5980 • The Journal of Neuroscience, April 3, 2013 • 33(14):5980–5991 Neurobiology of Disease Oligodendrocyte-Specific Activation of PERK Signaling Protects Mice against Experimental Autoimmune Encephalomyelitis Wensheng Lin,1,2 Yifeng Lin,1 Jin Li,1 Ali G. Fenstermaker,3 Sharon W. Way,3 Benjamin Clayton,3 Stephanie Jamison,1 Heather P. Harding,4 David Ron,4 and Brian Popko3 1Department of Cell Biology & Neuroscience, University of South Alabama College of Medicine, Mobile, Alabama 36688, 2Department of Comparative Medicine, University of South Alabama College of Medicine, Mobile, Alabama 36688, 3Department of Neurology, University of Chicago, Chicago, Illinois 60637, and 4University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge CB20QQ, United Kingdom There is compelling evidence that oligodendrocyte apoptosis, in response to CNS inflammation, contributes significantly to the develop- ment of the demyelinating disorder multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). There- fore, approaches designed to protect oligodendrocytes would likely have therapeutic value. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum (ER) stress increases cell survival under various cytotoxic conditions. Moreover, there is evidence that PERK signaling is activated in oligodendrocytes within demyelinating lesions in multiple sclerosis and EAE. Our previous study demonstrated that CNS delivery of the inflammatory cytokine interferon-␥ before EAE onset protected mice against EAE, and this protection was dependent on PERK signaling. In our current study, we sought to elucidate the role ofPERKsignalinginoligodendrocytesduringEAE.WegeneratedtransgenicmicethatallowfortemporallycontrolledactivationofPERK signaling, in the absence of ER stress, specifically in oligodendrocytes. We demonstrated that persistent activation of PERK signaling was not deleterious to oligodendrocyte viability or the myelin of adult animals. Importantly, we found that enhanced activation of PERK signaling specifically in oligodendrocytes significantly attenuated EAE disease severity, which was associated with reduced oligodendro- cyte apoptosis, demyelination, and axonal degeneration. This effect was not the result of an altered degree of the inflammatory response in EAE mice. Our results provide direct evidence that activation of PERK signaling in oligodendrocytes is cytoprotective, protecting mice against EAE. Introduction characterized by CNS inflammation, demyelination, oligoden- Multiple sclerosis (MS) and its animal model, experimental au- drocyte death, and axonal degeneration (Frohman et al., 2006; toimmune encephalomyelitis (EAE), are T cell-mediated auto- Trapp and Nave, 2008). Recent studies suggest that oligodendro- immune demyelinating diseases of the CNS. These disorders are cytes are the target of immune attack in MS and EAE and that oligodendrocyte apoptosis contributes significantly to the devel- Received April 3, 2012; revised Feb. 14, 2013; accepted Feb. 22, 2013. opment of these diseases (Matute and Pe´rez-Cerda´, 2005; Author contributions: W.L. and B.P. designed research; W.L., Y.L., J.L., A.G.F., S.W.W., B.C., and S.J. performed McGuire et al., 2010). Profound oligodendrocyte apoptosis has research; H.P.H. and D.R. contributed unpublished reagents/analytic tools; W.L., Y.L., S.W.W., H.P.H., D.R., and B.P. been observed in newly forming MS lesions in the absence of analyzed data; W.L., B.C., and B.P. wrote the paper. infiltrating lymphocytes or myelin phagocytes (Barnett and This work was supported by National Multiple Sclerosis Society Grants TA 3026-A-1 and RG-4813-A-2 to W.L., National Institutes of Health Grant NS34939 to B.P., and the Myelin Repair Foundation to B.P. This work used the Prineas, 2004). Both oligodendrocyte-targeted expression of an- Nikon A1 confocal microscope funded by National Institutes of Health Grant S10RR027535. We thank Dr. Wendy tiapoptotic proteins and oligodendrocyte-targeted deletion of a Macklin (University of Colorado Denver, Aurora, Colorado) for providing the proteolipid protein-based expression proapoptotic protein protect oligodendrocytes from EAE- construct and Dr. M.A. Aryan Namboodiri (Uniformed Services University of the Health Sciences, Bethesda, Mary- land) for providing the antibody against aspartoacylase. induced apoptosis, resulting in the attenuation of demyelination The authors declare no competing financial interests. and axonal degeneration in EAE lesions (Hisahara et al., 2000; Correspondence should be addressed to either of the following: Dr. Wensheng Lin, Department of Neuroscience, McGuire et al., 2010). University of Minnesota, 2101 6th Street SE, WMBB4-410, Minneapolis, MN 55455, E-mail: [email protected]; or Dr. Endoplasmic reticulum (ER) stress, the stress of accumulating Brian Popko, Center for Peripheral Neuropathy, Department of Neurology, University of Chicago, 5841 South Mary- land Avenue, MC2030, Chicago, IL 60637, E-mail: [email protected]. unfolded or misfolded proteins in the ER, activates pancreatic ER W. Lin’s, Y. Lin’s, and S. Jamison’s present address: Department of Neuroscience and Institute for Translational kinase (PERK), which coordinates an adaptive program known Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455. as the integrated stress response (ISR) by phosphorylating trans- J. Li’s present address: Department of Ophthalmology, 9th Hospital, Shanghai Jiaotong University School of lation initiation factor 2␣ (eIF2␣)(Harding et al., 2003; Proud, Medical Science, Shanghai, China. ␣ DOI:10.1523/JNEUROSCI.1636-12.2013 2005; Marciniak and Ron, 2006). Phosphorylation of eIF2 pro- Copyright © 2013 the authors 0270-6474/13/335980-12$15.00/0 motes a stress-resistant state through the global attenuation of Lin et al. • PERK Protects Oligodendrocyte against Immune Attack J. Neurosci., April 3, 2013 • 33(14):5980–5991 • 5981 protein biosynthesis and the induction of stress-induced cyto- complete compliance with the National Institutes of Health Guide for the protective genes. Accumulating evidence suggests that the ISR is Care and Use of Laboratory Animals and were approved by the Institu- involved in the pathogenesis of MS and EAE (Lin and Popko, tional Animal Care and Use Committees of the University of South Ala- 2009). Activation of the ISR was observed in multiple cell types bama, the University of Chicago, and the University of Minnesota. within MS and EAE demyelinating lesions, including oligo- Mixed glial cultures and oligodendrocyte cultures. Mixed glial cultures were prepared from neonatal PLP/Fv2E-PERK mice as described previ- dendrocytes, T cells, microglia/macrophages, and astrocytes ously (Baerwald et al., 2000). The cells were cultured in DMEM (Invit- (Chakrabarty et al., 2004; Mha´ille et al., 2008; Cunnea et al., rogen) with 10% FBS at 37°C with 5% CO2. After 12 d, the cells were 2011). Additionally, it has been shown that the ISR is a potent switched to a differentiation medium containing 5 ␮l/ml insulin, 50 regulator of inflammatory T-cell differentiation (Scheu et al., ␮g/ml transferrin, 30 nM selenium, 10 nM biotin, 10 nM progesterone, 2006; Sundrud et al., 2009) and that it also regulates the function and 15 nM triiodothyronine, and 0.1% BSA. After5dofdifferentiation, of macrophages in inflammatory diseases (Woo et al., 2009). Our the cells were treated with AP20187 or vehicle for 24 h. RNA was isolated previous study demonstrated that CNS delivery of the inflammatory from the cells using Trizol reagent (Invitrogen). cytokine IFN-␥ before EAE onset protected against EAE-induced Oligodendrocyte progenitor cells (OPCs) were isolated from PLP/ oligodendrocyte death and demyelination in a PERK-dependent Fv2E-PERK and wild-type littermate mice using the sequential immuno- manner, as the protective effects of IFN-␥ were associated with panning protocol described by Dugas et al. (2006). Briefly, whole brains PERK activation in oligodendrocytes and were abrogated in from 6-day-old pups were dissected and enzymatically dissociated with papain at 37°C. After enzymatic dissociation and gentle trituration, the PERK haploinsufficient animals (Lin et al., 2007). Nevertheless, single-cell suspension was sequentially incubated on dishes coated with ␥ Ϫ Ϫ ϩ because IFN- is a pleiotropic immune cytokine that regulates antibodies against Ran-2, GalC, and O4. Ran-2 , GalC ,O4 OPCs T-cell differentiation and microglia/macrophage activation were trypsinized from the O4 panning plate and seeded onto poly-D- (Boehm et al., 1997), it is not clear whether abrogation of the lysine coated 12 mm glass coverslips. Cells were cultured in DMEM, protective effects of IFN-␥ in PERK haploinsufficient animals is supplemented with human transferrin (100 ␮g/ml), BSA (100 ␮g/ml), the result of the impairment of ISR signaling in oligodendrocytes putrescine (16 ␮g/ml), progesterone (60 ng/ml), sodium selenite (40 or in inflammatory cells. Collectively, the role of the ISR in oli- ng/ml), N-acetyl-L-cysteine (5 ␮g/ml), D- biotin (10 ng/ml), insulin godendrocytes during MS and EAE remains unclear. (5 ␮g/ml), glutamine (2 mM), sodium pyruvate (1 mM), penicillin- ϫ ϫ In the current study, we sought to dissect the precise role of the streptomycin (100 U each), B-27 (1 ; Invitrogen), Trace Elements (1 ; ␮ ISR in oligodendrocytes during EAE pathogenesis. We generated Mediatech), growth factors for differentiation: forskolin (4.2 g/ml),