Enhanced Integrated Stress Response Promotes Myelinating Oligodendrocyte Survival in Response to Interferon-γ
2008; Elsevier BV; Volume: 173; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.080449
ISSN1525-2191
AutoresWensheng Lin, Phillip E. Kunkler, Heather P. Harding, David Ron, Richard P. Kraig, Brian Popko,
Tópico(s)Immune Cell Function and Interaction
ResumoThe T-cell-derived, pleiotropic cytokine interferon (IFN)-γ is believed to play a key regulatory role in immune-mediated demyelinating disorders of the central nervous system, including multiple sclerosis and experimental autoimmune encephalomyelitis. Our previous work has demonstrated that the endoplasmic reticulum (ER) stress response modulates the response of oligodendrocytes to this cytokine. The ER stress response activates the pancreatic ER kinase, which coordinates an adaptive program known as the integrated stress response by phosphorylating translation initiation factor 2α (eIF2α). In this study, we found that growth arrest and DNA damage 34 (GADD34), a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2α, was selectively up-regulated in myelinating oligodendrocytes in mice that ectopically expressed IFN-γ in the central nervous system. We also found that a GADD34 mutant strain of mice displayed increased levels of phosphorylated eIF2α (p-eIF2α) in myelinating oligodendrocytes when exposure to IFN-γ, as well as diminished oligodendrocyte loss and hypomyelination. Furthermore, treatment with salubrinal, a small chemical compound that specifically inhibits protein phosphatase 1(PP1)-GADD34 phosphatase activity, increased the levels of p-eIF2α and ameliorated hypomyelination and oligodendrocyte loss in cultured hippocampal slices exposed to IFN-γ. Thus, our data provide evidence that an enhanced integrated stress response could promote oligodendrocyte survival in immune-mediated demyelination diseases. The T-cell-derived, pleiotropic cytokine interferon (IFN)-γ is believed to play a key regulatory role in immune-mediated demyelinating disorders of the central nervous system, including multiple sclerosis and experimental autoimmune encephalomyelitis. Our previous work has demonstrated that the endoplasmic reticulum (ER) stress response modulates the response of oligodendrocytes to this cytokine. The ER stress response activates the pancreatic ER kinase, which coordinates an adaptive program known as the integrated stress response by phosphorylating translation initiation factor 2α (eIF2α). In this study, we found that growth arrest and DNA damage 34 (GADD34), a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2α, was selectively up-regulated in myelinating oligodendrocytes in mice that ectopically expressed IFN-γ in the central nervous system. We also found that a GADD34 mutant strain of mice displayed increased levels of phosphorylated eIF2α (p-eIF2α) in myelinating oligodendrocytes when exposure to IFN-γ, as well as diminished oligodendrocyte loss and hypomyelination. Furthermore, treatment with salubrinal, a small chemical compound that specifically inhibits protein phosphatase 1(PP1)-GADD34 phosphatase activity, increased the levels of p-eIF2α and ameliorated hypomyelination and oligodendrocyte loss in cultured hippocampal slices exposed to IFN-γ. Thus, our data provide evidence that an enhanced integrated stress response could promote oligodendrocyte survival in immune-mediated demyelination diseases. In response to cellular stresses, a family of protein kinases phosphorylate the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on serine 51 to adapt cells to the stresses, which has been referred to as the integrated stress response (ISR).1Harding HP Zhang Y Zeng H Novoa I Lu PD Calfon M Sadri N Yun C Popko B Paules R Stojdl DF Bell JC Hettmann T Leiden JM Ron D An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2528) Google Scholar, 2Proud CG eIF2 and the control of cell physiology.Semin Cell Dev Biol. 2005; 16: 3-12Crossref PubMed Scopus (309) Google Scholar, 3Lu PD Jousse C Marciniak SJ Zhang Y Novoa I Scheuner D Kaufman RJ Ron D Harding HP Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2.EMBO J. 2004; 23: 169-179Crossref PubMed Scopus (316) Google Scholar Phosphorylation of eIF2α attenuates global protein translation, which allows cells to conserve resources, and activates a transcriptional program that promotes the expression of numerous cytoprotective genes. Endoplasmic reticulum (ER) stress, which is elicited by the accumulation of unfolded or misfolded proteins in the ER, activates adaptive coordinated responses including the activation of pancreatic ER kinase (PERK), an ER-localized eIF2α kinase. PERK-eIF2α-mediated ISR protects cells against ER stress and other cellular stresses.1Harding HP Zhang Y Zeng H Novoa I Lu PD Calfon M Sadri N Yun C Popko B Paules R Stojdl DF Bell JC Hettmann T Leiden JM Ron D An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2528) Google Scholar, 3Lu PD Jousse C Marciniak SJ Zhang Y Novoa I Scheuner D Kaufman RJ Ron D Harding HP Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2.EMBO J. 2004; 23: 169-179Crossref PubMed Scopus (316) Google Scholar, 4Boyce M Yuan J Cellular response to endoplasmic reticulum stress: a matter of life or death.Cell Death Differ. 2006; 13: 363-373Crossref PubMed Scopus (594) Google Scholar Nevertheless, ER stress that cannot be resolved by the adaptive responses ultimately leads to the apoptotic death of the cells.4Boyce M Yuan J Cellular response to endoplasmic reticulum stress: a matter of life or death.Cell Death Differ. 2006; 13: 363-373Crossref PubMed Scopus (594) Google Scholar, 5Schröder M Kaufman RJ Divergent roles of IRE1alpha and PERK in the unfolded protein response.Curr Mol Med. 2006; 6: 5-36Crossref PubMed Scopus (104) Google Scholar To recover from inhibition of global protein biosynthesis induced by the ISR, eIF2α is quickly dephosphorylated by a complex containing the enzyme phosphatase (PP1) and its essential nonenzymatic co-factor, growth arrest and DNA damage 34 (GADD34).6Connor JH Weiser DC Li S Hallenbeck JM Shenolikar S Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1.Mol Cell Biol. 2001; 21: 6841-6850Crossref PubMed Scopus (234) Google Scholar, 7Novoa I Zeng H Harding HP Ron D Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha.J Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1088) Google Scholar, 8Novoa I Zhang Y Zeng H Jungreis R Harding HP Ron D Stress-induced gene expression requires programmed recovery from translational repression.EMBO J. 2003; 22: 1180-1187Crossref PubMed Scopus (369) Google Scholar Interestingly, the expression of GADD34 is regulated by induction of the cytosolic transcription factor ATF4, the translation of which is up-regulated in the presence of eIF2α phosphorylation, creating a tight autofeedback loop.8Novoa I Zhang Y Zeng H Jungreis R Harding HP Ron D Stress-induced gene expression requires programmed recovery from translational repression.EMBO J. 2003; 22: 1180-1187Crossref PubMed Scopus (369) Google Scholar, 9Jiang HY Wek SA McGrath BC Lu D Hai T Harding HP Wang X Ron D Cavener DR Wek RC Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response.Mol Cell Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (408) Google Scholar Importantly, it has been demonstrated that GADD34 inactivation increases the levels of phosphorylated eIF2α (p-eIF2α) in ER-stressed cells and protects cells from the stress.10Jousse C Oyadomari S Novoa I Lu P Zhang Y Harding HP Ron D Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells.J Cell Biol. 2003; 163: 767-775Crossref PubMed Scopus (252) Google Scholar, 11Marciniak SJ Yun CY Oyadomari S Novoa I Zhang Y Jungreis R Nagata K Harding HP Ron D CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1582) Google Scholar Salubrinal (sal), a small chemical compound, has been identified that specifically inhibits PP1-GADD34 phosphatase activity; resulting in sustained eIF2α phosphorylation in ER-stressed cells. Treatment with sal protects cells from ER stress and viral infection.12Boyce M Bryant KF Jousse C Long K Harding HP Scheuner D Kaufman RJ Ma D Coen DM Ron D Yuan J A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress.Science. 2005; 307: 935-939Crossref PubMed Scopus (1226) Google Scholar, 13Wiseman RL Balch WE A new pharmacology—drugging stressed folding pathways.Trends Mol Med. 2005; 11: 347-350Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Studies have suggested that myelinating cells, oligodendrocytes in the central nervous system (CNS), and Schwann cells in the peripheral nervous system, are highly sensitive to the disruption of homeostasis in the ER.14Southwood CM Garbern J Jiang W Gow A The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease.Neuron. 2002; 36: 585-596Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar, 16Pennuto M Tinelli E Malaguti M Del Carro U D'Antonio M Ron D Quattrini A Feltri ML Wrabetz L Ablation of the UPR-mediator CHOP restores motor function and reduces demyelination in Charcot-Marie-Tooth 1B mice.Neuron. 2008; 57: 393-405Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar During active phases of myelination these cells produce a vast amount of myelin as an extension of their plasma membrane, which may contribute to their sensitivity to disruptions in the secretory pathway. For example, Southwood and colleagues14Southwood CM Garbern J Jiang W Gow A The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease.Neuron. 2002; 36: 585-596Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar have demonstrated that oligodendrocyte apoptosis in proteolipid protein mutant mice is associated with ER stress in these cells. Moreover, a recent report has indicated that ER stress in Schwann cells contributes to demyelination in the peripheral nervous system of P0 glycoprotein mutant mice.16Pennuto M Tinelli E Malaguti M Del Carro U D'Antonio M Ron D Quattrini A Feltri ML Wrabetz L Ablation of the UPR-mediator CHOP restores motor function and reduces demyelination in Charcot-Marie-Tooth 1B mice.Neuron. 2008; 57: 393-405Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar We have also shown that the ER stress response in oligodendrocytes is a factor in immune-mediated demyelinating disorders.15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar The T-cell-derived pleiotropic cytokine interferon (IFN)-γ, which becomes detectable in the CNS in the symptomatic phase of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), is believed to play a crucial role in these immune-mediated demyelinating diseases.17Popko B Corbin JG Baerwald KD Dupree J Garcia AM The effects of interferon-gamma on the central nervous system.Mol Neurobiol. 1997; 14: 19-35Crossref PubMed Scopus (150) Google Scholar, 18Imitola J Chitnis T Khoury SJ Cytokines in multiple sclerosis: from bench to bedside.Pharmacol Ther. 2005; 106: 163-177Crossref PubMed Scopus (176) Google Scholar, 19Lees JR Cross AH A little stress is good: IFN-gamma, demyelination, and multiple sclerosis.J Clin Invest. 2007; 117: 297-299Crossref PubMed Scopus (42) Google Scholar Nevertheless, the data concerning its roles, beneficial or detrimental, in these disorders are at times contradictory.20Mühl H Pfeilschifter J Anti-inflammatory properties of pro-inflammatory interferon-gamma.Int Immunopharmacol. 2003; 3: 1247-1255Crossref PubMed Scopus (215) Google Scholar, 21Wheeler RD Owens T The changing face of cytokines in the brain: perspectives from EAE.Curr Pharm Des. 2005; 11: 1031-1037Crossref PubMed Scopus (33) Google Scholar In previous reports, we have shown that myelinating oligodendrocyte death during development or remyelinating oligodendrocyte death in cuprizone-induced demyelinated lesions elicited by IFN-γ is associated with a severe ER stress response, and that PERK is essential for oligodendrocyte survival during ER stress.15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar, 22Lin W Kemper A Dupree JL Harding HP Ron D Popko B Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress.Brain. 2006; 129: 1306-1318Crossref PubMed Scopus (177) Google Scholar In contrast, we have also demonstrated that the protective effect of IFN-γ on EAE-induced demyelination is associated with a modest ER stress response in mature oligodendrocytes, and PERK is also essential for the protective role of IFN-γ in EAE.23Lin W Bailey SL Ho H Harding HP Ron D Miller SD Popko B The integrated stress response prevents demyelination by protecting oligodendrocytes against immune-mediated damage.J Clin Invest. 2007; 117: 448-456Crossref PubMed Scopus (158) Google Scholar Here we demonstrate that GADD34 blockage, via genetic mutation or sal treatment, ameliorated IFN-γ-induced oligodendrocyte loss and hypomyelination. Line110 GFAP/tTA mice and line184 TRE/IFN-γ mice on the C57BL/6 background24Lin W Kemper A McCarthy KD Pytel P Wang JP Campbell IL Utset MF Popko B Interferon-gamma induced medulloblastoma in the developing cerebellum.J Neurosci. 2004; 24: 10074-10083Crossref PubMed Scopus (98) Google Scholar were mated with GADD34 mutant mice11Marciniak SJ Yun CY Oyadomari S Novoa I Zhang Y Jungreis R Nagata K Harding HP Ron D CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1582) Google Scholar on the C57BL/6 background, and the resulting progeny were intercrossed to obtain GFAP/tTA; TRE/IFN-γ; GADD34 WT mice, and GFAP/tTA; TRE/IFN-γ; GADD34 mutant mice. To prevent transcriptional activation of the TRE/IFN-γ transgene by tTA, 0.05 mg/ml of doxycycline was added to the drinking water and was provided ad libitum. All animal procedures were conducted in complete compliance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Chicago. Hippocampal slice cultures were prepared from postnatal day 6 rat pups and maintained until their use as previously described25Kunkler PE Kraig RP Reactive astrocytosis from excitotoxic injury in hippocampal organ culture parallels that seen in vivo.J Cereb Blood Flow Metab. 1997; 17: 26-43Crossref PubMed Scopus (59) Google Scholar, 26Kunkler PE Hulse RE Schmitt MW Nicholson C Kraig RP Optical current source density analysis in hippocampal organotypic culture shows that spreading depression occurs with uniquely reversing currents.J Neurosci. 2005; 25: 3952-3961Crossref PubMed Scopus (31) Google Scholar, 27Kunkler PE Briant J Hulse RE Kraig RP Neural activity-dependent modulation of myelination. (abstract).Soc Neurosci. 2006; 32: 87.5Google Scholar except that they were transferred to serum-free media at 4 days in vitro. Serum-free media consisted of Neurobasal (96%; Invitrogen, Carlsbad, CA) supplemented to 42 mmol/L glucose, 1 mmol/L glutamine (Sigma-Aldrich, St. Louis, MO), B27 supplement (2%, Invitrogen), gentamicin (10 μg/ml), and Fungizone (250 μg/ml, Invitrogen). Cultures were fed twice a week with freshly prepared serum-free media containing rat IFN-γ (EMB Biosciences, San Diego, CA) and sal (EMB Biosciences) as indicated. RNA samples were isolated from mouse brains using TRIzol reagent (Invitrogen) and were treated with DNaseI (Invitrogen) to eliminate genomic DNA. Reverse transcription was performed using a SuperScript first-strand synthesis system for RT-PCR kit (Invitrogen). Real-time PCR was performed with iQ Supermix (Bio-Rad Laboratories, Hercules, CA) on a real-time PCR detection system (model iQ, Bio-Rad Laboratories) as described.22Lin W Kemper A Dupree JL Harding HP Ron D Popko B Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress.Brain. 2006; 129: 1306-1318Crossref PubMed Scopus (177) Google Scholar The following primers and probes for real-time PCR were used: glyceraldehyde phosphate dehydrogenase (GAPDH) sense primer (5′-CTCAACTACATGGTCTACATGTTCCA-3′); GAPDH antisense primer (5′-CCATTCTCGGCCTTGACTGT-3′); GAPDH probe (5′-TGAC TCCACTCACGGCAAATTCAACG-3′); GADD34 sense primer (5′-CCCTCCAACTCTCCTTCTTCAG-3′); GADD34 antisense primer (5′-CAGCCTCAGCATTCCGACAA-3′); GADD34 probe (vCGTGCCCTGCCCTGTGTGCCTCTA-3′); tumor necrosis factor-α (TNF-α) sense primer (5′-GGCAGGTTCTGTCCCTTTCA-3′); TNF-α antisense primer (5′-ACCGCCTGGAGTTCTGGAA); TNF-α probe (5′-CCCAAGGCGCCACATCTCCCT-3′); inducible nitric oxide synthase (iNOs) sense primer (5′-GCTGGGCTGTACAAACCTTCC-3′); iNOs antisense primer (5′-TTGAGGTCTAAAGGCTCCGG-3′); iNOs probe (5′-TGTCCG AAGCAAACATCACATTCAGATCC-3′); IFN-γ sense primer (5′-GATATCTGGAGGAACTGGCAAAA-3′); IFN-γ antisense primer (5′-CTTCAAAGAGTCTGAGGTAGAAAGAGATA AT-3′); IFN-γ probe (5′-TGGTGACATGAAAATCCTGC AGAGCCA-3′). Anesthetized mice were perfused through the left cardiac ventricle with 4% paraformaldehyde in phosphate-buffered saline (PBS). The half saggital brains were removed, postfixed with paraformaldehyde, cryopreserved in 30% sucrose, embedded in optimal cutting temperature compound, and frozen on dry-ice. Frozen sections were cut in a cryostat at a thickness of 10 μm. For immunohistochemistry, the sections were treated with −20°C acetone, blocked with PBS containing 10% goat serum and 0.1% Triton X-100, and incubated overnight with the primary antibody diluted in blocking solution. Fluorescein, Texas Red, or enzyme-labeled secondary antibodies (Vector Laboratories, Burlingame, CA) were used for detection. Immunohistochemistry for CC1 (APC7, 1:50; EMB Biosciences), GADD34 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), p-eIF-2α (1:50; Cell Signaling Technology, Beverly, MA), myelin basic protein (MBP, 1:1000; Sternberger Monoclonals, Lutherville, MD), CD3 (1:50, Santa Cruz Biotechnology), CD11b (1:50; Chemicon, Temecula, CA), and Rip (1:1000, Chemicon) was performed and analyzed as previously described.22Lin W Kemper A Dupree JL Harding HP Ron D Popko B Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress.Brain. 2006; 129: 1306-1318Crossref PubMed Scopus (177) Google Scholar, 23Lin W Bailey SL Ho H Harding HP Ron D Miller SD Popko B The integrated stress response prevents demyelination by protecting oligodendrocytes against immune-mediated damage.J Clin Invest. 2007; 117: 448-456Crossref PubMed Scopus (158) Google Scholar Hippocampal slice cultures were prepared and processed for immunohistochemistry as described previously.25Kunkler PE Kraig RP Reactive astrocytosis from excitotoxic injury in hippocampal organ culture parallels that seen in vivo.J Cereb Blood Flow Metab. 1997; 17: 26-43Crossref PubMed Scopus (59) Google Scholar, 26Kunkler PE Hulse RE Schmitt MW Nicholson C Kraig RP Optical current source density analysis in hippocampal organotypic culture shows that spreading depression occurs with uniquely reversing currents.J Neurosci. 2005; 25: 3952-3961Crossref PubMed Scopus (31) Google Scholar Mice were anesthetized and perfused with 4% paraformaldehyde and 2.5% glutaraldehyde. White matter of the corpus callosum was processed. Thin sections were cut, stained with uranyl acetate and lead citrate, and analyzed as previously described.22Lin W Kemper A Dupree JL Harding HP Ron D Popko B Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress.Brain. 2006; 129: 1306-1318Crossref PubMed Scopus (177) Google Scholar, 28Coetzee T Fujita N Dupree J Shi R Blight A Suzuki K Suzuki K Popko B Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability.Cell. 1996; 86: 209-219Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar Six cultured hippocampal slices per each condition were rinsed in ice-cold PBS, pooled, and homogenized using a motorized homogenizer as previously described.22Lin W Kemper A Dupree JL Harding HP Ron D Popko B Interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress.Brain. 2006; 129: 1306-1318Crossref PubMed Scopus (177) Google Scholar After incubating on ice for 15 minutes, the extracts were cleared by centrifugation at 14,000 rpm twice for 30 minutes each. The protein content of each extract was determined by protein assay (Bio-Rad Laboratories). The extracts (40 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose. The blots were incubated with primary antibody (see below), and the signal was revealed by chemiluminescence after reacting with horseradish peroxidase-conjugated second antibody. The following primary antibodies were used: eIF-2α (1:500, Santa Cruz Biotechnology), p-eIF-2α (1:1000, Cell Signaling Technology), ATF4 (1:500, Santa Cruz Biotechnology), MBP (1:5000, Sternberger Monoclonals), and actin (1:1000, Sigma-Aldrich). The density of immunoblotting was quantified with Kodak 1D Image Analysis software (Kodak, Hercules, New Haven, CT). Data are expressed as mean ± SD. Multiple comparisons were statistically evaluated by one-way ANOVA test followed by Holm-Sidak test using Sigmastat 3.1 software (Hearne Scientific Software, Chicago, IL). Differences were considered statistically significant if P < 0.05. GADD34, which is expressed at very low levels in unstressed cells, is a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2α.7Novoa I Zeng H Harding HP Ron D Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha.J Cell Biol. 2001; 153: 1011-1022Crossref PubMed Scopus (1088) Google Scholar, 8Novoa I Zhang Y Zeng H Jungreis R Harding HP Ron D Stress-induced gene expression requires programmed recovery from translational repression.EMBO J. 2003; 22: 1180-1187Crossref PubMed Scopus (369) Google Scholar We have generated transgenic mice that allow for the temporally controlled delivery of IFN-γ to the CNS using the tetracycline-controllable system.24Lin W Kemper A McCarthy KD Pytel P Wang JP Campbell IL Utset MF Popko B Interferon-gamma induced medulloblastoma in the developing cerebellum.J Neurosci. 2004; 24: 10074-10083Crossref PubMed Scopus (98) Google Scholar The expression of the IFN-γ transgene is repressed in GFAP/tTA; TRE/IFN-γ double-transgenic mice treated with doxycycline solution from conception (IFN-γCNS− mice). The levels of IFN-γ become detectable at approximately postnatal day 10 (P10) in the CNS of GFAP/tTA; TRE/IFN-γ double-transgenic mice that are released from doxycycline solution at embryonic day 14 (E14, IFN-γCNS+ mice).15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar We have shown that activation of PERK-eIF2α pathway plays a crucial role in oligodendrocyte survival in IFN-γCNS+ mice.15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar Real-time PCR analysis revealed that the presence of IFN-γ in the CNS significantly increased the expression of GADD34 in the brain (1.24 ± 0.07 versus 1.0 ± 0.11, P < 0.05; Figure 1A). We further determined the cellular expression pattern of GADD34 in the CNS using CC1, a marker for oligodendrocytes, and GADD34 double immunostaining. We found that GADD34 was undetectable in oligodendrocytes in the CNS of 21-day-old control IFN-γCNS− mice (Figure 1B), but became detectable in these cells primarily in the corpus callosum of IFN-γCNS+ mice (Figure 1C). Moreover, 48 ± 7% of the CC1-positive oligodendrocytes in the corpus callosum were GADD34-positive, and 89 ± 11% of GADD34-positive cells were CC1-positive cells. Myelination in the corpus callosum begins at approximately P14, and becomes robust at approximately P21, a time point when myelination in other areas of the CNS is largely complete.29Vincze A Mázló M Seress L Komoly S Abrahám H A correlative light and electron microscopic study of postnatal myelination in the murine corpus callosum.Int J Dev Neurosci. 2008; 26: 575-584Crossref PubMed Scopus (60) Google Scholar Thus, these data are consistent with our previous report that oligodendrocytes at the active stage of myelination are most sensitive to IFN-γ.15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar We pursued a genetic approach to assess the protective effects of GADD34 blockage on oligodendrocyte death elicited by IFN-γ. GADD34 mutant mice, whose inactive GADD34 allele encodes a C-terminally truncated protein that lacks the phosphatase domain, appear healthy and display resistance to cell death induced by ER stress.11Marciniak SJ Yun CY Oyadomari S Novoa I Zhang Y Jungreis R Nagata K Harding HP Ron D CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1582) Google Scholar We crossed GFAP/tTA and TRE/IFN-γ mice with GADD34 mutant mice, and the resulting progeny were intercrossed to obtain GFAP/tTA; TRE/IFN-γ; GADD34 WT mice and GFAP/tTA; TRE/IFN-γ; GADD34 mutant mice. GFAP/tTA; TRE/IFN-γ; GADD34 WT mice released from doxycycline at E14 (IFN-γCNS+ GADD34 WT mice) showed tremor and ataxia as previously described.15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar In contrast, the tremoring phenotype was much milder in GFAP/tTA; TRE/IFN-γ; GADD34 mutant mice released from doxycycline at E14 (IFN-γCNS+ GADD34 mutant mice; Table 1). Nevertheless, a number of GFAP/tTA; TRE/IFN-γ; GADD34 mutant mice died by P 28. In previous reports, we have shown that the presence of IFN-γ in the CNS during development induces cerebellar dysplasia or medulloblastoma.24Lin W Kemper A McCarthy KD Pytel P Wang JP Campbell IL Utset MF Popko B Interferon-gamma induced medulloblastoma in the developing cerebellum.J Neurosci. 2004; 24: 10074-10083Crossref PubMed Scopus (98) Google Scholar, 30Corbin JG Kelly D Rath EM Baerwald KD Suzuki K Popko B Targeted CNS expression of interferon-gamma in transgenic mice leads to hypomyelination, reactive gliosis, and abnormal cerebellar development.Mol Cell Neurosci. 1996; 7: 354-370Crossref PubMed Scopus (180) Google Scholar, 31Wang J Lin W Popko B Campbell IL Inducible production of interferon-gamma in the developing brain causes cerebellar dysplasia with activation of the Sonic hedgehog pathway.Mol Cell Neurosci. 2004; 27: 489-496Crossref PubMed Scopus (59) Google Scholar Our preliminary data suggest that the premature death of these mice might be attributable to enhanced medulloblastoma formation (unpublished data).Table 1GADD34 Inactivation Alleviated Tremoring Phenotype in Mice Expressing IFN-γ in the CNSNo tremor (percentage)Minor tremor (percentage)Tremor (percentage)IFN-γCNS− GADD34 WT mice (n = 20)10000IFN-γCNS− GADD34 mutant mice (n = 20)10000IFN-γCNS+ GADD34 WT mice (n = 20)102070IFN-γCNS+ GADD34 mutant mice (n = 24)334225 Open table in a new tab We next examined the correlation between the severities of the tremoring phenotype and the levels of p-eIF-2α in oligodendrocytes in these animals. CC1 and p-eIF2α double labeling showed modest activation of eIF2α in oligodendrocytes in the corpus callosum of 21-day-old IFN-γCNS+ GADD34 WT mice, and GADD34 inactivation further increased the levels of p-eIF2α in these cells in IFN-γCNS+ GADD34 mutant mice (Figure 1, D and E). Collectively, these data indicate that GADD34 inactivation elevated the levels of p-eIF2α in oligodendrocytes in response to IFN-γ and attenuated the severity of the tremoring phenotype. In our previous report, we showed that the tremoring phenotype in mice expressing IFN-γ in the CNS during development was primarily caused by the death of myelinating oligodendrocytes and subsequent hypomyelination.15Lin W Harding HP Ron D Popko B Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-gamma.J Cell Biol. 2005; 169: 603-612Crossref PubMed Scopus (172) Google Scholar Importantly, in the absence of IFN-γ, GADD34 inactivation did not significant
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