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INVOLVEMENT OF ENDOPLASMIC RETICULUM STRESS IN THE NECROPTOSIS OF MICROGLIA/MACROPHAGES AFTER SPINAL CORD INJURY
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H. FAN, a H.-B. TANG, b J. KANG, a L. SHAN, c H. SONG, d K. ZHU, e J. WANG, a G. JU a* AND Y.-Z. WANG a*
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a Department of Neurobiology and Collaborative Innovation Center for Brain Science, Institute of Neurosciences, Fourth Military Medical University, 169 Chang Le Xi Road, Xi’an 710032, China
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b Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University, 127 Chang Le Xi Road, Xi’an 710003, China
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c Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xin Si Road, Xi’an, Shaanxi 710038, China
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Department of Occupational and Environmental Health, School of Public Health, Fourth Military Medical University, Xi’an 710032, China
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e Zhejiang University China Brain Bank, Department of Pathology and Pathophysiology, Department of Neuroscience, 866 Yu-Hang-Tang Road, Zhejiang University Zi-Jin-Gang Campus, Hangzhou, Zhejiang 310058, China
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Abstract—Microglia/macrophages play a crucial role in inflammation after spinal cord injury (SCI). Although extensive studies have been performed on the mechanisms of microglia/macrophage activation and recruitment, how microglia/macrophages are eliminated remains unclear. In the present study, we observed a high- level expression of mixed lineage kinase domain-like protein (MLKL), a key molecule in the execution of necroptosis, in microglia/macrophages after SCI in mice. In vivo PI-labeling and Necrostatin-1 treatment confirmed the necroptosis of microglia/macrophages. Interestingly, our electronic microscopic (EM) study revealed that MLKL localized not only at the membrane but also on the endoplasmic reticulum (ER) of necroptotic microglia/macrophages. Furthermore, receptor-interacting protein 3 (RIP3), another necrosome component, was also found on the ER of necroptotic microglia/macrophages. And Glucose-regulated protein 78 (GRP78), an ER stress sensor, was up-regulated in MLKL-positive microglia/macrophages after SCI, suggesting
a possible link between necroptosis and ER stress. In vitro, oxygen–glucose deprivation (OGD) stress induced ER stress and necroptosis in microglia. Inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly blocked the OGD-induced necroptosis of microglia. In the end, our data showed that, GRP78 and phosphorylated MLKL were coexpressed by the microglia/macrophages in the injured human spinal cord. Taken together, these results suggested that microglia/macrophages undergo an ER-stress involved necroptosis after SCI, implying that ER stress and necroptosis could be manipulated for modulating inflammation postSCI. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.
Key words: spinal cord injury, microglia/macrophages, necroptosis, ER stress. 23
*Corresponding authors. Tel: +86-29-84774565; fax: +86-2983246270 (G. Ju). Tel: +86-29-84774562; fax: +86-29-83246270 (Y.Z. Wang). E-mail addresses: [email protected] (G. Ju), yazhouw@fmmu. edu.cn (Y.-Z. Wang). Abbreviations: 4-PBA, 4-phenylbutyrate; CC3, cleaved Caspase-3; CXCR4, chemokine (C-X-C motif) receptor 4; EM, electronic microscopic; ER, endoplasmic reticulum; HMGB1, high-mobility group box protein 1; MLKL, mixed lineage kinase domain-like protein; Nec-1, Necrostatin-1; NGS, normal goat serum; OGD, oxygen–glucose deprivation; pMLKL, phosphorylated MLKL; RIP1, receptor-interacting protein 1; RIP3, receptor-interacting protein 3; SCI, spinal cord injury; EDTA, ethylenediaminetetraacetic acid; GRP78, Glucose-regulated protein 78; DMEM, Dulbecco’s modified Eagle medium; PI, Propidium Iodide; PBS, phosphate-buffered saline. http://dx.doi.org/10.1016/j.neuroscience.2015.10.049 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1
INTRODUCTION
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Spinal contusion is the major type of spinal cord injury (SCI) in the clinic, which often results in permanent loss of motor and sensory functions. Although several clinical trials have been tested, it still lacks effective treatment. A unique pathological change following SCI is the secondary injury which is characterized by gradual enlargement of lesion area and the presence of chronic inflammation (Allison and Ditor, 2015). The spinal microglia, together with recruited macrophages which exhibited an almost identical phenotype with activated microglia, plays a key role in the initiation and development of inflammation (David and Kroner, 2011). After SCI, the function of microglia/macrophages has been thought to be mainly destructive, possibly due to the dominance of their M1 sub-population (Hu et al., 2015). Many studies have been carried out to elucidate the mechanisms of their proliferation, migration and M1/M2 phenotype conversion, with the aim of biasing the post-SCI inflammation toward better repair (Mabon et al., 2000; Zai and Wrathall, 2005; Kroner et al., 2014). However, how microglia/macrophages are eliminated from the injured spinal cord remains unclear. Necrosis and apoptosis are the two major types of cell death after SCI (Beattie et al., 2002). Extensive studies have revealed the cellular and molecular mechanisms of apoptosis after SCI (Yong et al., 1998). However, necrosis has long been thought to be uncontrollable, and the mechanism of necrosis after SCI remains largely unexplored. Recent studies have identified a type of programed necrosis (necroptosis) and uncovered its
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underlying mechanisms which involves an intracellular signaling cascade transduced by receptor-interacting protein 1/3 (RIP1/3) and mixed lineage kinase domain-like protein (MLKL) (Sun et al., 2012), thus offering new molecular tools for re-examining necrosis after SCI. Endoplasmic reticulum stress (ER stress) is a cellular response to multiple injury conditions, which usually showed accumulation of unfolded proteins in the cytoplasm (Hoozemans and Scheper, 2012). Many studies have demonstrated that severe ER stress can activate the intracellular signaling that finally leads to apoptosis or autophagy (Gorman et al., 2012; Liu et al., 2015).Whether ER stress is also involved in the activation of necroptosis has been poorly studied. In the present study, we investigated the necroptosis of microglia/macrophages after SCI, and the involvement of ER stress in the necroptosis of microglia/macrophages.
EXPERIMENTAL PROCEDURES
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Human samples
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Snap-frozen normal human spinal cord tissues were obtained from the human brain bank of the school of medicine at Zhejiang University. Biopsy of injured spinal cord tissues were performed with informed consent obtained from each patient prior to surgery and experiments involving human spinal tissues were approved by the Institutional Review Board of Tangdu Hospital, Fourth Military Medical University.
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Spinal cord contusion and in vivo treatment
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Male C57BL/6 mice were purchased from the Laboratory Animal Center of Fourth Military Medical University and all the protocols of animal experiments were approved by the Animal Care and Use Committee of the Fourth Military Medical University. Mice (6–8 w) were anesthetized with 1% sodium pentobarbital (80 mg/kg), followed by bilateral laminectomy of vertebrae T8–T9. Spinal cord lateral crushing was made at the T8 vertebra by using a forceps with a tip-gap set at 0.2 mm for 15 s. Manual bladder expression was performed once a day after surgery. Sham SCI was made by performing a dorsal laminectomy without crushing the spinal cord.
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Necrostain-1 (Nec-1) administration
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Nec-1 (7.8 mg/kg) was administrated (i.v.) twice a day for 5 days for examining its effect on the necroptosis after SCI.
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Primary microglia and N9 cells culture
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Microglial cells were isolated from the brain tissue of neonatal C57BL/6 mice. Briefly, the skin and skull were peeled away and the meningeal lining was then gently removed under a microscope. The cerebral cortical tissue was then minced into a fine slurry with scissors, followed by digestion with 0.125% trypsin/0.02% EDTA. Cells were suspended in 12 ml of Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS, 100 lg/ml penicillin–streptomycin and 2 mM L-glutamine, seeded
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onto poly-D-lysine coated T-75 flasks and maintained at 37 °C in an incubator with humidified 5% CO2 atmosphere. After 9 days culture, when the cells were approximately 90% confluent, and purified by shaking at 260 rpm/min for 1 h. Immunostaining of calcium-binding adapter molecule 1 (Iba-1) was adopted to ensure that over 95% of the cells were Iba-1- positive. Microglial cells were then seeded in 6-well or 24-well plates at a density of 2 106 cells or 0.5 106 per well. Mouse microglial N9 cells were maintained in DMEM supplemented with 5% FBS, 100 lg/ml penicillin–streptomycin and 2 mM L-glutamine. For experiments, cells were plated onto 6-well or 24-well plates at a density of 3 106 cells or 1 106 cells per well.
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Oxygen–glucose deprivation (OGD) injury and Nec-1, 4-phenylbutyrate (4-PBA) treatment
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OGD in N9 cells and primary microglia was performed by replacing the medium with glucose-free DMEM (Gibco, USA) and cultures were incubated in a hypoxic chamber (95% N2 and 5% CO2) at 37 °C for 12 h. After OGD treatment, cells were transferred back to normal condition with fresh culture medium for 4, 16, 24, 36 and 48 h for N9 cells and 36 h for microglia respectively. Control cells were maintained in regular DMEM under normoxic conditions. For Nec-1 and 4-PBA treatment, 20 lM Nec-1 and 4 mM 4-PBA were added into the culture medium when OGD was begun, until the end of the experiment. Immediately at the end of treatments, total cellular proteins were isolated for Western-blotting analysis, or cells were stained with Propidium Iodide (PI) for death detection.
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PI staining
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In vitro PI labeling. N9 cells and primary microglia were exposed to OGD for 12 h and subsequently placed back to normal culture medium for 36 h. PI (5 lM, Sigma) and Hoechst 33342 (5 lg/ml, Sigma) were added into the culture medium and incubated for 30 min at 37 °C. Cells were then washed three times with 0.01 M phosphate- buffered saline (PBS) and fixed with 4% (w/v) paraformaldehyde in phosphate buffer (PB) (4% PFA) for 10 min at room temperature and then imaged under an inverted fluorescence microscope (IX71, Olympus) equipped with an Olympus DP72 digital camera. For each of triplicate experiments, pictures were taken from eight random fields. All cells in the images were analyzed. Image Tool (University of Texas Health Sciences Center at San Antonio) was used for quantification. In vivo PI staining. PI (10 mg/ml) was diluted in 0.9% NaCl. Twenty milligram per kilogram of PI in a total volume of not more than 100 ll was administered (i.p.) to mice 1 h before sacrifice as described (Ito et al., 1997; Oerlemans et al., 2012).
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Immunohistochemistry
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Five days after SCI, mice were anesthetized and perfused intracardially with 20 ml of saline followed by 40 ml of 4% PFA. Subsequently, 1.5-cm spinal cord segments containing the lesion site were removed and post-fixed for 2 h, then kept in 25% sucrose at 4 °C until they sank. For immunostaining, serial sagittal sections (12 lm thick) were cut on a cryostat. Then the slides were blocked with 0.01 M PBS containing 3% BSA and 0.3% Triton X-100 for 30 min, and incubated with rabbit anti-Iba-1 (1:1000, Wako), rat anti-MLKL (1:200, Millipore), rabbit anti-phosphorylated MLKL (pMLKL) (1:300, Abcam), mouse anti-CD11b (1:200, Abcam), goat anti-chemokine (C-X-C motif) receptor 4 (CXCR4)
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(1:200, Abcam), rabbit anti-CC3 (1:400, CST), rabbit anti-GFAP (1:1000, Dako), rabbit anti-NeuN (1:800, Milipore), rabbit anti-CC1 (1:600, Milipore) or goat anti-Glucose-regulated protein 78 (GRP78) (1:200, Santa Cruz Biotechnology) at room temperature overnight. After three washings in 0.01 M PBS, the sections were incubated with the corresponding secondary antibodies conjugated with Alexa Fluor 594 (donkey anti-rabbit IgG, 1:1000, Jackson ImmunoResearch), Alexa Fluor 488 (donkey anti-rat IgG, 1:1000, Jackson ImmunoResearch) or DyLight 649 (donkey anti-goat IgG, 1:1000, Jackson ImmunoResearch) for 4 h at room temperature in the dark. All sections were counterstained with Hoechst 33342 to label nuclei. Sections were examined and
Fig. 1. Double-immunostaining of MLKL and neural cell markers in injured spinal cord at 5 dpi. (A–C) Double-immunostaining of MLKL and Iba-1. (D–F) Double-immunostaining of MLKL and GFAP, CC1 or NeuN. (G) Quantification of MLKL-positive cells. Notice that MLKL was mainly expressed in Iba-1-positive cells, and that MLKL-immunoreacitivities were localized both in somas (asterisk in B and C) and on membrane (arrows in B and C) of Iba-1-positive cells. (H) The bilateral areas 300 lm rostral and caudal to the lesion epicenter on which quantification was performed. Scale bars = 20 lm (A–F), 100 lm (H). Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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photographed under a confocal microscope (FV1000, Olympus). Image Tool was used for quantification. Cell counting was performed on bilateral areas 300um rostral and caudal to the lesion center on areas (Fig. 1H).
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Western blot
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N9 cells and microglia were lysed with ice-cold lysis buffer (50 mM Tris–HCl, 5 mM EDTA, 150 mM NaCl, 0.5 % deoxysodiumcholate, 0.1 % sodium dodecyl sulfate, 1 mM dithiothreitol and 1% Triton X-100, pH 8.0) containing protease inhibitor mixture of 0.1 mg/ml aprotinin, 1 mM PMSF, 5 mM sodium fluoride, and 1 mM sodium orthovanadate at indicated time points. Proteins were separated in 10 % SDS–PAGE gel, and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore), followed by blocking with 5 % nonfat milk for 1 h at room temperature. Membranes were incubated with primary antibodies for GRP78 (1:200, Santa Cruz Biotechnology), MLKL (1:400, Millipore), receptor-interacting protein 3 (RIP3) (1:500, ENZO), p-eIF2a (1:500, CST), b-actin (1:5000, Sigma) or high- mobility group box protein 1 (HMGB1) (1:500, Proteintech) at 4 °C overnight. After washing three times with Tris-buffered saline containing 0.1% Tween 20 (0.1 % TBST), membranes were incubated with secondary antibodies conjugated with horse-radish peroxidase (1:8000, Jackson ImmunoResearch) for 1 h at room temperature. Bands were developed by using enhanced Electrochemical Luminescence (ECL) solution (Millipore) and detected with the Bio-Rad Image Lab system.
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Image J (NIH) was used for measurement of the densities of specific bands.
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Immunoelectron microscopic study
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Mice were deeply anesthetized with 1% sodium pentobarbital (80 mg/kg) and perfused slowly with 20 mL saline, followed by 50 mL 4% PFA containing 0.05% glutaraldehyde and 15% (v/v) saturated picric acid. Then the injured spinal cord segment was removed and post-fixed in the same fixative solution for 4 h at 4 °C. Sections 50-lm-thick were prepared with a vibratome (VT 1000S, Leica), then placed in 0.01 M PBS containing 25% sucrose and 10% glycerol for 2 h for cryoprotection. Following a freeze–thaw treatment and blocking in 0.01 M PBS containing 5% BSA and 5% normal goat serum (NGS) for 4 h, sections were incubated with rat anti-MLKL (1:200, Millipore), or rabbit ant-RIP3 (1:200, ENZO) diluted in 0.01 M PBS containing 1% BSA and 1% NGS overnight at room temperature. They were then washed in 0.01 M PBS and incubated overnight with anti-rat or anti-rabbit IgG conjugated to 1.4 nm gold particles (1:100, Nanoprobes). After another fixing in 2% glutaraldehyde in 0.01 M PBS for 45 min, the silver enhancement was performed with HQ Silver Kit (Nanoprobes) in the dark. Immunolabeled sections were then immersed in 0.5% osmium tetroxide for 1 h, dehydrated in graded ethanol series, then in propylene oxide, and finally flat-embedded in Epon812 (SPI-CHEM). After polymerization, sections were examined under a light
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Fig. 2. Membrane localization of MLKL in microglia/macrophages. (A–D) Triple-immunostaining of MLKL/Iba-1/CXCR4. Notice the overlap of MLKL- and CXCR4-immunoreactivity at the membrane of Iba-1-positive cells (arrows in A-D), and the cytoplasmic distribution of MLKL-immunoreactivity (arrowhead in A–D). Scale bars = 20 lm. (E) An EM image of a microglia/macrophage in the normal spinal cord. (F, G) Immuno-electronic study of MLKL in spinal cord at 5 dpi. G is the higher magnified image boxed in F. MLKL-immunoreactivities are mainly localized on the membrane of microglia/macrophages (arrowheads in F), especially the ruptured membrane (arrows in F, G). Asterisk in G points the lysed cytoplasm. Scale bars = 1 lm (F), 500 nm (E and G). Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Fig. 3. Necroptosis of microglia/macrophages after SCI. (A–C) Combination of in vivo PI-labeling with double-staining of MLKL/Iba-1. Notice the PI-labeled MLKL/Iba-1 double-positive cell. Scale bars = 20 lm. (D–F) Combination of in vivo PI-labeling with immunohistochemistry of Iba-1 in Nec-1 treated and control mice at 5 dpi. Notice the decrease of PI-labeled Iba-1-positive cells in Nec-1 treated mice. Scale bars = 20 lm. *p < 0.05. (G–L) Triple-immunostaining of Iba-1/cleaved Caspase-3 (CC3)/MLKL and quantification of staining. Notice the separate immunoreactivity of CC3 and MLKL in Iba-1-positive cells. Scale bar = 30 lm.
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microscope. Five sections containing MLKL or RIP3 immunoreactivity surrounding the lesion site were selected from each mouse, trimmed under a stereomicroscope and mounted onto blank resin stubs.
With an ultramicrotome (EM UC6, Leica), ultrathin sections were cut and then mounted on mesh grids. The sections were then counterstained with uranyl acetate and lead citrate, observed and photographed by
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Fig. 4. ER localization of MLKL and RIP3 in microglia/macrophages after SCI. (A, B) Immuno-electron microscopic study of MLKL. Notice the ER localization of MLKL (box in A and arrows in B) and membrane localization of MLKL (arrowheads in A). (C–E) Immuno-electron microscopic study of RIP3. Notice the ER localization of RIP3 (box in C and arrows in D and E). Arrowhead in C points to RIP3-immunoreactivity on mitochondria. Scale bars = 500 nm (A), 200 nm (B, D and E), 1 lm (C).
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a digital camera (832 SC1000, Gatan) under an electron microscope (JEM-1230, JEOL Ltd).
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Statistical analysis
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All data were presented as mean ± SEM. A one-way ANOVA was adopted for comparison between groups. Statistical analyses were performed using the GraphPad Prism software (San Diego, USA). P < 0.05 was considered statistically significant.
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RESULTS Expression of MLKL and necroptosis of microglia/macrophages after SCI We first examined the expression of MLKL, the key molecule in the intracellular signaling of necroptosis (Sun et al., 2012; Zhao et al., 2012; Murphy et al., 2013; Wu et al., 2013), in normal and injured spinal cords by immunohistochemistry. No MLKL-immunoreactivity was observed in the normal spinal tissue (data not shown). However, numerous MLKL-positive cells were detected within and surrounding the lesion center at 5 dpi. Double-immunostaining of MLKL with different neural cell markers was carried out to analyze the cell types that express MLKL after SCI. Quantification in bilateral areas 300 lm rostral and dorsal to lesion epicenter
(Fig. 1H) showed that approximately 57.6% of the MLKL-positive cells expressed Iba-1, a marker for microglia/macrophages (Fig. 1A–C, G). Approximately 34.2% of MLKL-positive cells expressed GFAP, a marker of astrocytes (Fig. 1D, G), 5.7% of MLKL-positive cells expressed oligodendrocyte marker CC1, and 2.5% expressed the neuronal marker NeuN, respectively (Fig. 1E–G). These data showed that MLKL were mainly expressed in microglia/macrophages within and around the lesion site after SCI, suggesting that necroptosis may occur in microglia/macrophages. Because the membrane localization of MLKL was thought to trigger necroptosis by initiating ion influx (Cai et al., 2014; Chen et al., 2014; Dondelinger et al., 2014; Hildebrand et al., 2014; Wang et al., 2014a), we examined the distribution of MLKL in microglia/macrophages by both immunohistochemistry and immune-electron microscopic study. Triple-immunostaining of MLKL with Iba-1 and CXCR4, a chemokine receptor that is highly expressed on the membrane of microglia/macrophages, revealed that MLKL-immunoreactivity was distributed both in the cytoplasm and on the membrane of Iba-1 - positive cells (Fig. 2A–D). Under the electron microscope, cells with apoptotic features (such as chromatin aggregation, nuclear and cytoplasmic condensation, and an intact cell membrane) could be occasionally found (data not shown). Microglia/macrophages were rec-
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Fig. 5. Expression of GRP78 in microglia/macrophages in normal and injured spinal cords. (A–C) Triple-immunostaining of GRP78/Iba-1/MLKL in the normal spinal cord. (D–F) Triple-immunostaining of GRP78/Iba-1/MLKL at 5 dpi. Scale bar = 20 lm. (G) Quantification of GRP78-, Iba-1- and MLKL-positive cells in injured spinal cord. Notice the triple-staining of GRP78/Iba-1/MLKL in injured spinal cord. 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331
ognized by their characteristic oval or round nuclei with dense and highly clumped heterochromatin (Fig. 2E). However, due to the morphological changes of apoptosis, it is hard to determine the apoptotic microglia/macrophages. In contrast, microglia/macrophages with necrotic features, such as lysis of cytoplasmic content and breakdown of cell membrane were frequently observed (Fig. 2F, G). Intensive MLKL immunoreactivities, visualized by immunogold silver-enhanced particles, were found along the outer surface of the cell membrane (arrowheads in Fig. 2F and Fig. 4A), with some part of membrane ruptured (arrows in Fig. 2F, G) and adjacent cytoplasm swollen and translucent (asterisk in Fig. 2G), indicating a leakage or lysis of cytoplasmic material. The membrane localization of MLKL was consisting with its role in increasing membrane permeability. To further examine the membrane integrity of MLKL-positive microglia, we performed in vivo PI labeling at 5 dpi and triple-stained MLKL/PI/Iba-1. PI-labeled MLKL/Iba-1 double-positive cells were frequently found within and around the lesion center (Fig. 3A–C), implying the necroptosis of microglia/macrophages. To confirm the occurrence of necroptosis, we administered Necrostatin1 (Nec-1), an inhibitor of necroptosis, to examine its effects on the PI-labeling of microglia/macrophages. Five
successive days of Nec-1 administration significantly decreased the percent of PI-labeled Iba-1-positive cells in bilateral areas 300 lm rostral and caudal to the lesion center as defined in Fig. 1H (Fig. 3D–F). Because the activation of necroptotic signaling is thought to be coupled with inhibition of apoptosis, we then examined the relation between Caspase-3 activation and MLKL expression. Triple-immunostaining of Iba-1/cleaved caspase-3 (CC3)/MLKL showed a separate expression of CC3 and MLKL in Iba-1-positive cells, suggesting the inhibition of apoptotic machinery in necroptotic microglia/macrophages (Fig. 3G–K). Quantification showed that approximately 11% Iba-1- positive cells in bilateral areas 300 lm rostral and caudal to the lesion center were labeled by CC3. In the same region, approximately 25% Iba-1- positive cells expressed MLKL (Fig. 3L). Taken together, these data indicated that necroptosis contributed to the death of microglia/macrophages after SCI.
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Localization of MLKL and RIP3 on the ER of necroptotic microglia/macrophages after SCI
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In our electron-microscope study, MLKL immunoreactivity was surprisingly observed on ER (box in Fig. 4A, arrows in
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Fig. 6. ER stress and necroptosis of OGD treated N9 cells. (A) Western-blotting of GRP78 and p-eIF2a at different time points after 12 h-OGD exposure. (B) Western-blotting of RIP3, MLKL and HMGB1 at different time points after exposure to OGD. Notice the increase of ER stress markers (GRP78, p-eIF2a) and necroptotic markers (RIP3, MLKL and HMGB1) after OGD treatment. (C–F) Effects of Nec-1 on PI-labeling. Notice that Nec1 can significantly prevent the increase of PI-labeling induced by OGD. ***p < 0.001, *p < 0.05. Scale bars = 200 lm. (G–J) Effects of Nec-1 on the expression of RIP3, MLKL and HMGB1. Notice that Nec-1 can significantly decrease the expression of RIP3, MLKL and HMGB1 induced by OGD. ** p < 0.01, *p < 0.05. 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369
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Fig. 4B) with even more immunogold particles than on mitochondria (Fig. 4A) where MLKL reportedly interact with PGAM5 for the execution of cell death (Wang et al., 2012). Because translocation of MLKL to mitochondrial associated membranes is mediated by phosphorylated RIP3 (Chen et al., 2013), we then performed immuneelectron microscopic study of RIP3 to test whether RIP3 could translocate to ER. In RIP3-positive microglia/macrophages, cytoplasmic lysis, the ultrastructural change of necrosis, was observed (Fig. 4C). Similar to MLKL, many RIP3 immunoreactivities were also found on ER (Fig. 4C, and arrows in Fig. 4D, E). These results confirmed the idea that microglia/macrophages died through necroptosis, and additionally indicated a possible involvement of ER in the necroptosis of microglia/macrophages. Expression of ER sensor GRP78 in necroptotic microglia/macrophages after SCI The finding that MLKL and RIP3 were localized at ER prompted us to examine whether ER stress could be
induced in necroptotic microglia/macrophages after SCI. Triple immunostaining of MLKL, Iba-1 and ER stress sensor GRP78 was performed (Fig. 5D–F). Similar to MLKL, GRP78 immuoreactivity was highly expressed in cells surrounding the lesion center. Approximately 44.6% of GRP78-positive cells expressed Iba-1 (Fig. 5D, G). Importantly, among the MLKL-positive cells, 70.2% cells were GRP78-positive (Fig. 5E, G). Further, approximately 68.2% of MLKL/Iba-1 double-positive cells were GRP78-positive (Fig. 5F, G). These data showed a well co-expression of GRP78 and MLKL in microglia/macrophages, and supported the implication from EM study that ER stress may be induced in necroptotic microglia/macrophages.
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Because it has been reported that ER stress could be induced by ischemia (Doroudgar et al., 2009) and ischemia is an important factor in the secondary SCI, we then
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Fig. 7. Effects of the ER stress inhibitor on the necroptosis of primary microglia after OGD treatment. (A–B) Western-blotting of GRP78 and p-eIF2a in control cells, or cells treated by 4-PBA alone, OGD, or OGD plus 4-PBA. Notice that 4-PBA significantly decreased the expression of GRP78 and p-eIF2a induced by OGD. **p < 0.01, *p < 0.05. (C–F) Western-blotting of MLKL, RIP3 and HMGB1 in control cells, or cells treated by 4-PBA alone, OGD, or OGD plus 4-PBA. Notice that 4-PBA significantly decreased the expression of MLKL, RIP3 and HMGB1 induced by OGD. **p < 0.01, * p < 0.05. (G–K) PI staining and quantification of PI-positive cells in control cells, or cells treated by 4-PBA alone, OGD, or OGD plus 4-PBA. Notice that 4-PBA significantly decreased the number of PI-labeled cells induced by OGD. **p < 0.01, ***p < 0.001. Scale bar = 200 lm.
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tested whether OGD treatment could induce necroptosis of microglia in vitro. N9 cells, a mouse microglia cell line, were exposed to OGD for 12 h, and then were replenished with normal oxygen and glucose for different time points (4, 16, 24, 36 and 48 h). ER stress was evaluated by immunoblotting of GRP78 and p-eIF2a, two markers of ER stress. Data showed that GRP78 and p-eIF2a were up-regulated from 16 h and peaked at 36 h after reoxygenation (Fig. 6A). Interestingly, the expression of necroptotic proteins, MLKL, RIP3 and HMGB1, was also increased with a similar time course after reoxygenation (Fig. 6B). PI staining was further performed to assess cell death. At 36 h after OGD exposure, the peak of ER stress as showed by GRP78 and p-eIF2a expression, the number of PI-positive cells was markedly increased by about eightfold (p < 0.001) in comparison with control (Fig. 6C–F). Addition of Nec-1 (20 lM), an inhibitor of necroptosis in culture medium, significantly attenuated the OGD-induced increase of MLKL, RIP3, HMGB1 and the number of PI-labeled cells (Fig. 6C–J). Therefore, OGD treatment not only induced ER stress, but also necroptosis in N9 microglial cells, suggesting a possible role of ER stress in the necroptosis of microglia.
Suppression of necroptosis of primary microglia by inhibition of ER stress
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We further cultured primary microglia, induced necroptosis by OGD, and added an ER stress inhibitor, 4-PBA (de Almeida et al., 2007) to evaluate the role of ER stress in the necroptosis of microglia. Based on the results of N9 cells, we examined ER stress and necroptosis of microglia at 36 h after reoxygenation. The expression of GRP78 and p-eIF2a increased 3.4- and 2.9-folds respectively (Fig. 7A, B). This increase was effectively blocked by 4-PBA (Fig. 7A, B). At 36 h postreoxygenation, the expression of MLKL, HMGB1 and RIP3 increased 2.8-, 2.4- and 2.3-folds respectively (Fig. 7C–F). PI staining showed an 8.3-fold increase of cell death relative to normal controls (Fig. 7G–K). Addition of 4-PBA significantly attenuated the above increase of MLKL, HMGB1, RIP3 and the percent of PI-labeled cells (Fig. 7C–K). 4-PBA alone has no significant effects on the ER stress and necroptosis of microglia (Fig. 7A–K). These data suggested that ER stress was actively involved in the necroptosis of primary microglia induced by OGD exposure.
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Fig. 8. Expression of pMLKL and GRP78 in microglia/macrophages in human SCI. (A–D) Triple-immunostaining of MLKL, GRP78 and CD11b in the normal human spinal cord. (E–H) Represent images of triple-immunostaining of MLKL, GRP78 and CD11b in the injured human spinal cord at 5 dpi. Arrows point to the double or triple-stained cells. Scale bar = 50 lm.
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Co-expression of phosphorylated MLKL and GRP78 by microglia/macrophages in the injured human spinal cord We next examined whether ER stress and necroptosis could occur in the microglia/macrophages after human SCI . Because phosphorylation of MLKL was required for the necroptotic function of MLKL (Sun et al., 2012; Rodriguez et al., 2015), we examined the expression of pMLKL in human spinal tissue. In the normal spinal cord, there is no expression of pMLKL and GRP78 (Fig. 8B, C). After SCI, triple-immunostaining of pMLKL/GRP78/ CD11b (a marker of microglia/macrophages) in one patient at 5d after SCI showed that pMLKL and GRP78 were highly expressed in CD11b-positive cells (Fig. 8E, F). Noticeably, pMLKL- and GRP78-immunoreactivities overlapped well with each other (Fig. 8G, H). Similar expression pattern of pMLKL, GRP78 and CD11b were observed in another patient at 15 dpi (data not shown). These results indicated the co-occurrence of ER stress and necroptosis in microglia/macrophages in human after SCI.
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DISCUSSION
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Persistent and excessive activation of microglia/macrophages have been suggested to be detrimental to the functional recovery after SCI (Zhou et al., 2014). Previous studies have reported Caspase-dependent apoptosis of microglia/macrophages after SCI (Beattie et al., 2000). Recently, more and more evidence have suggested that necroptosis is closely associated with inflammation (Newton, 2015; Pasparakis and Vandenabeele, 2015; Silke et al., 2015). A recent study reported necroptosis of microglia in vitro upon Toll-like receptor (TLR) activation in combination with
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Caspase inhibition (Kim and Li, 2013). Considering the chronic inflammation after SCI, it would be important to explore the necroptosis of microglia/macrophages after SCI, which, however, still remains poorly investigated. Previous studies have demonstrated that Nec-1 had beneficial effects on SCI and suggested that necroptosis might contribute to the early cell damage post-SCI (Liu et al., 2014; Wang et al., 2014b, 2015). In the present study, by light and electronic microscopic studies, we first demonstrated an induction of MLKL, a key molecule in necroptosis, on the membrane of microglia/macrophages after SCI. Then, by in vivo PI-labeling and pharmacological manipulation, we confirmed the occurrence of necroptosis in microglia/macrophages after SCI. It thus appears that not only apoptosis, but necroptosis may also be exploited to regulate the process of microglia/macrophage-mediated inflammation after SCI. ER, which normally functions as an intracellular Ca2+ store and protein folding platform (Di Sano et al., 2012), is actively involved in apoptosis when stressed (Liu et al., 2013). ER stress induced apoptosis has been demonstrated to happen in various neurological conditions (Lindholm et al., 2006; Zhang et al., 2013). After SCI, insufficient vascularization in the contusion lesion site induced ER stress and corresponding cell death (Yamauchi et al., 2007). The localization of MLKL- and RIP3-immunoreactivity at ER indicates a possible involvement of ER in necroptosis. Whether MLKL/RIP3 complex could induce calcium release from ER during necroptosis is of interest to be further probed. Our data further showed that GRP78, a widely adopted ER stress sensor, was induced in MLKL-positive microglia/macrophages after SCI. In addition, the expression of GRP78 and p-eIF2a, two ER stress markers, increased in parallel with the expression of
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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necroptotic proteins upon OGD treatment in vitro. Importantly, inhibition of ER stress could attenuate the necroptosis of microglia cells induced by OGD treatment. These data indicate an involvement of ER stress in the necroptosis of microglia/macrophages, and are in consistence with a recent report that ER stress can induce necroptosis of L929 cells independent of TNFR1 (Saveljeva et al., 2015). The low-level expression of MLKL and GRP78 detected by Western-blot in vitro and the negative immunostaining of MLKL and GRP78 in the normal spinal cord may be due to the difference of methods or the ‘‘active” status of microglia/macrophages in vitro. Co-expression of pMLKL and GRP78 in microglia/macrophages in the injured human spinal cord suggested that there may be a conserved relationship between ER stress and necroptosis, which is to be further explored for detail. Nevertheless, our data provided a novel point for understanding of the mechanisms that regulate the death of microglia/macrophages after SCI, which could be utilized for modulating post-SCI inflammation.
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CONFLICT OF INTEREST
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The authors declare no conflict of interest.
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Acknowledgments—This work was supported by grants from the National Natural Science Foundation of China (NSFC, 31271583, 81571224) to Dr. W.Y, and NSFC (81371364).to Dr. J.G., and research fellowship for outstanding Ph.D. candidates of Fourth Military Medical University (2013D09) to Dr. H.F. We appreciate the technical assistances from Drs. Jie Xu, Kun Chen and HaiFeng Zhang, and the kind gifts of antibodies from Dr. Heng Ma. We thank Dr. Biswas Sangita (University of California at Davis) for editing the language.
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REFERENCES
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Allison DJ, Ditor DS (2015) Immune dysfunction and chronic inflammation following spinal cord injury. Spinal Cord 53:14–18. Beattie MS, Farooqui AA, Bresnahan JC (2000) Review of current evidence for apoptosis after spinal cord injury. J Neurotrauma 17:915–925. Beattie MS, Hermann GE, Rogers RC, Bresnahan JC (2002) Cell death in models of spinal cord injury. Prog Brain Res 137:37–47. Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, Ward Y, Wu LG, Liu ZG (2014) Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16:55–65. Chen W, Zhou Z, Li L, Zhong CQ, Zheng X, Wu X, Zhang Y, Ma H, Huang D, Li W, Xia Z, Han J (2013) Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem 288:16247–16261. Chen X, Li W, Ren J, Huang D, He WT, Song Y, Yang C, Li W, Zheng X, Chen P, Han J (2014) Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res 24:105–121. David S, Kroner A (2011) Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci 12:388–399. de Almeida SF, Picarote G, Fleming JV, Carmo-Fonseca M, Azevedo JE, de Sousa M (2007) Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation. J Biol Chem 282:27905–27912.
529 530 531 532 533 534 535
11
Di Sano F, Bernardoni P, Piacentini M (2012) The reticulons: guardians of the structure and function of the endoplasmic reticulum. Exp Cell Res 318:1201–1207. Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I, Hulpiau P, Weber K, Sehon CA, Marquis RW, Bertin J, Gough PJ, Savvides S, Martinou JC, Bertrand MJ, Vandenabeele P (2014) MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 7:971–981. Doroudgar S, Thuerauf DJ, Marcinko MC, Belmont PJ, Glembotski CC (2009) Ischemia activates the ATF6 branch of the endoplasmic reticulum stress response. J Biol Chem 284:29735–29745. Gorman AM, Healy SJ, Jager R, Samali A (2012) Stress management at the ER: regulators of ER stress-induced apoptosis. Pharmacol Ther 134:306–316. Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P, Pierotti C, Garnier JM, Dobson RC, Webb AI, Tripaydonis A, Babon JJ, Mulcair MD, Scanlon MJ, Alexander WS, Wilks AF, Czabotar PE, Lessene G, Murphy JM, Silke J (2014) Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc Natl Acad Sci USA 111:15072–15077. Hoozemans JJ, Scheper W (2012) Endoplasmic reticulum: the unfolded protein response is tangled in neurodegeneration. Int J Biochem Cell Biol 44:1295–1298. Hu X, Leak RK, Shi Y, Suenaga J, Gao Y, Zheng P, Chen J (2015) Microglial and macrophage polarization-new prospects for brain repair. Nat Rev Neurol 11:56–64. Ito WD, Schaarschmidt S, Klask R, Hansen S, Scha¨fer HJ, Mathey D, Bhakdi S (1997) Infarct size measurement by triphenyltetrazolium chloride staining versus in vivo injection of propidium iodide. J Mol Cell Cardiol 29:2169–2175. Kim SJ, Li J (2013) Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia. Cell Death Dis 4:e716. Kroner A, Greenhalgh AD, Zarruk JG, Passos Dos Santos R, Gaestel M, David S (2014) TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord. Neuron 83:1098–1116. Lindholm D, Wootz H, Korhonen L (2006) ER stress and neurodegenerative diseases. Cell Death Differ 13:385–392. Liu D, Zhang M, Yin H (2013) Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis. Int J Neurosci 123:155–162. Liu M, Wu W, Li H, Li S, Huang LT, Yang YQ, Sun Q, Wang CX, Yu Z, Hang CH (2014) Necroptosis, a novel type of programmed cell death, contributes to early neural cells damage after spinal cord injury in adult mice. J Spinal Cord Med. Liu S, Sarkar C, Dinizo M, Faden AI, Koh EY, Lipinski MM, Wu J (2015) Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death. Cell Death Dis 6:e1582. Mabon PJ, Weaver LC, Dekaban GA (2000) Inhibition of monocyte/macrophage migration to a spinal cord injury site by an antibody to the integrin alphaD: a potential new antiinflammatory treatment. Exp Neurol 166:52–64. Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S, Lewis R, Lalaoui N, Metcalf D, Webb AI, Young SN, Varghese LN, Tannahill GM, Hatchell EC, Majewski IJ, Okamoto T, Dobson RC, Hilton DJ, Babon JJ, Nicola NA, Strasser A, Silke J, Alexander WS (2013) The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39:443–453. Newton K (2015) RIPK1 and RIPK3: critical regulators of inflammation and cell death. Trends Cell Biol 25:347–353. Oerlemans MI, Liu J, Arslan F, den Ouden K, van Middelaar BJ, Doevendans PA, Sluijter JP (2012) Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo. Basic Res Cardiol 107:270. Pasparakis M, Vandenabeele P (2015) Necroptosis and its role in inflammation. Nature 517:311–320.
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Rodriguez DA, Weinlich R, Brown S, Guy C, Fitzgerald P, Dillon CP, Oberst A, Quarato G, Low J, Cripps JG, Chen T, Green DR (2015) Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell Death Differ. Saveljeva S, Mc Laughlin SL, Vandenabeele P, Samali A, Bertrand MJ (2015) Endoplasmic reticulum stress induces ligandindependent TNFR1-mediated necroptosis in L929 cells. Cell Death Dis 6:e1587. Silke J, Rickard JA, Gerlic M (2015) The diverse role of RIP kinases in necroptosis and inflammation. Nat Immunol 16:689–697. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148:213–227. Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF, Wang FS, Wang X (2014a) Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 54:133–146. Wang Y, Wang H, Tao Y, Zhang S, Wang J, Feng X (2014b) Necroptosis inhibitor necrostatin-1 promotes cell protection and physiological function in traumatic spinal cord injury. Neuroscience 266:91–101. Wang Y, Wang J, Yang H, Zhou J, Feng X, Wang H, Tao Y (2015) Necrostatin-1 mitigates mitochondrial dysfunction post-spinal cord injury. Neuroscience 289:224–232. Wang Z, Jiang H, Chen S, Du F, Wang X (2012) The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 148:228–243.
Wu J, Huang Z, Ren J, Zhang Z, He P, Li Y, Ma J, Chen W, Zhang Y, Zhou X, Yang Z, Wu SQ, Chen L, Han J (2013) Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res 23:994–1006. Yamauchi T, Sakurai M, Abe K, Matsumiya G, Sawa Y (2007) Impact of the endoplasmic reticulum stress response in spinal cord after transient ischemia. Brain Res 1169:24–33. Yong C, Arnold PM, Zoubine MN, Citron BA, Watanabe I, Berman NE, Festoff BW (1998) Apoptosis in cellular compartments of rat spinal cord after severe contusion injury. J Neurotrauma 15:459–472. Zai LJ, Wrathall JR (2005) Cell proliferation and replacement following contusive spinal cord injury. Glia 50:247–257. Zhang HY, Zhang X, Wang ZG, Shi HX, Wu FZ, Lin BB, Xu XL, Wang XJ, Fu XB, Li ZY, Shen CJ, Li XK, Xiao J (2013) Exogenous basic fibroblast growth factor inhibits ER stress-induced apoptosis and improves recovery from spinal cord injury. CNS Neurosci Ther 19:20–29. Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J, Liu ZG (2012) Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 109:5322–5327. Zhou X, He X, Ren Y (2014) Function of microglia and macrophages in secondary damage after spinal cord injury. Neural Regen Res 9:87–95.
(Accepted 24 October 2015) (Available online xxxx)
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INVOLVEMENT OF ENDOPLASMIC RETICULUM STRESS IN THE NECROPTOSIS OF MICROGLIA/MACROPHAGES AFTER SPINAL CORD INJURY
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H. FAN, a H.-B. TANG, b J. KANG, a L. SHAN, c H. SONG, d K. ZHU, e J. WANG, a G. JU a* AND Y.-Z. WANG a*
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a Department of Neurobiology and Collaborative Innovation Center for Brain Science, Institute of Neurosciences, Fourth Military Medical University, 169 Chang Le Xi Road, Xi’an 710032, China
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b Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University, 127 Chang Le Xi Road, Xi’an 710003, China
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c Department of Orthopedics, Tangdu Hospital, Fourth Military Medical University, Xin Si Road, Xi’an, Shaanxi 710038, China
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Department of Occupational and Environmental Health, School of Public Health, Fourth Military Medical University, Xi’an 710032, China
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e Zhejiang University China Brain Bank, Department of Pathology and Pathophysiology, Department of Neuroscience, 866 Yu-Hang-Tang Road, Zhejiang University Zi-Jin-Gang Campus, Hangzhou, Zhejiang 310058, China
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Abstract—Microglia/macrophages play a crucial role in inflammation after spinal cord injury (SCI). Although extensive studies have been performed on the mechanisms of microglia/macrophage activation and recruitment, how microglia/macrophages are eliminated remains unclear. In the present study, we observed a high- level expression of mixed lineage kinase domain-like protein (MLKL), a key molecule in the execution of necroptosis, in microglia/macrophages after SCI in mice. In vivo PI-labeling and Necrostatin-1 treatment confirmed the necroptosis of microglia/macrophages. Interestingly, our electronic microscopic (EM) study revealed that MLKL localized not only at the membrane but also on the endoplasmic reticulum (ER) of necroptotic microglia/macrophages. Furthermore, receptor-interacting protein 3 (RIP3), another necrosome component, was also found on the ER of necroptotic microglia/macrophages. And Glucose-regulated protein 78 (GRP78), an ER stress sensor, was up-regulated in MLKL-positive microglia/macrophages after SCI, suggesting
a possible link between necroptosis and ER stress. In vitro, oxygen–glucose deprivation (OGD) stress induced ER stress and necroptosis in microglia. Inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly blocked the OGD-induced necroptosis of microglia. In the end, our data showed that, GRP78 and phosphorylated MLKL were coexpressed by the microglia/macrophages in the injured human spinal cord. Taken together, these results suggested that microglia/macrophages undergo an ER-stress involved necroptosis after SCI, implying that ER stress and necroptosis could be manipulated for modulating inflammation postSCI. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.
Key words: spinal cord injury, microglia/macrophages, necroptosis, ER stress. 23
*Corresponding authors. Tel: +86-29-84774565; fax: +86-2983246270 (G. Ju). Tel: +86-29-84774562; fax: +86-29-83246270 (Y.Z. Wang). E-mail addresses: [email protected] (G. Ju), yazhouw@fmmu. edu.cn (Y.-Z. Wang). Abbreviations: 4-PBA, 4-phenylbutyrate; CC3, cleaved Caspase-3; CXCR4, chemokine (C-X-C motif) receptor 4; EM, electronic microscopic; ER, endoplasmic reticulum; HMGB1, high-mobility group box protein 1; MLKL, mixed lineage kinase domain-like protein; Nec-1, Necrostatin-1; NGS, normal goat serum; OGD, oxygen–glucose deprivation; pMLKL, phosphorylated MLKL; RIP1, receptor-interacting protein 1; RIP3, receptor-interacting protein 3; SCI, spinal cord injury; EDTA, ethylenediaminetetraacetic acid; GRP78, Glucose-regulated protein 78; DMEM, Dulbecco’s modified Eagle medium; PI, Propidium Iodide; PBS, phosphate-buffered saline. http://dx.doi.org/10.1016/j.neuroscience.2015.10.049 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1
INTRODUCTION
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Spinal contusion is the major type of spinal cord injury (SCI) in the clinic, which often results in permanent loss of motor and sensory functions. Although several clinical trials have been tested, it still lacks effective treatment. A unique pathological change following SCI is the secondary injury which is characterized by gradual enlargement of lesion area and the presence of chronic inflammation (Allison and Ditor, 2015). The spinal microglia, together with recruited macrophages which exhibited an almost identical phenotype with activated microglia, plays a key role in the initiation and development of inflammation (David and Kroner, 2011). After SCI, the function of microglia/macrophages has been thought to be mainly destructive, possibly due to the dominance of their M1 sub-population (Hu et al., 2015). Many studies have been carried out to elucidate the mechanisms of their proliferation, migration and M1/M2 phenotype conversion, with the aim of biasing the post-SCI inflammation toward better repair (Mabon et al., 2000; Zai and Wrathall, 2005; Kroner et al., 2014). However, how microglia/macrophages are eliminated from the injured spinal cord remains unclear. Necrosis and apoptosis are the two major types of cell death after SCI (Beattie et al., 2002). Extensive studies have revealed the cellular and molecular mechanisms of apoptosis after SCI (Yong et al., 1998). However, necrosis has long been thought to be uncontrollable, and the mechanism of necrosis after SCI remains largely unexplored. Recent studies have identified a type of programed necrosis (necroptosis) and uncovered its
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underlying mechanisms which involves an intracellular signaling cascade transduced by receptor-interacting protein 1/3 (RIP1/3) and mixed lineage kinase domain-like protein (MLKL) (Sun et al., 2012), thus offering new molecular tools for re-examining necrosis after SCI. Endoplasmic reticulum stress (ER stress) is a cellular response to multiple injury conditions, which usually showed accumulation of unfolded proteins in the cytoplasm (Hoozemans and Scheper, 2012). Many studies have demonstrated that severe ER stress can activate the intracellular signaling that finally leads to apoptosis or autophagy (Gorman et al., 2012; Liu et al., 2015).Whether ER stress is also involved in the activation of necroptosis has been poorly studied. In the present study, we investigated the necroptosis of microglia/macrophages after SCI, and the involvement of ER stress in the necroptosis of microglia/macrophages.
EXPERIMENTAL PROCEDURES
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Human samples
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Snap-frozen normal human spinal cord tissues were obtained from the human brain bank of the school of medicine at Zhejiang University. Biopsy of injured spinal cord tissues were performed with informed consent obtained from each patient prior to surgery and experiments involving human spinal tissues were approved by the Institutional Review Board of Tangdu Hospital, Fourth Military Medical University.
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Spinal cord contusion and in vivo treatment
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Male C57BL/6 mice were purchased from the Laboratory Animal Center of Fourth Military Medical University and all the protocols of animal experiments were approved by the Animal Care and Use Committee of the Fourth Military Medical University. Mice (6–8 w) were anesthetized with 1% sodium pentobarbital (80 mg/kg), followed by bilateral laminectomy of vertebrae T8–T9. Spinal cord lateral crushing was made at the T8 vertebra by using a forceps with a tip-gap set at 0.2 mm for 15 s. Manual bladder expression was performed once a day after surgery. Sham SCI was made by performing a dorsal laminectomy without crushing the spinal cord.
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Necrostain-1 (Nec-1) administration
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Nec-1 (7.8 mg/kg) was administrated (i.v.) twice a day for 5 days for examining its effect on the necroptosis after SCI.
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Primary microglia and N9 cells culture
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Microglial cells were isolated from the brain tissue of neonatal C57BL/6 mice. Briefly, the skin and skull were peeled away and the meningeal lining was then gently removed under a microscope. The cerebral cortical tissue was then minced into a fine slurry with scissors, followed by digestion with 0.125% trypsin/0.02% EDTA. Cells were suspended in 12 ml of Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS, 100 lg/ml penicillin–streptomycin and 2 mM L-glutamine, seeded
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onto poly-D-lysine coated T-75 flasks and maintained at 37 °C in an incubator with humidified 5% CO2 atmosphere. After 9 days culture, when the cells were approximately 90% confluent, and purified by shaking at 260 rpm/min for 1 h. Immunostaining of calcium-binding adapter molecule 1 (Iba-1) was adopted to ensure that over 95% of the cells were Iba-1- positive. Microglial cells were then seeded in 6-well or 24-well plates at a density of 2 106 cells or 0.5 106 per well. Mouse microglial N9 cells were maintained in DMEM supplemented with 5% FBS, 100 lg/ml penicillin–streptomycin and 2 mM L-glutamine. For experiments, cells were plated onto 6-well or 24-well plates at a density of 3 106 cells or 1 106 cells per well.
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Oxygen–glucose deprivation (OGD) injury and Nec-1, 4-phenylbutyrate (4-PBA) treatment
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OGD in N9 cells and primary microglia was performed by replacing the medium with glucose-free DMEM (Gibco, USA) and cultures were incubated in a hypoxic chamber (95% N2 and 5% CO2) at 37 °C for 12 h. After OGD treatment, cells were transferred back to normal condition with fresh culture medium for 4, 16, 24, 36 and 48 h for N9 cells and 36 h for microglia respectively. Control cells were maintained in regular DMEM under normoxic conditions. For Nec-1 and 4-PBA treatment, 20 lM Nec-1 and 4 mM 4-PBA were added into the culture medium when OGD was begun, until the end of the experiment. Immediately at the end of treatments, total cellular proteins were isolated for Western-blotting analysis, or cells were stained with Propidium Iodide (PI) for death detection.
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PI staining
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In vitro PI labeling. N9 cells and primary microglia were exposed to OGD for 12 h and subsequently placed back to normal culture medium for 36 h. PI (5 lM, Sigma) and Hoechst 33342 (5 lg/ml, Sigma) were added into the culture medium and incubated for 30 min at 37 °C. Cells were then washed three times with 0.01 M phosphate- buffered saline (PBS) and fixed with 4% (w/v) paraformaldehyde in phosphate buffer (PB) (4% PFA) for 10 min at room temperature and then imaged under an inverted fluorescence microscope (IX71, Olympus) equipped with an Olympus DP72 digital camera. For each of triplicate experiments, pictures were taken from eight random fields. All cells in the images were analyzed. Image Tool (University of Texas Health Sciences Center at San Antonio) was used for quantification. In vivo PI staining. PI (10 mg/ml) was diluted in 0.9% NaCl. Twenty milligram per kilogram of PI in a total volume of not more than 100 ll was administered (i.p.) to mice 1 h before sacrifice as described (Ito et al., 1997; Oerlemans et al., 2012).
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Immunohistochemistry
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Five days after SCI, mice were anesthetized and perfused intracardially with 20 ml of saline followed by 40 ml of 4% PFA. Subsequently, 1.5-cm spinal cord segments containing the lesion site were removed and post-fixed for 2 h, then kept in 25% sucrose at 4 °C until they sank. For immunostaining, serial sagittal sections (12 lm thick) were cut on a cryostat. Then the slides were blocked with 0.01 M PBS containing 3% BSA and 0.3% Triton X-100 for 30 min, and incubated with rabbit anti-Iba-1 (1:1000, Wako), rat anti-MLKL (1:200, Millipore), rabbit anti-phosphorylated MLKL (pMLKL) (1:300, Abcam), mouse anti-CD11b (1:200, Abcam), goat anti-chemokine (C-X-C motif) receptor 4 (CXCR4)
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(1:200, Abcam), rabbit anti-CC3 (1:400, CST), rabbit anti-GFAP (1:1000, Dako), rabbit anti-NeuN (1:800, Milipore), rabbit anti-CC1 (1:600, Milipore) or goat anti-Glucose-regulated protein 78 (GRP78) (1:200, Santa Cruz Biotechnology) at room temperature overnight. After three washings in 0.01 M PBS, the sections were incubated with the corresponding secondary antibodies conjugated with Alexa Fluor 594 (donkey anti-rabbit IgG, 1:1000, Jackson ImmunoResearch), Alexa Fluor 488 (donkey anti-rat IgG, 1:1000, Jackson ImmunoResearch) or DyLight 649 (donkey anti-goat IgG, 1:1000, Jackson ImmunoResearch) for 4 h at room temperature in the dark. All sections were counterstained with Hoechst 33342 to label nuclei. Sections were examined and
Fig. 1. Double-immunostaining of MLKL and neural cell markers in injured spinal cord at 5 dpi. (A–C) Double-immunostaining of MLKL and Iba-1. (D–F) Double-immunostaining of MLKL and GFAP, CC1 or NeuN. (G) Quantification of MLKL-positive cells. Notice that MLKL was mainly expressed in Iba-1-positive cells, and that MLKL-immunoreacitivities were localized both in somas (asterisk in B and C) and on membrane (arrows in B and C) of Iba-1-positive cells. (H) The bilateral areas 300 lm rostral and caudal to the lesion epicenter on which quantification was performed. Scale bars = 20 lm (A–F), 100 lm (H). Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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photographed under a confocal microscope (FV1000, Olympus). Image Tool was used for quantification. Cell counting was performed on bilateral areas 300um rostral and caudal to the lesion center on areas (Fig. 1H).
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Western blot
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N9 cells and microglia were lysed with ice-cold lysis buffer (50 mM Tris–HCl, 5 mM EDTA, 150 mM NaCl, 0.5 % deoxysodiumcholate, 0.1 % sodium dodecyl sulfate, 1 mM dithiothreitol and 1% Triton X-100, pH 8.0) containing protease inhibitor mixture of 0.1 mg/ml aprotinin, 1 mM PMSF, 5 mM sodium fluoride, and 1 mM sodium orthovanadate at indicated time points. Proteins were separated in 10 % SDS–PAGE gel, and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore), followed by blocking with 5 % nonfat milk for 1 h at room temperature. Membranes were incubated with primary antibodies for GRP78 (1:200, Santa Cruz Biotechnology), MLKL (1:400, Millipore), receptor-interacting protein 3 (RIP3) (1:500, ENZO), p-eIF2a (1:500, CST), b-actin (1:5000, Sigma) or high- mobility group box protein 1 (HMGB1) (1:500, Proteintech) at 4 °C overnight. After washing three times with Tris-buffered saline containing 0.1% Tween 20 (0.1 % TBST), membranes were incubated with secondary antibodies conjugated with horse-radish peroxidase (1:8000, Jackson ImmunoResearch) for 1 h at room temperature. Bands were developed by using enhanced Electrochemical Luminescence (ECL) solution (Millipore) and detected with the Bio-Rad Image Lab system.
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Image J (NIH) was used for measurement of the densities of specific bands.
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Immunoelectron microscopic study
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Mice were deeply anesthetized with 1% sodium pentobarbital (80 mg/kg) and perfused slowly with 20 mL saline, followed by 50 mL 4% PFA containing 0.05% glutaraldehyde and 15% (v/v) saturated picric acid. Then the injured spinal cord segment was removed and post-fixed in the same fixative solution for 4 h at 4 °C. Sections 50-lm-thick were prepared with a vibratome (VT 1000S, Leica), then placed in 0.01 M PBS containing 25% sucrose and 10% glycerol for 2 h for cryoprotection. Following a freeze–thaw treatment and blocking in 0.01 M PBS containing 5% BSA and 5% normal goat serum (NGS) for 4 h, sections were incubated with rat anti-MLKL (1:200, Millipore), or rabbit ant-RIP3 (1:200, ENZO) diluted in 0.01 M PBS containing 1% BSA and 1% NGS overnight at room temperature. They were then washed in 0.01 M PBS and incubated overnight with anti-rat or anti-rabbit IgG conjugated to 1.4 nm gold particles (1:100, Nanoprobes). After another fixing in 2% glutaraldehyde in 0.01 M PBS for 45 min, the silver enhancement was performed with HQ Silver Kit (Nanoprobes) in the dark. Immunolabeled sections were then immersed in 0.5% osmium tetroxide for 1 h, dehydrated in graded ethanol series, then in propylene oxide, and finally flat-embedded in Epon812 (SPI-CHEM). After polymerization, sections were examined under a light
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Fig. 2. Membrane localization of MLKL in microglia/macrophages. (A–D) Triple-immunostaining of MLKL/Iba-1/CXCR4. Notice the overlap of MLKL- and CXCR4-immunoreactivity at the membrane of Iba-1-positive cells (arrows in A-D), and the cytoplasmic distribution of MLKL-immunoreactivity (arrowhead in A–D). Scale bars = 20 lm. (E) An EM image of a microglia/macrophage in the normal spinal cord. (F, G) Immuno-electronic study of MLKL in spinal cord at 5 dpi. G is the higher magnified image boxed in F. MLKL-immunoreactivities are mainly localized on the membrane of microglia/macrophages (arrowheads in F), especially the ruptured membrane (arrows in F, G). Asterisk in G points the lysed cytoplasm. Scale bars = 1 lm (F), 500 nm (E and G). Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Fig. 3. Necroptosis of microglia/macrophages after SCI. (A–C) Combination of in vivo PI-labeling with double-staining of MLKL/Iba-1. Notice the PI-labeled MLKL/Iba-1 double-positive cell. Scale bars = 20 lm. (D–F) Combination of in vivo PI-labeling with immunohistochemistry of Iba-1 in Nec-1 treated and control mice at 5 dpi. Notice the decrease of PI-labeled Iba-1-positive cells in Nec-1 treated mice. Scale bars = 20 lm. *p < 0.05. (G–L) Triple-immunostaining of Iba-1/cleaved Caspase-3 (CC3)/MLKL and quantification of staining. Notice the separate immunoreactivity of CC3 and MLKL in Iba-1-positive cells. Scale bar = 30 lm.
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microscope. Five sections containing MLKL or RIP3 immunoreactivity surrounding the lesion site were selected from each mouse, trimmed under a stereomicroscope and mounted onto blank resin stubs.
With an ultramicrotome (EM UC6, Leica), ultrathin sections were cut and then mounted on mesh grids. The sections were then counterstained with uranyl acetate and lead citrate, observed and photographed by
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Fig. 4. ER localization of MLKL and RIP3 in microglia/macrophages after SCI. (A, B) Immuno-electron microscopic study of MLKL. Notice the ER localization of MLKL (box in A and arrows in B) and membrane localization of MLKL (arrowheads in A). (C–E) Immuno-electron microscopic study of RIP3. Notice the ER localization of RIP3 (box in C and arrows in D and E). Arrowhead in C points to RIP3-immunoreactivity on mitochondria. Scale bars = 500 nm (A), 200 nm (B, D and E), 1 lm (C).
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a digital camera (832 SC1000, Gatan) under an electron microscope (JEM-1230, JEOL Ltd).
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Statistical analysis
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All data were presented as mean ± SEM. A one-way ANOVA was adopted for comparison between groups. Statistical analyses were performed using the GraphPad Prism software (San Diego, USA). P < 0.05 was considered statistically significant.
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RESULTS Expression of MLKL and necroptosis of microglia/macrophages after SCI We first examined the expression of MLKL, the key molecule in the intracellular signaling of necroptosis (Sun et al., 2012; Zhao et al., 2012; Murphy et al., 2013; Wu et al., 2013), in normal and injured spinal cords by immunohistochemistry. No MLKL-immunoreactivity was observed in the normal spinal tissue (data not shown). However, numerous MLKL-positive cells were detected within and surrounding the lesion center at 5 dpi. Double-immunostaining of MLKL with different neural cell markers was carried out to analyze the cell types that express MLKL after SCI. Quantification in bilateral areas 300 lm rostral and dorsal to lesion epicenter
(Fig. 1H) showed that approximately 57.6% of the MLKL-positive cells expressed Iba-1, a marker for microglia/macrophages (Fig. 1A–C, G). Approximately 34.2% of MLKL-positive cells expressed GFAP, a marker of astrocytes (Fig. 1D, G), 5.7% of MLKL-positive cells expressed oligodendrocyte marker CC1, and 2.5% expressed the neuronal marker NeuN, respectively (Fig. 1E–G). These data showed that MLKL were mainly expressed in microglia/macrophages within and around the lesion site after SCI, suggesting that necroptosis may occur in microglia/macrophages. Because the membrane localization of MLKL was thought to trigger necroptosis by initiating ion influx (Cai et al., 2014; Chen et al., 2014; Dondelinger et al., 2014; Hildebrand et al., 2014; Wang et al., 2014a), we examined the distribution of MLKL in microglia/macrophages by both immunohistochemistry and immune-electron microscopic study. Triple-immunostaining of MLKL with Iba-1 and CXCR4, a chemokine receptor that is highly expressed on the membrane of microglia/macrophages, revealed that MLKL-immunoreactivity was distributed both in the cytoplasm and on the membrane of Iba-1 - positive cells (Fig. 2A–D). Under the electron microscope, cells with apoptotic features (such as chromatin aggregation, nuclear and cytoplasmic condensation, and an intact cell membrane) could be occasionally found (data not shown). Microglia/macrophages were rec-
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Fig. 5. Expression of GRP78 in microglia/macrophages in normal and injured spinal cords. (A–C) Triple-immunostaining of GRP78/Iba-1/MLKL in the normal spinal cord. (D–F) Triple-immunostaining of GRP78/Iba-1/MLKL at 5 dpi. Scale bar = 20 lm. (G) Quantification of GRP78-, Iba-1- and MLKL-positive cells in injured spinal cord. Notice the triple-staining of GRP78/Iba-1/MLKL in injured spinal cord. 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331
ognized by their characteristic oval or round nuclei with dense and highly clumped heterochromatin (Fig. 2E). However, due to the morphological changes of apoptosis, it is hard to determine the apoptotic microglia/macrophages. In contrast, microglia/macrophages with necrotic features, such as lysis of cytoplasmic content and breakdown of cell membrane were frequently observed (Fig. 2F, G). Intensive MLKL immunoreactivities, visualized by immunogold silver-enhanced particles, were found along the outer surface of the cell membrane (arrowheads in Fig. 2F and Fig. 4A), with some part of membrane ruptured (arrows in Fig. 2F, G) and adjacent cytoplasm swollen and translucent (asterisk in Fig. 2G), indicating a leakage or lysis of cytoplasmic material. The membrane localization of MLKL was consisting with its role in increasing membrane permeability. To further examine the membrane integrity of MLKL-positive microglia, we performed in vivo PI labeling at 5 dpi and triple-stained MLKL/PI/Iba-1. PI-labeled MLKL/Iba-1 double-positive cells were frequently found within and around the lesion center (Fig. 3A–C), implying the necroptosis of microglia/macrophages. To confirm the occurrence of necroptosis, we administered Necrostatin1 (Nec-1), an inhibitor of necroptosis, to examine its effects on the PI-labeling of microglia/macrophages. Five
successive days of Nec-1 administration significantly decreased the percent of PI-labeled Iba-1-positive cells in bilateral areas 300 lm rostral and caudal to the lesion center as defined in Fig. 1H (Fig. 3D–F). Because the activation of necroptotic signaling is thought to be coupled with inhibition of apoptosis, we then examined the relation between Caspase-3 activation and MLKL expression. Triple-immunostaining of Iba-1/cleaved caspase-3 (CC3)/MLKL showed a separate expression of CC3 and MLKL in Iba-1-positive cells, suggesting the inhibition of apoptotic machinery in necroptotic microglia/macrophages (Fig. 3G–K). Quantification showed that approximately 11% Iba-1- positive cells in bilateral areas 300 lm rostral and caudal to the lesion center were labeled by CC3. In the same region, approximately 25% Iba-1- positive cells expressed MLKL (Fig. 3L). Taken together, these data indicated that necroptosis contributed to the death of microglia/macrophages after SCI.
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Localization of MLKL and RIP3 on the ER of necroptotic microglia/macrophages after SCI
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In our electron-microscope study, MLKL immunoreactivity was surprisingly observed on ER (box in Fig. 4A, arrows in
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Fig. 6. ER stress and necroptosis of OGD treated N9 cells. (A) Western-blotting of GRP78 and p-eIF2a at different time points after 12 h-OGD exposure. (B) Western-blotting of RIP3, MLKL and HMGB1 at different time points after exposure to OGD. Notice the increase of ER stress markers (GRP78, p-eIF2a) and necroptotic markers (RIP3, MLKL and HMGB1) after OGD treatment. (C–F) Effects of Nec-1 on PI-labeling. Notice that Nec1 can significantly prevent the increase of PI-labeling induced by OGD. ***p < 0.001, *p < 0.05. Scale bars = 200 lm. (G–J) Effects of Nec-1 on the expression of RIP3, MLKL and HMGB1. Notice that Nec-1 can significantly decrease the expression of RIP3, MLKL and HMGB1 induced by OGD. ** p < 0.01, *p < 0.05. 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369
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Fig. 4B) with even more immunogold particles than on mitochondria (Fig. 4A) where MLKL reportedly interact with PGAM5 for the execution of cell death (Wang et al., 2012). Because translocation of MLKL to mitochondrial associated membranes is mediated by phosphorylated RIP3 (Chen et al., 2013), we then performed immuneelectron microscopic study of RIP3 to test whether RIP3 could translocate to ER. In RIP3-positive microglia/macrophages, cytoplasmic lysis, the ultrastructural change of necrosis, was observed (Fig. 4C). Similar to MLKL, many RIP3 immunoreactivities were also found on ER (Fig. 4C, and arrows in Fig. 4D, E). These results confirmed the idea that microglia/macrophages died through necroptosis, and additionally indicated a possible involvement of ER in the necroptosis of microglia/macrophages. Expression of ER sensor GRP78 in necroptotic microglia/macrophages after SCI The finding that MLKL and RIP3 were localized at ER prompted us to examine whether ER stress could be
induced in necroptotic microglia/macrophages after SCI. Triple immunostaining of MLKL, Iba-1 and ER stress sensor GRP78 was performed (Fig. 5D–F). Similar to MLKL, GRP78 immuoreactivity was highly expressed in cells surrounding the lesion center. Approximately 44.6% of GRP78-positive cells expressed Iba-1 (Fig. 5D, G). Importantly, among the MLKL-positive cells, 70.2% cells were GRP78-positive (Fig. 5E, G). Further, approximately 68.2% of MLKL/Iba-1 double-positive cells were GRP78-positive (Fig. 5F, G). These data showed a well co-expression of GRP78 and MLKL in microglia/macrophages, and supported the implication from EM study that ER stress may be induced in necroptotic microglia/macrophages.
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Necroptosis of mouse microglial N9 cells in oxygenglucose deprivation (OGD)-induced ER stress
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Because it has been reported that ER stress could be induced by ischemia (Doroudgar et al., 2009) and ischemia is an important factor in the secondary SCI, we then
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Fig. 7. Effects of the ER stress inhibitor on the necroptosis of primary microglia after OGD treatment. (A–B) Western-blotting of GRP78 and p-eIF2a in control cells, or cells treated by 4-PBA alone, OGD, or OGD plus 4-PBA. Notice that 4-PBA significantly decreased the expression of GRP78 and p-eIF2a induced by OGD. **p < 0.01, *p < 0.05. (C–F) Western-blotting of MLKL, RIP3 and HMGB1 in control cells, or cells treated by 4-PBA alone, OGD, or OGD plus 4-PBA. Notice that 4-PBA significantly decreased the expression of MLKL, RIP3 and HMGB1 induced by OGD. **p < 0.01, * p < 0.05. (G–K) PI staining and quantification of PI-positive cells in control cells, or cells treated by 4-PBA alone, OGD, or OGD plus 4-PBA. Notice that 4-PBA significantly decreased the number of PI-labeled cells induced by OGD. **p < 0.01, ***p < 0.001. Scale bar = 200 lm.
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tested whether OGD treatment could induce necroptosis of microglia in vitro. N9 cells, a mouse microglia cell line, were exposed to OGD for 12 h, and then were replenished with normal oxygen and glucose for different time points (4, 16, 24, 36 and 48 h). ER stress was evaluated by immunoblotting of GRP78 and p-eIF2a, two markers of ER stress. Data showed that GRP78 and p-eIF2a were up-regulated from 16 h and peaked at 36 h after reoxygenation (Fig. 6A). Interestingly, the expression of necroptotic proteins, MLKL, RIP3 and HMGB1, was also increased with a similar time course after reoxygenation (Fig. 6B). PI staining was further performed to assess cell death. At 36 h after OGD exposure, the peak of ER stress as showed by GRP78 and p-eIF2a expression, the number of PI-positive cells was markedly increased by about eightfold (p < 0.001) in comparison with control (Fig. 6C–F). Addition of Nec-1 (20 lM), an inhibitor of necroptosis in culture medium, significantly attenuated the OGD-induced increase of MLKL, RIP3, HMGB1 and the number of PI-labeled cells (Fig. 6C–J). Therefore, OGD treatment not only induced ER stress, but also necroptosis in N9 microglial cells, suggesting a possible role of ER stress in the necroptosis of microglia.
Suppression of necroptosis of primary microglia by inhibition of ER stress
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We further cultured primary microglia, induced necroptosis by OGD, and added an ER stress inhibitor, 4-PBA (de Almeida et al., 2007) to evaluate the role of ER stress in the necroptosis of microglia. Based on the results of N9 cells, we examined ER stress and necroptosis of microglia at 36 h after reoxygenation. The expression of GRP78 and p-eIF2a increased 3.4- and 2.9-folds respectively (Fig. 7A, B). This increase was effectively blocked by 4-PBA (Fig. 7A, B). At 36 h postreoxygenation, the expression of MLKL, HMGB1 and RIP3 increased 2.8-, 2.4- and 2.3-folds respectively (Fig. 7C–F). PI staining showed an 8.3-fold increase of cell death relative to normal controls (Fig. 7G–K). Addition of 4-PBA significantly attenuated the above increase of MLKL, HMGB1, RIP3 and the percent of PI-labeled cells (Fig. 7C–K). 4-PBA alone has no significant effects on the ER stress and necroptosis of microglia (Fig. 7A–K). These data suggested that ER stress was actively involved in the necroptosis of primary microglia induced by OGD exposure.
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Fig. 8. Expression of pMLKL and GRP78 in microglia/macrophages in human SCI. (A–D) Triple-immunostaining of MLKL, GRP78 and CD11b in the normal human spinal cord. (E–H) Represent images of triple-immunostaining of MLKL, GRP78 and CD11b in the injured human spinal cord at 5 dpi. Arrows point to the double or triple-stained cells. Scale bar = 50 lm.
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Co-expression of phosphorylated MLKL and GRP78 by microglia/macrophages in the injured human spinal cord We next examined whether ER stress and necroptosis could occur in the microglia/macrophages after human SCI . Because phosphorylation of MLKL was required for the necroptotic function of MLKL (Sun et al., 2012; Rodriguez et al., 2015), we examined the expression of pMLKL in human spinal tissue. In the normal spinal cord, there is no expression of pMLKL and GRP78 (Fig. 8B, C). After SCI, triple-immunostaining of pMLKL/GRP78/ CD11b (a marker of microglia/macrophages) in one patient at 5d after SCI showed that pMLKL and GRP78 were highly expressed in CD11b-positive cells (Fig. 8E, F). Noticeably, pMLKL- and GRP78-immunoreactivities overlapped well with each other (Fig. 8G, H). Similar expression pattern of pMLKL, GRP78 and CD11b were observed in another patient at 15 dpi (data not shown). These results indicated the co-occurrence of ER stress and necroptosis in microglia/macrophages in human after SCI.
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DISCUSSION
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Persistent and excessive activation of microglia/macrophages have been suggested to be detrimental to the functional recovery after SCI (Zhou et al., 2014). Previous studies have reported Caspase-dependent apoptosis of microglia/macrophages after SCI (Beattie et al., 2000). Recently, more and more evidence have suggested that necroptosis is closely associated with inflammation (Newton, 2015; Pasparakis and Vandenabeele, 2015; Silke et al., 2015). A recent study reported necroptosis of microglia in vitro upon Toll-like receptor (TLR) activation in combination with
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Caspase inhibition (Kim and Li, 2013). Considering the chronic inflammation after SCI, it would be important to explore the necroptosis of microglia/macrophages after SCI, which, however, still remains poorly investigated. Previous studies have demonstrated that Nec-1 had beneficial effects on SCI and suggested that necroptosis might contribute to the early cell damage post-SCI (Liu et al., 2014; Wang et al., 2014b, 2015). In the present study, by light and electronic microscopic studies, we first demonstrated an induction of MLKL, a key molecule in necroptosis, on the membrane of microglia/macrophages after SCI. Then, by in vivo PI-labeling and pharmacological manipulation, we confirmed the occurrence of necroptosis in microglia/macrophages after SCI. It thus appears that not only apoptosis, but necroptosis may also be exploited to regulate the process of microglia/macrophage-mediated inflammation after SCI. ER, which normally functions as an intracellular Ca2+ store and protein folding platform (Di Sano et al., 2012), is actively involved in apoptosis when stressed (Liu et al., 2013). ER stress induced apoptosis has been demonstrated to happen in various neurological conditions (Lindholm et al., 2006; Zhang et al., 2013). After SCI, insufficient vascularization in the contusion lesion site induced ER stress and corresponding cell death (Yamauchi et al., 2007). The localization of MLKL- and RIP3-immunoreactivity at ER indicates a possible involvement of ER in necroptosis. Whether MLKL/RIP3 complex could induce calcium release from ER during necroptosis is of interest to be further probed. Our data further showed that GRP78, a widely adopted ER stress sensor, was induced in MLKL-positive microglia/macrophages after SCI. In addition, the expression of GRP78 and p-eIF2a, two ER stress markers, increased in parallel with the expression of
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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necroptotic proteins upon OGD treatment in vitro. Importantly, inhibition of ER stress could attenuate the necroptosis of microglia cells induced by OGD treatment. These data indicate an involvement of ER stress in the necroptosis of microglia/macrophages, and are in consistence with a recent report that ER stress can induce necroptosis of L929 cells independent of TNFR1 (Saveljeva et al., 2015). The low-level expression of MLKL and GRP78 detected by Western-blot in vitro and the negative immunostaining of MLKL and GRP78 in the normal spinal cord may be due to the difference of methods or the ‘‘active” status of microglia/macrophages in vitro. Co-expression of pMLKL and GRP78 in microglia/macrophages in the injured human spinal cord suggested that there may be a conserved relationship between ER stress and necroptosis, which is to be further explored for detail. Nevertheless, our data provided a novel point for understanding of the mechanisms that regulate the death of microglia/macrophages after SCI, which could be utilized for modulating post-SCI inflammation.
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CONFLICT OF INTEREST
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The authors declare no conflict of interest.
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Acknowledgments—This work was supported by grants from the National Natural Science Foundation of China (NSFC, 31271583, 81571224) to Dr. W.Y, and NSFC (81371364).to Dr. J.G., and research fellowship for outstanding Ph.D. candidates of Fourth Military Medical University (2013D09) to Dr. H.F. We appreciate the technical assistances from Drs. Jie Xu, Kun Chen and HaiFeng Zhang, and the kind gifts of antibodies from Dr. Heng Ma. We thank Dr. Biswas Sangita (University of California at Davis) for editing the language.
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REFERENCES
538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564
Allison DJ, Ditor DS (2015) Immune dysfunction and chronic inflammation following spinal cord injury. Spinal Cord 53:14–18. Beattie MS, Farooqui AA, Bresnahan JC (2000) Review of current evidence for apoptosis after spinal cord injury. J Neurotrauma 17:915–925. Beattie MS, Hermann GE, Rogers RC, Bresnahan JC (2002) Cell death in models of spinal cord injury. Prog Brain Res 137:37–47. Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, Ward Y, Wu LG, Liu ZG (2014) Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16:55–65. Chen W, Zhou Z, Li L, Zhong CQ, Zheng X, Wu X, Zhang Y, Ma H, Huang D, Li W, Xia Z, Han J (2013) Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem 288:16247–16261. Chen X, Li W, Ren J, Huang D, He WT, Song Y, Yang C, Li W, Zheng X, Chen P, Han J (2014) Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res 24:105–121. David S, Kroner A (2011) Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci 12:388–399. de Almeida SF, Picarote G, Fleming JV, Carmo-Fonseca M, Azevedo JE, de Sousa M (2007) Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation. J Biol Chem 282:27905–27912.
529 530 531 532 533 534 535
11
Di Sano F, Bernardoni P, Piacentini M (2012) The reticulons: guardians of the structure and function of the endoplasmic reticulum. Exp Cell Res 318:1201–1207. Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I, Hulpiau P, Weber K, Sehon CA, Marquis RW, Bertin J, Gough PJ, Savvides S, Martinou JC, Bertrand MJ, Vandenabeele P (2014) MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 7:971–981. Doroudgar S, Thuerauf DJ, Marcinko MC, Belmont PJ, Glembotski CC (2009) Ischemia activates the ATF6 branch of the endoplasmic reticulum stress response. J Biol Chem 284:29735–29745. Gorman AM, Healy SJ, Jager R, Samali A (2012) Stress management at the ER: regulators of ER stress-induced apoptosis. Pharmacol Ther 134:306–316. Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P, Pierotti C, Garnier JM, Dobson RC, Webb AI, Tripaydonis A, Babon JJ, Mulcair MD, Scanlon MJ, Alexander WS, Wilks AF, Czabotar PE, Lessene G, Murphy JM, Silke J (2014) Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc Natl Acad Sci USA 111:15072–15077. Hoozemans JJ, Scheper W (2012) Endoplasmic reticulum: the unfolded protein response is tangled in neurodegeneration. Int J Biochem Cell Biol 44:1295–1298. Hu X, Leak RK, Shi Y, Suenaga J, Gao Y, Zheng P, Chen J (2015) Microglial and macrophage polarization-new prospects for brain repair. Nat Rev Neurol 11:56–64. Ito WD, Schaarschmidt S, Klask R, Hansen S, Scha¨fer HJ, Mathey D, Bhakdi S (1997) Infarct size measurement by triphenyltetrazolium chloride staining versus in vivo injection of propidium iodide. J Mol Cell Cardiol 29:2169–2175. Kim SJ, Li J (2013) Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia. Cell Death Dis 4:e716. Kroner A, Greenhalgh AD, Zarruk JG, Passos Dos Santos R, Gaestel M, David S (2014) TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord. Neuron 83:1098–1116. Lindholm D, Wootz H, Korhonen L (2006) ER stress and neurodegenerative diseases. Cell Death Differ 13:385–392. Liu D, Zhang M, Yin H (2013) Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis. Int J Neurosci 123:155–162. Liu M, Wu W, Li H, Li S, Huang LT, Yang YQ, Sun Q, Wang CX, Yu Z, Hang CH (2014) Necroptosis, a novel type of programmed cell death, contributes to early neural cells damage after spinal cord injury in adult mice. J Spinal Cord Med. Liu S, Sarkar C, Dinizo M, Faden AI, Koh EY, Lipinski MM, Wu J (2015) Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death. Cell Death Dis 6:e1582. Mabon PJ, Weaver LC, Dekaban GA (2000) Inhibition of monocyte/macrophage migration to a spinal cord injury site by an antibody to the integrin alphaD: a potential new antiinflammatory treatment. Exp Neurol 166:52–64. Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S, Lewis R, Lalaoui N, Metcalf D, Webb AI, Young SN, Varghese LN, Tannahill GM, Hatchell EC, Majewski IJ, Okamoto T, Dobson RC, Hilton DJ, Babon JJ, Nicola NA, Strasser A, Silke J, Alexander WS (2013) The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39:443–453. Newton K (2015) RIPK1 and RIPK3: critical regulators of inflammation and cell death. Trends Cell Biol 25:347–353. Oerlemans MI, Liu J, Arslan F, den Ouden K, van Middelaar BJ, Doevendans PA, Sluijter JP (2012) Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo. Basic Res Cardiol 107:270. Pasparakis M, Vandenabeele P (2015) Necroptosis and its role in inflammation. Nature 517:311–320.
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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Rodriguez DA, Weinlich R, Brown S, Guy C, Fitzgerald P, Dillon CP, Oberst A, Quarato G, Low J, Cripps JG, Chen T, Green DR (2015) Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell Death Differ. Saveljeva S, Mc Laughlin SL, Vandenabeele P, Samali A, Bertrand MJ (2015) Endoplasmic reticulum stress induces ligandindependent TNFR1-mediated necroptosis in L929 cells. Cell Death Dis 6:e1587. Silke J, Rickard JA, Gerlic M (2015) The diverse role of RIP kinases in necroptosis and inflammation. Nat Immunol 16:689–697. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148:213–227. Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF, Wang FS, Wang X (2014a) Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 54:133–146. Wang Y, Wang H, Tao Y, Zhang S, Wang J, Feng X (2014b) Necroptosis inhibitor necrostatin-1 promotes cell protection and physiological function in traumatic spinal cord injury. Neuroscience 266:91–101. Wang Y, Wang J, Yang H, Zhou J, Feng X, Wang H, Tao Y (2015) Necrostatin-1 mitigates mitochondrial dysfunction post-spinal cord injury. Neuroscience 289:224–232. Wang Z, Jiang H, Chen S, Du F, Wang X (2012) The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 148:228–243.
Wu J, Huang Z, Ren J, Zhang Z, He P, Li Y, Ma J, Chen W, Zhang Y, Zhou X, Yang Z, Wu SQ, Chen L, Han J (2013) Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res 23:994–1006. Yamauchi T, Sakurai M, Abe K, Matsumiya G, Sawa Y (2007) Impact of the endoplasmic reticulum stress response in spinal cord after transient ischemia. Brain Res 1169:24–33. Yong C, Arnold PM, Zoubine MN, Citron BA, Watanabe I, Berman NE, Festoff BW (1998) Apoptosis in cellular compartments of rat spinal cord after severe contusion injury. J Neurotrauma 15:459–472. Zai LJ, Wrathall JR (2005) Cell proliferation and replacement following contusive spinal cord injury. Glia 50:247–257. Zhang HY, Zhang X, Wang ZG, Shi HX, Wu FZ, Lin BB, Xu XL, Wang XJ, Fu XB, Li ZY, Shen CJ, Li XK, Xiao J (2013) Exogenous basic fibroblast growth factor inhibits ER stress-induced apoptosis and improves recovery from spinal cord injury. CNS Neurosci Ther 19:20–29. Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J, Liu ZG (2012) Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 109:5322–5327. Zhou X, He X, Ren Y (2014) Function of microglia and macrophages in secondary damage after spinal cord injury. Neural Regen Res 9:87–95.
(Accepted 24 October 2015) (Available online xxxx)
Please cite this article in press as: Fan H et al. Involvement of endoplasmic reticulum stress in the necroptosis of microglia/macrophages after spinal cord injury. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.10.049
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