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Apolipoprotein E-deficient mice are more vulnerable to ER stress after transient forebrain ischemia
Neurochemistry International 54 (2009) 403–409
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Apolipoprotein E-deficient mice are more vulnerable to ER stress after transient forebrain ischemia Nobuhiro Osada, Yasuhiro Kosuge, Tetsuroh Kihara, Kumiko Ishige, Yoshihisa Ito * Research Unit of Pharmacology, Department of Clinical Pharmacy, College 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 November 2008 Received in revised form 15 January 2009 Accepted 19 January 2009 Available online 6 February 2009
Apolipoprotein E-deficient (apoE/) mice have been shown to have increased vulnerability to neuronal damage induced by cerebral ischemia; however, the mechanism of this increased vulnerability remains unclear. In order to define the role of the apoE protein against ischemia-induced ER stress and cell death, experiments were performed to compare ER stress-associated chaperones and signal proteins in the hippocampus of apoE/ mice to those of WT mice after being subjected to forebrain ischemia and reperfusion. Although neuronal loss in area CA1–CA3 of the hippocampus was observed 3 days after ischemia in both types of mice, the damage in apoE/ mice was more severe. In apoE/ mice, a more extensive increase in 78-kDa glucose-regulated protein (GRP78) was observed after the insult, whereas the level of GRP94 was not changed. The expression of both C/EBP homologous protein (CHOP) and caspase-12 was increased in the hippocampus in both WT and apoE/ mice after ischemia. The increased levels of CHOP in apoE/ mice were significantly higher than those in WT mice, whereas the levels of caspase-12 in the two were comparable. Furthermore, whereas the levels of c-Jun N-terminal kinase (JNK), p-JNK1 and p-JNK2 in WT mice were unchanged after ischemia, they were significantly increased in apoE/ mice 24 h and 48 h after ischemia. These results suggest that increased vulnerability of the hippocampus to forebrain ischemia and reperfusion in apoE/ mice is at least partly attributable to perturbed induction of an ER chaperone, GRP 94, and enhancement of the CHOP- and JNKdependent apoptotic pathway in the hippocampus. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: apoE/ mice Forebrain ischemia ER stress Hippocampus
1. Introduction Apoptosis plays a pivotal role in neuronal cell death resulting from ischemic stroke. Several lines of evidence suggest that apoptosis is regulated by multiple pathways. The two most wellstudied pathways are the cell surface death receptor pathway and the mitochondria-initiated pathway (Budihardjo et al., 1999). Recently, another apoptosis-regulatory pathway involved in ER stress has been receiving attention. The condition in which ER function is impaired, so-called ER stress, can lead to an accumulation of unfolded or malfolded proteins in the ER lumen (Kaufman, 1999). In the case of mild ER stress, cells develop a selfprotective 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 (Cudna and Dickson, 2003), thus relieving cells from the stress. However, if the damage is too severe to repair, the UPR ultimately initiates the apoptotic pathway (Oyadomari and Mori, 2004; Cudna and Dickson, 2003). Several
* Corresponding author. Tel.: +81 474 65 5832; fax: +81 474 65 5832. E-mail address: [email protected] (Y. Ito). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.01.010
proteins have been implicated in this apoptotic pathway, including a transcription factor, C/EBP homologous protein (CHOP) (Wang et al., 1996), and the ER-resident caspase, caspase-12 (Nakagawa et al., 2000). In humans, ER stress has been shown to be involved in not only ischemia but also some neuronal diseases, such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and prion-related disorders (LaFerla, 2002; Ryu et al., 2002; Imai et al., 2001; Nishitoh et al., 2008; Hetz et al., 2003). Recent evidence indicates that apolipoprotein E (apoE) plays a central role in CNS injury, ALS–parkinsonism dementia complex (Wilson and Shaw, 2007) and the interactions of herpes simplex products (Carter, 2008). Following brain injury, the synthesis of apoE is markedly increased, and it is secreted, predominantly from astrocytes, into the extracellular space (Poirier, 1994). The expression of apoE increased in the selectively vulnerable hippocampus following transient forebrain ischemia (Hall et al., 1995). Neuronal apoE immunoreactivity was significantly increased in all hippocampal areas (CA1, CA2, CA3/4, dentate fascia) of the human brain following global ischemia (Horsburgh et al., 1999a). It is thought that intraneuronal apoE plays a pivotal role in a protective response following injury by providing lipids for neuronal repair and remodeling. Insight into the role of apoE in brain injury has been provided by the development of genetically
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modified mice. It has been shown that apoE/ mice have a poorer outcome in a focal model of ischemia (Laskowitz et al., 1997), and that this response may be isoform-specific (Sheng et al., 1998). These findings support the suggestion that apoE has a protective effect against ischemic neuronal damage; however, the molecular mechanism underlying the apoE-mediated protection against ischemic neuronal damage remains unclear. Brain ischemia in mice has been shown to cause ER-stress-mediated apoptosis of cells in the striatum and hippocampus, and CHOP plays a crucial role in this cell death (Tajiri et al., 2004). More recent studies have demonstrated that markers of ER stress and UPR activation are induced dramatically in macrophages at all stages of atherosclerosis development in apoE/ mice (Zhou et al., 2005). Taken together, these findings suggest that apoE protects neurons against ischemic cell death by attenuating ER stress during the injury. In order to define the role of the apoE protein against ischemiainduced ER stress and cell death in the hippocampus, experiments were performed to compare ER stress-associated signal proteins including ER chaperones, GRP 78 and 94, CHOP, caspase-12 and cJun N-terminal kinase (JNK) activity in the hippocampus in apoE/ mice and wild type (WT) mice after being subjected to forebrain ischemia and reperfusion. We found that the increased vulnerability of hippocampal neurons to ischemic damage was at least partly attributable to disturbed induction of ER chaperones and higher UPR activation. 2. Experimental procedures 2.1. Animals All experimental procedures were approved by the Institutional Animal Center Use Committee of Graduate School of Pharmacy, Nihon University. ApoE/ mice, originally produced by Zhang et al. (1992), were purchased from the Jackson Laboratory (Bar Harbor, ME) and back-crossed to WT C57BL/6 mice (Charles River Inc., Yokohama, Japan) to have genetic backgrounds of the homozygote. Both mice were bred in this study. Animals were fed standard laboratory chow and given free access to water prior to surgery. After mating of heterozygote, we selected the homozygous and WT mice by polymerase chain reaction (PCR) amplification of genomic DNA extracted from tails. Male mice (10–12 week old) were used in the present study.
staining that indicated apoptotic cells, paraffin sections were processed immunohistochemically. Briefly, 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 (DAKO, Japan) antibodies (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 immunoreactivities were detected with 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. For cell-counting procedure, the number of morphologically damaged ssDNA positive neurons and viable neurons in hippocampal CA1–CA3 regions were manually counted in four brain slices in each animal and the percentage of ssDNA positive neurons was calculated. The results in each group were presented as the mean S.E.M. 2.4. Western blotting Following transient forebrain ischemia, the mice were sacrificed under the sodium pentobarbital anesthesia at 24 h and 48 h after reperfusion. Sham-operated animals served as controls. Brains were rapidly microdissected on an ice-chilled plate. The hippocampus was homogenized using homogenizer (Homogenizer, Subsonic, IWAKI GLASS CO., LTD) in 800 mL 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, phosphatase 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. Aliquots from each sample (10 mg protein/lane) were separated by 7.5–12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) paper (Millipore, MA, USA) (100 V, 90 min). The membranes were blocked in blocking buffer containing 20 mM Tris– HCl (pH 7.6), 137 mM NaCl, 5% skim milk for 1 h at room temperature and then treated with anti-caspase-12 (SIGMA, USA) antibody (diluted 1:1000), anti-CHOP (Santa Cruz Biotechnology, USA) antibody (diluted 1:1000), anti-KDEL (StressGen, Canada) antibody (diluted 1:2000) or JNK antibody (diluted 1:1000), Phospho-JNK (Cell Signaling Technology, Boston, MA, USA) antibody (diluted 1:1000) overnight at 4 8C. The membranes were washed repeatedly in TTBS containing 20 mM Tris– HCl (pH 7.6), 137 mM NaCl, 0.05% Tween 20, then HRP-conjugated secondary antibody (diluted 1:20,000) was added for 1 h. Immunoreactive bands were detected by electrochemiluminescence (ECL, Amersham Pharmacia Biotech). Optical density on the blots was measured with Scion imaging software (www.scioncorp.com).
2.2. Transient forebrain ischemia 2.5. Immunofluoresence Transient forebrain ischemia was created by bilateral common carotid artery (BCCA) occlusion as described previously (Kitagawa et al., 1998b). Surgical procedures were performed under chloral hydrate anesthesia (450 mg/kg, i.p.) and the rectal temperature was kept at 37 1.5 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). In order to measure cortical perfusion by laser Doppler blood-flowmetry, a polyacrylamide column for measurement was attached to the intact skull with dental cement, 3.5 mm lateral bregma. For procedures involving BCCA occlusion, the arteries were approached through a midline cervical incision. Both arteries were identified and ligated with small arterial clips for 20 min, after which time the clips were removed, the return of these arteries blood flow was visually confirmed, the incision was closed, and reperfusion was continued for the indicated time. Sham-operated mice received similar operative procedures; however, the arteries, after isolation, were not occluded and incision was closed. All the mice were killed with a lethal dose of the sodium pentobarbital before recovery of tissues.
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 anti-CHOP antibody (diluted 1:200) for 24 h at 4 8C. After washing with PBS, the sections were incubated for 1 h with Alexa Fluor 488-conjugated goat IgG (diluted 1:500, Molecular Probes, USA). After rinsing with PBS, the sections were analyzed using a confocal laser microscope (Zeiss LSM-410, Germany). Negative controls were prepared by omitting the primary antibody. 2.6. Statistics Statistical significance was assessed by a paired Student’s t-test when 2 groups were compared, and one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple test when multiple groups were compared.
3. Results 2.3. Histological assessment and immunohistochemistry 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). For semiquantitative evaluation, the degree of damage was assessed in the CA1–CA3 area by percentage of damaged cells: grade 0, no cell damage visible; grade 1, <50% of cells damaged; grade 2, >50% of cells damaged. The length of the CA1–CA3 area with each degree of damage was measured, and the mean histological score was calculated as described previously (Kitagawa et al., 1998a), by the following formula: (1 length with grade 1 + 2 length with grade 2)/(total length from the CA1 to the CA3 area). For single-stranded DNA (ssDNA)
3.1. Hippocampal neurons in apoE/ mice are more vulnerable to ischemia/reperfusion injury than those in WT mice. There were no significant differences in any of the parameters of cerebral blood flow and rectal temperature between the WT and apoE/ mice during and after transient forebrain ischemia (Table 1). Delayed neuronal death in the hippocampus was compared between WT and apoE/ mice. Histological evaluation of hippocampal neurons by hematoxylin and eosin staining (Fig. 1) and ssDNA staining (Fig. 2) revealed that neurons in the CA regions
N. Osada et al. / Neurochemistry International 54 (2009) 403–409 Table 1 Cerebral blood flow (CBF) and rectal temperature (RT) profiles 20 min after ischemia in WT and apoE/ mice. Values are expressed as the mean S.E.M. for six independent experiments. Ischemic CBF are mean values obtained at 20 min during ischemia. Post-ischemia data for CBF and RT were measured 5 min after reperfusion. No significant differences were observed between the WT and apoE/mice. Cerebral blood flow (%) Pre-ischemia Ischemia WT 100 apoE/ 100
Rectal temperature (8C)
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apoE/ mice was significantly higher than that in WT mice (Fig. 1I). Moreover, many neurons in the apoE/ mice showed condensed nuclei; however, most of the neurons in WT mice showed preservation of apparently normal nuclei. Cell-counting analysis of area CA1–CA3 also showed that 19% and 65% of pyramidal neurons were ssDNA-positive in WT and apoE/ mice, respectively, after transient forebrain ischemia (Fig. 2G).
Post-ischemia Pre-ischemia Post-ischemia
35.8 3.8 65.8 6.1 35.3 7.5 70.7 7.8
37.8 0.29 37.9 0.10
37.9 0.27 37.9 0.13
had degenerated, becoming ssDNA-positive 3 days after ischemia and reperfusion in both groups. However, in the apoE/ mice, the numbers of degenerated neurons and ssDNA-positive neurons were higher in areas CA1–CA3. Then, semiquantitative analysis of CA1–CA3 area showed that the grade of neuronal injury in the
3.2. Increased induction of ER stress-associated chaperones and signal proteins in the hippocampus of apoE/ mice Experiments were performed to investigate the induction of the ER chaperones GRP78 and 94 after ischemia and reperfusion in WT and apoE/ mice. A significant and transient increase of both GRP78 and 94 was observed 24 h after ischemia and reperfusion in WT mice (Fig. 3). In apoE/ mice, the increase in the level of GRP78 was time-dependent, and the level was still elevated at 48 h after
Fig. 1. Histological evaluation of the hippocampus after transient forebrain ischemia in WT and apoE/ mice. Coronal sections of sham-operated WT (A and B) and apoE/ mice (E and F). Sections prepared 3 days after transient forebrain ischemia for 20 min in WT (C and D) and apoE/ mice (G and H) are shown. Photos B, D, F and H show magnified views of the areas indicated in A, C, E and G, respectively. Semiquantitative assessment of ischemic neuronal damage is shown in (I). The histological grade was calculated by measuring the length of the CA1–CA3 subfields with the damaged cells. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05 compared with the WT group, paired t-test.
Fig. 2. Histological evaluation of ssDNA staining in the hippocampus after transient forebrain ischemia in WT (A–C) and apoE/ mice (D–F). Panels A and D are coronal sections of sham-operated animals. Panels B, C, E and F are coronal sections prepared 3 days after transient forebrain ischemia for 20 min. Panels C and F show magnified views of the areas indicated in B and E, respectively. Cell-counting analysis of ssDNA-positive neurons in the CA1–CA3 subfields is shown in (G). Values are expressed as the mean S.E.M. (n = 4). *p < 0.01 compared with the WT group, paired t-test.
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Fig. 3. Expression of 78-kDa glucose-regulated protein (GRP78) and GRP94 in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. (A) Western blot analysis of GRP78 and GRP94 in the hippocampus of WT (left panel) and apoE/ (right panel) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of GRP78 and GRP94 in WT (left panel) and apoE/ (right panel) mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05, **p < 0.001 compared with the sham group, one-way ANOVA followed by Tukey’s test.
ischemia (Fig. 3). In contrast, no such increase of GRP94 immunoreactivity was observed in apoE/ mice either 24 h or 48 h after ischemia. Western blot analysis also revealed that CHOP protein, a transcription factor involved in ER stress-induced gene expression, was barely detectable in the sham-operated WT and apoE/ mice (Fig. 4A). A transient and significant increase in CHOP protein was observed 24 h after ischemia/reperfusion in the WT mice (Fig. 4A and B). ApoE/ mice showed a more drastic increase in the protein, and the increase was still evident 48 h after ischemia/reperfusion (Fig. 4A and B). Distribution of CHOP immunoreactivity in the CA1 subfields of the hippocampus was also compared using coronal sections prepared 24 h after transient forebrain ischemia in WT and
apoE/ mice. CHOP protein showed higher immunoreactivity in area CA1 in apoE/ mice than in that of WT mice (Fig. 4C). 3.3. Induction of phosphorylated c-Jun N-terminal kinases and the cleaved form of caspase-12 by ischemia/reperfusion We investigated c-Jun N-terminal kinases in the hippocampus of WT and apoE/ mice after transient forebrain ischemia. Western blot analysis of p-JNK revealed that p-JNK1 and p-JNK2 levels were significantly increased in apoE/ mice 24 h and 48 h after transient forebrain ischemia, whereas no such increase in pJNK levels was observed in WT mice (Fig. 5). In contrast, there was no change in the levels of JNK1 and 2 in either WT or apoE/ mice.
Fig. 4. Expression of CHOP in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. Western blot analysis of CHOP in the hippocampus of WT (A, left) and apoE/ (A, right) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of CHOP in WT (left panel) and apoE/ (right panel) mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.01, **p < 0.001 compared with the sham group, one-way ANOVA followed by Tukey’s test. #p < 0.01 compared with the WT group at the same time point, paired t-test. (C) Distribution of CHOP immunoreactivity in the CA1 subfields of the hippocampus after transient forebrain ischemia in WT (a–c) and apoE/ (d–f) mice. a and d are coronal sections of sham-operated animals. b, c, e and f are coronal sections prepared 24 h after transient forebrain ischemia for 20 min. c and f are magnified views of the indicated regions in b and e, respectively.
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Fig. 5. Expression of c-Jun N-terminal kinases (JNK) in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. (A) Western blot analysis of P-JNK and JNK in the hippocampus of WT (left panel) and apoE/ (right panel) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of p-JNKs in WT and apoE/ mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05, **p < 0.01 compared with the sham group, one-way ANOVA followed by Tukey’s test.
Fig. 6. Expression of caspase-12 in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. (A) Western blot analysis of caspase-12 in the hippocampus of WT (left panel) and apoE/ (right panel) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of caspase-12 in WT (left panel) and apoE/ (right panel) mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05, **p < 0.01 compared with the sham group, one-way ANOVA followed by Tukey’s test.
Next, the expression of the cleaved form of caspase-12 in the hippocampus 24 h and 48 h after ischemia/reperfusion in WT and apoE/ mice was also compared using Western blot analysis. Ischemia/reperfusion significantly increased the level of the cleaved form of caspase-12 in both WT and apoE/ mice 48 h after transient forebrain ischemia (Fig. 6). There was no obvious difference in the level of the protein between the two groups. 4. Discussion ApoE is a plasma protein that serves as a ligand for low density lipoprotein receptors and, through its interaction with these receptors, participates in the transport of cholesterol and other
lipids (Mahley, 1988). A role for apoE in modulating neuronal toxicity is supported by findings indicating that apoE receptors modulate various signaling cascades (Herz and Beffert, 2000; Hoe et al., 2005). The present study clearly demonstrated that apoE/ mice have increased susceptibility to transient forebrain ischemia, i.e. show increased numbers of degenerated and ssDNA-positive neurons in the hippocampus, in comparison with WT mice. These results are comparable to earlier reports demonstrating that apoE deficiency worsened histological outcome after transient focal and global ischemia (Horsburgh et al., 1999b; Sheng et al., 1999; Laskowitz et al., 1997). In contrast, cerebral blood flow measured before, during, and 30 min after ischemia in cohort animals has been found to be similar in the apoE/ and wild-type groups (Sheng et al., 1999). One explanation is that apoE protects against secondary oxidative damage to injured neurons following cerebral ischemia and head trauma. Brain anti-oxidant levels are more severely depleted in apoE/ mice as compared to genetically matched wild types following closed head injury (Lomnitski et al., 1997). There is growing evidence that ER stress plays an important role in neuronal death caused by brain ischemia (Paschen et al., 1998; Kumar et al., 2003; Tajiri et al., 2004; Qi et al., 2004). It has also been shown that ischemia/reperfusion injury up-regulates the expression of ER chaperones such as GRP78 and GRP94 (Kudo et al., 2007; Bando et al., 2003). Consistently, our data showed that the expression of GRP78 and GRP94 was increased after forebrain ischemia/reperfusion in WT mice. The present results clearly showed that the expression of ER chaperones in apoE/ mice after ischemia/reperfusion was somewhat different from that in WT mice. Although the level of GRP78 in apoE/ mice was increased and lasted longer than that in WT mice, and was maintained at its highest level 48 h after ischemia/reperfusion, GRP94, which has been shown to suppress ischemia/reperfusion-associated neuronal cell death (Bando et al., 2003), was not induced in apoE/ mice 24 h and 48 h after transient forebrain ischemia, suggesting disturbance of ER chaperone induction in the hippocampus after the insult. Prolonged or severe ER stress has been shown to result in apoptotic cell death though activation of multiple ER-specific
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proapoptotic factors including CHOP, caspase-12 and JNK. CHOP is expressed at very low levels under physiological conditions, but strongly induced in response to ER stress by mechanisms that still remain to be determined (Oyadomari and Mori, 2004). It has been shown that levels of CHOP mRNA in the cerebral cortex do not rise significantly after cerebral ischemia, whereas in the hippocampus they increase markedly relative to controls, and that perturbations of ER function play a more prominent role in the hippocampus than they do in the cortex after transient cerebral ischemia (Paschen et al., 1998). One possible explanation is that induced CHOP activates the ER-specific caspase, caspase-12, and/or the MAP kinase signaling pathway, thus playing a critical role in apoptosis regulation. The present study also showed that the level of CHOP was transiently elevated 24 h after forebrain ischemia/reperfusion in WT mice. In apoE/ mice, a more drastic increase in the protein level was observed, which was still maintained at 48 h after ischemia/reperfusion, whereas the level in WT mice returned to the level in sham-operated animals. The pronounced increase of CHOP in the hippocampus of apoE/ mice implies that ischemiainduced disturbances of hippocampal ER function are more marked in apoE/ than in WT mice, thus worsening neuronal damage in the hippocampus in apoE/ mice. Inositol-requiring kinase 1 (IRE1), one of the crucial transmembrane ER signaling proteins, and TNF receptor-associated factor 2 (TRAF2) have been shown to collaborate to activate JNKs through apoptosis signal-regulating kinase 1 (ASK1) activation (Urano et al., 2000). Ab-induced apoptosis also involves activation of the ER stress response, which recruits the JNK as well as CHOP (Ghribi et al., 2004). Treatment with apoE in vitro as well as in vivo has been shown to decrease JNK activation. ApoE treatment of primary neurons decreased activation of JNK (Hoe et al., 2005). Similar to the observations in primary neurons, in vivo injections of apoE-derived peptide into the rat hippocampus decreased JNK activation (Hoe et al., 2006). These results suggest that apoE exerts an antiapoptotic effect. Moreover, activation of JNK has been shown to play a pivotal role following cerebral ischemia in rat hippocampus. JNK3 activation in response to cerebral ischemia was mediated by GluR6 activation (Tian et al., 2005). In the present study, the levels of both p-JNK1 and p-JNK2 were not significantly increased in WT mice after transient forebrain ischemia/reperfusion. In apoE/ mice, the p-JNK levels in the sham-operated animals were lower than those in WT mice; however, ischemia/reperfusion dramatically increased the levels of p-JNK1 and 2 in a time-dependent manner with no change in the JNK1 and 2 levels, suggesting that activation of JNK1 and 2 is also involved in the worsened histological outcome in the hippocampus after transient focal and global ischemia. We have shown that exposure of cultured hippocampal neurons to Ab or TM, an ER stress inducer, resulted in a timedependent increase in the expression of GRP78 and caspase-12, which play a pivotal role in inducing apoptosis (Kosuge et al., 2003; Ishige et al., 2007). In organotypic hippocampal cultures (OHC), we have also shown that Ab alone did not affect UPR itself, but potentiated the TM-induced stimulation of the calpain-, caspase12- and caspase-3-dependent cell death pathway, thus augmenting TM-induced neuronal death in OHC cultured for 3 weeks (Kosuge et al., 2008; Imai et al., 2007). Although the expression of the cleaved form of caspase-12 increased in a time-dependent manner in the hippocampus after ischemia/reperfusion in both WT and apoE/ mice, there was no obvious difference between the two groups. These results suggest that although the caspase-12dependent apoptotic pathway plays a crucial role in the hippocampus after transient ischemia/reperfusion, this signaling pathway does not contribute to the increased vulnerability of hippocampal neurons in apoE/ mice. It has been suggested that one possible mechanism by which apoE ameliorates the effects of focal ischemia is down-regulation
of the CNS inflammatory response (Laskowitz et al., 1997). Another possible explanation is that apoE might protect against ischemic insults by acting as an antioxidant (Laskowitz et al., 1997). However, the mechanism underlying the apoE-mediated protection against ischemic neuronal damage has yet to be determined. The present data clearly indicated that deficiency of apoE led to increased ischemic neuronal cell death through severe ER stress and UPR in the hippocampus. Moreover, CHOP and JNK appear to play a pivotal role in the enhancement of neuronal apoptosis induced by ischemia/reperfusion. ApoE plays a role in the protection of neurons from ER stress caused by brain ischemia/ reperfusion, and may provide a novel therapeutic strategy for ER stress-related insults and diseases. Acknowledgements We thank Drs. Y. Edagawa and M. Arakawa for valuable discussions. This work was supported by the ‘‘Academic Frontier’’ project for private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology, 2007–2009. References Bando, Y., Katayama, T., Kasai, K., Taniguchi, M., Tamatani, M., Tohyama, M., 2003. GRP94 (94 kDa glucose-regulated protein) suppresses ischemic neuronal cell death against ischemia/reperfusion injury. Eur. J. Neurosci. 18, 829–840. 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. Budihardjo, I., Oliver, H., Lutter, M., Luo, X., Wang, X., 1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell. Dev. Biol. 15, 269–290. Carter, C.J., 2008. Interactions between the products of the Herpes simplex genome and Alzheimer’s disease susceptibility genes: relevance to pathological-signalling cascades. Neurochem. Int. 52, 920–934. Cudna, R.E., Dickson, A.J., 2003. Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol. Bioeng. 81, 56–65. Ghribi, O., Herman, M.M., Pramoonjago, P., Spaulding, N.K., Savory, J., 2004. GDNF regulates the A beta-induced endoplasmic reticulum stress response in rabbit hippocampus by inhibiting the activation of gadd 153 and the JNK and ERK kinases. Neurobiol. Dis. 16, 417–427. Hall, E.D., Oostveen, J.A., Dunn, E., Carter, D.B., 1995. Increased amyloid protein precursor and apolipoprotein E immunoreactivity in the selectively vulnerable hippocampus following transient forebrain ischemia in gerbils. Exp. Neurol. 135, 17–27. Herz, J., Beffert, U., 2000. Apolipoprotein E receptors: linking brain development and Alzheimer’s disease. Nat. Rev. Neurosci. 1, 51–58. 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. Hoe, H.S., Pocivavsek, A., Dai, H., Chakraborty, G., Harris, D.C., Rebeck, G.W., 2006. Effects of apoE on neuronal signaling and APP processing in rodent brain. Brain Res. 1112, 70–79. Hoe, H.S., Harris, D.C., Rebeck, W., 2005. Multiple pathways of apolipoprotein E signaling in primary neurons. J. Neurochem. 93, 145–155. Horsburgh, K., Graham, D.I., Stewart, J., Nicoll, J.A., 1999a. Influence of apolipoprotein E genotype on neuronal damage and apoE immunoreactivity in human hippocampus following global ischemia. J. Neuropathol. Exp. Neurol. 58, 227– 234. Horsburgh, K., Kelly, S., McCulloch, J., Higgins, G.A., Roses, A.D., Nicoll, J.A., 1999b. Increased neuronal damage in apolipoprotein E-deficient mice following global ischaemia. Neuroreport 10, 837–841. Imai, T., Kosuge, Y., Ishige, K., Ito, Y., 2007. Amyloid beta-protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures. Neuroscience 147, 639–651. Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y., Takahashi, R., 2001. An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell 105, 891–902. 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. Kaufman, R.J., 1999. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13, 1211–1233. Kitagawa, K., Matsumoto, M., Tsujimoto, Y., Ohtsuki, T., Kuwabara, K., Matsushita, K., Yang, G., Tanabe, H., Martinou, J.C., Hori, M., Yanagihara, T., 1998a. Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of BCL-2 in transgenic mice. Stroke 29, 2616–2621.
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Apolipoprotein E-deficient mice are more vulnerable to ER stress after transient forebrain ischemia Nobuhiro Osada, Yasuhiro Kosuge, Tetsuroh Kihara, Kumiko Ishige, Yoshihisa Ito * Research Unit of Pharmacology, Department of Clinical Pharmacy, College 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 November 2008 Received in revised form 15 January 2009 Accepted 19 January 2009 Available online 6 February 2009
Apolipoprotein E-deficient (apoE/) mice have been shown to have increased vulnerability to neuronal damage induced by cerebral ischemia; however, the mechanism of this increased vulnerability remains unclear. In order to define the role of the apoE protein against ischemia-induced ER stress and cell death, experiments were performed to compare ER stress-associated chaperones and signal proteins in the hippocampus of apoE/ mice to those of WT mice after being subjected to forebrain ischemia and reperfusion. Although neuronal loss in area CA1–CA3 of the hippocampus was observed 3 days after ischemia in both types of mice, the damage in apoE/ mice was more severe. In apoE/ mice, a more extensive increase in 78-kDa glucose-regulated protein (GRP78) was observed after the insult, whereas the level of GRP94 was not changed. The expression of both C/EBP homologous protein (CHOP) and caspase-12 was increased in the hippocampus in both WT and apoE/ mice after ischemia. The increased levels of CHOP in apoE/ mice were significantly higher than those in WT mice, whereas the levels of caspase-12 in the two were comparable. Furthermore, whereas the levels of c-Jun N-terminal kinase (JNK), p-JNK1 and p-JNK2 in WT mice were unchanged after ischemia, they were significantly increased in apoE/ mice 24 h and 48 h after ischemia. These results suggest that increased vulnerability of the hippocampus to forebrain ischemia and reperfusion in apoE/ mice is at least partly attributable to perturbed induction of an ER chaperone, GRP 94, and enhancement of the CHOP- and JNKdependent apoptotic pathway in the hippocampus. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: apoE/ mice Forebrain ischemia ER stress Hippocampus
1. Introduction Apoptosis plays a pivotal role in neuronal cell death resulting from ischemic stroke. Several lines of evidence suggest that apoptosis is regulated by multiple pathways. The two most wellstudied pathways are the cell surface death receptor pathway and the mitochondria-initiated pathway (Budihardjo et al., 1999). Recently, another apoptosis-regulatory pathway involved in ER stress has been receiving attention. The condition in which ER function is impaired, so-called ER stress, can lead to an accumulation of unfolded or malfolded proteins in the ER lumen (Kaufman, 1999). In the case of mild ER stress, cells develop a selfprotective 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 (Cudna and Dickson, 2003), thus relieving cells from the stress. However, if the damage is too severe to repair, the UPR ultimately initiates the apoptotic pathway (Oyadomari and Mori, 2004; Cudna and Dickson, 2003). Several
* Corresponding author. Tel.: +81 474 65 5832; fax: +81 474 65 5832. E-mail address: [email protected] (Y. Ito). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.01.010
proteins have been implicated in this apoptotic pathway, including a transcription factor, C/EBP homologous protein (CHOP) (Wang et al., 1996), and the ER-resident caspase, caspase-12 (Nakagawa et al., 2000). In humans, ER stress has been shown to be involved in not only ischemia but also some neuronal diseases, such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and prion-related disorders (LaFerla, 2002; Ryu et al., 2002; Imai et al., 2001; Nishitoh et al., 2008; Hetz et al., 2003). Recent evidence indicates that apolipoprotein E (apoE) plays a central role in CNS injury, ALS–parkinsonism dementia complex (Wilson and Shaw, 2007) and the interactions of herpes simplex products (Carter, 2008). Following brain injury, the synthesis of apoE is markedly increased, and it is secreted, predominantly from astrocytes, into the extracellular space (Poirier, 1994). The expression of apoE increased in the selectively vulnerable hippocampus following transient forebrain ischemia (Hall et al., 1995). Neuronal apoE immunoreactivity was significantly increased in all hippocampal areas (CA1, CA2, CA3/4, dentate fascia) of the human brain following global ischemia (Horsburgh et al., 1999a). It is thought that intraneuronal apoE plays a pivotal role in a protective response following injury by providing lipids for neuronal repair and remodeling. Insight into the role of apoE in brain injury has been provided by the development of genetically
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modified mice. It has been shown that apoE/ mice have a poorer outcome in a focal model of ischemia (Laskowitz et al., 1997), and that this response may be isoform-specific (Sheng et al., 1998). These findings support the suggestion that apoE has a protective effect against ischemic neuronal damage; however, the molecular mechanism underlying the apoE-mediated protection against ischemic neuronal damage remains unclear. Brain ischemia in mice has been shown to cause ER-stress-mediated apoptosis of cells in the striatum and hippocampus, and CHOP plays a crucial role in this cell death (Tajiri et al., 2004). More recent studies have demonstrated that markers of ER stress and UPR activation are induced dramatically in macrophages at all stages of atherosclerosis development in apoE/ mice (Zhou et al., 2005). Taken together, these findings suggest that apoE protects neurons against ischemic cell death by attenuating ER stress during the injury. In order to define the role of the apoE protein against ischemiainduced ER stress and cell death in the hippocampus, experiments were performed to compare ER stress-associated signal proteins including ER chaperones, GRP 78 and 94, CHOP, caspase-12 and cJun N-terminal kinase (JNK) activity in the hippocampus in apoE/ mice and wild type (WT) mice after being subjected to forebrain ischemia and reperfusion. We found that the increased vulnerability of hippocampal neurons to ischemic damage was at least partly attributable to disturbed induction of ER chaperones and higher UPR activation. 2. Experimental procedures 2.1. Animals All experimental procedures were approved by the Institutional Animal Center Use Committee of Graduate School of Pharmacy, Nihon University. ApoE/ mice, originally produced by Zhang et al. (1992), were purchased from the Jackson Laboratory (Bar Harbor, ME) and back-crossed to WT C57BL/6 mice (Charles River Inc., Yokohama, Japan) to have genetic backgrounds of the homozygote. Both mice were bred in this study. Animals were fed standard laboratory chow and given free access to water prior to surgery. After mating of heterozygote, we selected the homozygous and WT mice by polymerase chain reaction (PCR) amplification of genomic DNA extracted from tails. Male mice (10–12 week old) were used in the present study.
staining that indicated apoptotic cells, paraffin sections were processed immunohistochemically. Briefly, 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 (DAKO, Japan) antibodies (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 immunoreactivities were detected with 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. For cell-counting procedure, the number of morphologically damaged ssDNA positive neurons and viable neurons in hippocampal CA1–CA3 regions were manually counted in four brain slices in each animal and the percentage of ssDNA positive neurons was calculated. The results in each group were presented as the mean S.E.M. 2.4. Western blotting Following transient forebrain ischemia, the mice were sacrificed under the sodium pentobarbital anesthesia at 24 h and 48 h after reperfusion. Sham-operated animals served as controls. Brains were rapidly microdissected on an ice-chilled plate. The hippocampus was homogenized using homogenizer (Homogenizer, Subsonic, IWAKI GLASS CO., LTD) in 800 mL 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, phosphatase 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. Aliquots from each sample (10 mg protein/lane) were separated by 7.5–12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) paper (Millipore, MA, USA) (100 V, 90 min). The membranes were blocked in blocking buffer containing 20 mM Tris– HCl (pH 7.6), 137 mM NaCl, 5% skim milk for 1 h at room temperature and then treated with anti-caspase-12 (SIGMA, USA) antibody (diluted 1:1000), anti-CHOP (Santa Cruz Biotechnology, USA) antibody (diluted 1:1000), anti-KDEL (StressGen, Canada) antibody (diluted 1:2000) or JNK antibody (diluted 1:1000), Phospho-JNK (Cell Signaling Technology, Boston, MA, USA) antibody (diluted 1:1000) overnight at 4 8C. The membranes were washed repeatedly in TTBS containing 20 mM Tris– HCl (pH 7.6), 137 mM NaCl, 0.05% Tween 20, then HRP-conjugated secondary antibody (diluted 1:20,000) was added for 1 h. Immunoreactive bands were detected by electrochemiluminescence (ECL, Amersham Pharmacia Biotech). Optical density on the blots was measured with Scion imaging software (www.scioncorp.com).
2.2. Transient forebrain ischemia 2.5. Immunofluoresence Transient forebrain ischemia was created by bilateral common carotid artery (BCCA) occlusion as described previously (Kitagawa et al., 1998b). Surgical procedures were performed under chloral hydrate anesthesia (450 mg/kg, i.p.) and the rectal temperature was kept at 37 1.5 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). In order to measure cortical perfusion by laser Doppler blood-flowmetry, a polyacrylamide column for measurement was attached to the intact skull with dental cement, 3.5 mm lateral bregma. For procedures involving BCCA occlusion, the arteries were approached through a midline cervical incision. Both arteries were identified and ligated with small arterial clips for 20 min, after which time the clips were removed, the return of these arteries blood flow was visually confirmed, the incision was closed, and reperfusion was continued for the indicated time. Sham-operated mice received similar operative procedures; however, the arteries, after isolation, were not occluded and incision was closed. All the mice were killed with a lethal dose of the sodium pentobarbital before recovery of tissues.
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 anti-CHOP antibody (diluted 1:200) for 24 h at 4 8C. After washing with PBS, the sections were incubated for 1 h with Alexa Fluor 488-conjugated goat IgG (diluted 1:500, Molecular Probes, USA). After rinsing with PBS, the sections were analyzed using a confocal laser microscope (Zeiss LSM-410, Germany). Negative controls were prepared by omitting the primary antibody. 2.6. Statistics Statistical significance was assessed by a paired Student’s t-test when 2 groups were compared, and one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple test when multiple groups were compared.
3. Results 2.3. Histological assessment and immunohistochemistry 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). For semiquantitative evaluation, the degree of damage was assessed in the CA1–CA3 area by percentage of damaged cells: grade 0, no cell damage visible; grade 1, <50% of cells damaged; grade 2, >50% of cells damaged. The length of the CA1–CA3 area with each degree of damage was measured, and the mean histological score was calculated as described previously (Kitagawa et al., 1998a), by the following formula: (1 length with grade 1 + 2 length with grade 2)/(total length from the CA1 to the CA3 area). For single-stranded DNA (ssDNA)
3.1. Hippocampal neurons in apoE/ mice are more vulnerable to ischemia/reperfusion injury than those in WT mice. There were no significant differences in any of the parameters of cerebral blood flow and rectal temperature between the WT and apoE/ mice during and after transient forebrain ischemia (Table 1). Delayed neuronal death in the hippocampus was compared between WT and apoE/ mice. Histological evaluation of hippocampal neurons by hematoxylin and eosin staining (Fig. 1) and ssDNA staining (Fig. 2) revealed that neurons in the CA regions
N. Osada et al. / Neurochemistry International 54 (2009) 403–409 Table 1 Cerebral blood flow (CBF) and rectal temperature (RT) profiles 20 min after ischemia in WT and apoE/ mice. Values are expressed as the mean S.E.M. for six independent experiments. Ischemic CBF are mean values obtained at 20 min during ischemia. Post-ischemia data for CBF and RT were measured 5 min after reperfusion. No significant differences were observed between the WT and apoE/mice. Cerebral blood flow (%) Pre-ischemia Ischemia WT 100 apoE/ 100
Rectal temperature (8C)
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apoE/ mice was significantly higher than that in WT mice (Fig. 1I). Moreover, many neurons in the apoE/ mice showed condensed nuclei; however, most of the neurons in WT mice showed preservation of apparently normal nuclei. Cell-counting analysis of area CA1–CA3 also showed that 19% and 65% of pyramidal neurons were ssDNA-positive in WT and apoE/ mice, respectively, after transient forebrain ischemia (Fig. 2G).
Post-ischemia Pre-ischemia Post-ischemia
35.8 3.8 65.8 6.1 35.3 7.5 70.7 7.8
37.8 0.29 37.9 0.10
37.9 0.27 37.9 0.13
had degenerated, becoming ssDNA-positive 3 days after ischemia and reperfusion in both groups. However, in the apoE/ mice, the numbers of degenerated neurons and ssDNA-positive neurons were higher in areas CA1–CA3. Then, semiquantitative analysis of CA1–CA3 area showed that the grade of neuronal injury in the
3.2. Increased induction of ER stress-associated chaperones and signal proteins in the hippocampus of apoE/ mice Experiments were performed to investigate the induction of the ER chaperones GRP78 and 94 after ischemia and reperfusion in WT and apoE/ mice. A significant and transient increase of both GRP78 and 94 was observed 24 h after ischemia and reperfusion in WT mice (Fig. 3). In apoE/ mice, the increase in the level of GRP78 was time-dependent, and the level was still elevated at 48 h after
Fig. 1. Histological evaluation of the hippocampus after transient forebrain ischemia in WT and apoE/ mice. Coronal sections of sham-operated WT (A and B) and apoE/ mice (E and F). Sections prepared 3 days after transient forebrain ischemia for 20 min in WT (C and D) and apoE/ mice (G and H) are shown. Photos B, D, F and H show magnified views of the areas indicated in A, C, E and G, respectively. Semiquantitative assessment of ischemic neuronal damage is shown in (I). The histological grade was calculated by measuring the length of the CA1–CA3 subfields with the damaged cells. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05 compared with the WT group, paired t-test.
Fig. 2. Histological evaluation of ssDNA staining in the hippocampus after transient forebrain ischemia in WT (A–C) and apoE/ mice (D–F). Panels A and D are coronal sections of sham-operated animals. Panels B, C, E and F are coronal sections prepared 3 days after transient forebrain ischemia for 20 min. Panels C and F show magnified views of the areas indicated in B and E, respectively. Cell-counting analysis of ssDNA-positive neurons in the CA1–CA3 subfields is shown in (G). Values are expressed as the mean S.E.M. (n = 4). *p < 0.01 compared with the WT group, paired t-test.
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Fig. 3. Expression of 78-kDa glucose-regulated protein (GRP78) and GRP94 in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. (A) Western blot analysis of GRP78 and GRP94 in the hippocampus of WT (left panel) and apoE/ (right panel) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of GRP78 and GRP94 in WT (left panel) and apoE/ (right panel) mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05, **p < 0.001 compared with the sham group, one-way ANOVA followed by Tukey’s test.
ischemia (Fig. 3). In contrast, no such increase of GRP94 immunoreactivity was observed in apoE/ mice either 24 h or 48 h after ischemia. Western blot analysis also revealed that CHOP protein, a transcription factor involved in ER stress-induced gene expression, was barely detectable in the sham-operated WT and apoE/ mice (Fig. 4A). A transient and significant increase in CHOP protein was observed 24 h after ischemia/reperfusion in the WT mice (Fig. 4A and B). ApoE/ mice showed a more drastic increase in the protein, and the increase was still evident 48 h after ischemia/reperfusion (Fig. 4A and B). Distribution of CHOP immunoreactivity in the CA1 subfields of the hippocampus was also compared using coronal sections prepared 24 h after transient forebrain ischemia in WT and
apoE/ mice. CHOP protein showed higher immunoreactivity in area CA1 in apoE/ mice than in that of WT mice (Fig. 4C). 3.3. Induction of phosphorylated c-Jun N-terminal kinases and the cleaved form of caspase-12 by ischemia/reperfusion We investigated c-Jun N-terminal kinases in the hippocampus of WT and apoE/ mice after transient forebrain ischemia. Western blot analysis of p-JNK revealed that p-JNK1 and p-JNK2 levels were significantly increased in apoE/ mice 24 h and 48 h after transient forebrain ischemia, whereas no such increase in pJNK levels was observed in WT mice (Fig. 5). In contrast, there was no change in the levels of JNK1 and 2 in either WT or apoE/ mice.
Fig. 4. Expression of CHOP in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. Western blot analysis of CHOP in the hippocampus of WT (A, left) and apoE/ (A, right) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of CHOP in WT (left panel) and apoE/ (right panel) mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.01, **p < 0.001 compared with the sham group, one-way ANOVA followed by Tukey’s test. #p < 0.01 compared with the WT group at the same time point, paired t-test. (C) Distribution of CHOP immunoreactivity in the CA1 subfields of the hippocampus after transient forebrain ischemia in WT (a–c) and apoE/ (d–f) mice. a and d are coronal sections of sham-operated animals. b, c, e and f are coronal sections prepared 24 h after transient forebrain ischemia for 20 min. c and f are magnified views of the indicated regions in b and e, respectively.
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Fig. 5. Expression of c-Jun N-terminal kinases (JNK) in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. (A) Western blot analysis of P-JNK and JNK in the hippocampus of WT (left panel) and apoE/ (right panel) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of p-JNKs in WT and apoE/ mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05, **p < 0.01 compared with the sham group, one-way ANOVA followed by Tukey’s test.
Fig. 6. Expression of caspase-12 in the hippocampus after transient forebrain ischemia in WT and apoE/ mice. (A) Western blot analysis of caspase-12 in the hippocampus of WT (left panel) and apoE/ (right panel) mice after transient forebrain ischemia for 20 min followed by 24 h and 48 h of reperfusion. (B) Relative amounts of caspase-12 in WT (left panel) and apoE/ (right panel) mice were assessed by densitometric analysis. Values are expressed as the mean S.E.M. (n = 4). *p < 0.05, **p < 0.01 compared with the sham group, one-way ANOVA followed by Tukey’s test.
Next, the expression of the cleaved form of caspase-12 in the hippocampus 24 h and 48 h after ischemia/reperfusion in WT and apoE/ mice was also compared using Western blot analysis. Ischemia/reperfusion significantly increased the level of the cleaved form of caspase-12 in both WT and apoE/ mice 48 h after transient forebrain ischemia (Fig. 6). There was no obvious difference in the level of the protein between the two groups. 4. Discussion ApoE is a plasma protein that serves as a ligand for low density lipoprotein receptors and, through its interaction with these receptors, participates in the transport of cholesterol and other
lipids (Mahley, 1988). A role for apoE in modulating neuronal toxicity is supported by findings indicating that apoE receptors modulate various signaling cascades (Herz and Beffert, 2000; Hoe et al., 2005). The present study clearly demonstrated that apoE/ mice have increased susceptibility to transient forebrain ischemia, i.e. show increased numbers of degenerated and ssDNA-positive neurons in the hippocampus, in comparison with WT mice. These results are comparable to earlier reports demonstrating that apoE deficiency worsened histological outcome after transient focal and global ischemia (Horsburgh et al., 1999b; Sheng et al., 1999; Laskowitz et al., 1997). In contrast, cerebral blood flow measured before, during, and 30 min after ischemia in cohort animals has been found to be similar in the apoE/ and wild-type groups (Sheng et al., 1999). One explanation is that apoE protects against secondary oxidative damage to injured neurons following cerebral ischemia and head trauma. Brain anti-oxidant levels are more severely depleted in apoE/ mice as compared to genetically matched wild types following closed head injury (Lomnitski et al., 1997). There is growing evidence that ER stress plays an important role in neuronal death caused by brain ischemia (Paschen et al., 1998; Kumar et al., 2003; Tajiri et al., 2004; Qi et al., 2004). It has also been shown that ischemia/reperfusion injury up-regulates the expression of ER chaperones such as GRP78 and GRP94 (Kudo et al., 2007; Bando et al., 2003). Consistently, our data showed that the expression of GRP78 and GRP94 was increased after forebrain ischemia/reperfusion in WT mice. The present results clearly showed that the expression of ER chaperones in apoE/ mice after ischemia/reperfusion was somewhat different from that in WT mice. Although the level of GRP78 in apoE/ mice was increased and lasted longer than that in WT mice, and was maintained at its highest level 48 h after ischemia/reperfusion, GRP94, which has been shown to suppress ischemia/reperfusion-associated neuronal cell death (Bando et al., 2003), was not induced in apoE/ mice 24 h and 48 h after transient forebrain ischemia, suggesting disturbance of ER chaperone induction in the hippocampus after the insult. Prolonged or severe ER stress has been shown to result in apoptotic cell death though activation of multiple ER-specific
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proapoptotic factors including CHOP, caspase-12 and JNK. CHOP is expressed at very low levels under physiological conditions, but strongly induced in response to ER stress by mechanisms that still remain to be determined (Oyadomari and Mori, 2004). It has been shown that levels of CHOP mRNA in the cerebral cortex do not rise significantly after cerebral ischemia, whereas in the hippocampus they increase markedly relative to controls, and that perturbations of ER function play a more prominent role in the hippocampus than they do in the cortex after transient cerebral ischemia (Paschen et al., 1998). One possible explanation is that induced CHOP activates the ER-specific caspase, caspase-12, and/or the MAP kinase signaling pathway, thus playing a critical role in apoptosis regulation. The present study also showed that the level of CHOP was transiently elevated 24 h after forebrain ischemia/reperfusion in WT mice. In apoE/ mice, a more drastic increase in the protein level was observed, which was still maintained at 48 h after ischemia/reperfusion, whereas the level in WT mice returned to the level in sham-operated animals. The pronounced increase of CHOP in the hippocampus of apoE/ mice implies that ischemiainduced disturbances of hippocampal ER function are more marked in apoE/ than in WT mice, thus worsening neuronal damage in the hippocampus in apoE/ mice. Inositol-requiring kinase 1 (IRE1), one of the crucial transmembrane ER signaling proteins, and TNF receptor-associated factor 2 (TRAF2) have been shown to collaborate to activate JNKs through apoptosis signal-regulating kinase 1 (ASK1) activation (Urano et al., 2000). Ab-induced apoptosis also involves activation of the ER stress response, which recruits the JNK as well as CHOP (Ghribi et al., 2004). Treatment with apoE in vitro as well as in vivo has been shown to decrease JNK activation. ApoE treatment of primary neurons decreased activation of JNK (Hoe et al., 2005). Similar to the observations in primary neurons, in vivo injections of apoE-derived peptide into the rat hippocampus decreased JNK activation (Hoe et al., 2006). These results suggest that apoE exerts an antiapoptotic effect. Moreover, activation of JNK has been shown to play a pivotal role following cerebral ischemia in rat hippocampus. JNK3 activation in response to cerebral ischemia was mediated by GluR6 activation (Tian et al., 2005). In the present study, the levels of both p-JNK1 and p-JNK2 were not significantly increased in WT mice after transient forebrain ischemia/reperfusion. In apoE/ mice, the p-JNK levels in the sham-operated animals were lower than those in WT mice; however, ischemia/reperfusion dramatically increased the levels of p-JNK1 and 2 in a time-dependent manner with no change in the JNK1 and 2 levels, suggesting that activation of JNK1 and 2 is also involved in the worsened histological outcome in the hippocampus after transient focal and global ischemia. We have shown that exposure of cultured hippocampal neurons to Ab or TM, an ER stress inducer, resulted in a timedependent increase in the expression of GRP78 and caspase-12, which play a pivotal role in inducing apoptosis (Kosuge et al., 2003; Ishige et al., 2007). In organotypic hippocampal cultures (OHC), we have also shown that Ab alone did not affect UPR itself, but potentiated the TM-induced stimulation of the calpain-, caspase12- and caspase-3-dependent cell death pathway, thus augmenting TM-induced neuronal death in OHC cultured for 3 weeks (Kosuge et al., 2008; Imai et al., 2007). Although the expression of the cleaved form of caspase-12 increased in a time-dependent manner in the hippocampus after ischemia/reperfusion in both WT and apoE/ mice, there was no obvious difference between the two groups. These results suggest that although the caspase-12dependent apoptotic pathway plays a crucial role in the hippocampus after transient ischemia/reperfusion, this signaling pathway does not contribute to the increased vulnerability of hippocampal neurons in apoE/ mice. It has been suggested that one possible mechanism by which apoE ameliorates the effects of focal ischemia is down-regulation
of the CNS inflammatory response (Laskowitz et al., 1997). Another possible explanation is that apoE might protect against ischemic insults by acting as an antioxidant (Laskowitz et al., 1997). However, the mechanism underlying the apoE-mediated protection against ischemic neuronal damage has yet to be determined. The present data clearly indicated that deficiency of apoE led to increased ischemic neuronal cell death through severe ER stress and UPR in the hippocampus. Moreover, CHOP and JNK appear to play a pivotal role in the enhancement of neuronal apoptosis induced by ischemia/reperfusion. ApoE plays a role in the protection of neurons from ER stress caused by brain ischemia/ reperfusion, and may provide a novel therapeutic strategy for ER stress-related insults and diseases. Acknowledgements We thank Drs. Y. Edagawa and M. Arakawa for valuable discussions. This work was supported by the ‘‘Academic Frontier’’ project for private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology, 2007–2009. References Bando, Y., Katayama, T., Kasai, K., Taniguchi, M., Tamatani, M., Tohyama, M., 2003. GRP94 (94 kDa glucose-regulated protein) suppresses ischemic neuronal cell death against ischemia/reperfusion injury. Eur. J. Neurosci. 18, 829–840. 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. Budihardjo, I., Oliver, H., Lutter, M., Luo, X., Wang, X., 1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell. Dev. Biol. 15, 269–290. Carter, C.J., 2008. Interactions between the products of the Herpes simplex genome and Alzheimer’s disease susceptibility genes: relevance to pathological-signalling cascades. Neurochem. Int. 52, 920–934. Cudna, R.E., Dickson, A.J., 2003. Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol. Bioeng. 81, 56–65. Ghribi, O., Herman, M.M., Pramoonjago, P., Spaulding, N.K., Savory, J., 2004. GDNF regulates the A beta-induced endoplasmic reticulum stress response in rabbit hippocampus by inhibiting the activation of gadd 153 and the JNK and ERK kinases. Neurobiol. Dis. 16, 417–427. Hall, E.D., Oostveen, J.A., Dunn, E., Carter, D.B., 1995. Increased amyloid protein precursor and apolipoprotein E immunoreactivity in the selectively vulnerable hippocampus following transient forebrain ischemia in gerbils. Exp. Neurol. 135, 17–27. Herz, J., Beffert, U., 2000. Apolipoprotein E receptors: linking brain development and Alzheimer’s disease. Nat. Rev. Neurosci. 1, 51–58. 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. Hoe, H.S., Pocivavsek, A., Dai, H., Chakraborty, G., Harris, D.C., Rebeck, G.W., 2006. Effects of apoE on neuronal signaling and APP processing in rodent brain. Brain Res. 1112, 70–79. Hoe, H.S., Harris, D.C., Rebeck, W., 2005. Multiple pathways of apolipoprotein E signaling in primary neurons. J. Neurochem. 93, 145–155. Horsburgh, K., Graham, D.I., Stewart, J., Nicoll, J.A., 1999a. Influence of apolipoprotein E genotype on neuronal damage and apoE immunoreactivity in human hippocampus following global ischemia. J. Neuropathol. Exp. Neurol. 58, 227– 234. Horsburgh, K., Kelly, S., McCulloch, J., Higgins, G.A., Roses, A.D., Nicoll, J.A., 1999b. Increased neuronal damage in apolipoprotein E-deficient mice following global ischaemia. Neuroreport 10, 837–841. Imai, T., Kosuge, Y., Ishige, K., Ito, Y., 2007. Amyloid beta-protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures. Neuroscience 147, 639–651. Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y., Takahashi, R., 2001. An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell 105, 891–902. 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. Kaufman, R.J., 1999. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13, 1211–1233. Kitagawa, K., Matsumoto, M., Tsujimoto, Y., Ohtsuki, T., Kuwabara, K., Matsushita, K., Yang, G., Tanabe, H., Martinou, J.C., Hori, M., Yanagihara, T., 1998a. Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of BCL-2 in transgenic mice. Stroke 29, 2616–2621.
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