH at 37 °C overnight and measured by the bicinchoninic acid assay (Pierce). Measurement of System X -c Activity and of Other Amino Acid Uptake Systems—6 × 105 A/A and S/S MEF in parallel, 3 × 105 HT22, or PC12 cells with or without 30 μm salubrinal were plated in 24-well dishes and grown for 24, 24, and 48 h, respectively. For PC12 cells, the plates were coated with poly-l-lysine. Cells were washed three times with sodium-free HBSS. Uptake was performed in triplicate with 25 μm [35S]cystine (PerkinElmer Life Sciences) for 20 min at 37 °C. To specifically measure system X-c activity, 10 mm glutamate was added to parallel wells in triplicate. Cells were washed three times with ice-cold, sodium-free HBSS and lysed in 0.2 n NaOH. Radioactivity was measured by liquid scintillation counting and normalized to protein measured by the Bradford method (Pierce). Because [35S]cystine became unavailable during the course of the experiments, system X-c activity in HT22 cells treated with salubrinal was measured as sodium-insensitive, HCA-inhibitable uptake of [3H]glutamate (PerkinElmer Life Sciences). Cells in triplicate were incubated in 10 μm glutamate ([3H]glutamate/cold glutamate 1/1000) with or without 1 mm HCA adjusted to pH 7.4 in sodium-free HBSS. To measure cysteine uptake, [35S]cystine was preincubated with 10 μm cysteamine, and uptake was performed similarly as for system X-c but with sodium-containing HBSS. This results in glutamate-insensitive, cysteine-sensitive uptake of radioactivity reflecting the function of system ASC and others (34Maher P. Lewerenz J. Lozano C. Torres J.L. J. Neurochem. 2008; 107: 690-700Crossref PubMed Scopus (37) Google Scholar). To measure system L activity, 1 μm [3H]leucine was used in sodium-containing HBSS. Protein Preparation—For Western blotting, confluent A/A or S/S MEFs in 100-mm dishes, 3.3 × 105 HT22 cells per 60-mm dish grown for 24 h, or 1.2 × 106 wild-type PC12 cells and 6 × 105 PC12r7 cells per 100-mm dish grown for 48 h were used. For whole cell extracts, cells were rinsed twice with ice-cold phosphate-buffered saline and scraped into lysis buffer consisting of 50 mm HEPES, pH 7.5, 50 mm NaCl, 10 mm NaF, 10 mm sodium pyrophosphate, 5 mm EDTA, 1% Triton X-100, 1 mm sodium orthovanadate, 1× protease inhibitor, and phosphatase inhibitor mixtures (Sigma). Cells were sonicated on ice and centrifuged. Cystosolic, membrane, and nuclear fractions were prepared as described previously (35Cordey M. Pike C.J. J. Neurochem. 2006; 96: 204-217Crossref PubMed Scopus (28) Google Scholar). Protein in the different fractions was quantified by the bicinchoninic acid method (Pierce) and adjusted to equal concentrations. 5× Western blot sample buffer (74 mm Tris-HCl, pH 8.0, 6.25% SDS, 10% β-mercaptoethanol, 20% glycerol) was added to a final concentration of 2.5×, and samples were boiled for 5 min. Samples of midfrontal cortex from individuals with histologically confirmed Alzheimer disease and age-matched control brains were obtained from Dr. Carol Miller at the Alzheimer Disease Research Center, Los Angeles, CA, and were described in detail previously (29Soucek T. Cumming R. Dargusch R. Maher P. Schubert D. Neuron. 2003; 39: 43-56Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 30Cumming R.C. Dargusch R. Fischer W.H. Schubert D. J. Biol. Chem. 2007; 282: 30523-30534Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Frozen tissue samples were partially thawed, minced in 5× weight/volume extraction buffer (50 mm Tris-HCl, pH 7.5, 2% SDS, and protein inhibitor mixture), sonicated on ice, and centrifuged. Protein concentrations were determined using the Lowry assay (Pierce). Samples were boiled in 1× Western blot sample buffer. Western Blotting—For SDS-PAGE, similar amounts of cellular protein, typically 40 μg per lane were used. In experiments using human brain extracts, 40 μg per lane was loaded for detection of phospho-eIF2α and ATF4 and 20 μg/lane for detection of pan-eIF2α and actin. All samples were separated using 10% Criterion XT Precast BisTris gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes, and the quality of protein measurement, electrophoresis, and transfer was checked by staining with Ponceau S. Membranes were blocked with 5% skim milk in TBS-T (20 mm Tris buffer, pH 7.5, 0.5 m NaCl, 0.1% Tween 20) for 2 h at room temperature and incubated overnight at 4 °C in the primary antibody diluted in 5% bovine serum albumin in TBS, 0.05% Tween 20. The primary antibodies used were as follows: rabbit anti-phospho-Ser-51-eIF2α (catalog number 9721, 1/1000) and rabbit anti-eIF2α (catalog number 9722, 1/500) from Cell Signaling (Beverly, MA); rabbit anti-ATF4 (catalog number sc-200, 1/500) and rabbit anti-Nrf2, (catalog number sc-13032, 1/500) from Santa Cruz Biotechnology; mouse anti-actin (catalog number A5441, 1/200,000) from Sigma; and rabbit anti-xCT (1/1000, a generous gift from Dr. Sylvia Smith, Medical College of Georgia, Augusta, GA). Subsequently, blots were washed in TBS, 0.05% Tween 20 and incubated for 1 h at room temperature in horse-radish peroxidase-goat anti-rabbit or goat anti-mouse (Bio-Rad) diluted 1/5000 in 5% skim milk in TBS, 0.1% Tween 20. After additional washing, protein bands were detected by chemiluminescence using the Super Signal West Pico substrate (Pierce). For relative eIF2α phosphorylation, parallel blots were performed. For all other antibodies, the same membrane was re-probed for actin. Ponceau S-stained membranes and autoradiographs were scanned using a Bio-Rad GS800 scanner. Band density was measured using the manufacturer's software. Relative eIF2α phosphorylation was calculated as the ratio of band densities obtained by the phospho- and the pan-eIF2α antibody. Relative expression of the other proteins was normalized to actin band density, with the exception of the comparison of wild-type and resistant PC12 cells that express highly different amounts of nonmembrane-associated actin (30Cumming R.C. Dargusch R. Fischer W.H. Schubert D. J. Biol. Chem. 2007; 282: 30523-30534Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) that sediments with the nuclear fraction using our technique. Here relative expression was normalized to histone band density obtained by scanning the Ponceau S-stained membranes. Each Western blot was repeated at least three times with independent protein samples. ATF4 Promoter Analysis—Genomic sequence comparison was done using the NCBI genome data base and the reference sequence and the BLASTn algorithm (blast.ncbi.nlm.nih.gov). The reference sequences for the different genomic sequences are NT 039229.7/Mm3 39269 37 for murine chromosome 3, NW 047625.2/Rn2 WGA21474 for rat chromosome 2, NT 016354.18/Hs4 16510 for human chromosome 4, and NW 001493490.2/Bt17 WGA16084 for bovine chromosome 17. Statistical Analysis—Data from at least three independent experiments were normalized, pooled, and analyzed using Graph Pad Prism 4 software followed by appropriate statistical tests. For exclusion of outliers, the established definition of an extreme outlier was used as follows: values lower or higher than the 25% percentile (Q1) or 75% percentile (Q3) minus or plus three times the interquartile range (Q3–Q1), respectively. In our earlier study on eIF2α and GSH metabolism (12Tan S. Somia N. Maher P. Schubert P. J. Cell Biol. 2001; 152: 997-1006Crossref PubMed Scopus (61) Google Scholar), we showed that nerve cells engineered to express decreased levels of eIF2α were much less sensitive than wild-type cells to oxidative glutamate toxicity. This decrease in sensitivity to glutamate correlated with an increase in the levels of GSH that the cells were able to maintain in the presence of glutamate. Similar results were obtained following overexpression of a constitutively phosphorylated form of eIF2α (13Lu P.D. Jousse C. Marciniak S.J. Zhang Y. Novoa I. Scheuner D. Kaufman R.J. Ron D. Harding H.P. EMBO J. 2004; 23: 169-179Crossref PubMed Scopus (301) Google Scholar). To explore further the role of eIF2α phosphorylation in modulating the response of cells to toxic stress, we utilized MEFs derived from mice homozygously expressing a nonphosphorylatable form of eIF2α (A/A MEFs) and compared them with wild-type fibroblasts (S/S MEFs) (10Scheuner D. Song B. McEwan E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). When S/S and A/A MEFs were subjected to oxidative glutamate toxicity by exposure to increasing concentrations of glutamate and cell survival assessed after 24 h by the MTT assay, we observed a striking difference in sensitivity between the two cell lines (Fig. 1A). Whereas S/S MEFs were relatively resistant to glutamate treatment with an EC50 of ∼7.5 mm for cell death, A/A MEFs showed a significant enhancement of sensitivity with an EC50 of ∼1 mm (Fig. 1A). Similar results were obtained following treatment with HCA, another system X-c inhibitor (Fig. 1B). Of note, the sensitivity of both cell lines against inhibition of GSH synthesis by the glutamate cysteine ligase inhibitor, buthionine sulfoximine (36Griffith O.W. Meister A. J. Biol. Chem. 1979; 254: 7558-7560Abstract Full Text PDF PubMed Google Scholar), was similar (Fig. 1C). On the other hand, A/A MEFs were significantly more sensitive to a variety of other insults that decrease GSH levels in cells, including the peroxynitrite generator SIN-1 (37Burdo J. Schubert D. Maher P. Brain Res. 2008; 1189: 12-22Crossref PubMed Scopus (33) Google Scholar), BCNU (38Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3551) Google Scholar), ethacrynic acid (38Schafer F.Q. Buettner G.R. Free Radic.