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Characterization of neuronal and astroglial responses to ER stress in the hippocampal CA1 area in mice following transient forebrain ischemia
Neurochemistry International 57 (2010) 1–7
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Characterization of neuronal and astroglial responses to ER stress in the hippocampal CA1 area in mice following transient forebrain ischemia Nobuhiro Osada, Yasuhiro Kosuge, Kumiko Ishige, Yoshihisa Ito * Research Unit of Pharmacology, Department of Clinical Pharmacy, School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 February 2010 Received in revised form 10 March 2010 Accepted 24 March 2010 Available online 31 March 2010
Transient forebrain ischemia has been shown to cause neuronal injury in the CA1 area of the hippocampus in mice. In addition to neuronal injury, astrocytes in area CA1 undergo apoptosis under ischemic conditions. Although failure of impaired astrocytes to take up glutamate is thought to contribute to the pathogenesis of cerebral ischemia, the molecular mechanism underlying this phenomenon remains unexplored. In the present study, we investigated neuronal and astroglial responses to endoplasmic reticulum (ER) stress, which is an important sequela of transient forebrain ischemia in the hippocampus of mice. Cellular injury was observed in area CA1 of the hippocampus 72 h after reperfusion, and ssDNA positivity was detectable in some glial cells as well as neurons in this area. An increase of 78-kDa glucose-regulated protein (GRP78), an indicator of ER stress, was detected in pyramidal neurons and astrocytes in this area after the insult. Immunohistochemical analysis showed that caspase-12 was increased in pyramidal neurons and astrocytes located in the extrapyramidal cell layer. Immunoreactivity for C/EBP homologous protein (CHOP) was increased significantly in pyramidal cells but not in astrocytes. These results suggest that astrocytes as well as pyramidal neurons in area CA1 undergo apoptosis through an ER stress-dependent mechanism after ischemia. Unlike the situation in neuronal apoptosis, CHOP does not play a role in the cell death of astrocytes. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Endoplasmic reticulum stress Caspase-12 Astrocyte Forebrain ischemia Hippocampus
1. Introduction Transient forebrain ischemia causes delayed loss of pyramidal neurons in area CA1 of the hippocampus, whereas the neighboring CA3 area and dentate gyrus (DG), and most cortical neurons, remain essentially intact (Kirino, 1982; Osada et al., 2009). Transient cerebral ischemia elicits activation and proliferation of microglia and astrocytes (Petito et al., 1990; Gehrmann et al., 1992; Kato et al., 1995). Astrocytes are the predominant glial cell type in the brain and are essential for neuronal survival and synaptic function, as well as for neurogenesis and neural repair (Aschner et al., 2002; Swanson et al., 2004). Moreover, astrocytic death has been found after focal ischemia in the rat brain (Liu et al., 1999). A recent study has demonstrated that astrocytes might modulate the pathogenesis of many acute and chronic neurodegenerative disorders, such as cerebral ischemia and neurodegenerative
Abbreviations: BCCA, bilateral common carotid artery; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; DG, dentate gyrus; DTT, dithiothreitol; GRP78, 78-kDa glucose-regulated protein; H&E, hematoxylin and eosin; PBS, phosphatebuffered saline; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethane sulfonyl fluoride; SDS, sodium dodecyl sulfate; ssDNA, single-stranded DNA. * Corresponding author. Tel.: +81 47 465 5832; fax: +81 47 465 5832. E-mail address: [email protected] (Y. Ito). 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.03.017
conditions such as Alzheimer’s disease and Parkinson’s disease (Takuma et al., 2004). The classical function of astrocytes during ischemia is uptake of glutamate to minimize any excitotoxic insult to neighboring neurons (Dugan et al., 1995). Failure of impaired astrocytes to take up and release glutamate is thought to contribute to ischemia-induced neuronal death (Swanson et al., 2004). However, the mechanisms involved in this impairment have not been fully explained. Endoplasmic reticulum (ER) stress has been shown to be involved in some neuronal pathologies such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and prionrelated disorders (LaFerla, 2002; Ryu et al., 2002; Hetz et al., 2003; Nishitoh et al., 2008). The ER plays a critical role in a variety of processes, including protein synthesis and folding and maintenance of Ca2+ homeostasis. A number of studies have demonstrated that ER stress disrupts these normal functions, and thus the ER has become a focus of interest with regard to its role in cellular pathology (Lin et al., 2008). When mild ER stress occurs, cells develop a self-protective signal transduction pathway termed the unfolded protein response (UPR), which includes the induction of molecular chaperones in the ER, translational attenuation, and enhancement of ER-associated degradation, thus relieving cells from the stress. However, if the damage is too severe to repair, the UPR ultimately initiates the apoptosis pathway, and damaged cells
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are eliminated in order to protect the organism (Kozutsumi et al., 1988; Ron, 2002). Several proteins have been implicated in this apoptotic pathway, including a transcription factor, C/EBP homologous protein (CHOP), also known as gadd 153 (Oyadomari et al., 2002), the ER-resident caspase, caspase-12 (Nakagawa et al., 2000; Kosuge et al., 2003), and c-Jun N-terminal protein kinase (JNK) (Kadowaki et al., 2005). Caspase-12 is localized specifically on the cytoplasmic side of the ER and is thought to play a pivotal role in ER stress-induced cell death in the rodent brain (Nakagawa and Yuan, 2000; Nakagawa et al., 2000). Our previous studies showed that neuronal death induced by amyloid b-peptide and tunicamycin, an ER stress inducer, was mainly dependent upon the pathway mediated by caspase-12 in rat cultured hippocampal neurons (Kosuge et al., 2003, 2006) and organotypic hippocampal slice cultures (Imai et al., 2007; Ishige et al., 2007). CHOP is a proapoptotic transcription factor expressed at very low levels under physiological conditions but is induced extensively in response to ER stress, thus causing apoptosis (Wang et al., 1996; Oyadomari and Mori, 2004). Recent studies have also demonstrated that ER stress plays a key role in neuronal death resulting from ischemia (Paschen and Doutheil, 1999; DeGracia and Montie, 2004; Osada et al., 2009). However, the underlying mechanism responsible for ER stress in astrocytes after transient forebrain ischemia is yet to be clarified. In order to elucidate the pathophysiological mechanism of ischemia/reperfusion-induced ER stress in astrocytes, we performed experiments to compare the expressions of ER stressassociated signal proteins, including GRP 78, CHOP and caspase-12, in neurons and astrocytes in area CA1 of the hippocampus after transient forebrain ischemia. 2. Materials and methods 2.1. Animals Male C57BL/6 mice aged 9–10 weeks were used in this study. Animals used in the present study were fed standard laboratory chow and given free access to water prior to surgery. All efforts were made to minimize the number of animals used and their suffering. All experiments with animals have done in compliance with the Ethical Guidelines for Animal Experiments at Nihon University. 2.2. Transient forebrain ischemia Transient forebrain ischemia was produced by bilateral common carotid artery (BCCA) occlusion as described previously (Osada et al., 2009). Briefly, surgical procedures were performed under chloral hydrate anesthesia (450 mg/kg, i.p.) and the rectal temperature was maintained at 37.5 0.5 8C with a heating blanket (Animal Blanket Controller, Nihon Kohden, Tokyo, Japan). Measurement of cortical perfusion was performed by omega flow meter (OMEGAWAVE, Inc., Tokyo, Japan). BCCA were occluded using small arterial clips for 20 min, after which time the clips were removed, the restoration of blood flow was visually confirmed, the incision was closed, and reperfusion was continued for the indicated time. Sham-operated mice underwent exposure of vessels without withdrawal of blood or clamping of carotid arteries. 2.3. Histological assessment Anesthetized animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed for 24 h in the same fixative. Post-fixed brains were embedded in paraffin and sliced on a microtome at a 6 mm thickness. The sections between 1.5 mm and 2.5 mm posterior of the bregma were used for this study. For histological assessment of damage to the hippocampus, the paraffin-embedded brain sections were stained with hematoxylin and eosin (H&E) or single-stranded DNA (ssDNA) staining. The sections were pretreated with 0.1% trypsin in phosphate buffered saline (PBS) for 20 min at 37 8C, incubated with 0.3% H2O2 in methanol for 15 min, appropriate 1.5% blocking normal goat serum for 1 h at room temperature and exposed to the anti-ssDNA antibody (DAKO, Japan) at dilution 1:500 for 24 h at 4 8C. They were then incubated in the biotin-conjugated IgG (Vector Laboratories, Burlingame, CA) against the host of the primary antibody for 1 h and then incubated with avidin–biotin–peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories) for 60 min. Then the staining was visualized using 0.025% 3,30 -diaminobenzidine (DAB) and 0.075% H2O2 in Tris– HCl (pH 7.6). Negative controls were prepared identically expect for omission of the primary antibody.
2.4. Western blotting Following transient forebrain ischemia, mice were sacrificed under the sodium pentobarbital anesthesia at 1 h, 6 h, 12 h, 24 h and 48 h after ischemia. Shamoperated animals served as controls. Brains were rapidly microdissected on an icechilled plate. The hippocampus was homogenized using homogenizer (Homogenizer, Subsonic, Iwaki Glass Co., Ltd) in 800 mL homogenized buffer containing 10 mM HEPES-NaOH (pH 7.9), 10 mM potassium chloride, 0.1 mM EDTA, 0.1 mM EGTA, 1 M phenylmethane sulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 62.5 mL/mL protease inhibitor cocktail and 62.5 mL/mL 10% Nonidet P-40, centrifuged, and then supernatants were used. Protein concentrations were determined using the method of Bradford (1976). Protein extracts were mixed 1:3 in 4 sample buffer containing 125 mM Tris–HCl (pH 6.0), 3% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 10% glycerol and 0.2% bromophenol blue, separated by 5–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) paper (Millipore, MA, USA). The membranes were treated for 1 h with a blocking buffer containing 20 mM Tris–HCl (pH 7.6), 137 mM NaCl, 5% skim milk at room temperature and then incubated overnight at 4 8C with a 1:1000 dilution of anti-caspase-12 monoclonal antibody (Sigma, USA), a 1:1000 dilution of anti-CHOP polyclonal antibody (Santa Cruz Biotechnology, USA), or a 1:2000 dilution of anti-KDEL monoclonal antibody (StressGen, Canada). The membranes were washed repeatedly in TTBS containing 20 mM Tris–HCl (pH 7.6), 137 mM NaCl, 0.05% Tween 20, then incubated with a 1:10,000 dilution of HRP-conjugated secondary antibody for 1 h. Immunoreactive bands were detected by ECL (Amersham Pharmacia Biotech). To adjust for protein loading, membranes were also immunostained with anti-bactin monoclonal antibody (Sigma, USA). Optical density on the blots was measured with Scion imaging software (www.scioncorp.com). 2.5. Immunohistochemistry For immunohistochemistry, post-fixed brains were immersed for 24 h in PBS containing 30% sucrose and coronally sectioned on a cryostat at an 18 mm thickness. After blocking nonspecific binding with 1.5% normal goat serum, the sections were incubated primary antibodies for 24 h at 4 8C. The primary antibodies were used and the dilutions for each were anti-KDEL monoclonal antibody at 1:200, anti-GFAP polyclonal antibody (Chemicon International, USA) at 1:200, anti-CHOP polyclonal antibody at 1:200 and anti-caspase-12 polyclonal antibody (Exalpha Biologicals, USA) at 1:500. After washing with PBS, the sections were incubated for 1 h with a 1:500 dilution of Alexa Fluor 488-conjugated goat IgG, or Alexa Fluor 594-conjugated goat IgG (Molecular Probes, USA). Some sections were counterstained by incubation for 10 min with To-PRO3 (Molecular Probes, USA). After rinsing with PBS, the sections were analyzed using a confocal laser microscope (Zeiss LSM-510, Germany). Negative controls were prepared by omitting the primary antibodies. 2.6. Statistical analysis All the data are expressed as mean SEM. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple tests.
3. Results 3.1. Histological evaluation of area CA1 of the hippocampus after ischemia/reperfusion Neuronal injury in area CA1 was observed 72 h after transient forebrain ischemia. Histological evaluation of hippocampal neurons by hematoxylin and eosin staining (Fig. 1A and B) and ssDNA staining (Fig. 1C and D) revealed that pyramidal neurons in the CA1 region had degenerated (with pyknotic, shrunken nuclei in H&Estained sections, Fig. 1B). Most of the morphologically damaged neurons became positive for ssDNA staining (Fig. 1D). ssDNA positivity was also detectable in cells located in the vicinity of the pyramidal cell layer in area CA1 after ischemia/reperfusion (Fig. 1D). 3.2. Temporal profiles of GRP78 in the hippocampus after ischemia/ reperfusion In order to investigate the induction of the ER chaperone, GRP78, that contains a characteristic COOH-terminal Lys-Asp-GluLeu (KDEL) sequence, we examined GRP78 expression by Western blotting and immunohistochemistry using an anti-KDEL monoclonal antibody after ischemia/reperfusion. Western blot analysis
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Fig. 1. Histological assessment of area CA1 after ischemia/reperfusion. The upper panels (A and B) show hematoxylin and eosin (H&E) staining in area CA1 and the bottom panels (C and D) show ssDNA staining. Panels A and C are sham-operated coronal sections. Panels B and D are sections prepared 72 h after transient forebrain ischemia for 20 min. Arrows indicate ssDNA positivity in cells located in the vicinity of the pyramidal cell layer. Scale bar, 100 mm.
showed that GRP78 expression was increased in the hippocampus after transient forebrain ischemia, with a peak expression level at 12–48 h after reperfusion (Fig. 2). To identify the cell types in which GRP78 was up-regulated, we performed immunofluorescence staining for GRP78 in area CA1 after ischemia/reperfusion. Although very low immunoreactivity for KDEL was confirmed in the sham-operated groups (Fig. 3A), the immunoreactivity was strongly increased 24 h after ischemia/ reperfusion mainly in the pyramidal cells (Fig. 3B). The immunoreactivity for KDEL tended to decrease in the pyramidal cells 48 h after ischemia/reperfusion; however, the immunoreactivity was
clearly defined in non-neuronal cells in the vicinity of the pyramidal cell layer (Fig. 3C). Next, we performed double immunofluorescence staining for KDEL and GFAP, a marker of astrocytes, 48 h after ischemia/reperfusion. Confocal images revealed that the signal for KDEL was co-localized with the GFAP signal, indicating that most of the KDEL-positive cells were astrocytes (Fig. 4). 3.3. Induction of CHOP protein in pyramidal cells of the hippocampus after ischemia/reperfusion In order to determine the involvement of CHOP in these cell death pathways in neurons and astrocytes after ischemia/reperfusion, we investigated the induction of CHOP after transient forebrain ischemia. Western blot analysis showed that CHOP expression was barely detectable in the sham-operated group. In contrast, a transient and significant increase of CHOP protein was observed 24 h after ischemia/reperfusion in the hippocampus (Fig. 5A and B). Although immunohistochemical analysis for CHOP in area CA1 demonstrated almost no immunoreactivity in the sham-operated groups (Fig. 5C), the signal for CHOP was identified in pyramidal neurons 24 h after ischemia/reperfusion and disappeared after 48 h. However, induction of CHOP was not observed in the extrapyramidal cell layer where KDEL-positive astrocytes were located 48 h after ischemia/reperfusion. These results were consistent with the timewindow observed in Western blot analysis, suggesting that CHOP functions in neurons but not in astrocytes. 3.4. Expression of caspase-12 in astrocytes after ischemia/reperfusion
Fig. 2. Change in the level of GRP78 expression in the hippocampus after ischemia/ reperfusion. (A) Western blot analysis of GRP78 in the hippocampus after transient forebrain ischemia for 20 min followed by 1 h, 6 h, 12 h, 24 h and 48 h of reperfusion. (B) Relative amount of GRP78 was assessed by densitometric analysis. Values are expressed as the mean SEM for four independent experiments. *p < 0.05, **p < 0.01 compared with the sham-operated group, one-way ANOVA followed by Tukey’s test.
Next, we investigated the expression of the cleaved form of caspase-12 in the hippocampus after ischemia/reperfusion. Western blotting showed that the immunoreactivity of cleaved caspase-12 increased from 24 h and peaked at 48 h after ischemia/ reperfusion (Fig. 6A and B). We then determined the localization of caspase-12 in coronal sections by immunohistochemical staining. In the sham-operated mice, immunoreactivity for caspase-12 was very low in area CA1 (Fig. 6C). Caspase-12-positive cells were detected not only in the pyramidal cell layer but also in its vicinity 24 h after ischemia/reperfusion, and the number of caspase-12positive cells was increased at 48 h (Fig. 6C). Immunoreactivity for KDEL was identified only in the pyramidal cell layer 24 h after reperfusion; however, it was also identified in the vicinity of the
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Fig. 3. Immunofluorescence staining for KDEL (GRP78) in area CA1 after ischemia/reperfusion. Panel A is coronal sections of the sham-operated group. Panels B and C are coronal sections prepared 24 h or 48 h after transient forebrain ischemia for 20 min. Arrows indicated some glial cells. Scale bar, 20 mm.
Fig. 4. Double-labeling confocal immunofluorescence microscopy staining of KDEL (GRP78) and GFAP in area CA1 after ischemia/reperfusion. The hippocampus was taken for analysis at 48 h after ischemia/reperfusion. (A) Some glial cells were costained with KDEL (green) and GFAP (red) in area CA1 (arrows). Nuclei were counterstained with TOPRO3 (blue). Panels B–D show magnified views of the area indicated in panel A. Yellow indicates the co-localized of signals for KDEL (green, B) and GFAP (red, C) in area CA1. Scale bar, 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
pyramidal cell layer 48 h after reperfusion. KDEL and caspase-12 signals were co-localized not only in pyramidal cells but also in cells located outside the pyramidal cell layer. 4. Discussion Neuronal death in area CA1 of the hippocampus after forebrain ischemia has been shown to occur in a delayed manner (Kirino, 1982). There is a growing evidence that this neuronal death is at least partly due to apoptosis (MacManus et al., 1993; Nitatori et al., 1995; Osada et al., 2009). Recently, it has been demonstrated that astrocytes also undergo apoptosis under ischemic conditions (Giffard and Swanson, 2005). In the present study, we showed that not only neurons but also astrocytes located outside the pyramidal cell layer in area CA1 were injured, and that both types of cells showed positive ssDNA staining 72 h after ischemia/reperfusion. These results indicate that not only neurons but also non-neuronal cells in area CA1 undergo apoptosis after transient forebrain ischemia.
There is a growing evidence that ER stress plays an important role in neuronal death caused by brain ischemia (Paschen and Doutheil, 1999; Kumar et al., 2003; Tajiri et al., 2004; Osada et al., 2009). Consistently, our data showed that the level of GRP78 was significantly increased in the hippocampus, and immunoreactivity for GRP78 was detected in the pyramidal cell layer of area CA1 24 h after ischemia/reperfusion. We also found that immunoreactivity for KDEL appeared in astrocytes located in the vicinity of the pyramidal cell layer 48 h after ischemia/reperfusion. These results suggest that ER stress is a key pathological mechanism not only in neuronal, but also in astroglial, cell death after transient forebrain ischemia in area CA1 of the hippocampus. Although failure of impaired astrocytes to take up glutamate is thought to contribute to the pathogenesis of cerebral ischemia (Swanson et al., 2004), the molecular pathways responsible for astroglial cell death have not been adequately characterized (Takuma et al., 2004; Ouyang et al., 2007). CHOP is expressed at very low levels under physiological conditions, but strongly induced in response to ER stress through
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Fig. 5. Induction of CHOP protein in pyramidal cells of the hippocampus after ischemia/reperfusion. (A) Western blot analysis of CHOP in the hippocampus after transient forebrain ischemia for 20 min followed by 1 h, 6 h, 12 h, 24 h and 48 h of reperfusion. (B) Relative amount of CHOP was assessed by densitometric analysis. Values are expressed as the mean SEM for four independent experiments. *p < 0.001 compared with the sham-operated group, one-way ANOVA followed by Tukey’s test. (C) Distribution of CHOP immunoreactivity in area CA1 after transient forebrain ischemia. Panel (a) is coronal section of the sham-operated group. Panels (b) and (c) were coronal sections prepared 24 h or 48 h after transient forebrain ischemia for 20 min. Scale bar, 50 mm.
mechanisms that still remain to be determined (Wang et al., 1996; Oyadomari and Mori, 2004). It has been shown that the level of CHOP mRNA in the cerebral cortex does not increase significantly after cerebral ischemia, whereas in the hippocampus it increases markedly relative to controls, and that perturbation of ER function plays a more prominent role in the hippocampus than in the cortex after transient cerebral ischemia. In order to clarify the mechanisms underlying ischemia/reperfusion-induced ER stress in neuronal and astroglial death, we characterized the expression of CHOP in area CA1 after ischemia/reperfusion. We showed that the level of CHOP was markedly increased 24 h after ischemia/reperfusion only in pyramidal neurons of area CA1. Subsequently, the induced CHOP disappeared 48 h after ischemia/reperfusion. In contrast, no CHOP signal was observed outside the pyramidal cell layer, where KDEL-positive astrocytes were located. It has been shown that cultured hippocampal neurons from CHOP-knockout mice are more resistant to hypoxia/reoxygenation-induced cell death than those from wild-type animals (Tajiri et al., 2004). Furthermore, ischemia-induced loss of neurons has been shown to be decreased in CHOP-knockout mice (Tajiri et al., 2004). These findings indicate that ischemia-associated ER stress is induced predominantly through the CHOP-dependent signaling pathway in neurons of the hippocampus. CHOP has also been shown to play a pivotal role in astroglial cell death induced by oxygen and glucose deprivation,
a commonly used in vitro model for ischemic insult (Benavides et al., 2005). However, our data clearly showed no evident signal for CHOP in astrocytes of area CA1 after ischemia/reperfusion. These results suggest that the CHOP signaling pathway is not involved in astroglial cell death in area CA1 after ischemic insult in vivo. In order to investigate the contributions of caspase-12, another apoptosis-associated protein activated by ER stress, to ischemiainduced cell death in neurons and in astrocytes of area CA1, the expression of caspase-12 was assessed by Western blotting and immunohistochemistry. Western blot analysis showed that the expression of cleaved caspase-12 was increased in the hippocampus, with a peak at 48 h after ischemia/reperfusion. Furthermore, enhanced immunoreactivity of caspase-12 was apparent in both neurons of the pyramidal cell layer and astrocytes located in its vicinity in area CA1 48 h after ischemia/reperfusion. GRP78 has been shown to form a complex with caspase-12, thus preventing its release from the ER (Rao et al., 2002). During this ER stress, GRP78 binds to unfolded proteins in the ER lumen, thereby releasing caspase-12 from the ER membrane and inducing apoptotic cell death (Rao et al., 2002). In this study, the signal for caspase-12 was co-localized with the signal for KDEL in both neurons and astrocytes after injury. Cortical cell culture studies have shown that astrocytes can undergo apoptosis as a result of many factors relevant to ischemia, including acidosis, oxidative
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Fig. 6. Expression of caspase-12 in the hippocampal CA1 astrocytes after ischemia/reperfusion. Western blot analysis of caspase-12 in the hippocampus after transient forebrain ischemia for 20 min followed by 1 h, 6 h, 12 h, 24 h and 48 h of reperfusion. (B) Relative amount of cleaved-caspase-12 was assessed by densitometric analysis. Values are expressed as the mean SEM for four independent experiments. *p < 0.05 compared with the sham-operated group, one-way ANOVA followed by Tukey’s test. (C) Double staining for caspase-12 (red) and KDEL (green) signals in area CA1 after transient forebrain ischemia. Panel (a), (d), and (g) are coronal section of the sham-operated group. Panels (b, e, and h) and (c, f, and i) are prepared 24 h and 48 h after transient forebrain ischemia for 20 min, respectively. Yellow indicates the co-localized of signals for caspase-12 (red) and KDEL (green) in area CA1. Scale bar, 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
stress, glucose deprivation, and cytokines (Giffard and Swanson, 2005). Studies of cerebral ischemia in some animal models have confirmed that astroglial cell death induced by traumatic brain injury or ischemic acidosis is mediated by the caspase-12dependent pathway in the cortex and hippocampus (Larner et al., 2004; Aoyama et al., 2005). However, in the transient forebrain ischemia model, the contribution of caspase-12 to astroglial cell death has remained unclear. This is the first report involving a mouse model to show that expression of caspase-12 is increased in astrocytes in area CA1 following transient forebrain
ischemia. These results suggest that the caspase-12-dependent pathway is involved not only in neuronal, but also in astroglial, ER stress-dependent cell death in area CA1 after transient forebrain ischemia. In this study, caspase-12-positive cells were identified not only in the neurons but also in the astrocytes 24 h after ischemia/ reperfusion, but the signal for KDEL was detected only in the neurons 24 h after reperfusion (Fig. 6C). It has been reported that astrocytes have the unique ability of being able to tolerate under ischemic and hypoxic conditions that lead to ER stress. For
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example, in astrocytes subjected to hypoxia, oxygen-regulated protein 150 kD (ORP150) has been shown to play a crucial role in the resistance of these cells to ER stress (Kuwabara et al., 1996; Tamatani et al., 2001). These results suggest that astrocyte-specific proteins or pathway may play a role in the different consequences of the expression of GRP78 between neurons and astrocytes. In conclusion, the present data clearly indicate that the caspase12-dependent pathway is involved in both neuronal and astroglial cell death induced by ER stress resulting from an ischemic insult, whereas CHOP is involved only in neuronal, and not in astroglial, cell death in area CA1 in the hippocampus. These results suggest that CHOP plays a critical role in the regulation of apoptosis in pyramidal neurons after transient forebrain ischemia but is not an ER-specific proapoptotic factor in astrocytes in area CA1. Since long-term recovery involving neurite outgrowth and neuronal regeneration after brain injury has been shown to be influenced by astrocytes (Chen and Swanson, 2003), the physiological role of delayed astroglial cell death after transient cerebral ischemia should be examined further. Acknowledgements We would like to thank Mr. A. Suda, Nihon University, for technical assistance with confocal laser microscopy. This work was supported by the Academic Frontier Project for Private Universities, and a matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, 2007–2009. References Aoyama, K., Burns, D.M., Suh, S.W., Garnier, P., Matsumori, Y., Shiina, H., Swanson, R.A., 2005. Acidosis causes endoplasmic reticulum stress and caspase-12-mediated astrocyte death. J. Cereb. Blood Flow Metab. 25, 358–370. Aschner, M., Sonnewald, U., Tan, K.H., 2002. Astrocyte modulation of neurotoxic injury. Brain Pathol. 12, 475–481. Benavides, A., Pastor, D., Santos, P., Tranque, ., Calvo, S., 2005. CHOP plays a pivotal role in the astrocyte death induced by oxygen and glucose deprivation. Glia 52, 261–275. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Chen, Y., Swanson, R.A., 2003. Astrocytes and brain injury. J. Cereb. Blood Flow Metab. 23, 137–149. DeGracia, D.J., Montie, H.L., 2004. Cerebral ischemia and the unfolded protein response. J. Neurochem. 91, 1–8. Dugan, L.L., Bruno, V.M., Amagasu, S.M., Giffard, R.G., 1995. Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity. J. Neurosci. 15, 4545–4555. Gehrmann, J., Bonnekoh, P., Miyazawa, T., Oschlies, U., Dux, E., Hossmann, K.A., Kreutzberg, G.W., 1992. The microglial reaction in the rat hippocampus following global ischemia: immuno-electron microscopy. Acta Neuropathol. 84, 588– 595. Giffard, R.G., Swanson, R.A., 2005. Ischemia-induced programmed cell death in astrocytes. Glia 50, 299–306. Hetz, C., Russelakis-Carneiro, M., Maundrell, K., Castilla, J., Soto, C., 2003. Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. EMBO J. 22, 5435–5445. Imai, T., Kosuge, Y., Ishige, K., Ito, Y., 2007. Amyloid b-protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures. Neuroscience 147, 639–651. Ishige, K., Takagi, N., Imai, T., Rausch, W.D., Kosuge, Y., Kihara, T., Kusama-Eguchi, K., Ikeda, H., Cools, A.R., Waddington, J.L., Koshikawa, N., Ito, Y., 2007. Role of caspase-12 in amyloid b-peptide-induced toxicity in organotypic hippocampal slices cultured for long periods. J. Pharmacol. Sci. 104, 46–55. Kadowaki, H., Nishitoh, H., Urano, F., Sadamitsu, C., Matsuzawa, A., Takeda, K., Masutani, H., Yodoi, J., Urano, Y., Nagano, T., Ichijo, H., 2005. Amyloid b induces neuronal cell death through ROS-mediated ASK1 activation. Cell Death Differ. 12, 19–24. Kato, H., Kogure, K., Araki, T., Itoyama, Y., 1995. Graded expression of immunomolecules on activated microglia in the hippocampus following ischemia in a rat model of ischemic tolerance. Brain Res. 694, 85–93. Kirino, T., 1982. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57–69.
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Characterization of neuronal and astroglial responses to ER stress in the hippocampal CA1 area in mice following transient forebrain ischemia Nobuhiro Osada, Yasuhiro Kosuge, Kumiko Ishige, Yoshihisa Ito * Research Unit of Pharmacology, Department of Clinical Pharmacy, School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 February 2010 Received in revised form 10 March 2010 Accepted 24 March 2010 Available online 31 March 2010
Transient forebrain ischemia has been shown to cause neuronal injury in the CA1 area of the hippocampus in mice. In addition to neuronal injury, astrocytes in area CA1 undergo apoptosis under ischemic conditions. Although failure of impaired astrocytes to take up glutamate is thought to contribute to the pathogenesis of cerebral ischemia, the molecular mechanism underlying this phenomenon remains unexplored. In the present study, we investigated neuronal and astroglial responses to endoplasmic reticulum (ER) stress, which is an important sequela of transient forebrain ischemia in the hippocampus of mice. Cellular injury was observed in area CA1 of the hippocampus 72 h after reperfusion, and ssDNA positivity was detectable in some glial cells as well as neurons in this area. An increase of 78-kDa glucose-regulated protein (GRP78), an indicator of ER stress, was detected in pyramidal neurons and astrocytes in this area after the insult. Immunohistochemical analysis showed that caspase-12 was increased in pyramidal neurons and astrocytes located in the extrapyramidal cell layer. Immunoreactivity for C/EBP homologous protein (CHOP) was increased significantly in pyramidal cells but not in astrocytes. These results suggest that astrocytes as well as pyramidal neurons in area CA1 undergo apoptosis through an ER stress-dependent mechanism after ischemia. Unlike the situation in neuronal apoptosis, CHOP does not play a role in the cell death of astrocytes. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Endoplasmic reticulum stress Caspase-12 Astrocyte Forebrain ischemia Hippocampus
1. Introduction Transient forebrain ischemia causes delayed loss of pyramidal neurons in area CA1 of the hippocampus, whereas the neighboring CA3 area and dentate gyrus (DG), and most cortical neurons, remain essentially intact (Kirino, 1982; Osada et al., 2009). Transient cerebral ischemia elicits activation and proliferation of microglia and astrocytes (Petito et al., 1990; Gehrmann et al., 1992; Kato et al., 1995). Astrocytes are the predominant glial cell type in the brain and are essential for neuronal survival and synaptic function, as well as for neurogenesis and neural repair (Aschner et al., 2002; Swanson et al., 2004). Moreover, astrocytic death has been found after focal ischemia in the rat brain (Liu et al., 1999). A recent study has demonstrated that astrocytes might modulate the pathogenesis of many acute and chronic neurodegenerative disorders, such as cerebral ischemia and neurodegenerative
Abbreviations: BCCA, bilateral common carotid artery; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; DG, dentate gyrus; DTT, dithiothreitol; GRP78, 78-kDa glucose-regulated protein; H&E, hematoxylin and eosin; PBS, phosphatebuffered saline; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethane sulfonyl fluoride; SDS, sodium dodecyl sulfate; ssDNA, single-stranded DNA. * Corresponding author. Tel.: +81 47 465 5832; fax: +81 47 465 5832. E-mail address: [email protected] (Y. Ito). 0197-0186/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2010.03.017
conditions such as Alzheimer’s disease and Parkinson’s disease (Takuma et al., 2004). The classical function of astrocytes during ischemia is uptake of glutamate to minimize any excitotoxic insult to neighboring neurons (Dugan et al., 1995). Failure of impaired astrocytes to take up and release glutamate is thought to contribute to ischemia-induced neuronal death (Swanson et al., 2004). However, the mechanisms involved in this impairment have not been fully explained. Endoplasmic reticulum (ER) stress has been shown to be involved in some neuronal pathologies such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and prionrelated disorders (LaFerla, 2002; Ryu et al., 2002; Hetz et al., 2003; Nishitoh et al., 2008). The ER plays a critical role in a variety of processes, including protein synthesis and folding and maintenance of Ca2+ homeostasis. A number of studies have demonstrated that ER stress disrupts these normal functions, and thus the ER has become a focus of interest with regard to its role in cellular pathology (Lin et al., 2008). When mild ER stress occurs, cells develop a self-protective signal transduction pathway termed the unfolded protein response (UPR), which includes the induction of molecular chaperones in the ER, translational attenuation, and enhancement of ER-associated degradation, thus relieving cells from the stress. However, if the damage is too severe to repair, the UPR ultimately initiates the apoptosis pathway, and damaged cells
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are eliminated in order to protect the organism (Kozutsumi et al., 1988; Ron, 2002). Several proteins have been implicated in this apoptotic pathway, including a transcription factor, C/EBP homologous protein (CHOP), also known as gadd 153 (Oyadomari et al., 2002), the ER-resident caspase, caspase-12 (Nakagawa et al., 2000; Kosuge et al., 2003), and c-Jun N-terminal protein kinase (JNK) (Kadowaki et al., 2005). Caspase-12 is localized specifically on the cytoplasmic side of the ER and is thought to play a pivotal role in ER stress-induced cell death in the rodent brain (Nakagawa and Yuan, 2000; Nakagawa et al., 2000). Our previous studies showed that neuronal death induced by amyloid b-peptide and tunicamycin, an ER stress inducer, was mainly dependent upon the pathway mediated by caspase-12 in rat cultured hippocampal neurons (Kosuge et al., 2003, 2006) and organotypic hippocampal slice cultures (Imai et al., 2007; Ishige et al., 2007). CHOP is a proapoptotic transcription factor expressed at very low levels under physiological conditions but is induced extensively in response to ER stress, thus causing apoptosis (Wang et al., 1996; Oyadomari and Mori, 2004). Recent studies have also demonstrated that ER stress plays a key role in neuronal death resulting from ischemia (Paschen and Doutheil, 1999; DeGracia and Montie, 2004; Osada et al., 2009). However, the underlying mechanism responsible for ER stress in astrocytes after transient forebrain ischemia is yet to be clarified. In order to elucidate the pathophysiological mechanism of ischemia/reperfusion-induced ER stress in astrocytes, we performed experiments to compare the expressions of ER stressassociated signal proteins, including GRP 78, CHOP and caspase-12, in neurons and astrocytes in area CA1 of the hippocampus after transient forebrain ischemia. 2. Materials and methods 2.1. Animals Male C57BL/6 mice aged 9–10 weeks were used in this study. Animals used in the present study were fed standard laboratory chow and given free access to water prior to surgery. All efforts were made to minimize the number of animals used and their suffering. All experiments with animals have done in compliance with the Ethical Guidelines for Animal Experiments at Nihon University. 2.2. Transient forebrain ischemia Transient forebrain ischemia was produced by bilateral common carotid artery (BCCA) occlusion as described previously (Osada et al., 2009). Briefly, surgical procedures were performed under chloral hydrate anesthesia (450 mg/kg, i.p.) and the rectal temperature was maintained at 37.5 0.5 8C with a heating blanket (Animal Blanket Controller, Nihon Kohden, Tokyo, Japan). Measurement of cortical perfusion was performed by omega flow meter (OMEGAWAVE, Inc., Tokyo, Japan). BCCA were occluded using small arterial clips for 20 min, after which time the clips were removed, the restoration of blood flow was visually confirmed, the incision was closed, and reperfusion was continued for the indicated time. Sham-operated mice underwent exposure of vessels without withdrawal of blood or clamping of carotid arteries. 2.3. Histological assessment Anesthetized animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed for 24 h in the same fixative. Post-fixed brains were embedded in paraffin and sliced on a microtome at a 6 mm thickness. The sections between 1.5 mm and 2.5 mm posterior of the bregma were used for this study. For histological assessment of damage to the hippocampus, the paraffin-embedded brain sections were stained with hematoxylin and eosin (H&E) or single-stranded DNA (ssDNA) staining. The sections were pretreated with 0.1% trypsin in phosphate buffered saline (PBS) for 20 min at 37 8C, incubated with 0.3% H2O2 in methanol for 15 min, appropriate 1.5% blocking normal goat serum for 1 h at room temperature and exposed to the anti-ssDNA antibody (DAKO, Japan) at dilution 1:500 for 24 h at 4 8C. They were then incubated in the biotin-conjugated IgG (Vector Laboratories, Burlingame, CA) against the host of the primary antibody for 1 h and then incubated with avidin–biotin–peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories) for 60 min. Then the staining was visualized using 0.025% 3,30 -diaminobenzidine (DAB) and 0.075% H2O2 in Tris– HCl (pH 7.6). Negative controls were prepared identically expect for omission of the primary antibody.
2.4. Western blotting Following transient forebrain ischemia, mice were sacrificed under the sodium pentobarbital anesthesia at 1 h, 6 h, 12 h, 24 h and 48 h after ischemia. Shamoperated animals served as controls. Brains were rapidly microdissected on an icechilled plate. The hippocampus was homogenized using homogenizer (Homogenizer, Subsonic, Iwaki Glass Co., Ltd) in 800 mL homogenized buffer containing 10 mM HEPES-NaOH (pH 7.9), 10 mM potassium chloride, 0.1 mM EDTA, 0.1 mM EGTA, 1 M phenylmethane sulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 62.5 mL/mL protease inhibitor cocktail and 62.5 mL/mL 10% Nonidet P-40, centrifuged, and then supernatants were used. Protein concentrations were determined using the method of Bradford (1976). Protein extracts were mixed 1:3 in 4 sample buffer containing 125 mM Tris–HCl (pH 6.0), 3% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 10% glycerol and 0.2% bromophenol blue, separated by 5–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) paper (Millipore, MA, USA). The membranes were treated for 1 h with a blocking buffer containing 20 mM Tris–HCl (pH 7.6), 137 mM NaCl, 5% skim milk at room temperature and then incubated overnight at 4 8C with a 1:1000 dilution of anti-caspase-12 monoclonal antibody (Sigma, USA), a 1:1000 dilution of anti-CHOP polyclonal antibody (Santa Cruz Biotechnology, USA), or a 1:2000 dilution of anti-KDEL monoclonal antibody (StressGen, Canada). The membranes were washed repeatedly in TTBS containing 20 mM Tris–HCl (pH 7.6), 137 mM NaCl, 0.05% Tween 20, then incubated with a 1:10,000 dilution of HRP-conjugated secondary antibody for 1 h. Immunoreactive bands were detected by ECL (Amersham Pharmacia Biotech). To adjust for protein loading, membranes were also immunostained with anti-bactin monoclonal antibody (Sigma, USA). Optical density on the blots was measured with Scion imaging software (www.scioncorp.com). 2.5. Immunohistochemistry For immunohistochemistry, post-fixed brains were immersed for 24 h in PBS containing 30% sucrose and coronally sectioned on a cryostat at an 18 mm thickness. After blocking nonspecific binding with 1.5% normal goat serum, the sections were incubated primary antibodies for 24 h at 4 8C. The primary antibodies were used and the dilutions for each were anti-KDEL monoclonal antibody at 1:200, anti-GFAP polyclonal antibody (Chemicon International, USA) at 1:200, anti-CHOP polyclonal antibody at 1:200 and anti-caspase-12 polyclonal antibody (Exalpha Biologicals, USA) at 1:500. After washing with PBS, the sections were incubated for 1 h with a 1:500 dilution of Alexa Fluor 488-conjugated goat IgG, or Alexa Fluor 594-conjugated goat IgG (Molecular Probes, USA). Some sections were counterstained by incubation for 10 min with To-PRO3 (Molecular Probes, USA). After rinsing with PBS, the sections were analyzed using a confocal laser microscope (Zeiss LSM-510, Germany). Negative controls were prepared by omitting the primary antibodies. 2.6. Statistical analysis All the data are expressed as mean SEM. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple tests.
3. Results 3.1. Histological evaluation of area CA1 of the hippocampus after ischemia/reperfusion Neuronal injury in area CA1 was observed 72 h after transient forebrain ischemia. Histological evaluation of hippocampal neurons by hematoxylin and eosin staining (Fig. 1A and B) and ssDNA staining (Fig. 1C and D) revealed that pyramidal neurons in the CA1 region had degenerated (with pyknotic, shrunken nuclei in H&Estained sections, Fig. 1B). Most of the morphologically damaged neurons became positive for ssDNA staining (Fig. 1D). ssDNA positivity was also detectable in cells located in the vicinity of the pyramidal cell layer in area CA1 after ischemia/reperfusion (Fig. 1D). 3.2. Temporal profiles of GRP78 in the hippocampus after ischemia/ reperfusion In order to investigate the induction of the ER chaperone, GRP78, that contains a characteristic COOH-terminal Lys-Asp-GluLeu (KDEL) sequence, we examined GRP78 expression by Western blotting and immunohistochemistry using an anti-KDEL monoclonal antibody after ischemia/reperfusion. Western blot analysis
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Fig. 1. Histological assessment of area CA1 after ischemia/reperfusion. The upper panels (A and B) show hematoxylin and eosin (H&E) staining in area CA1 and the bottom panels (C and D) show ssDNA staining. Panels A and C are sham-operated coronal sections. Panels B and D are sections prepared 72 h after transient forebrain ischemia for 20 min. Arrows indicate ssDNA positivity in cells located in the vicinity of the pyramidal cell layer. Scale bar, 100 mm.
showed that GRP78 expression was increased in the hippocampus after transient forebrain ischemia, with a peak expression level at 12–48 h after reperfusion (Fig. 2). To identify the cell types in which GRP78 was up-regulated, we performed immunofluorescence staining for GRP78 in area CA1 after ischemia/reperfusion. Although very low immunoreactivity for KDEL was confirmed in the sham-operated groups (Fig. 3A), the immunoreactivity was strongly increased 24 h after ischemia/ reperfusion mainly in the pyramidal cells (Fig. 3B). The immunoreactivity for KDEL tended to decrease in the pyramidal cells 48 h after ischemia/reperfusion; however, the immunoreactivity was
clearly defined in non-neuronal cells in the vicinity of the pyramidal cell layer (Fig. 3C). Next, we performed double immunofluorescence staining for KDEL and GFAP, a marker of astrocytes, 48 h after ischemia/reperfusion. Confocal images revealed that the signal for KDEL was co-localized with the GFAP signal, indicating that most of the KDEL-positive cells were astrocytes (Fig. 4). 3.3. Induction of CHOP protein in pyramidal cells of the hippocampus after ischemia/reperfusion In order to determine the involvement of CHOP in these cell death pathways in neurons and astrocytes after ischemia/reperfusion, we investigated the induction of CHOP after transient forebrain ischemia. Western blot analysis showed that CHOP expression was barely detectable in the sham-operated group. In contrast, a transient and significant increase of CHOP protein was observed 24 h after ischemia/reperfusion in the hippocampus (Fig. 5A and B). Although immunohistochemical analysis for CHOP in area CA1 demonstrated almost no immunoreactivity in the sham-operated groups (Fig. 5C), the signal for CHOP was identified in pyramidal neurons 24 h after ischemia/reperfusion and disappeared after 48 h. However, induction of CHOP was not observed in the extrapyramidal cell layer where KDEL-positive astrocytes were located 48 h after ischemia/reperfusion. These results were consistent with the timewindow observed in Western blot analysis, suggesting that CHOP functions in neurons but not in astrocytes. 3.4. Expression of caspase-12 in astrocytes after ischemia/reperfusion
Fig. 2. Change in the level of GRP78 expression in the hippocampus after ischemia/ reperfusion. (A) Western blot analysis of GRP78 in the hippocampus after transient forebrain ischemia for 20 min followed by 1 h, 6 h, 12 h, 24 h and 48 h of reperfusion. (B) Relative amount of GRP78 was assessed by densitometric analysis. Values are expressed as the mean SEM for four independent experiments. *p < 0.05, **p < 0.01 compared with the sham-operated group, one-way ANOVA followed by Tukey’s test.
Next, we investigated the expression of the cleaved form of caspase-12 in the hippocampus after ischemia/reperfusion. Western blotting showed that the immunoreactivity of cleaved caspase-12 increased from 24 h and peaked at 48 h after ischemia/ reperfusion (Fig. 6A and B). We then determined the localization of caspase-12 in coronal sections by immunohistochemical staining. In the sham-operated mice, immunoreactivity for caspase-12 was very low in area CA1 (Fig. 6C). Caspase-12-positive cells were detected not only in the pyramidal cell layer but also in its vicinity 24 h after ischemia/reperfusion, and the number of caspase-12positive cells was increased at 48 h (Fig. 6C). Immunoreactivity for KDEL was identified only in the pyramidal cell layer 24 h after reperfusion; however, it was also identified in the vicinity of the
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Fig. 3. Immunofluorescence staining for KDEL (GRP78) in area CA1 after ischemia/reperfusion. Panel A is coronal sections of the sham-operated group. Panels B and C are coronal sections prepared 24 h or 48 h after transient forebrain ischemia for 20 min. Arrows indicated some glial cells. Scale bar, 20 mm.
Fig. 4. Double-labeling confocal immunofluorescence microscopy staining of KDEL (GRP78) and GFAP in area CA1 after ischemia/reperfusion. The hippocampus was taken for analysis at 48 h after ischemia/reperfusion. (A) Some glial cells were costained with KDEL (green) and GFAP (red) in area CA1 (arrows). Nuclei were counterstained with TOPRO3 (blue). Panels B–D show magnified views of the area indicated in panel A. Yellow indicates the co-localized of signals for KDEL (green, B) and GFAP (red, C) in area CA1. Scale bar, 20 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
pyramidal cell layer 48 h after reperfusion. KDEL and caspase-12 signals were co-localized not only in pyramidal cells but also in cells located outside the pyramidal cell layer. 4. Discussion Neuronal death in area CA1 of the hippocampus after forebrain ischemia has been shown to occur in a delayed manner (Kirino, 1982). There is a growing evidence that this neuronal death is at least partly due to apoptosis (MacManus et al., 1993; Nitatori et al., 1995; Osada et al., 2009). Recently, it has been demonstrated that astrocytes also undergo apoptosis under ischemic conditions (Giffard and Swanson, 2005). In the present study, we showed that not only neurons but also astrocytes located outside the pyramidal cell layer in area CA1 were injured, and that both types of cells showed positive ssDNA staining 72 h after ischemia/reperfusion. These results indicate that not only neurons but also non-neuronal cells in area CA1 undergo apoptosis after transient forebrain ischemia.
There is a growing evidence that ER stress plays an important role in neuronal death caused by brain ischemia (Paschen and Doutheil, 1999; Kumar et al., 2003; Tajiri et al., 2004; Osada et al., 2009). Consistently, our data showed that the level of GRP78 was significantly increased in the hippocampus, and immunoreactivity for GRP78 was detected in the pyramidal cell layer of area CA1 24 h after ischemia/reperfusion. We also found that immunoreactivity for KDEL appeared in astrocytes located in the vicinity of the pyramidal cell layer 48 h after ischemia/reperfusion. These results suggest that ER stress is a key pathological mechanism not only in neuronal, but also in astroglial, cell death after transient forebrain ischemia in area CA1 of the hippocampus. Although failure of impaired astrocytes to take up glutamate is thought to contribute to the pathogenesis of cerebral ischemia (Swanson et al., 2004), the molecular pathways responsible for astroglial cell death have not been adequately characterized (Takuma et al., 2004; Ouyang et al., 2007). CHOP is expressed at very low levels under physiological conditions, but strongly induced in response to ER stress through
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Fig. 5. Induction of CHOP protein in pyramidal cells of the hippocampus after ischemia/reperfusion. (A) Western blot analysis of CHOP in the hippocampus after transient forebrain ischemia for 20 min followed by 1 h, 6 h, 12 h, 24 h and 48 h of reperfusion. (B) Relative amount of CHOP was assessed by densitometric analysis. Values are expressed as the mean SEM for four independent experiments. *p < 0.001 compared with the sham-operated group, one-way ANOVA followed by Tukey’s test. (C) Distribution of CHOP immunoreactivity in area CA1 after transient forebrain ischemia. Panel (a) is coronal section of the sham-operated group. Panels (b) and (c) were coronal sections prepared 24 h or 48 h after transient forebrain ischemia for 20 min. Scale bar, 50 mm.
mechanisms that still remain to be determined (Wang et al., 1996; Oyadomari and Mori, 2004). It has been shown that the level of CHOP mRNA in the cerebral cortex does not increase significantly after cerebral ischemia, whereas in the hippocampus it increases markedly relative to controls, and that perturbation of ER function plays a more prominent role in the hippocampus than in the cortex after transient cerebral ischemia. In order to clarify the mechanisms underlying ischemia/reperfusion-induced ER stress in neuronal and astroglial death, we characterized the expression of CHOP in area CA1 after ischemia/reperfusion. We showed that the level of CHOP was markedly increased 24 h after ischemia/reperfusion only in pyramidal neurons of area CA1. Subsequently, the induced CHOP disappeared 48 h after ischemia/reperfusion. In contrast, no CHOP signal was observed outside the pyramidal cell layer, where KDEL-positive astrocytes were located. It has been shown that cultured hippocampal neurons from CHOP-knockout mice are more resistant to hypoxia/reoxygenation-induced cell death than those from wild-type animals (Tajiri et al., 2004). Furthermore, ischemia-induced loss of neurons has been shown to be decreased in CHOP-knockout mice (Tajiri et al., 2004). These findings indicate that ischemia-associated ER stress is induced predominantly through the CHOP-dependent signaling pathway in neurons of the hippocampus. CHOP has also been shown to play a pivotal role in astroglial cell death induced by oxygen and glucose deprivation,
a commonly used in vitro model for ischemic insult (Benavides et al., 2005). However, our data clearly showed no evident signal for CHOP in astrocytes of area CA1 after ischemia/reperfusion. These results suggest that the CHOP signaling pathway is not involved in astroglial cell death in area CA1 after ischemic insult in vivo. In order to investigate the contributions of caspase-12, another apoptosis-associated protein activated by ER stress, to ischemiainduced cell death in neurons and in astrocytes of area CA1, the expression of caspase-12 was assessed by Western blotting and immunohistochemistry. Western blot analysis showed that the expression of cleaved caspase-12 was increased in the hippocampus, with a peak at 48 h after ischemia/reperfusion. Furthermore, enhanced immunoreactivity of caspase-12 was apparent in both neurons of the pyramidal cell layer and astrocytes located in its vicinity in area CA1 48 h after ischemia/reperfusion. GRP78 has been shown to form a complex with caspase-12, thus preventing its release from the ER (Rao et al., 2002). During this ER stress, GRP78 binds to unfolded proteins in the ER lumen, thereby releasing caspase-12 from the ER membrane and inducing apoptotic cell death (Rao et al., 2002). In this study, the signal for caspase-12 was co-localized with the signal for KDEL in both neurons and astrocytes after injury. Cortical cell culture studies have shown that astrocytes can undergo apoptosis as a result of many factors relevant to ischemia, including acidosis, oxidative
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Fig. 6. Expression of caspase-12 in the hippocampal CA1 astrocytes after ischemia/reperfusion. Western blot analysis of caspase-12 in the hippocampus after transient forebrain ischemia for 20 min followed by 1 h, 6 h, 12 h, 24 h and 48 h of reperfusion. (B) Relative amount of cleaved-caspase-12 was assessed by densitometric analysis. Values are expressed as the mean SEM for four independent experiments. *p < 0.05 compared with the sham-operated group, one-way ANOVA followed by Tukey’s test. (C) Double staining for caspase-12 (red) and KDEL (green) signals in area CA1 after transient forebrain ischemia. Panel (a), (d), and (g) are coronal section of the sham-operated group. Panels (b, e, and h) and (c, f, and i) are prepared 24 h and 48 h after transient forebrain ischemia for 20 min, respectively. Yellow indicates the co-localized of signals for caspase-12 (red) and KDEL (green) in area CA1. Scale bar, 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
stress, glucose deprivation, and cytokines (Giffard and Swanson, 2005). Studies of cerebral ischemia in some animal models have confirmed that astroglial cell death induced by traumatic brain injury or ischemic acidosis is mediated by the caspase-12dependent pathway in the cortex and hippocampus (Larner et al., 2004; Aoyama et al., 2005). However, in the transient forebrain ischemia model, the contribution of caspase-12 to astroglial cell death has remained unclear. This is the first report involving a mouse model to show that expression of caspase-12 is increased in astrocytes in area CA1 following transient forebrain
ischemia. These results suggest that the caspase-12-dependent pathway is involved not only in neuronal, but also in astroglial, ER stress-dependent cell death in area CA1 after transient forebrain ischemia. In this study, caspase-12-positive cells were identified not only in the neurons but also in the astrocytes 24 h after ischemia/ reperfusion, but the signal for KDEL was detected only in the neurons 24 h after reperfusion (Fig. 6C). It has been reported that astrocytes have the unique ability of being able to tolerate under ischemic and hypoxic conditions that lead to ER stress. For
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example, in astrocytes subjected to hypoxia, oxygen-regulated protein 150 kD (ORP150) has been shown to play a crucial role in the resistance of these cells to ER stress (Kuwabara et al., 1996; Tamatani et al., 2001). These results suggest that astrocyte-specific proteins or pathway may play a role in the different consequences of the expression of GRP78 between neurons and astrocytes. In conclusion, the present data clearly indicate that the caspase12-dependent pathway is involved in both neuronal and astroglial cell death induced by ER stress resulting from an ischemic insult, whereas CHOP is involved only in neuronal, and not in astroglial, cell death in area CA1 in the hippocampus. These results suggest that CHOP plays a critical role in the regulation of apoptosis in pyramidal neurons after transient forebrain ischemia but is not an ER-specific proapoptotic factor in astrocytes in area CA1. Since long-term recovery involving neurite outgrowth and neuronal regeneration after brain injury has been shown to be influenced by astrocytes (Chen and Swanson, 2003), the physiological role of delayed astroglial cell death after transient cerebral ischemia should be examined further. Acknowledgements We would like to thank Mr. A. Suda, Nihon University, for technical assistance with confocal laser microscopy. This work was supported by the Academic Frontier Project for Private Universities, and a matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, 2007–2009. References Aoyama, K., Burns, D.M., Suh, S.W., Garnier, P., Matsumori, Y., Shiina, H., Swanson, R.A., 2005. Acidosis causes endoplasmic reticulum stress and caspase-12-mediated astrocyte death. J. Cereb. Blood Flow Metab. 25, 358–370. Aschner, M., Sonnewald, U., Tan, K.H., 2002. Astrocyte modulation of neurotoxic injury. Brain Pathol. 12, 475–481. Benavides, A., Pastor, D., Santos, P., Tranque, ., Calvo, S., 2005. CHOP plays a pivotal role in the astrocyte death induced by oxygen and glucose deprivation. Glia 52, 261–275. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Chen, Y., Swanson, R.A., 2003. Astrocytes and brain injury. J. Cereb. Blood Flow Metab. 23, 137–149. DeGracia, D.J., Montie, H.L., 2004. Cerebral ischemia and the unfolded protein response. J. Neurochem. 91, 1–8. Dugan, L.L., Bruno, V.M., Amagasu, S.M., Giffard, R.G., 1995. Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity. J. Neurosci. 15, 4545–4555. Gehrmann, J., Bonnekoh, P., Miyazawa, T., Oschlies, U., Dux, E., Hossmann, K.A., Kreutzberg, G.W., 1992. The microglial reaction in the rat hippocampus following global ischemia: immuno-electron microscopy. Acta Neuropathol. 84, 588– 595. Giffard, R.G., Swanson, R.A., 2005. Ischemia-induced programmed cell death in astrocytes. Glia 50, 299–306. Hetz, C., Russelakis-Carneiro, M., Maundrell, K., Castilla, J., Soto, C., 2003. Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. EMBO J. 22, 5435–5445. Imai, T., Kosuge, Y., Ishige, K., Ito, Y., 2007. Amyloid b-protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures. Neuroscience 147, 639–651. Ishige, K., Takagi, N., Imai, T., Rausch, W.D., Kosuge, Y., Kihara, T., Kusama-Eguchi, K., Ikeda, H., Cools, A.R., Waddington, J.L., Koshikawa, N., Ito, Y., 2007. Role of caspase-12 in amyloid b-peptide-induced toxicity in organotypic hippocampal slices cultured for long periods. J. Pharmacol. Sci. 104, 46–55. Kadowaki, H., Nishitoh, H., Urano, F., Sadamitsu, C., Matsuzawa, A., Takeda, K., Masutani, H., Yodoi, J., Urano, Y., Nagano, T., Ichijo, H., 2005. Amyloid b induces neuronal cell death through ROS-mediated ASK1 activation. Cell Death Differ. 12, 19–24. Kato, H., Kogure, K., Araki, T., Itoyama, Y., 1995. Graded expression of immunomolecules on activated microglia in the hippocampus following ischemia in a rat model of ischemic tolerance. Brain Res. 694, 85–93. Kirino, T., 1982. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57–69.
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