Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice

doi:10.1016/S0306-4522(02)00910-7

Neuroscience

Volume 118, Issue 2, 8 May 2003, Pages 491-499

https://doi.org/10.1016/S0306-4522(02)00910-7 Get rights and content

Abstract

We sought to clarify the involvement of caspase-12, a representative molecule related to endoplasmic reticulum (ER) stress-induced cell-death signaling pathways, in neuronal death resulting from ischemia/reperfusion in mice. Transient focal cerebral ischemia (1 h) was produced by intraluminal occlusion of the middle cerebral artery (MCA). We assessed the expression patterns of caspase-12, Bip/GRP78, an ER-resident molecular chaperone whose expression serves as a good marker of ER stress, and caspase-7 by Western blotting and/or immunohistochemistry. Double-fluorescent staining of caspase-12 immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of caspase-12 in cell death. We confirmed that ER stress was induced during reperfusion in our model, as witnessed by up-regulated Bip/GRP78 expression in the MCA territory. Western blot analysis revealed that caspase-12 activation occurred at 5–23 h of reperfusion, and immunoreactivity for caspase-12 was enhanced mainly in striatal neurons on the ischemic side at the same time points. We found the co-localization of caspase-12 immunoreactivity and DNA fragmentation detectable by the TUNEL method. We did not detect the presence of caspase-7 in the ER fraction at the period of caspase-12 cleavage. Our results imply that cerebral ischemia/reperfusion induces ER stress and that caspase-12 activation concurred with ER stress. Caspase-12 seems to be involved in neuronal death induced by ischemia/reperfusion. Caspase-7 is not likely to contribute to the cleavage of caspase-12 in our experimental model.

Section snippets

Animals

Thirty-four adult male C57/Black6 mice (body weight, 15.5–21.0 g) were purchased from Charles River Laboratory (Kanagawa, Japan). They were given food and water freely before and after surgery. All the experimental protocols pertinent to animals were given prior approval as meeting the Animal Experimentation Guidelines of the School of Medicine, Keio University, by the Laboratory Animals Care and Use Committee. We minimized the number of animals used, and used anesthesia appropriately to

Activation of caspase-12 following ischemia/reperfusion

To examine the activation of caspase-12, we performed Western blot analysis of caspase-12 using microsome fractions prepared from sham-operated mice and mice subjected to 1 h of ischemia followed by reperfusion. The relative band intensities of the non-cleaved form of caspase-12 were as follows: 100.0±0.0% in the normal state, 102.0±7.5% at 2 h of reperfusion, 112.0±8.5% at 5 h of reperfusion, 214.0±12.4% at 11 h of reperfusion, 296.5±27.9% at 23 h of reperfusion, 278.4±22.5% at 47 h of

Discussion

In the current study, we demonstrated that our cerebral ischemia/reperfusion model resulted in enhanced expression of Bip/GRP78, which strongly suggested that ER stress was induced in our experimental paradigm. Caspase-12, an ER-associated caspase, was activated during reperfusion, and the expression of caspase-12 was localized mainly in striatal neurons on the ischemic side. The activated caspase-12 was likely to contribute to neuronal death incurred from ischemia/reperfusion, because many

Acknowledgements

The authors thank Prof. Junying Yuan at the Department of Cell Biology, Harvard Medical School, for providing anti-caspase-12 antibody. This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (C-13670666), and a grant from the Medical Research Promotion Fund of School of Medicine, Keio University.

References (15)

M. Aoki et al.
Hypothermic treatment restores glucose regulated protein 78 (GRP78) expression in ischemic brain
Mol Brain Res
(2001)
Y. Ma et al.
The unfolding tale of the unfolded protein response
Cell
(2001)
J.A. Morris et al.
Immunoglobulin binding protein (BiP) function is requires to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins
J Biol Chem
(1997)
R.V. Rao et al.
Coupling endoplasmic reticulun stress to the cell death program
J Biol Chem
(2001)
T. Yoneda et al.
Activation of caspase-12, an endoplasmic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor-dependent mechanism in response to the ER stress
J Biol Chem
(2001)
Z. Yu et al.
The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosissuppression of oxidative stress and stabilization of calcium homeostasis
Exp Neurol
(1999)
D.J. DeGarcia et al.
Molecular pathways of protein synthesis inhibition during brain reperfusionimplication for neuronal survival or death
J Cereb Blood Flow Metab
(2002)

There are more references available in the full text version of this article.

Cited by (122)

Cerebral conditioning: Mechanisms and potential clinical implications
2022, Brain Hemorrhages
Citation Excerpt :
Evidence showed that ER stress plays dual roles during lethal ischemia and preconditioning. Lethal ischemia induces ER stress characterized by activation of CHOP and caspase-12,166,167 whereas pre- or post- conditioning might upregulate ER molecular chaperons or HSPs to abate severe ER stress occurring in lethal ischemia.168–171 Our results showed that pre-activation of autophagy by IPC could upregulate molecular chaperons including GRP78, HSP70 and HSP60 and hence reduce excessive ER stress-dependent apoptosis associated with lethal ischemia, as evidenced by downregulation of CHOP and caspase-12.157
Cerebral conditioning is defined as the mild harmful stimuli applied to the brain before ischemia (preconditioning), during ischemia (perconditioning), immediately, a few hours or days after reperfusion (postconditioning). Various noxious stimuli can precondition, percondition or postcondition the brain, including ischemia, hypoxia or anesthetics to induce tolerance to lethal ischemia/reperfusion injury. The mechanisms underlying cerebral conditioning range from sensors and transducers including extracellular neurotransmitters and receptors, intracellular signals, transcription factors and epigenetic regulation, to effectors involving physiological protection, stem cells, heat shock proteins, Bcl-2, mitochondria, ubiquitin - proteasome pathway and autophagy pathway. Based on the large number of studies conducted in animal models, researchers explore the clinical application of cerebral conditioning. Remote ischemic conditioning is considered the most applicable and clinically relevant form of cerebral conditioning. The translational research also involves development of the biomarkers of conditioning and optimization of organ transplantation. The present review summarized the recent progress in signaling pathways and clinical applications of cerebral conditioning to explore the possibility to develop a potential therapeutic strategy for the prevention and treatment of ischemic stroke.
Role of sorbitol-mediated cellular stress response in obesity-associated retinal degeneration
2020, Archives of Biochemistry and Biophysics
Citation Excerpt :
The ER stress is emerging as a key contributor to a growing list of human diseases, including diabetes [46] and neurodegeneration [25,47,48]. Neuronal ER stress has been studied in both in vivo, [49,50] and in vitro [51]. Recently, we reported that prolonged ER stress could trigger neuronal apoptosis in diabetic rat retina [52].
Obesity is a global health problem associated with several diseases including ocular complications. Earlier we reported progressive retinal degeneration because of obesity in a spontaneous obese rat (WNIN/Ob) model. In the current study, we examined the molecular mechanisms leading to retinal degeneration in WNIN/Ob rat.
Sorbitol was estimated by the fluorometric method in the retina of WNIN/Ob rats at different age (3-, 6- and 12- months), along with their respective lean rats. Immunoblotting was performed in the retina to assess the status of the insulin signaling pathway, ER stress and cellular stress (p38MAPK and ERK1/2). Human SK-N-SH cells were treated with 0.5 and 1.0 M sorbitol for 30 min to study insulin signaling, ER stress, and cellular stress. TUNEL assay was done to measure apoptosis. The retinal function in the rats was determined by electroretinogram.
A gradual but significantly higher intracellular sorbitol accumulation was observed in the retina of obese rats from 3- to 12-months. The cellular osmotic stress has activated the insulin signaling mechanism without activating AKT and also triggered ER stress. Both the stresses activated the ERK and p38MAPK signaling causing apoptosis in the retina leading to retinal degeneration. Retinal dysfunction was confirmed by altered scotopic and photopic electroretinogram responses. These in vivo results were mimicked in SK-N-SH cells when exposed to sorbitol in vitro.
These results suggest cellular stress due to sorbitol accumulation impairing the ER function, thereby leading to progressive retinal degeneration under obese conditions.
Orexin-A protects against oxygen-glucose deprivation/reoxygenation-induced cell damage by inhibiting endoplasmic reticulum stress-mediated apoptosis via the Gi and PI3K signaling pathways
2019, Cellular Signalling
The neuropeptide orexin-A (OXA) has a neuroprotective effect, acting as an anti-apoptotic factor in response to multiple stimuli. Apoptosis induced by endoplasmic reticulum stress (ERS) underlies oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell damage, an in vitro model of ischemia/reperfusion injury. However, that OXA inhibits ERS-induced apoptosis in the OGD/R model has not been reported. In the present study, we investigated the neuroprotective effect of OXA (0.1 μM) on OGD/R-induced damage in the human neuroblastoma cell line SH-SY5Y. After OXA treatment following 4 h oxygen-glucose deprivation (OGD) and then 4 h reoxygenation (R), cell morphology, viability, and apoptosis were analyzed by histology, Cell Counting Kit-8 assay, and flow cytometry, respectively. Western blotting was used to measure expression levels of ERS- and apoptosis-related proteins. To determine signaling pathways involved in OXA-mediated neuroprotection, the Gi pathway inhibitor pertussis toxin (PTX; 100 ng/mL) and PI3K inhibitor LY294002 (LY; 10 μM) were added. In addition, in order to prove the specificity of these characteristics, the OXA antagonist Suvorexant (DORA; Ki of 0.55 nM and 0.35 nM for OX1R and OX2R) was used for intervention. Our results showed that OGD/R induced cell damage, manifested as morphological changes and a significant decrease in viability. Furthermore, Western blotting detected an increase in ERS-related proteins GRP78, p-IRE1α, p-JNK, and Cleaved caspase-12, as well as apoptosis-related proteins Cleaved caspase-3 and Bax, and a decrease in the anti-apoptosis factor Bcl-2. OXA intervention alleviated the degree of cellular damage, and protein expression was also reversed. In addition, the protective effect of OXA was reduced by adding PTX and LY. Meanwhile, after the use of DORA, changes in the expression of related proteins were detected, and it was found that the protective effect of OXA was weakened. Collectively, our results indicate that OXA has a neuroprotective effect on OGD/R-induced cell damage by inhibiting ERS-induced apoptosis through the combined action of Gi and PI3K signaling pathways. These findings help to clarify the mechanism underlying the neuroprotective action of OXA, which should aid the development of further candidate drugs, and provide a new therapeutic direction for the treatment of ischemic stroke.
Salubrinal offers neuroprotection through suppressing endoplasmic reticulum stress, autophagy and apoptosis in a mouse traumatic brain injury model
2019, Neurobiology of Learning and Memory
Traumatic brain injury (TBI) is a complex injury that can cause severe disabilities and even death. TBI can induce secondary injury cascades, including but not limited to endoplasmic reticulum (ER) stress, apoptosis and autophagy. Although the investigators has previously shown that salubrinal, the selective phosphatase inhibitor of p-eIF2α, ameliorated neurologic deficits in murine TBI model, the neuroprotective mechanisms of salubrinal need further research to warrant the preclinical value. This study was undertaken to characterize the effects of salubrinal on cell death and neurological outcomes following TBI in mice and the potential mechanisms. In the current study, ER stress-related proteins including p-eIF2α, GRP78 and CHOP showed peak expressions both in the cortex and hippocampus from day 2 to day 3 after TBI, indicating ER stress was activated in our TBI model. Immunofluorescence staining showed that CHOP co-located NeuN-positive neuron, GFAP-positive astrocyte, Iba-1-positive microglia, CD31-positive vascular endothelial cell and PDGFR-β-positive pericyte in the cortex on day 2 after TBI, and these cells mentioned above constitute the neurovascular unit (NVU). We also found TBI-induced plasmalemma permeability, motor dysfunction, spatial learning and memory deficits and brain lesion volume were alleviated by continuous intraperitoneal administration of salubrinal post TBI. To investigate the underlying mechanisms further, we determined that salubrinal suppressed the expression of ER stress, autophagy and apoptosis related proteins on day 2 after TBI. In addition, salubrinal administration decreased the number of CHOP+/TUNEL+ and CHOP+/LC3+ cells on day 2 after TBI, detected by immunofluorescence. In conclusion, these data imply that salubrinal treatment improves morphological and functional outcomes caused by TBI in mice and these neuroprotective effects may be associated with inhibiting apoptosis, at least in part by suppressing ER stress-autophagy pathway.
Protein disulfide-isomerase A3 significantly reduces ischemia-induced damage by reducing oxidative and endoplasmic reticulum stress
2019, Neurochemistry International
Ischemia causes oxidative stress in the endoplasmic reticulum (ER), accelerates the accumulation of unfolded and misfolded proteins, and may ultimately lead to neuronal cell apoptosis. In the present study, we investigated the effects of protein disulfide-isomerase A3 (PDIA3), an ER-resident chaperone that catalyzes disulfide-bond formation in a subset of glycoproteins, against oxidative damage in the hypoxic HT22 cell line and against ischemic damage in the gerbil hippocampus. We also confirmed the neuroprotective effects of PDIA3 by using PDIA3-knockout HAP1 cells. The HT22 and HAP1 cell lines showed effective (dose-dependent and time-dependent) penetration and stable expression of the Tat-PDIA3 fusion protein 24 h after Tat-PDIA3 treatment compared to that in the control-PDIA3-treated group. We observed that the fluorescence for both 2′,7′-dichlorofluorescein diacetate (DCF-DA) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), which are markers for the formation of hydrogen peroxide (H₂O₂)-induced reactive oxygen species and apoptosis, respectively, was higher in HAP1 cells than in HT22 cells. The administration of Tat-PDIA3 significantly reduced the (1) DCF-DA and TUNEL fluorescence in HT22 and HAP1 cells, (2) ischemia-induced hyperactivity that was observed 1 day after ischemia/reperfusion, (3) ischemia-induced neuronal damage and glial (astrocytes and microglia) activation that was observed in the hippocampal CA1 region 4 days after ischemia/reperfusion, and (4) lipid peroxidation and nitric oxide generation in the hippocampal homogenates 3–12 h after ischemia/reperfusion. Transient forebrain ischemia significantly elevated the immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP) mRNA levels in the hippocampus at 12 h and 4 days after ischemia, relative to those in the time-matched sham-operated group. Administration of Tat-PDIA3 ameliorated the ischemia-induced upregulation of BiP mRNA levels versus the Tat peptide- or control-PDIA3-treated groups, and significantly reduced the induction of CHOP mRNA levels, at 12 h or 4 days after ischemia. Collectively, these results suggest that Tat-PDIA3 acts as a neuroprotective agent against ischemia by attenuating oxidative damage and blocking the apoptotic pathway that is related to the unfolded protein response in the ER.
Granulocyte-colony stimulating factor protects against endoplasmic reticulum stress in an experimental model of stroke
2018, Brain Research
Citation Excerpt :
It has been reported to be proteolytically cleaved to it active form, cleaved caspase-12, during prolonged ER stress (Yoneda et al., 2001). Caspase-12 was upregulated in transient MCAO and is associated with ER stress apoptosis (Shibata et al., 2003; Zhao et al., 2018). While these studies have advanced the field of ER stress in ischemia, the assessment was either done in vitro (Yoneda et al., 2001) or analysis was done on procaspase-12 and not the active form, cleaved caspase-12 (Shibata et al., 2003; Zhao et al., 2018).
Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exhibits a diverse range of neuroprotective mechanisms against a variety of neurological disorders including ischemic stroke. We investigated the anti-apoptotic mechanisms of G-CSF against endoplasmic reticulum (ER) stress induced apoptosis. Sprague-Dawley rats were subjected to transient occlusion of the middle cerebral artery (MCAO) for 90 min. Rats were injected with G-CSF (n = 15; 50 μg/kg body weight s.c.) for 4 days, starting 24 h post-MCAO and brains were harvested after 4 days reperfusion (n = 16). Key proteins in ER stress apoptosis were analyzed by immunoblotting. G-CSF reduced infarct volume to 53% and improved neurological deficits. G-CSF treatment significantly (P < .05) attenuated the expression of proteins involved in ER stress apoptosis pathway; ATF4, ATF6, p-p38MAPK, pJNK and CHOP. G-CSF treatment also re-established ER homeostasis evident by the reduction of the intraluminal ER stress sensor, GRP78 as well as reducing the overall cellular stress level protein, HSP27. G-CSF also up-regulated anti-apoptotic proteins pAKT and Bcl-2 while down-regulated the pro-apoptotic protein Bax. G-CSF exerts neuroprotection from cerebral ischemia through the preservation of the ER, resulting in the attenuation of pro-apoptotic proteins and the potentiation of anti-apoptotic proteins.

View all citing articles on Scopus

View full text

Original ContributionActivation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice

Abstract

Section snippets

Animals

Activation of caspase-12 following ischemia/reperfusion

Discussion

Acknowledgements

Mol Brain Res

Cell

J Biol Chem

J Biol Chem

J Biol Chem

Exp Neurol

Molecular pathways of protein synthesis inhibition during brain reperfusionimplication for neuronal survival or death

J Cereb Blood Flow Metab

Original Contribution
Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice