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Involvement of endoplasmic reticulum stress after middle cerebral artery occlusion in mice
Neuroscience 147 (2007) 957–967
INVOLVEMENT OF ENDOPLASMIC RETICULUM STRESS AFTER MIDDLE CEREBRAL ARTERY OCCLUSION IN MICE N. MORIMOTO,a Y. OIDA,a M. SHIMAZAWA,a M. MIURA,b T. KUDO,c K. IMAIZUMId AND H. HARAa*
homeostasis and in the folding and processing of newly synthesized proteins (Paschen and Doutheil, 1999). Various conditions such as alterations in calcium homeostasis, glucose deprivation, and hypoxia lead to the accumulation of unfolded proteins in the ER, leading to ER stress (Kaufman, 1999). In response to ER stress, cells exhibit a self-protective signal-transcription pathway termed the unfolded-protein response (UPR) (Cudna and Dickson, 2003). However, when the ER stress is very severe, the cells activate an apoptotic pathway. Under ER-stress conditions, three ER-transmembrane proteins [activating transcription factor-6 (ATF-6), inositol requiring enzyme 1 (IRE1), and double-stranded RNAdependent protein kinase-like endoplasmic reticulum kinase (PERK)] carry out the two main functions of the UPR (Ron and Harding, 2000). First, protein synthesis is inhibited, thus preventing further accumulation of unfolded protein in the ER. Indeed, PERK phosphorylates eukaryotic initiation factor 2␣ (eIF2␣) and induces various cellular responses, including protein synthesis inhibition (Mori, 2000; Ron and Harding, 2000). Second, up-regulation of ER-stress genes is induced, leading to the repair of unfolded proteins. Activating transcription factor-4 (ATF-4), following its activation by the phosphorylation of eIF2␣, induces C/EBP-homologous protein (CHOP) (DeGracia et al., 2002). CHOP is a transcription factor (Ubeda et al., 1999), and its expression is generally associated with apoptosis (Zinszner et al., 1998; McCullough et al., 2001). Endonuclease IRE1 induces processing of X-box protein 1 (XBP-1) mRNA (Shen et al., 2001; Yoshida et al., 2001; Calfon et al., 2002), with activated IRE1 cleaving a sequence of 26 bases from the coding region of XBP-1 mRNA (Gonzalez et al., 1999). The processed XBP-1 mRNA is then translated into the mature XBP-1 protein, an active transcription factor specific for ER-stress genes such as immunoglobulin binding protein (BiP)/glucose-regulated protein (GRP) 78 and GRP94 (Calfon et al., 2002). Various experimental models and observational studies have shown that cerebral ischemia induces an ERstress response, suggesting that ER plays an important role in the pathogenesis of neuronal cell injury during and after cerebral ischemia (Ito et al., 2001; Mouw et al., 2003; Paschen et al., 2003). Furthermore, ORP150, an ERassociated chaperon, is enhanced after stroke in the human brain (Tamatani et al., 2001). However, to our knowledge, most studies have been performed on cerebral ischemia and reperfusion models, but not a permanent cerebral ischemia model. Further, the roles of ER stress in the periphery of the middle cerebral artery (MCA) have not been fully evaluated. Clinically, permanent cerebral isch-
a
Department of Biofunctional Molecules, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan
b
Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
c
Department of Psychiatry, Osaka University Graduate School of Medicine, D3, 2-2, Yamadaoka, Suita 565-0871, Japan
d
Department of Anatomy, Faculty of Medicine, University of Miyazaki, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan
Abstract—The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. ER stress-related markers [immunoglobulin binding protein (BiP)/glucose-regulated protein (GRP) 78, activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP)] in the striatum and the cortex were investigated after permanent middle cerebral artery occlusion (MCAO) in mice. Using endoplasmic reticulum stress-activated indicator (ERAI) transgenic mice, which show splicing of X-box protein 1 (XBP-1) mRNA as green fluorescence, we monitored the regional changes in fluorescence after MCAO. BiP mRNA (by reverse-transcription polymerase chain reaction [RT-PCR] analysis) was increased in the cortex at 6 h. In immunohistochemical and/or Western blot analysis, the expressions of ER stress-related markers (BiP, ATF-4, and CHOP) were increased in the infarct region, more strongly in the cortex than in the striatum. ERAI fluorescence was observed in the ischemic area starting from 6 and 12 h, respectively, after MCAO, with the peaks at 1 day and the fluorescence co-localized with the 2,3,5-triphenyltetrazolium chloride (TTC)–visible extension of brain infarction. These findings suggest that permanent MCAO induces expression of ER-stress related genes mainly in the periphery of the MCA territory. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ER-stress, mouse, permanent middle cerebral artery occlusion, stroke.
The endoplasmic reticulum (ER) is an organelle that plays important roles in the maintenance of intracellular calcium *Corresponding author. Tel: ⫹81-58-237-8596; fax: ⫹81-58-2378596. E-mail address: [email protected] (H. Hara). Abbreviations: ATF-4, activating transcription factor-4; BiP, immunoglobulin binding protein; CHOP, C/EBP-homologous protein; DAB, diamino benzidine; eIF2␣, eukaryotic initiation factor 2␣; ER, endoplasmic reticulum; ERAI, endoplasmic reticulum stress-activated indicator; GFAP, glial fibrillary acidic protein; GRP, glucose-regulated protein; IRE1, inositol requiring enzyme 1; MAP2, microtubule-associated protein 2; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; PB, phosphate buffer; PERK, double-stranded RNAdependent protein kinase-like endoplasmic reticulum kinase; RT-PCR, reverse-transcription polymerase chain reaction; TTC, 2,3,5-triphenyltetrazolium chloride; UPR, unfolded-protein response; XBP-1, X-box protein 1.
0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.04.017
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emia happens frequently, most often without reperfusion. Therefore, in the present study we investigated the regional and time-dependent changes in ER stress-related markers (BiP, ATF-4, and CHOP) after permanent middle cerebral artery occlusion (MCAO) in mice. In addition, using endoplasmic reticulum stress-activated indicator (ERAI) transgenic mice (Iwawaki et al., 2004a), which display splicing of XBP-1 mRNA as green fluorescence, we evaluated both regional and time-related changes in the splicing of XBP-1 mRNA after MCAO. ERAI mice would be expected to provide valuable information as to the dynamics of ER stress-induced neuronal damage.
EXPERIMENTAL PROCEDURES Experimental materials The drugs used and their sources were as follows. Paraformaldehyde (Wako Pure Chemical Industries, Osaka, Japan), sucrose (Wako), sodium hydrogen phosphate 12-water (Nacalai tesque, Kyoto, Japan), sodium dihydrogen phosphate dihydrate (Nacalai tesque), potassium chloride (Wako), sodium chloride (Kishida Chemical, Osaka, Japan), Nembutal (Dainippon Pharmaceutical, Osaka, Japan), Triton X-100 (Bio-Rad, Hercules, CA, USA), O.C.T. compound (Sakura Fine Technical, Tokyo, Japan), Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA, USA), M.O.M. immunodetection kit (Vector Laboratories), diamino benzidine (DAB) peroxidase substrate kit (Vector Laboratories), mouse anti-KDEL monoclonal antibody (Stressgen Bioreagents, Victoria, BC, Canada), mouse anti-BiP monoclonal antibody (Transduction Laboratories, Lexington, KY, USA), mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (YLEM, San Francisco, CA, USA), mouse anti-microtubule-associated protein 2 (MAP2) monoclonal antibody (Sigma, St. Louis, MO, USA), rabbit anti-cyclic AMP response element-binding protein (CREB)-2 (ATF-4) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-growth arrest and DNA damage-inducible gene (GADD) 153 (the protein has also been designated CHOP) monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-actin polyclonal antibody (Santa Cruz Biotechnology), Alexa fluor 546 F(ab=)2 fragment of goat anti-mouse IgG (H⫹L) (Molecular Probes, Eugene, OR, USA), SuperSignal West Femto Maximum Sensitivity Substrate Antibodies (Pierce, Rockford, IL, USA), Running Buffer Solution (Wako), Owl’s Electroblot Buffer Kit (Owl, Portsmouth, NH, USA), SuperSep 10% (Wako), BCA Protein Assay Kit (Pierce), Immobilon-P (Millipore, Bedford, MA, USA), Gel Blot Paper (Schleicher & Schuell, Keene, NH, USA), Sample buffer (Wako), TSA Biotin system (PerkinElmer Life Sciences Inc., Boston, MA, USA), Precision Plus Protein Standards Dual Color (Bio-Rad), Block Ace (Dainippon Pharmaceutical), Can Get Signal (Toyobo, Osaka, Japan), dimethyl sulfoxide (DMSO; Koso Chemical, Tokyo, Japan), RIPA buffer (Sigma), protease inhibitor cocktail (Sigma), phosphatase inhibitor cocktail I, II (Sigma), ProLong Gold antifade reagent (Molecular Probes), and 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma).
Focal cerebral ischemia model in mice Male adult C57BL/6J mice weighing 23–27 g (Japan SLC, Hamamatsu, Japan) and adult male ERAI mice (Iwawaki et al., 2004a) weighing 21–30 g were used for the experiments, and were kept under standard lighting conditions (12-h light/dark cycle). Anesthesia was induced by means of 2.0% isoflurane, and maintained with 1% isoflurane in 70% N2O and 30% O2 using an animal general anesthesia machine (Soft Lander; Sin-ei Industry Co. Ltd., Saitama, Japan). Body temperature was maintained between 36.5 and 37.0 °C with the aid of a heating pad and
heating lamp. Filament occlusion of the left MCA was carried out as previously described (Hara et al., 1996, 1997). Briefly, the left MCA was occluded with an 8-0 nylon monofilament (Ethicon, Somerville, NJ, USA) coated with a mixture of silicone resin (Xantopren; Bayer Dental, Osaka, Japan).
Reverse-transcription polymerase chain reaction (RT-PCR) C57BL/6J mice were deeply anesthetized and decapitated at the indicated times after MCAO, and the brains were quickly removed and cut 5-mm coronal section (between 2 and 7 mm from frontal forebrain). Striatum and cortex of the left (ischemic side) MCA territory and contralateral hemisphere as control were carefully separated from the brains on ice under stereoscopic microscope, and then quickly frozen in dry ice and stored at ⫺80 °C. Total RNA was isolated using an RNeasy kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer’s protocol. RNA concentrations were determined spectrophotometrically at 260 nm. First-strand cDNA was synthesized in a 20 l reaction volume using a random primer (Takara, Otsu, Japan) and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA). PCR was performed in a total volume of 30 l containing 0.8 M of each primer, 0.2 mM dNTPs, 3 U TaqDNA polymerase (Promega, Madison, WI, USA), 2.5 mM MgCl2, and 10⫻ PCR buffer. The amplification conditions for semi-quantitative RT-PCR analysis were as follows: an initial denaturation step of 95 °C for 5 min), 22 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, with a final extension step of 72 °C for 7 min. The numbers of amplification cycles for detection of BiP and -actin were 18 and 15, respectively. The primers used for amplification were as follows: BiP, 5=-GTTTGCTGAGGAAGACAAAAAGCTC-3= and 5=-CACTTCCATAGAGTTTGCTGATAATTG-3=; -actin, 5=-TCCTCCCTGGAGAAGAGCTAC-3= and 5=-TCCTGCTTGCTGATCCACAT-3=. The PCR products were resolved by electrophoresis through a 4.8% (w/v) polyacrylamide gel. The density of each band was quantified using the Scion Image Program (Scion Corporation, Frederick, MD, USA).
Immunohistochemistry At 2 h, 6 h, 12 h, 1 day, and 3 days after MCAO, C57BL/6J and ERAI mice were injected with sodium pentobarbital (Nembutal; 50 mg/kg, i.p.), then perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Brains were removed after 15 min perfusion fixation at 4 °C, then immersed in the same fixative solution overnight at 4 °C. They were then immersed in 25% sucrose in 0.1 M PB for 24 h, and finally frozen in powdered dry ice. Coronal sections (10 m thick) were cut on a cryostat at ⫺20 °C, and stored at ⫺80 °C until use. After re-hydration, endogenous peroxidase activity was quenched using 0.3% hydrogen peroxidase in methanol. Next, brain sections were blocked with M.O.M. blocking reagent (M.O.M. immunodetection kit), then incubated overnight at 4 °C either with mouse anti-KDEL monoclonal antibody (1:400 dilution) or with mouse antiCHOP monoclonal antibody (1:100 dilution). They were washed and then incubated with biotinylated anti-mouse IgG before being incubated for 30 min at room temperature with avidin– biotin–peroxidase complex and then developed using DAB peroxidase substrate. CHOP signals were amplified using a TSA Biotin System. CHOPstained cells were counted in the striatum and the cortex. The results were expressed as positive cells per 1 mm2.
Western blot analysis C57BL/6J mice were deeply anesthetized and decapitated at the indicated times after MCAO, and the brains were quickly removed and cut into 5-mm coronal section (between 2 and 7 mm from frontal forebrain). Striatum and cortex of the left (ischemic side) MCA territory and contralateral hemisphere as control were carefully sepa-
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Fig. 1. Change in levels of BiP expression in the striatum and cortex after MCAO. (A) Quantitative analysis of RT-PCR showed that the expression level of BiP mRNA was significantly increased in the cortex at 6 h (n⫽6 –13; ** P⬍0.01 vs. Control). (B) Immunostaining for BiP in the striatum and cortex at 0 h, 2 h, 6 h, 12 h, 1 day, and 3 days after MCAO. Expression of BiP increased gradually from 2 h onwards in both striatum and cortex. Scale bar⫽20 m. (C) Western blot analysis showed that BiP was detectable. (D) Quantitative analysis of Western blotting showed that the expression level of BiP was significantly increased in the striatum at 1 day and in the cortex at both 12 h and 1 day. n⫽3–7; * P⬍0.05, ** P⬍0.01 vs. Control.
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rated from the brains on ice under stereoscopic microscope, and then quickly frozen in dry ice. For protein extraction, the tissue was homogenized in RIPA buffer containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktails. The homogenate was centrifuged at 11,000⫻g for 20 min, and the supernatant was collected for this study. Assays were performed to determine the protein concentration, comparisons being made with a known concentration of bovine serum albumin (BCA Protein Assay Kit). An aliquot of 5 g of protein was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, with the separated protein being transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). For immunoblotting, the following primary antibodies were used: mouse anti-BiP monoclonal antibody (1:250 dilution), rabbit anti-ATF-4 polyclonal antibody (1:200 dilution), and a rabbit anti-actin polyclonal antibody (1:2000 dilution). The secondary antibodies were anti-mouse HRP-conjugated IgG (1:2000 dilution) and goat anti-rabbit HRP-conjugated IgG (1:2000 dilution). The immunoreactive bands were visualized using SuperSignal west femto maximum sensitivity substrate. The band intensity was measured using a Lumino Imaging Analyzer (FAS-1000; Toyobo) and Gel Pro Analyzer (Media Cybernetics, Atlanta, GA, USA).
Observation of fluorescent tissues and TTC staining ERAI mice were deeply anesthetized and decapitated at the indicated times after MCAO, and the brains were quickly removed. Next, the forebrains were divided into 5 or 10 coronal slices (thickness 1 or 2 mm) using a mouse brain matrix (RBM-2000C; Activational Systems, Warren, MI, USA). A stereoscopic microscope fitted with on epifluorescence attachment (Olympus, Tokyo, Japan) was used to detect fluorescence from these tissues. For 2% TTC staining, brains were divided into five coronal (2 mm) slices and stained with TTC. The infract regions were recorded as images using a digital camera (Coolpix 4500, Nikon, Tokyo, Japan).
Fluorescent staining To confirm the localization of ERAI fluorescence, we carried out fluorescent staining for cell-phenotype markers. For immunofluorescence staining, the sections were incubated overnight at 4 °C with mouse anti-MAP2 monoclonal antibody (1:500 dilution) or mouse anti-GFAP monoclonal antibody. They were then washed and subsequently incubated for 3 h at room temperature with Alexa fluor 546 F(ab=)2 fragment of goat anti-mouse IgG (H⫹L). After fixation in 4% paraformaldehyde in PBS, we mounted each section in ProLong Gold antifade reagent.
Statistical analysis Data are presented as the means⫾S.E.M. Statistical comparisons were made using a one-way ANOVA followed by a Student’s t-test or Dunnett’s test using StatView version 5.0 (SAS Institute Inc., Cary, NC, USA), with P⬍0.05 being considered to indicate statistical significance.
RESULTS Induction of BiP mRNA and BiP protein in the striatum and cortex after MCAO BiP is induced under ER stress, and indeed is known to be a major marker of such stress. To examine the induction of BiP protein and BiP mRNA after permanent MCAO, we performed RT-PCR, immunohistostaining, and Western blot analysis (Fig. 1). The results of the RT-PCR analysis (Fig. 1A) revealed that in the striatum, BiP mRNA tended to be raised at 12 h after MCAO, the level being 1.2 times control. In the cortex, BiP mRNA was significantly raised
Fig. 2. Change in levels of ATF-4 expression in the striatum and cortex after MCAO. (A) Western blot analysis showed that ATF-4 was detectable. (B) Quantitative analysis of Western blotting showed that the expression level of ATF-4 was significantly increased in the striatum at 12 h after MCAO and in the cortex at 12 h, 1 day, and 3 days after MCAO. n⫽3–7; * P⬍0.05, ** P⬍0.01 vs. Control.
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(to 1.8 times control) at 6 h, but had recovered to near the control level at 12 h. Fig. 1B shows that after MCAO, immunoreactivity for BiP increased gradually from 2 h, with the peak at 12 h, in both striatum and cortex. At 1 and 3 days, most of the neurons in the striatum had degenerated, with the remaining neurons showing strong immunoreactivity. The immunoreactivity was stronger in the cortex than in the striatum. The results of Western analysis of BiP expression a (Fig. 1C and D) show that in the striatum, BiP tended to be
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raised at 12 h, was significantly raised at 1 day, and was back to near the control level at 3 days. Its level was 1.2 and 1.4 times control at 12 h and 1 day, respectively. In the cortex, BiP was significantly raised at 12 h and 1 day, with the peak at 1 day, and had declined to near control level at 3 days. Its level was 1.3 and 1.8 times control at 12 h and 1 day after MCAO, respectively. BiP expression was stronger in the cortex than in the striatum. These Western blot results were consistent with those from immunohistochemistry.
Fig. 3. Change in levels of CHOP expression in the striatum and cortex after MCAO. (A) Immunostaining for CHOP in the striatum and cortex at 0 h, 2 h, 6 h, 12 h, 1 day, and 3 days after MCAO. Expression of CHOP increased gradually from 6 h onwards in both striatum and cortex. Scale bar⫽1 m. (B) The number of CHOP-positive cells in the striatum and cortex. CHOP-positive cells were significantly increased at 12 h, 1 day, and 3 days in both areas. n⫽3; * P⬍0.05, ** P⬍0.01 vs. Control.
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The above results indicate that a major marker of ER stress (BiP) is induced in the early phase after permanent MCAO in mice. Induction of ATF-4 and CHOP in the striatum and cortex after MCAO CHOP is a key molecule linking ER stress to apoptotic cell death, and ATF-4 plays an active role in the induction of this molecule. We therefore investigated changes in ATF-4
expression by Western blot analysis (Fig. 2) and those in CHOP expression by immunohistochemical analysis (Fig. 3A). In the striatum, ATF-4 was significantly increased (to 1.4 times control) at 12 h after MCAO, but had declined to near control level at 1 day. In the cortex, ATF-4 was significantly increased at 12 h, 1 day, and 3 days, with the peak at 1 day (2.4, 3.3, and 1.7 times control, respectively). ATF-4 expression appeared to be stronger in the cortex than in the striatum.
Fig. 4. Coronal brain sections of TTC staining and fluorescence activity at 1 day after permanent MCAO in ERAI mice. TTC staining sections; A, C, E, and G. After TTC staining, damaged tissue (core region) is shown as a white region (E), whereas viable tissue is shown as a red region (C). Penumbra is shown as a pinkish region (G). Fluorescence sections; B, D, F, and H. Green fluorescence was observed in the infarct region of core (F) and penumbra (H). Fluorescence was measured after TTC staining and photographing using the same slices. A, C, E, and G were identical with B, D, F, and H, respectively. Figures C, E, and G are enlarged box-shaped C, E, and G in figure A. Similarly, figures D, F, and H are enlarged box-shaped D, F, and H in figure B. Scale bar⫽1 mm (A and B) and 200 m (C–H).
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Immunohistochemical analysis showed that in the striatum, CHOP expression increased gradually from 6 h after MCAO (Fig. 3A). At 1 day and 3 days, most of the neurons in the striatum had degenerated, with the remaining neurons displaying strong immunoreactivity. In the cortex, CHOP expression increased from 12 h, with the peak at 1 day. Quantitative analyses of CHOP-positive cells are shown in Fig. 3B. In the striatum, CHOP was significantly increased at 12 h, 1 day, and 3 days, with the peak at 1 day (3.6, 5.8, and 5.2 times control, respectively). In the cortex, CHOP was significantly increased at 12 h, 1 day, and 3 days, with the peak at 1 day (4.5, 7.1, and 6.1 times control, respectively). CHOP expression appeared to be stronger in the cortex than in the striatum. ATF-4 and CHOP expressions were each elevated in the infarct region in the early phase after MCAO, although there were no obvious morphological abnormalities. Fluorescence activity in ERAI mice after MCAO To monitor ER stress after MCAO, we used ERAI transgenic mice. Under ER-stress conditions, ERAI mice
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express green fluorescence [the explanation being that ER stress triggers splicing of XBP-1 mRNA and the subsequent translation of an indicator fusion protein, which is detectable by fluorescence (Iwawaki et al., 2004a)]. As shown in Fig. 4, we observed fluorescence at 1 day after MCAO on the ipsilateral side to the occlusion, but not on the contralateral side. To investigate the ERAI fluorescence-expressing region in more detail, we carried out TTC staining, after which the infarct region appears white, whereas the viable region appears red. As shown in Fig. 4, the green area of ERAI fluorescence overlapped with the white region (infarction), but not with the red region. We confirmed the presence of ERAI fluorescence in 5 (3–7 mm from the rostral pole) of the 10 coronal sections of forebrain at 1 day after MCAO (Fig. 5A), with the fluorescence being observed in the infarct region in each section. ERAI fluorescence first became apparent at 6 and 12 h after MCAO in the striatum and cortex, respectively (Fig. 5B), with the maximal level being detected in both regions at 1 day after MCAO.
Fig. 5. Fluorescence activity in brain of ERAI mice after MCAO. (A) Upper and lower figures are shown in normal pictures and the fluorescence activity, respectively, in brain coronal sections (3–7 mm from the rostral pole) at 1 day after MCAO. ERAI fluorescence was detected in the infarct region. (B) Fluorescence activity at 2 h to 3 days after MCAO. ERAI fluorescence was weak in the striatum at 6 h after MCAO and in the cortex at 12 h after MCAO. Strong fluorescence was detected in both regions at 1 and 3 days after MCAO. Scale bar⫽1 mm.
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Fig. 6. Fluorescence activity and immunofluorescence staining of dendrites (MAP2) in the cortex at 12 h and 1 day after MCAO. ERAI fluorescence co-localized with MAP2 (arrows) at 12 h after MCAO. Scale bar⫽20 m.
Localization of ERAI fluorescence Finally, we attempted to determine the cellular localization of ERAI fluorescence by means of immunofluorescence staining. The signals for ERAI fluorescence at 12 h after MCAO overlapped with that for MAP2 (Fig. 6), but not with that for Hoechst 33342 (data not shown). These findings which indicate that the fluorescence was localized to the cytosol and dendrites, and not was present in nuclei are consistent with the report that the XBP-1⌬DBD-Venus construct expressed by ERAI mice is an ER-stress indicator that is localized to the cytosol, not the nuclei (Iwawaki et al., 2004a; see Discussion). The signals for ERAI fluorescence at 1 and 3 days after MCAO overlapped with that for GFAP (Fig. 7), suggesting that glial cells also showed a pronounced ER-stress response.
DISCUSSION In the present study, we examined the regional and timerelated changes in three ER stress-related markers (BiP, ATF-4, and CHOP) after permanent MCAO in mice. Further, using ERAI transgenic mice we monitored the splicing of XBP-1 mRNA (as green fluorescence) after MCAO. These ER stress-related markers and ERAI fluorescence were all induced by MCAO, and indeed were rapidly upregulated in the ischemic region, even when no obvious morphological changes were detectable.
The UPR is mediated by PERK and IRE1. BiP binds to unfolded proteins in the ER and regulates the activation of these ER transducers (Bertolotti et al., 2000). Under normal conditions, BiP binds the luminal domains of PERK and IRE1, and prevents them from being activated. Under ER-stress conditions, BiP dissociates from PERK and IRE1 to bind unfolded proteins, and thereby renders each of these transducers liable to activation. Our results showed that (a) the expressions of BiP mRNA and BiP protein were enhanced after MCAO, (b) the expression of ER stress was stronger in the periphery of the MCA territory (mainly cortex) than in the core region (mainly striatum), and (c) BiP expression peaked at 12–24 h, before recovering to near control level at 3 days (Fig. 1). It has been reported that induction of BiP prevents neuronal damage being induced by ER stress (Wang et al., 1996; Rao et al., 2002; Reddy et al., 2003), and the increase in BiP expression may correlate with the degree of neuroprotection. Hence, a therapeutic approach leading to the induction of UPR activation may be effective for the treatment of cerebral ischemia. However, further studies will be needed to test this hypothesis. Activated PERK causes phosphorylation of eIF2␣, thereby inhibiting most protein synthesis (Bertolotti et al., 2000). When ER stress is very severe and prolonged, p-eIF2␣ becomes a key molecule in cell death-signal activation. ATF-4 is a transcription factor and is responsible for CHOP transcription by p-eIF2␣-mediated bypass scan-
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Fig. 7. Fluorescence activity and immunofluorescence staining of glial cells (GFAP) in the cortex at 1 and 3 days after MCAO. ERAI fluorescence co-localized with GFAP. Scale bar⫽20 m.
ning (Fawcett et al., 1999; Harding et al., 2000). In the present study, ATF-4 was significantly elevated at 12 h after MCAO in the striatum, and at 12 h, 1 day, and 3 days (with its peak at 1 day) in the cortex (Fig. 2), while CHOP was induced at 6 h in the ischemic region (Fig. 3). There is ample evidence that CHOP participates in apoptosis signaling (Kumar et al., 2003), and CHOP⫺/⫺ mice have smaller infarcts than wild-type mice after transient cerebral ischemia (Tajiri et al., 2004). Moreover, overexpression of BiP attenuates the induction of CHOP in ER stress and reduces ER stress-induced apoptosis (Wang et al., 1996). We noted that in the late phase of MCAO, BiP expression was reduced while CHOP expression was increased. These results indicate that a dysfunction of the protective mechanism leads to the induction of apoptosis by CHOP, and that CHOP expression may be closely related to the neuronal cell death occurring during permanent cerebral ischemia. Activated IRE1 is known to splice XBP-1 mRNA under ER-stress conditions (Yoshida et al., 2001). ERAI mice carry a human XBP-1 and venus (a variant of green fluorescence protein) fusion gene (Iwawaki et al., 2004a). Under normal conditions, the mRNA of the fusion gene is
not spliced, but under ER stress it is spliced, and then a XBP-1 and venus fusion protein is induced. ERAI fluorescence was observed from 6 h after MCAO, and it was expressed in a region more or less coincident with the infarct region. Paschen et al. (2003) have reported that XBP-1 mRNA was induced at 6 h after cerebral ischemia and reperfusion. In addition, we noted that ERAI fluorescence was expressed in glial cells at 1 day and 3 days after MCAO (Figs. 4, 5, and 7). This result suggests that in the late phase of the cerebral ischemia, glial cells also express a UPR, thus forming part of the protective mechanism against ER stress. Moreover, ERAI fluorescence could be detected in the cytosol and dendrites at 12 h after the start of ischemia (Fig. 6) and in the glia at 1 day and 3 days (Fig. 7), but not in the nuclei (Morimoto et al., unpublished observations). The finding in the ischemic region of an expression of ERAI fluorescence from the early phase of cerebral ischemia was quite similar to our findings for such other markers of ER stress as BiP and CHOP. Hence, ERAI mice may be useful for studies of the changes that occur during ER stress, and for elucidation of the relationship between neuronal cell death and ER stress. However, we should know the possibility that components of the ER
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response may be activated independently of ER stress in development and pathophysiological processes (Iwawaki et al., 2004b). In studies of ER stress after cerebral ischemia and reperfusion; (a) BiP mRNA was found to be increased at 6 –24 h after a 30 min ischemia and reperfusion in both the cortex and striatum (Qi et al., 2004), (b) BiP expression was increased at 5–23 h, then decreased at 47 h, after a 1 h ischemia and reperfusion affecting the MCA territory (Shibata et al., 2003), and (c) CHOP and ATF-4 expressions were raised in the striatum at 4 –24 h after a 30 min ischemia and reperfusion (Hayashi et al., 2005). In the present study, in contrast, BiP and CHOP tended to show similar patterns of change in expression, whereas BiP mRNA and ATF-4 expressions showed different patterns. This discrepancy is probably due to the use of different ischemia models (reperfusion vs. permanent). Compared with the reperfusion model, the permanent model exhibits a continued low blood flow condition, especially in the striatum. The levels of expression of the above ER-stress markers in the ischemic core region would appear to be lower in the permanent model than in the reperfusion model.
CONCLUSION In conclusion, ER stress plays an important causal role in permanent ischemic damage, with its participation perhaps being greater in the periphery of the MCA territory than in the core region.
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(Accepted 5 April 2007) (Available online 27 June 2007)
INVOLVEMENT OF ENDOPLASMIC RETICULUM STRESS AFTER MIDDLE CEREBRAL ARTERY OCCLUSION IN MICE N. MORIMOTO,a Y. OIDA,a M. SHIMAZAWA,a M. MIURA,b T. KUDO,c K. IMAIZUMId AND H. HARAa*
homeostasis and in the folding and processing of newly synthesized proteins (Paschen and Doutheil, 1999). Various conditions such as alterations in calcium homeostasis, glucose deprivation, and hypoxia lead to the accumulation of unfolded proteins in the ER, leading to ER stress (Kaufman, 1999). In response to ER stress, cells exhibit a self-protective signal-transcription pathway termed the unfolded-protein response (UPR) (Cudna and Dickson, 2003). However, when the ER stress is very severe, the cells activate an apoptotic pathway. Under ER-stress conditions, three ER-transmembrane proteins [activating transcription factor-6 (ATF-6), inositol requiring enzyme 1 (IRE1), and double-stranded RNAdependent protein kinase-like endoplasmic reticulum kinase (PERK)] carry out the two main functions of the UPR (Ron and Harding, 2000). First, protein synthesis is inhibited, thus preventing further accumulation of unfolded protein in the ER. Indeed, PERK phosphorylates eukaryotic initiation factor 2␣ (eIF2␣) and induces various cellular responses, including protein synthesis inhibition (Mori, 2000; Ron and Harding, 2000). Second, up-regulation of ER-stress genes is induced, leading to the repair of unfolded proteins. Activating transcription factor-4 (ATF-4), following its activation by the phosphorylation of eIF2␣, induces C/EBP-homologous protein (CHOP) (DeGracia et al., 2002). CHOP is a transcription factor (Ubeda et al., 1999), and its expression is generally associated with apoptosis (Zinszner et al., 1998; McCullough et al., 2001). Endonuclease IRE1 induces processing of X-box protein 1 (XBP-1) mRNA (Shen et al., 2001; Yoshida et al., 2001; Calfon et al., 2002), with activated IRE1 cleaving a sequence of 26 bases from the coding region of XBP-1 mRNA (Gonzalez et al., 1999). The processed XBP-1 mRNA is then translated into the mature XBP-1 protein, an active transcription factor specific for ER-stress genes such as immunoglobulin binding protein (BiP)/glucose-regulated protein (GRP) 78 and GRP94 (Calfon et al., 2002). Various experimental models and observational studies have shown that cerebral ischemia induces an ERstress response, suggesting that ER plays an important role in the pathogenesis of neuronal cell injury during and after cerebral ischemia (Ito et al., 2001; Mouw et al., 2003; Paschen et al., 2003). Furthermore, ORP150, an ERassociated chaperon, is enhanced after stroke in the human brain (Tamatani et al., 2001). However, to our knowledge, most studies have been performed on cerebral ischemia and reperfusion models, but not a permanent cerebral ischemia model. Further, the roles of ER stress in the periphery of the middle cerebral artery (MCA) have not been fully evaluated. Clinically, permanent cerebral isch-
a
Department of Biofunctional Molecules, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan
b
Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
c
Department of Psychiatry, Osaka University Graduate School of Medicine, D3, 2-2, Yamadaoka, Suita 565-0871, Japan
d
Department of Anatomy, Faculty of Medicine, University of Miyazaki, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan
Abstract—The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. ER stress-related markers [immunoglobulin binding protein (BiP)/glucose-regulated protein (GRP) 78, activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP)] in the striatum and the cortex were investigated after permanent middle cerebral artery occlusion (MCAO) in mice. Using endoplasmic reticulum stress-activated indicator (ERAI) transgenic mice, which show splicing of X-box protein 1 (XBP-1) mRNA as green fluorescence, we monitored the regional changes in fluorescence after MCAO. BiP mRNA (by reverse-transcription polymerase chain reaction [RT-PCR] analysis) was increased in the cortex at 6 h. In immunohistochemical and/or Western blot analysis, the expressions of ER stress-related markers (BiP, ATF-4, and CHOP) were increased in the infarct region, more strongly in the cortex than in the striatum. ERAI fluorescence was observed in the ischemic area starting from 6 and 12 h, respectively, after MCAO, with the peaks at 1 day and the fluorescence co-localized with the 2,3,5-triphenyltetrazolium chloride (TTC)–visible extension of brain infarction. These findings suggest that permanent MCAO induces expression of ER-stress related genes mainly in the periphery of the MCA territory. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ER-stress, mouse, permanent middle cerebral artery occlusion, stroke.
The endoplasmic reticulum (ER) is an organelle that plays important roles in the maintenance of intracellular calcium *Corresponding author. Tel: ⫹81-58-237-8596; fax: ⫹81-58-2378596. E-mail address: [email protected] (H. Hara). Abbreviations: ATF-4, activating transcription factor-4; BiP, immunoglobulin binding protein; CHOP, C/EBP-homologous protein; DAB, diamino benzidine; eIF2␣, eukaryotic initiation factor 2␣; ER, endoplasmic reticulum; ERAI, endoplasmic reticulum stress-activated indicator; GFAP, glial fibrillary acidic protein; GRP, glucose-regulated protein; IRE1, inositol requiring enzyme 1; MAP2, microtubule-associated protein 2; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; PB, phosphate buffer; PERK, double-stranded RNAdependent protein kinase-like endoplasmic reticulum kinase; RT-PCR, reverse-transcription polymerase chain reaction; TTC, 2,3,5-triphenyltetrazolium chloride; UPR, unfolded-protein response; XBP-1, X-box protein 1.
0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.04.017
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emia happens frequently, most often without reperfusion. Therefore, in the present study we investigated the regional and time-dependent changes in ER stress-related markers (BiP, ATF-4, and CHOP) after permanent middle cerebral artery occlusion (MCAO) in mice. In addition, using endoplasmic reticulum stress-activated indicator (ERAI) transgenic mice (Iwawaki et al., 2004a), which display splicing of XBP-1 mRNA as green fluorescence, we evaluated both regional and time-related changes in the splicing of XBP-1 mRNA after MCAO. ERAI mice would be expected to provide valuable information as to the dynamics of ER stress-induced neuronal damage.
EXPERIMENTAL PROCEDURES Experimental materials The drugs used and their sources were as follows. Paraformaldehyde (Wako Pure Chemical Industries, Osaka, Japan), sucrose (Wako), sodium hydrogen phosphate 12-water (Nacalai tesque, Kyoto, Japan), sodium dihydrogen phosphate dihydrate (Nacalai tesque), potassium chloride (Wako), sodium chloride (Kishida Chemical, Osaka, Japan), Nembutal (Dainippon Pharmaceutical, Osaka, Japan), Triton X-100 (Bio-Rad, Hercules, CA, USA), O.C.T. compound (Sakura Fine Technical, Tokyo, Japan), Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA, USA), M.O.M. immunodetection kit (Vector Laboratories), diamino benzidine (DAB) peroxidase substrate kit (Vector Laboratories), mouse anti-KDEL monoclonal antibody (Stressgen Bioreagents, Victoria, BC, Canada), mouse anti-BiP monoclonal antibody (Transduction Laboratories, Lexington, KY, USA), mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (YLEM, San Francisco, CA, USA), mouse anti-microtubule-associated protein 2 (MAP2) monoclonal antibody (Sigma, St. Louis, MO, USA), rabbit anti-cyclic AMP response element-binding protein (CREB)-2 (ATF-4) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-growth arrest and DNA damage-inducible gene (GADD) 153 (the protein has also been designated CHOP) monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-actin polyclonal antibody (Santa Cruz Biotechnology), Alexa fluor 546 F(ab=)2 fragment of goat anti-mouse IgG (H⫹L) (Molecular Probes, Eugene, OR, USA), SuperSignal West Femto Maximum Sensitivity Substrate Antibodies (Pierce, Rockford, IL, USA), Running Buffer Solution (Wako), Owl’s Electroblot Buffer Kit (Owl, Portsmouth, NH, USA), SuperSep 10% (Wako), BCA Protein Assay Kit (Pierce), Immobilon-P (Millipore, Bedford, MA, USA), Gel Blot Paper (Schleicher & Schuell, Keene, NH, USA), Sample buffer (Wako), TSA Biotin system (PerkinElmer Life Sciences Inc., Boston, MA, USA), Precision Plus Protein Standards Dual Color (Bio-Rad), Block Ace (Dainippon Pharmaceutical), Can Get Signal (Toyobo, Osaka, Japan), dimethyl sulfoxide (DMSO; Koso Chemical, Tokyo, Japan), RIPA buffer (Sigma), protease inhibitor cocktail (Sigma), phosphatase inhibitor cocktail I, II (Sigma), ProLong Gold antifade reagent (Molecular Probes), and 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma).
Focal cerebral ischemia model in mice Male adult C57BL/6J mice weighing 23–27 g (Japan SLC, Hamamatsu, Japan) and adult male ERAI mice (Iwawaki et al., 2004a) weighing 21–30 g were used for the experiments, and were kept under standard lighting conditions (12-h light/dark cycle). Anesthesia was induced by means of 2.0% isoflurane, and maintained with 1% isoflurane in 70% N2O and 30% O2 using an animal general anesthesia machine (Soft Lander; Sin-ei Industry Co. Ltd., Saitama, Japan). Body temperature was maintained between 36.5 and 37.0 °C with the aid of a heating pad and
heating lamp. Filament occlusion of the left MCA was carried out as previously described (Hara et al., 1996, 1997). Briefly, the left MCA was occluded with an 8-0 nylon monofilament (Ethicon, Somerville, NJ, USA) coated with a mixture of silicone resin (Xantopren; Bayer Dental, Osaka, Japan).
Reverse-transcription polymerase chain reaction (RT-PCR) C57BL/6J mice were deeply anesthetized and decapitated at the indicated times after MCAO, and the brains were quickly removed and cut 5-mm coronal section (between 2 and 7 mm from frontal forebrain). Striatum and cortex of the left (ischemic side) MCA territory and contralateral hemisphere as control were carefully separated from the brains on ice under stereoscopic microscope, and then quickly frozen in dry ice and stored at ⫺80 °C. Total RNA was isolated using an RNeasy kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer’s protocol. RNA concentrations were determined spectrophotometrically at 260 nm. First-strand cDNA was synthesized in a 20 l reaction volume using a random primer (Takara, Otsu, Japan) and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA). PCR was performed in a total volume of 30 l containing 0.8 M of each primer, 0.2 mM dNTPs, 3 U TaqDNA polymerase (Promega, Madison, WI, USA), 2.5 mM MgCl2, and 10⫻ PCR buffer. The amplification conditions for semi-quantitative RT-PCR analysis were as follows: an initial denaturation step of 95 °C for 5 min), 22 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, with a final extension step of 72 °C for 7 min. The numbers of amplification cycles for detection of BiP and -actin were 18 and 15, respectively. The primers used for amplification were as follows: BiP, 5=-GTTTGCTGAGGAAGACAAAAAGCTC-3= and 5=-CACTTCCATAGAGTTTGCTGATAATTG-3=; -actin, 5=-TCCTCCCTGGAGAAGAGCTAC-3= and 5=-TCCTGCTTGCTGATCCACAT-3=. The PCR products were resolved by electrophoresis through a 4.8% (w/v) polyacrylamide gel. The density of each band was quantified using the Scion Image Program (Scion Corporation, Frederick, MD, USA).
Immunohistochemistry At 2 h, 6 h, 12 h, 1 day, and 3 days after MCAO, C57BL/6J and ERAI mice were injected with sodium pentobarbital (Nembutal; 50 mg/kg, i.p.), then perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Brains were removed after 15 min perfusion fixation at 4 °C, then immersed in the same fixative solution overnight at 4 °C. They were then immersed in 25% sucrose in 0.1 M PB for 24 h, and finally frozen in powdered dry ice. Coronal sections (10 m thick) were cut on a cryostat at ⫺20 °C, and stored at ⫺80 °C until use. After re-hydration, endogenous peroxidase activity was quenched using 0.3% hydrogen peroxidase in methanol. Next, brain sections were blocked with M.O.M. blocking reagent (M.O.M. immunodetection kit), then incubated overnight at 4 °C either with mouse anti-KDEL monoclonal antibody (1:400 dilution) or with mouse antiCHOP monoclonal antibody (1:100 dilution). They were washed and then incubated with biotinylated anti-mouse IgG before being incubated for 30 min at room temperature with avidin– biotin–peroxidase complex and then developed using DAB peroxidase substrate. CHOP signals were amplified using a TSA Biotin System. CHOPstained cells were counted in the striatum and the cortex. The results were expressed as positive cells per 1 mm2.
Western blot analysis C57BL/6J mice were deeply anesthetized and decapitated at the indicated times after MCAO, and the brains were quickly removed and cut into 5-mm coronal section (between 2 and 7 mm from frontal forebrain). Striatum and cortex of the left (ischemic side) MCA territory and contralateral hemisphere as control were carefully sepa-
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Fig. 1. Change in levels of BiP expression in the striatum and cortex after MCAO. (A) Quantitative analysis of RT-PCR showed that the expression level of BiP mRNA was significantly increased in the cortex at 6 h (n⫽6 –13; ** P⬍0.01 vs. Control). (B) Immunostaining for BiP in the striatum and cortex at 0 h, 2 h, 6 h, 12 h, 1 day, and 3 days after MCAO. Expression of BiP increased gradually from 2 h onwards in both striatum and cortex. Scale bar⫽20 m. (C) Western blot analysis showed that BiP was detectable. (D) Quantitative analysis of Western blotting showed that the expression level of BiP was significantly increased in the striatum at 1 day and in the cortex at both 12 h and 1 day. n⫽3–7; * P⬍0.05, ** P⬍0.01 vs. Control.
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rated from the brains on ice under stereoscopic microscope, and then quickly frozen in dry ice. For protein extraction, the tissue was homogenized in RIPA buffer containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktails. The homogenate was centrifuged at 11,000⫻g for 20 min, and the supernatant was collected for this study. Assays were performed to determine the protein concentration, comparisons being made with a known concentration of bovine serum albumin (BCA Protein Assay Kit). An aliquot of 5 g of protein was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, with the separated protein being transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). For immunoblotting, the following primary antibodies were used: mouse anti-BiP monoclonal antibody (1:250 dilution), rabbit anti-ATF-4 polyclonal antibody (1:200 dilution), and a rabbit anti-actin polyclonal antibody (1:2000 dilution). The secondary antibodies were anti-mouse HRP-conjugated IgG (1:2000 dilution) and goat anti-rabbit HRP-conjugated IgG (1:2000 dilution). The immunoreactive bands were visualized using SuperSignal west femto maximum sensitivity substrate. The band intensity was measured using a Lumino Imaging Analyzer (FAS-1000; Toyobo) and Gel Pro Analyzer (Media Cybernetics, Atlanta, GA, USA).
Observation of fluorescent tissues and TTC staining ERAI mice were deeply anesthetized and decapitated at the indicated times after MCAO, and the brains were quickly removed. Next, the forebrains were divided into 5 or 10 coronal slices (thickness 1 or 2 mm) using a mouse brain matrix (RBM-2000C; Activational Systems, Warren, MI, USA). A stereoscopic microscope fitted with on epifluorescence attachment (Olympus, Tokyo, Japan) was used to detect fluorescence from these tissues. For 2% TTC staining, brains were divided into five coronal (2 mm) slices and stained with TTC. The infract regions were recorded as images using a digital camera (Coolpix 4500, Nikon, Tokyo, Japan).
Fluorescent staining To confirm the localization of ERAI fluorescence, we carried out fluorescent staining for cell-phenotype markers. For immunofluorescence staining, the sections were incubated overnight at 4 °C with mouse anti-MAP2 monoclonal antibody (1:500 dilution) or mouse anti-GFAP monoclonal antibody. They were then washed and subsequently incubated for 3 h at room temperature with Alexa fluor 546 F(ab=)2 fragment of goat anti-mouse IgG (H⫹L). After fixation in 4% paraformaldehyde in PBS, we mounted each section in ProLong Gold antifade reagent.
Statistical analysis Data are presented as the means⫾S.E.M. Statistical comparisons were made using a one-way ANOVA followed by a Student’s t-test or Dunnett’s test using StatView version 5.0 (SAS Institute Inc., Cary, NC, USA), with P⬍0.05 being considered to indicate statistical significance.
RESULTS Induction of BiP mRNA and BiP protein in the striatum and cortex after MCAO BiP is induced under ER stress, and indeed is known to be a major marker of such stress. To examine the induction of BiP protein and BiP mRNA after permanent MCAO, we performed RT-PCR, immunohistostaining, and Western blot analysis (Fig. 1). The results of the RT-PCR analysis (Fig. 1A) revealed that in the striatum, BiP mRNA tended to be raised at 12 h after MCAO, the level being 1.2 times control. In the cortex, BiP mRNA was significantly raised
Fig. 2. Change in levels of ATF-4 expression in the striatum and cortex after MCAO. (A) Western blot analysis showed that ATF-4 was detectable. (B) Quantitative analysis of Western blotting showed that the expression level of ATF-4 was significantly increased in the striatum at 12 h after MCAO and in the cortex at 12 h, 1 day, and 3 days after MCAO. n⫽3–7; * P⬍0.05, ** P⬍0.01 vs. Control.
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(to 1.8 times control) at 6 h, but had recovered to near the control level at 12 h. Fig. 1B shows that after MCAO, immunoreactivity for BiP increased gradually from 2 h, with the peak at 12 h, in both striatum and cortex. At 1 and 3 days, most of the neurons in the striatum had degenerated, with the remaining neurons showing strong immunoreactivity. The immunoreactivity was stronger in the cortex than in the striatum. The results of Western analysis of BiP expression a (Fig. 1C and D) show that in the striatum, BiP tended to be
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raised at 12 h, was significantly raised at 1 day, and was back to near the control level at 3 days. Its level was 1.2 and 1.4 times control at 12 h and 1 day, respectively. In the cortex, BiP was significantly raised at 12 h and 1 day, with the peak at 1 day, and had declined to near control level at 3 days. Its level was 1.3 and 1.8 times control at 12 h and 1 day after MCAO, respectively. BiP expression was stronger in the cortex than in the striatum. These Western blot results were consistent with those from immunohistochemistry.
Fig. 3. Change in levels of CHOP expression in the striatum and cortex after MCAO. (A) Immunostaining for CHOP in the striatum and cortex at 0 h, 2 h, 6 h, 12 h, 1 day, and 3 days after MCAO. Expression of CHOP increased gradually from 6 h onwards in both striatum and cortex. Scale bar⫽1 m. (B) The number of CHOP-positive cells in the striatum and cortex. CHOP-positive cells were significantly increased at 12 h, 1 day, and 3 days in both areas. n⫽3; * P⬍0.05, ** P⬍0.01 vs. Control.
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The above results indicate that a major marker of ER stress (BiP) is induced in the early phase after permanent MCAO in mice. Induction of ATF-4 and CHOP in the striatum and cortex after MCAO CHOP is a key molecule linking ER stress to apoptotic cell death, and ATF-4 plays an active role in the induction of this molecule. We therefore investigated changes in ATF-4
expression by Western blot analysis (Fig. 2) and those in CHOP expression by immunohistochemical analysis (Fig. 3A). In the striatum, ATF-4 was significantly increased (to 1.4 times control) at 12 h after MCAO, but had declined to near control level at 1 day. In the cortex, ATF-4 was significantly increased at 12 h, 1 day, and 3 days, with the peak at 1 day (2.4, 3.3, and 1.7 times control, respectively). ATF-4 expression appeared to be stronger in the cortex than in the striatum.
Fig. 4. Coronal brain sections of TTC staining and fluorescence activity at 1 day after permanent MCAO in ERAI mice. TTC staining sections; A, C, E, and G. After TTC staining, damaged tissue (core region) is shown as a white region (E), whereas viable tissue is shown as a red region (C). Penumbra is shown as a pinkish region (G). Fluorescence sections; B, D, F, and H. Green fluorescence was observed in the infarct region of core (F) and penumbra (H). Fluorescence was measured after TTC staining and photographing using the same slices. A, C, E, and G were identical with B, D, F, and H, respectively. Figures C, E, and G are enlarged box-shaped C, E, and G in figure A. Similarly, figures D, F, and H are enlarged box-shaped D, F, and H in figure B. Scale bar⫽1 mm (A and B) and 200 m (C–H).
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Immunohistochemical analysis showed that in the striatum, CHOP expression increased gradually from 6 h after MCAO (Fig. 3A). At 1 day and 3 days, most of the neurons in the striatum had degenerated, with the remaining neurons displaying strong immunoreactivity. In the cortex, CHOP expression increased from 12 h, with the peak at 1 day. Quantitative analyses of CHOP-positive cells are shown in Fig. 3B. In the striatum, CHOP was significantly increased at 12 h, 1 day, and 3 days, with the peak at 1 day (3.6, 5.8, and 5.2 times control, respectively). In the cortex, CHOP was significantly increased at 12 h, 1 day, and 3 days, with the peak at 1 day (4.5, 7.1, and 6.1 times control, respectively). CHOP expression appeared to be stronger in the cortex than in the striatum. ATF-4 and CHOP expressions were each elevated in the infarct region in the early phase after MCAO, although there were no obvious morphological abnormalities. Fluorescence activity in ERAI mice after MCAO To monitor ER stress after MCAO, we used ERAI transgenic mice. Under ER-stress conditions, ERAI mice
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express green fluorescence [the explanation being that ER stress triggers splicing of XBP-1 mRNA and the subsequent translation of an indicator fusion protein, which is detectable by fluorescence (Iwawaki et al., 2004a)]. As shown in Fig. 4, we observed fluorescence at 1 day after MCAO on the ipsilateral side to the occlusion, but not on the contralateral side. To investigate the ERAI fluorescence-expressing region in more detail, we carried out TTC staining, after which the infarct region appears white, whereas the viable region appears red. As shown in Fig. 4, the green area of ERAI fluorescence overlapped with the white region (infarction), but not with the red region. We confirmed the presence of ERAI fluorescence in 5 (3–7 mm from the rostral pole) of the 10 coronal sections of forebrain at 1 day after MCAO (Fig. 5A), with the fluorescence being observed in the infarct region in each section. ERAI fluorescence first became apparent at 6 and 12 h after MCAO in the striatum and cortex, respectively (Fig. 5B), with the maximal level being detected in both regions at 1 day after MCAO.
Fig. 5. Fluorescence activity in brain of ERAI mice after MCAO. (A) Upper and lower figures are shown in normal pictures and the fluorescence activity, respectively, in brain coronal sections (3–7 mm from the rostral pole) at 1 day after MCAO. ERAI fluorescence was detected in the infarct region. (B) Fluorescence activity at 2 h to 3 days after MCAO. ERAI fluorescence was weak in the striatum at 6 h after MCAO and in the cortex at 12 h after MCAO. Strong fluorescence was detected in both regions at 1 and 3 days after MCAO. Scale bar⫽1 mm.
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Fig. 6. Fluorescence activity and immunofluorescence staining of dendrites (MAP2) in the cortex at 12 h and 1 day after MCAO. ERAI fluorescence co-localized with MAP2 (arrows) at 12 h after MCAO. Scale bar⫽20 m.
Localization of ERAI fluorescence Finally, we attempted to determine the cellular localization of ERAI fluorescence by means of immunofluorescence staining. The signals for ERAI fluorescence at 12 h after MCAO overlapped with that for MAP2 (Fig. 6), but not with that for Hoechst 33342 (data not shown). These findings which indicate that the fluorescence was localized to the cytosol and dendrites, and not was present in nuclei are consistent with the report that the XBP-1⌬DBD-Venus construct expressed by ERAI mice is an ER-stress indicator that is localized to the cytosol, not the nuclei (Iwawaki et al., 2004a; see Discussion). The signals for ERAI fluorescence at 1 and 3 days after MCAO overlapped with that for GFAP (Fig. 7), suggesting that glial cells also showed a pronounced ER-stress response.
DISCUSSION In the present study, we examined the regional and timerelated changes in three ER stress-related markers (BiP, ATF-4, and CHOP) after permanent MCAO in mice. Further, using ERAI transgenic mice we monitored the splicing of XBP-1 mRNA (as green fluorescence) after MCAO. These ER stress-related markers and ERAI fluorescence were all induced by MCAO, and indeed were rapidly upregulated in the ischemic region, even when no obvious morphological changes were detectable.
The UPR is mediated by PERK and IRE1. BiP binds to unfolded proteins in the ER and regulates the activation of these ER transducers (Bertolotti et al., 2000). Under normal conditions, BiP binds the luminal domains of PERK and IRE1, and prevents them from being activated. Under ER-stress conditions, BiP dissociates from PERK and IRE1 to bind unfolded proteins, and thereby renders each of these transducers liable to activation. Our results showed that (a) the expressions of BiP mRNA and BiP protein were enhanced after MCAO, (b) the expression of ER stress was stronger in the periphery of the MCA territory (mainly cortex) than in the core region (mainly striatum), and (c) BiP expression peaked at 12–24 h, before recovering to near control level at 3 days (Fig. 1). It has been reported that induction of BiP prevents neuronal damage being induced by ER stress (Wang et al., 1996; Rao et al., 2002; Reddy et al., 2003), and the increase in BiP expression may correlate with the degree of neuroprotection. Hence, a therapeutic approach leading to the induction of UPR activation may be effective for the treatment of cerebral ischemia. However, further studies will be needed to test this hypothesis. Activated PERK causes phosphorylation of eIF2␣, thereby inhibiting most protein synthesis (Bertolotti et al., 2000). When ER stress is very severe and prolonged, p-eIF2␣ becomes a key molecule in cell death-signal activation. ATF-4 is a transcription factor and is responsible for CHOP transcription by p-eIF2␣-mediated bypass scan-
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Fig. 7. Fluorescence activity and immunofluorescence staining of glial cells (GFAP) in the cortex at 1 and 3 days after MCAO. ERAI fluorescence co-localized with GFAP. Scale bar⫽20 m.
ning (Fawcett et al., 1999; Harding et al., 2000). In the present study, ATF-4 was significantly elevated at 12 h after MCAO in the striatum, and at 12 h, 1 day, and 3 days (with its peak at 1 day) in the cortex (Fig. 2), while CHOP was induced at 6 h in the ischemic region (Fig. 3). There is ample evidence that CHOP participates in apoptosis signaling (Kumar et al., 2003), and CHOP⫺/⫺ mice have smaller infarcts than wild-type mice after transient cerebral ischemia (Tajiri et al., 2004). Moreover, overexpression of BiP attenuates the induction of CHOP in ER stress and reduces ER stress-induced apoptosis (Wang et al., 1996). We noted that in the late phase of MCAO, BiP expression was reduced while CHOP expression was increased. These results indicate that a dysfunction of the protective mechanism leads to the induction of apoptosis by CHOP, and that CHOP expression may be closely related to the neuronal cell death occurring during permanent cerebral ischemia. Activated IRE1 is known to splice XBP-1 mRNA under ER-stress conditions (Yoshida et al., 2001). ERAI mice carry a human XBP-1 and venus (a variant of green fluorescence protein) fusion gene (Iwawaki et al., 2004a). Under normal conditions, the mRNA of the fusion gene is
not spliced, but under ER stress it is spliced, and then a XBP-1 and venus fusion protein is induced. ERAI fluorescence was observed from 6 h after MCAO, and it was expressed in a region more or less coincident with the infarct region. Paschen et al. (2003) have reported that XBP-1 mRNA was induced at 6 h after cerebral ischemia and reperfusion. In addition, we noted that ERAI fluorescence was expressed in glial cells at 1 day and 3 days after MCAO (Figs. 4, 5, and 7). This result suggests that in the late phase of the cerebral ischemia, glial cells also express a UPR, thus forming part of the protective mechanism against ER stress. Moreover, ERAI fluorescence could be detected in the cytosol and dendrites at 12 h after the start of ischemia (Fig. 6) and in the glia at 1 day and 3 days (Fig. 7), but not in the nuclei (Morimoto et al., unpublished observations). The finding in the ischemic region of an expression of ERAI fluorescence from the early phase of cerebral ischemia was quite similar to our findings for such other markers of ER stress as BiP and CHOP. Hence, ERAI mice may be useful for studies of the changes that occur during ER stress, and for elucidation of the relationship between neuronal cell death and ER stress. However, we should know the possibility that components of the ER
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response may be activated independently of ER stress in development and pathophysiological processes (Iwawaki et al., 2004b). In studies of ER stress after cerebral ischemia and reperfusion; (a) BiP mRNA was found to be increased at 6 –24 h after a 30 min ischemia and reperfusion in both the cortex and striatum (Qi et al., 2004), (b) BiP expression was increased at 5–23 h, then decreased at 47 h, after a 1 h ischemia and reperfusion affecting the MCA territory (Shibata et al., 2003), and (c) CHOP and ATF-4 expressions were raised in the striatum at 4 –24 h after a 30 min ischemia and reperfusion (Hayashi et al., 2005). In the present study, in contrast, BiP and CHOP tended to show similar patterns of change in expression, whereas BiP mRNA and ATF-4 expressions showed different patterns. This discrepancy is probably due to the use of different ischemia models (reperfusion vs. permanent). Compared with the reperfusion model, the permanent model exhibits a continued low blood flow condition, especially in the striatum. The levels of expression of the above ER-stress markers in the ischemic core region would appear to be lower in the permanent model than in the reperfusion model.
CONCLUSION In conclusion, ER stress plays an important causal role in permanent ischemic damage, with its participation perhaps being greater in the periphery of the MCA territory than in the core region.
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(Accepted 5 April 2007) (Available online 27 June 2007)