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1.
Juan A. Martinez Zhiqun Zhang Stanislav I. Svetlov Ronald L. Hayes Kevin K. Wang Stephen F. Larner 《Apoptosis : an international journal on programmed cell death》2010,15(12):1480-1493
Neuronal cell death after traumatic brain injury, Alzheimer’s disease and ischemic stroke may in part be mediated through
endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR results in induction of molecular chaperone GRP78
and the ER-resident caspase-12, whose activation has been proposed to be mediated by calpain and caspase processing, although
their relative contribution remains unclear. In this study we induced ER stress with thapsigargin (TG), and determined the
activation profile of calpain-2, caspase-3, caspase-7, and caspase-12 by analyses of protein levels, corresponding substrates
and breakdown products (BDP). Specific calpain and caspase activity was assessed by analysis of αII-spectrin BDP of 145 kDa
(SBDP145), BDP of 150 kDa (SBDP150) and BDP of 120 kDa (SBDP120). Decrease in pro-calpain-2 protein and increased SBDP145
levels by 3 h after TG treatment indicated early calpain activity. Active caspase-7 (p20) increase occurred after 8 h, followed
by concomitant up-regulation of active caspase-3 and SBDP120 after 24 h. In vitro digestion experiments supported that SBDP120
was exclusively generated by active caspase-3 and validated that kinectin and co-chaperone p23 were calpain and caspase-7
substrates, respectively. Pro-caspase-12 protein processing by the specific action of calpain and caspase-3/7 was observed
in a time-dependent manner. N-terminal pro-domain processing of pro-caspase-12 by calpain generated a 38 kDa fragment, while
caspase-3/7 generated a 35 kDa fragment. Antibody developed specifically against the caspase-3/7 C-terminal cleavage site
D341 detected the presence of large subunit (p20) containing 23 kDa fragment that increased after 24 h of TG treatment. Significant
caspase-12 enzyme activity was only detected after 24 h of TG treatment and was completely inhibited by caspase 3/7 inhibitor
DEVD-fmk and partially by calpain inhibitor SNJ-1945. ER-stress-induced cell death pathway in TG-treated PC12 cells was characterized
by up-regulation of GRP-78 and processing and activation of caspase-12 by the orchestrated proteolytic activity of calpain-2
and caspase-3/7. 相似文献
2.
Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of specificity for defining neuropathological cascades. Identification of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Non-erythroid alpha II-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of alpha II-spectrin by calpain and caspase-3 results in accumulation of protease-specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of alpha II-spectrin and alpha II-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native alpha II-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-specific 145 kDa SBDP in CSF were 244%, 530% and 665% of sham-injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase-3-specific SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of alpha II-spectrin and alpha II-SBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase-3. 相似文献
3.
Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings. 相似文献
4.
Nath R Huggins M Glantz SB Morrow JS McGinnis K Nadimpalli R Wanga KK 《Neurochemistry international》2000,37(4):351-361
Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo. 相似文献
5.
Evidence for Activation of Caspase-3-Like Protease in Excitotoxin- and Hypoxia/Hypoglycemia-Injured Neurons 总被引:2,自引:0,他引:2
Rathna Nath Albert Probert Jr. †Kim M. McGinnis † Kevin K. W. Wang 《Journal of neurochemistry》1998,71(1):186-195
Abstract: Caspase activation has been shown to be a critical step in several models of neuronal apoptosis such as staurosporine treatment of human neuroblastoma SH-SY5Y cells and potassium deprivation of rat cerebellar granule neurons. One common event is the appearance of caspase-mediated 120-kDa nonerythroid α-spectrin breakdown product (SBDP120). Second, inhibitors of the caspase family are effective blockers of such neuronal death. In this study, we report the appearance of caspase-mediated SBDP120 in excitotoxin-challenged fetal rat cerebrocortical neurons [ N -methyl- d -aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate] and rat cerebellar granule neurons (NMDA and kainate). A general caspase inhibitor, carbobenzoxy-Asp-CH2 OC(O)-2,6-dichlorobenzene (Z-D-DCB), blocked the formation of SBDP120 under these conditions and attenuated the observed NMDA-induced lactate dehydrogenase (LDH) release in both cell types. Furthermore, hydrolytic activity toward a caspase-3-preferred synthetic peptide substrate, acetyl-DEVD-7-amido-4-methylcoumarin, was significantly elevated in NMDA-treated granule neurons. Lastly, oxygen-glucose deprivation (OGD)-challenged cerebrocortical cultures also showed the appearance of SBDP120. Again, Z-D-DCB blocked the SBDP120 formation as well as attenuated the LDH release from the OGD-challenged neurons. Taken together, the presence of caspase-specific SBDP120 and the neuroprotective effects of Z-D-DCB strongly suggest that caspase activation contributes at least in part to excitotoxin- and OGD-induced neuronal death. 相似文献
6.
Glioblastoma is the most malignant human brain tumor that shows poor response to existing therapeutic agents. Search continues
for an effective therapy for controlling this deadliest brain tumor. Curcumin (CCM), a polyphenolic compound from Curcuma longa, possesses anti-cancer properties in both in vitro and in vivo. In the present investigation, we evaluated the therapeutic
efficacy of CCM against human malignant glioblastoma U87MG cells. Trypan blue dye exclusion test showed decreased viability
of U87MG cells with increasing dose of CCM. Wright staining and ApopTag assay, respectively, showed the morphological and
biochemical features of apoptosis in U87MG cells treated with 25 μM and 50 μM of CCM for 24 h. Western blotting showed activation
of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, and release of cytochrome c from mitochondria followed by activation of caspase-9 and caspase-3 for apoptosis. Also, CCM treatments increased cytosolic
level of Smac/Diablo to suppress the inhibitor-of-apoptosis proteins and down regulated anti-apoptotic nuclear factor kappa
B (NFκB), favoring the apoptosis. Increased activities of calpain and caspase-3 cleaved 270 kDa α-spectrin at specific sites
generating 145 kDa spectrin break down product (SBDP) and 120 kDa SBDP, respectively, leading to apoptosis in U87MG cells.
Results show that CCM is an effective therapeutic agent for suppression of anti-apoptotic factors and activation of calpain
and caspase proteolytic cascades for apoptosis in human malignant glioblastoma cells.
Special issue in honor of Naren Banik. 相似文献
7.
Ramunas Rolius Chloe Antoniou Lidia A. Nazarova Stephen H. Kim Garrett Cobb Pooja Gala Priyanka Rajaram Qufei Li Leslie W.-M. Fung 《Cellular & molecular biology letters》2010,15(3):395-405
Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains
are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid
(brain) α- and β-spectrin and erythroid β-spectrin. The cleavage of erythroid α-spectrin by calpain has not been reported.
Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid
but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid
and non-erythroid spectrin fragments used were from the N-terminal region of α-spectrin, and C-terminal region of β-spectrin,
both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited
a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are
warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that
calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether
the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to
the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides
with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity. 相似文献
8.
Karmakar S Banik NL Patel SJ Ray SK 《Apoptosis : an international journal on programmed cell death》2007,12(11):2077-2087
Glioblastoma is the most prevalent and highly malignant brain tumor that continues to defy current treatment strategies. This
investigation used all-trans retinoic acid (ATRA) and taxol (TXL) as a combination therapy for controlling the growth of human glioblastoma T98G xenografted
in athymic nude mice. Histopathological examination revealed that ATRA induced differentiation and combination of ATRA and
TXL caused more apoptosis than either treatment alone. Combination therapy decreased expression of telomerase, nuclear factor
kappa B (NFκВ), and inhibitor-of-apoptosis proteins (IAPs) indicating suppression of survival factors while upregulated Smac/Diablo.
Combination therapy also changed expression of Bax and Bcl-2 proteins leading to increased Bax:Bcl-2 ratio, mitochondrial
release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9. Increased activities of calpain and caspase-3 degraded
270 kD α-spectrin at the specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively.
Further, increased activity of caspase-3 cleaved inhibitor-of-caspase-activated DNase (ICAD). In situ double immunofluorescent
labelings showed overexpression of calpain, caspase-12, caspase-3, and AIF during apoptosis, suggesting involvement of both
caspase-dependent and caspase-independent pathways for apoptosis. Our investigation revealed that treatment of glioblastoma
T98G xenografts with the combination of ATRA and TXL induced differentiation and multiple molecular mechanisms for apoptosis. 相似文献
9.
Aims Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective
effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3
actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death. Methods Male Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine
was administrated intravenously 1 h after MCAo. The dose–responses of taurine to MCAo were determined. Next, the effects of
taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2
(MAP-2) and αII-spectrin, and the apoptotic cell death in penumbra were evaluated. Results Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment
with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain
and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on μ-calpain activity. Moreover,
taurine significantly increased the MAP-2 and αII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL
staining positive score within penumbra 24 h after MCAo. Conclusions Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest
that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic
cell death pathways. 相似文献
10.
Neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions as well as neurodegenerative diseases. In the present study, we have characterized calpain activation in cytosolic extract of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Pathology of FMCM resulted in the increase in activity of calpains in both cerebral cortex and cerebellum. Western blot analysis revealed an increase in the levels of mu-calpain (calpain-1) in the cytosolic fraction of infected cerebral cortex and cerebellum although a decrease in the level of m-calpain was observed in the cytosolic fraction of infected cerebellum and cerebral cortex. Calpain activation was further confirmed by monitoring the formation of calpain-specific spectrin breakdown products (SBDP). Protease-specific SBDP revealed the formation of calpain-generated 150kDa product in the infected cerebral cortex and cerebellum. The specific signature fragment of calpain activation and spectrin breakdown after Plasmodium berghei ANKA infection provide a strong evidence of the role of calpains during the cell death in cerebral cortex and cerebellum. Given the role of calpains in neurodegeneration and cell death, our results strongly suggest that calpains are important mediators of cell injury and neurological sequelae associated with FMCM. 相似文献
11.
Neurotoxicity in rat cortex and hippocampus following acute methamphetamine administration was characterized and compared to changes following traumatic brain injury. Doses of 10, 20, and 40 mg/kg of methamphetamine produced significant increases in calpain- and caspase-cleaved alpha II-spectrin and tau protein fragments, suggesting cell injury or death. Changes in proteolytic products were significantly increased over vehicle controls. Use of fragment specific biomarkers detected prominent calpain-mediated protein fragments in the cortex and hippocampus while caspase-mediated protein fragments were also detected in the hippocampus. Remarkably, proteolytic product increases at the 40 mg/kg dose after 24 h were as high as those observed in experimental traumatic brain injury. Use of calpain and caspase proteolytic inhibitors may be useful in preventing methamphetamine-induced neurotoxicity. 相似文献
12.
Glioblastoma is the deadliest brain tumor in humans. Current therapies are mostly ineffective and new agents need to be explored
for controlling this devastating disease. Inositol hexaphosphate (IP6) is a phytochemical that is widely found in corns, cereals,
nuts, and high fiber-content foods. Previous studies demonstrated anti-cancer properties of IP6 in several in vitro and in
vivo tumor models. However, therapeutic efficacy of IP6 has not yet been evaluated in glioblastoma. Here, we explored the
molecular mechanism of action of IP6 in human malignant glioblastoma T98G cells. The viability of T98G cells decreased following
treatment with increasing doses of IP6. T98G cells exposed to 0.25, 0.5, and 1 mM IP6 for 24 h showed morphological and biochemical
features of apoptosis. Western blotting indicated changes in expression of Bax and Bcl-2 proteins resulting in an increase
in Bax:Bcl-2 ratio and upregulation of cytosolic levels of cytochrome c and Smac/Diablo, suggesting involvement of mitochondria-dependent
caspase cascade in apoptosis. IP6 downregulated cell survival factors such as baculovirus inhibitor-of-apoptosis repeat containing-2
(BIRC-2) protein and telomerase to promote apoptosis. Upregulation of calpain and caspase-9 occurred in course of apoptosis.
Increased activities of calpain and caspase-3 cleaved 270 kD α-spectrin at specific sites generating 145 kD spectrin break
down product (SBDP) and 120 kD SBDP, respectively. Increased caspase-3 activity also cleaved inhibitor of caspase-3-activated
DNase and poly(ADP-ribose) polymerase. Collectively, our results demonstrated that IP6 down regulated the survival factors
BIRC-2 and telomerase and upregulated calpain and caspase-3 activities for apoptosis in T98G cells.
Special issue in honor of Naren Banik. 相似文献
13.
Hwang IK Ahn JH Yoo DY Lee CH Yoo KY Choi JH Moon SM Shin HC Won MH 《Neurochemical research》2012,37(3):480-486
The activation of caspase-3 is considered to be a reliable marker for apoptotic cell death, and a 120-kDa fragment of αII-spectrin
is generated by caspase-3 mediated cleavage of this structural protein. In the present study, we compared cleaved αII-spectrin
(120-kDa) and cleaved caspase-3-immunoreactive cells and their protein levels in the cervical (C5–C6) and lumbar (L3–L4) levels of the spinal cord in adult (1–2 year-old) and aged (10–12 year-old) dogs (German shepherds). Weak cleaved αII-spectrin
and cleaved caspase-3 immunoreactivity was found in neurons of the adult group; however, their immunoreactivity was distinctively
increased in the neuronal cytoplasm in the aged group compared to those in the adult group, although the distribution pattern
of their neurons was similar between the adult and age group. In addition, cleaved αII-spectrin and cleaved caspase-3 levels
in the aged spinal cord were markedly increased compared to those in the adult group. These findings suggest that the increases
of cleaved αII-spectrin and cleaved caspase-3 immunoreactivity may be related to aging of the spinal cord in dogs. 相似文献
14.
Calbindin-D28K protects against apoptotic and necrotic cell death; these effects have been attributed to its ability to buffer calcium. In this study, we investigated the mechanisms underlying the neuroprotective effects of calbindin-D28K in staurosporine (STS)-induced apoptosis and 1-methyl-4-phenylpyridinium (MPP+)-induced necrosis. Treatment of the dopaminergic neuronal cell line MN9D with STS or MPP+ induced cell death that was associated with increased levels of free intracellular calcium. However, only MPP+-induced death was inhibited by co-treatment of the cells with a calcium chelator or a sodium/calcium antiporter inhibitor. Overexpression of calbindin-D28K prevented MPP+-induced MN9D cell death, which occurs in the absence of any detectable caspase activation. These pro-survival effects of calbindin-D28K were associated with the inhibition of calcium-mediated calpain activation, as determined by processing of Bax. Overexpression of calbindin-D28K also blocked STS-induced MN9D death. However, this effect was accompanied by the inhibition of capase-3 cleavage, poly(ADP-ribose)polymerase cleavage, and caspase activity. These findings suggest that calbindin-D28K protects against both types of cell death by inhibiting caspase- or calcium-mediated death signaling pathway. 相似文献
15.
Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells 总被引:12,自引:1,他引:11
Karmakar S Banik NL Patel SJ Ray SK 《Apoptosis : an international journal on programmed cell death》2007,12(4):671-684
Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl
sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability
of human primary neurons was unaffected after 24 h treatment with 50 and 100 μM DAS and 50 μM DADS but slightly affected with
100 μM DADS. Treatment with 50 and 100 μM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining
showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 μM DAS or DADS for 24 h. ApopTag assay
demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca2+]i, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFκB).
Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities
produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor
of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic
factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells. 相似文献
16.
Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells 总被引:12,自引:0,他引:12
Nath R Davis M Probert AW Kupina NC Ren X Schielke GP Wang KK 《Biochemical and biophysical research communications》2000,274(1):16-21
Recently, it was shown that conversion of cdk5 activator protein p35 to a C-terminal fragment p25 promotes a deregulation of cdk5 activity, which may contribute to neurodegeneration in Alzheimer's disease. In this study, we present evidence that calpain is a protease involved in the conversion of p35 to p25. To activate calpain, rat cerebellar granule neurons were treated with maitotoxin (MTX). A C-terminus-directed anti-p35 antibody detected that p35 conversion to p25 paralleled the formation of calpain-generated alpha-spectrin (alpha-fodrin) breakdown products (SBDP's) in a maitotoxin-dose-dependent manner. Two calpain inhibitors (MDl28170 and SJA6017) reduced p35 processing but were unchanged when exposed to the caspase inhibitor carbobenzoxy-Asp-CH(2)OC(=O)-2, 6-dichlorobenzene or the proteasome inhibitors (lactacystin and Z-Ile-Glu(OtBu)Ala-Leu-CHO). p35 protein was also degraded to p25 when rat brain lysate was subjected to in vitro digestion with purified mu- and m-calpains. Additionally, in a rat temporary middle cerebral artery occlusion model, p35 processing to p25 again paralleled SBDP formation in the ischemic core. Lastly, in malonate-injured rat brains, the ipsilateral side showed a striking correlation of SBDP formation with p35 to p25 conversion and tau phosphorylation (at Ser202 and Thr205) increase. These data suggest that calpain is a major neuronal protease capable of converting p35 to p25 and might play a pathological role of activating cdk5 and its phosphorylation of tau in Alzheimer's disease. 相似文献
17.
Tamada Y Fukiage C Daibo S Yoshida Y Azuma M Shearer TR 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2002,131(2):221-225
Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N2/5% CO2. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate alpha-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of mu- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa alpha-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro. 相似文献
18.
Yap YW Whiteman M Bay BH Li Y Sheu FS Qi RZ Tan CH Cheung NS 《Journal of neurochemistry》2006,98(5):1597-1609
3-Chlorotyrosine, a bio-marker of hypochlorous acid (HOCl) in vivo, was reported to be substantially elevated in the Alzheimer's disease (AD) brains. Thus, HOCl might be implicated in the development of AD. However, its effect and mechanism on neuronal cell death have not been investigated. Here, we report for the first time that HOCl treatment induces an apoptotic-necrotic continuum of concentration-dependent cell death in cultured cortical neurons. Neurotoxicity caused by an intermediate concentration of HOCl (250 microm) exhibited several biochemical markers of apoptosis in the absence of caspase activation. However, the involvement of calpains was demonstrated by data showing that calpain inhibitors protect cortical neurons from apoptosis and the formation of 145/150 kDa alpha-fodrin fragments. Moreover, an increase in cytosolic Ca2+ concentration was associated with HOCl neurotoxicity and Ca2+ channel antagonists, and Ca2+ chelators prevented cleavage of alpha-fodrin and the induction of apoptosis. Finally, we found that calpain activation ruptured lysosomes. Stabilization of lysosomes by calpain inhibitors or imidazoline drugs, as well as inhibition of cathepsin protease activities, rescued cells from HOCl-induced neurotoxicity. Our results showed for the first time that HOCl induces apoptosis in cortical neurons, and that the cell death process involves calpain activation and rupture of lysosomes. 相似文献
19.
Iyer Aarti Kamindla Rajesh Kolluru V. A. Ramaiah 《Apoptosis : an international journal on programmed cell death》2010,15(6):679-692
An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2α) involved in
translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of
BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in
non-ER stress-induced eIF2α phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER
stress-induced eIF2α phosphorylation. Infection of Sf9 cells by wt and a mutant Δpk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced
eIF2α phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Δpk2 baculovirus that lacks
pk2, an inhibitor of eIF2α kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER
stress-induced eIF2α phosphorylation, thereby suggesting that eIF2α phosphorylation is a cause and consequence of caspase
activation. The importance of BiP affecting the delicate balance between eIF2α phosphorylation-mediated cell survival and
death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2α phosphorylation-mediated
cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2α, stimulates caspase activation
in cell extracts devoid of BiP. These findings therefore suggest that eIF2α phosphorylation is primarily a stress signal and
evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling
activities, and inter-protein interactions. 相似文献
20.
W S Choi E H Lee C W Chung Y K Jung B K Jin S U Kim T H Oh T C Saido Y J Oh 《Journal of neurochemistry》2001,77(6):1531-1541
Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells. 相似文献