Differential induction of apoptosis by antidepressants in glioma and neuroblastoma cell lines

Several antidepressants, mainly selective serotonin-reuptake inhibitors (SSRIs) and some tricyclic antidepressants (TCAs), have been shown to possess potent apoptotic activity in different cell lines. Our aim was to screen and select those agents with significant activity and elucidate the molecular pathway underlying this process in rat glioma and human neuroblastoma cell lines. We studied the effect of different antidepressants on apoptotic markers, including: cell viability, DNA fragmentation, cytochrome c (Cyt c) release from mitochondria, and caspase-3- like activity. In addition, the involvement of MAPK genes, c-Jun, and ERK was determined. Paroxetine and fluoxetine, SSRIs, clomipramine, a TCA, but not imipramine or mianserin (an atypical antidepressant), caused apoptosis in both cell lines, as assessed by flow cytometry of propidium iodide-stained C6 cells and typical fluorescence microscopy in glioma cells. These apoptotic changes were preceded by rapid increase in p-c-Jun levels, Cyt c release from mitochondria, and increased caspase-3-like activity. Assessment of paroxetine cytotoxicity in primary mouse brain and neuronal cultures showed significantly lower sensitivity to the drug's proapoptotic activity. These results strongly suggest that selected antidepressants induce apoptosis in neuronal and glial cell lines. Activation of p-c-Jun and subsequent increased Cyt c mitochondrial release participate in the apoptotic mechanism of the antidepressant. The high sensitivity to these drugs of the cancer cell, compared with primary brain tissue, suggests the potential use of these agents in the treatment of brain-derived tumors.     

Intracerebral injection of caspase-3 inhibitor prevents neuronal apoptosis after kainic acid-evoked status epilepticus

In the aftermath of prolonged continuous seizure activity (status epilepticus, SE), neuronal cell death occurs in the brain regions through which the seizure propagates. Recent studies have implicated apoptotic processes in this seizure-related injury. Because activation of caspase-3-like cysteine proteases plays a crucial role in mammalian neuronal apoptosis, we explored the possibility that activation of caspase-3 is involved in the neuronal apoptotic cell death that occurs in rat brain following SE induced by systemic kainic acid. Caspase-3 activity was determined immunocytochemically using CM1 antibodies specific for catalytically active subunit (p17) of the enzyme. We found an induction of caspase-3 activity in rhinal cortex and amygdala at 24 h after SE. To determine whether activation of caspase-3-like proteases is a necessary component of the injury process, we delivered a caspase-3 inhibitor, z-DEVD-fmk, into the lateral ventricle prior to, and following SE. z-DEVD-fmk treatment substantially attenuated apoptotic cell death after SE, both in hippocampus and rhinal cortex, as evaluated by analysis of internucleosomal DNA fragmentation and neuronal nuclear morphology. Our findings implicate caspase-3 cysteine protease in the neurodegenerative response to SE and suggest that this degeneration can be attenuated by inhibition of caspase-3-like enzyme activity.     

The Role of Zinc in the Modulation of Neuronal Proliferation and Apoptosis

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G₀/G₁ phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.     

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm)

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH_m). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.     

Cytochrome c release and caspase activation after traumatic brain injury

Experimental traumatic brain injury (TBI) results in a rapid and significant necrosis of cortical tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the primary damage resulting in significant neurological dysfunction. The identification of cell death pathways that mediate this secondary traumatic injury have not been elucidated, however recent studies have implicated a role for apoptosis in the neuropathology of traumatic brain injury. The present study utilized a controlled cortical impact model of brain injury to assess the involvement of apoptotic pathways: release of cytochrome c from mitochondria and the activation of caspase-1- and caspase-3-like proteases in the injured cortex at 6, 12 and 24 h post-injury. Collectively, these results demonstrate cytochrome c release from mitochondria and its redistribution into the cytosol occurs in a time-dependent manner following TBI. The release of cytochrome c is accompanied by a time-dependent increase in caspase-3-like protease activity with no apparent increase in caspase-1-like activity. However, pretreatment with a general caspase inhibitor had no significant effect on the amount of cortical damage observed at 7 days post-injury. Our data suggest that several pro-apoptotic events occur following TBI, however the translocation of cytochrome c itself and/or other events upstream of caspase activation/inhibition may be sufficient to induce neuronal cell death.     

Varicella-zoster virus—induced apoptosis in MeWo cells is accompanied by down-regulation of Bcl-2 expression

Varicella-zoster virus infects multiple human and monkey cells in culture. The mode of cell death appears to be autophagy or apoptosis. Analysis of VZV-infected human melanoma (MeWo) cells revealed that Bcl-2 mRNA and protein levels were decreased significantly 64 and 72 hpi (hours post infection), accompanied by the release of cytochrome c from mitochondria into the cytoplasm. Western blot analysis of virus-infected cells revealed activation of caspase-8, a marker for the extrinsic pathway of apoptosis, and caspase-9, a marker for the intrinsic pathway of apoptosis at 64 and 72 hpi. Significant increases in the levels of cleaved caspase-3 and cleaved poly (ADP) ribose polymerase (PARP) were also seen at the height of cytopathic effect. Thus VZV induces apoptosis in MeWo cells in which Bcl-2 is down-regulated. Future studies will determine differences in the cascade of apoptotic events in non-neuronal cells compared to neurons that allow VZV to become latent.     

Polyamines prevent apoptotic cell death in cultured cerebellar granule neurons

Polyamines play critical roles during the development of brain neurons. In the present study we examined the effects of polyamines on neuronal apoptotic death. Rat cerebellar granule neurons were cultured in the presence of a depolarizing concentration of KCl (25 mM) in the medium. Apoptotic neuronal death was induced by changing the medium to that containing 5.6 mM KCl without serum. Spermine as well as spermidine and putrescine prevented cell death in a concentration-dependent manner with the order of potency being spermine>spermidine>putrescine. The effect of spermine was partially blocked by several NMDA-type glutamate receptor antagonists including (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801). MK-801-sensitive neuroprotection by spermine depended on cell density. Activation of CPP32 (caspase-3/Yama/apopain)-like proteolytic activity, a key mediator of apoptosis, precedes neuronal death, and polyamines prevented an increase in this activity. These results demonstrate that polyamines protect neurons from apoptotic cell death through both NMDA receptor-dependent and -independent mechanisms, acting upstream from the activation of CPP32-like protease(s). © 1997 Elsevier Science B.V. All rights reserved.     

BDNF regulates BIM expression levels in 3-nitropropionic acid-treated cortical neurons

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase that has been used to explore the primary mechanisms of cell death associated with mitochondrial dysfunction and neurodegeneration in Huntington's disease. In this study we investigated the ability of brain-derived neurotrophic factor (BDNF) to suppress mitochondrial-dependent cell death induced by 3-NP in primary cortical neurons. This neurotrophin prevented 3-NP-induced release of cytochrome c and Smac/Diablo, caspase-3-like activity and nuclear condensation/fragmentation. Furthermore, it greatly increased phosphorylation of Akt and MAPK, suggesting the involvement of these signalling pathways in BDNF neuroprotection. Interestingly, BDNF decreased the levels of the pro-apoptotic protein Bim in mitochondrial and total cell lysates through the activation of the MEK1/2 pathway. This effect was due to an increase in the degradation rates of Bim. Our data support an important role for BDNF, in protecting cortical neurons against apoptotic cell death caused by inhibition of mitochondrial complex II.     

Estradiol protects PC12 cells against CoCl2-induced apoptosis

In hypoxic/ischemic conditions, neuronal apoptotic events are occurred, resulting in neuronal diseases. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway leading to the inhibitory effects of estradiol against cobalt chloride (CoCl₂)-mediated hypoxic death in PC12 cells. Estradiol inhibits CoCl₂-induced cell death with genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. Pre-incubation of estradiol prior to CoCl₂ treatment attenuated CoCl₂-mediated the reactive oxygen species (ROS) production and limited the activities of the caspase cascades, such as caspase-8, -9 and -3. Furthermore, estradiol downregulated the Bax:Bcl-2 ratio and decreased the release of cytochrome c from the mitochondria into the cytosol in CoCl₂-treated cells, indicating that estradiol affect on mitochondrial pathway. Estradiol attenuated also CoCl₂-induced upregulation of Fas-ligand (Fas-L) and truncated of Bid in sequence of death receptor-mediated pathway. In addition, estradiol increased the phosphorylation of Akt in CoCl₂-treated cells, demonstrating that estradiol has no affect on upstream signaling through the PI3K/Akt in inhibition of CoCl₂-induced apoptosis in PC12 cells.Taken together, estradiol was found to have a neuroprotective effect against CoCl₂-induced apoptosis of PC12 cells by the attenuating ROS production and the modulating apoptotic signal pathway through Bcl-2 family, cytochrome c, Fas/Fas-L as well as PI3K/Akt pathway.     

Amyloid beta peptide induces cytochrome C release from isolated mitochondria

Amyloid beta peptide (Abeta) is a neurotoxic metabolic product of the amyloid precursor protein (APP). Abeta is strongly implicated in the pathology of Alzheimer's disease (AD) and can be formed intracellularly. In this study, we show that the addition of Abeta to isolated mouse brain mitochondria can directly induce cytochrome c (Cyt c) release and mitochondrial swelling, which were partially inhibited by cyclosporin A (CsA). These results suggest that the Abetaaccumulated intracellularly by APP processing might exert neurotoxicity by interacting with mitochondria and inducing mitochondrial swelling and release of Cyt c, which activates caspase-3 and finally can lead to apoptosis in neuronal cells and to neurodegeneration in AD.     

白藜芦醇通过Fas-线粒体双途径诱导U-87脑胶质瘤细胞凋亡

目的研究白藜芦醇（Res）对人脑胶质瘤细胞系U-87细胞增殖抑制和诱导凋亡作用,并探讨其分子机制。方法以人脑胶质瘤细胞系U-87细胞为靶细胞,应用四氮唑蓝（MTT）比色法测定细胞增殖活性;AnnexinV/碘化丙啶（PI）双标记和细胞形态学法检测U-87细胞凋亡;流式细胞仪（FCM）检测细胞Fas蛋白表达水平、线粒体跨膜电位（Δψm）、细胞色素C（Cytc）释放和Caspase-3活性变化。结果Res明显抑制U-87细胞的增殖,呈浓度及时间依赖性（P〈0.01）;20μmol/L和40μmol/LRes处理U-87细胞,AnnexinV/PI染色显示凋亡细胞明显增多,细胞凋亡率分别为42.57%和62%,同时细胞出现典型的凋亡形态改变;FCM检测显示Fas蛋白表达增高1.6～2.2倍,线粒体Δψm降低15%～63%,胞浆Cytc含量增加2.5～7.3倍,Caspase-3被激活,活性增高25%～112%。结论Res体外明显抑制U-87细胞的增殖,通过Fas-线粒体双途径诱导U-87细胞凋亡。     

Involvement of calcineurin in the neurotoxic effects induced by amyloid-beta and prion peptides

It is usually accepted that prion and amyloid-beta (A beta) peptides induce apoptotic cell death. However, the mechanisms that trigger neuronal death, induced by these amyloidogenic peptides, remain to be clarified. In the present study we analysed the neurotoxic effects of the synthetic prion and A beta peptides, PrP106-126 and A beta 25-35, in primary cultures of rat brain cortical cells. PrP106-126 and A beta 25-35 incubated at a concentration of 25 micro m for 24 h, did not affect cell membrane integrity, but decreased the metabolic capacity of the cells. The intracellular free Ca2+ concentration and reactive oxygen species levels increased significantly after 24 h treatment with PrP106-126 and A beta 25-35. Furthermore, these peptides (after 24 h exposure) also induced cytochrome c release from mitochondria and increased caspase-3-like activity. FK506, an inhibitor of the Ca2+/calmodulin-dependent phosphatase, calcineurin, was able to prevent cytochrome c release, caspase-3 activation and cell death induced by A beta 25-35 or PrP106-126 peptides. Taken together these data suggest that calcineurin is involved in A beta 25-35 and PrP106-126 neurotoxicity.     

boc-Aspartyl(OMe)-fluoromethylketone attenuates mitochondrial release of cytochrome c and delays brain tissue loss after traumatic brain injury in rats.

The pathobiology of traumatic brain injury (TBI) includes activation of multiple caspases followed by cell death with a spectrum of apoptotic phenotypes. There are initiator (e.g. caspase-2, -8, and -9) and effector (e.g. caspase-3 and -7) caspases. Recently, caspase-2 and -8 have been shown to regulate cell death via provoking cytochrome c release from the mitochondria upstream of caspase-9. Here, we show that an intracerebral injection of the pan-caspase inhibitor boc-Aspartyl(OMe)-fluoromethylketone (BAF; 1 micromol) 1 min after TBI in rats reduces caspase-3-like activity, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and tissue damage, and cytochrome c release in ipsilateral cortex at 24 h versus vehicle. To investigate whether either caspase-2 and/or caspase-8 activation may contribute to cytochrome release, the effect of BAF treatment on caspase-2 and caspase-8 proteolysis was also examined. boc-aspartyl(OMe)-fluoromethylketone treatment inhibited proteolysis of caspase-2 but not caspase-8 24 h after TBI in rats versus vehicle. However, BAF with or without nerve growth factor (12.5 ng/h x 14 days intracerebrally via osmotic pump) did not result in differences in motor function, Morris water maze performance, hippocampal neuron survival, nor contusion volume at 14 days. These data suggest that BAF treatment reduces acute cell death after TBI by inhibiting mitochondrial release of cytochrome c, possibly via a mechanism involving initiator caspases; however, BAF appears to delay cell death, rather than result in permanent protection.     

Inhibition of cysteine cathepsin B and L activation in astrocytes contributes to neuroprotection against cerebral ischemia via blocking the tBid‐mitochondrial apoptotic signaling pathway

The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here, we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid‐mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA‐074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP₂ and GFAP levels. In OGD‐induced astrocyte injury, CA‐074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD‐induced astrocytes injury, Clik148 or CA‐074Me reversed pMCAO or OGD‐induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial cytochrome‐c (Cyt‐c) and increase in cytoplastic Cyt‐c and active caspase‐3 at 12–24 h of the late stage of pMCAO or OGD. CA‐074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt‐c and decreased cytoplastic Cyt‐c and active caspase‐3 at 6 h of the early stage of Bid activation. CA‐074Me or Clik148 blocked the pMCAO‐induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase‐3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD‐induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid‐mitochondrial apoptotic signaling pathway. GLIA 2014;62:855–880     

Activation of cell death pathway after a brief period of global ischemia in diabetic and non-diabetic animals

Mitochondria play a critical role in the pathogenesis of cerebral ischemia. Acute hyperglycemia has been shown to activate the mitochondria-initiated cell death pathway after an intermediate period of ischemia. The objective of the present study was to determine if diabetic hyperglycemia induced by streptozotocin activates the cell death pathway after a brief period of global ischemia. Five minutes of global ischemia was induced in nondiabetic and diabetic rats. Brain samples were collected after 30 min, 6 h, 1, 3, and 7 days of recirculation as well as from sham-operated controls. Histopathological examination in the hippocampal CA1, CA3, hilus, and dentate gyrus regions, as well as in the cortical and thalamic areas, showed that neuronal death in diabetic animals increased compared to nondiabetic ischemic controls. Neuronal damage maturation occurred after 7 days of recovery in nondiabetic rats, while it was shortened to 3 days of recovery in diabetic animals. Western blot analyses revealed that release of cytochrome c markedly increased after 1 and 3 days of reperfusion in diabetic rats. Caspase-3 activation was evident in the nuclear fraction of the cortex of diabetic rats after 3 days recovery and it was preceded by activation of caspase-9, but not activation of caspase-8. Electron microscopy demonstrated that chromatin condensation and mitochondrial swelling were features of the diabetes-mediated ischemic neuronal damage. However, no apoptotic bodies were observed in any sections examined. These results suggest that a brief period of global ischemia in diabetic animals activates a neuronal cell death pathway involving cytochrome c release, caspase-9 activation, and caspase-3 cleavage, all of which are most likely initiated by early mitochondria damage.     

Novel mitochondrial proteins and decreased intrinsic activity of cytochrome- C Oxidase

Swayback disease (SD), a fatal neurological disorder affecting lambs and kids, is characterized by abnormal mitochondria and low cytochrome-c oxidase activity. The cytochrome-c oxidase content and activity and the protein composition of mitochondria isolated from the brains of SD lambs were investigated. Difference spectra analysis indicated that the cytochrome-c oxidase content of mitochondria from SD animals was lower than normal, and electrophoresis showed that when compared to mitochondria from normal animals, lipid-depleted mitochondria from SD lambs had a different protein composition, particularly, in the 40–55 kDa region. Polarographic studies, using cytochrome-c as substrate, confirmed low intrinsic activity of cytochrome-c oxidase within the mitochondria of SD lambs. These studies also showed that at low ionic strength, such mitochondria did not yield the expected characteristic biphasic Eadie-Hofstee plots.     

Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice

In experimental models of cerebral ischemia, cells within the damaged territory die by necrosis and by apoptosis that contributes to the expansion of the insult. Apoptotic machinery mobilizes intracellular processes such as induction of Bcl-2 family members, activation of the proteolytic cascade including the caspases, and cleavage of caspase substrates, such as poly(ADP–ribose) polymerase or PARP. Mitochondria play a pivotal role in controlling apoptosis by releasing cytochrome c and modulating redox state, both under the regulation of manganese superoxide dismutase (Mn SOD) via superoxide anion detoxification. The implication and the kinetics of such events in apoptosis induced after focal permanent ischemia in mice remains to be studied. In a paradigm of ischemic insult induced by occlusion of the middle cerebral artery (MCAO) in mice, we showed by immunohistochemistry a constitutive expression of caspase-3 that is enhanced after MCAO in neurons localized within the infarcted zone. As a function of time intervals after MCAO, the cytochrome c amount increased in the cytosolic fraction of ischemic cortical extracts. The kinetics of the release was in concordance with the expression of caspase-3 and the subsequent cleavage of PARP appearing before the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. When the apoptotic markers progressively appeared, no changes of Mn SOD activity or Mn SOD expression were detected after MCAO. We can therefore speculate that the recruitment of Mn SOD did not participate per se in the release of cytochrome c elicited after permanent focal ischemia.     

Cadmium exposure induces mitochondria-dependent apoptosis in oligodendrocytes

Cadmium toxicity has been associated with learning disabilities and Parkinsonian symptoms in humans. We have previously shown that cultured oligodendrocytes are directly damaged by cadmium exposure. Here, we characterized the molecular mechanisms underlying cadmium-induced cell death in oligodendrocyte progenitors (OLP). Cadmium caused a concentration-dependent decrease in cell viability as assessed by mitochondrial dehydrogenase activity and by the cellular release of lactate dehydrogenase (LDH). A short exposure (1 h) to cadmium (25–100 μM), followed by several hours of recovery, produced a predominant apoptotic mechanism of cell death, involving the mitochondrial intrinsic pathway, as evidenced by nuclear condensation, DNA fragmentation, bax integration into the outer mitochondrial membrane, cytochrome c release, and activation of caspases-9 and -3. Pretreatment of OLPs with the pan-caspase inhibitor, zVAD-fmk, prevented caspase-3 activation but only slightly reduced cell death 11 h after cadmium exposure and failed to prevent cadmium-induced bax insertion into the mitochondrial membrane. In contrast, the anti-oxidant N-acetyl cysteine blocked caspase-3 activation and significantly protected OLPs from cadmium-induced cell death. Continuous exposure (18–48 h) of OLPs to low micromolar concentrations (0.001–25 μM) of cadmium significantly decreased mitochondrial metabolic activity, increased LDH leakage starting at 5 μM and maximally activated caspase-3. These results suggest that cadmium induces OLP cell death mainly by apoptosis, and at higher concentrations or with prolonged exposure to the heavy metal there is an increase in cytoplasmic membrane damage, an index of necrosis. More importantly, transient exposure to cadmium is sufficient to damage OLPs and could in principle impair myelination in the neonate.     

Temporal profile of cytochrome c and caspase-3 immunoreactivities and TUNEL staining after permanent middle cerebral artery occlusion in rats

Abstract

Although apoptotic pathways play important roles in ischemic neuronal injury, exact mechanism of apoptotic enzyme cascade has not been fully studied. Immunohistochemical stainings for cytochrome c and caspase-3, and histochemical staining for a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling method (TUNED were examined in a rat model of permanent middle cerebral artery (MCA) occlusion. Cytochrome c was strongly induced in neurons of the ischemic penumbra from 3 h after MCA occlusion, and caspase-3 began to be induced in the same area from 3 h with a peak at 8 h. Neuronal cells in MCA area became TUNEL positive at delayed time, reaching a peak at 24 h. Thus, the peak of induction of cytochrome c preceded that of caspase-3, and these two peaks were also precedence of the peak of DNA-fragmentation. Western blot analysis showed cytosolic expression of cytochrome c from mitochondria. This study demonstrated 7. Rapid release of cytochrome c from mitochondria to the cytosol, mainly in neurons of the cortex at 3 h after ischemia. 2. Subsequent peaks of caspase-3 and TUNEL in this order. These temporal profiles suggest a serial cascadic activation of apoptotic pathways in neuronal death after permanent MCA occlusion of rats. [Neurol Res 2000; 22: 223-228]     

Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death.

Activation of terminal caspases such as caspase-3 plays an important role in the execution of neuronal cell death after transient cerebral ischemia. Although the precise mechanism by which terminal caspases are activated in ischemic neurons remains elusive, recent studies have postulated that the mitochondrial cell death-signaling pathway may participate in this process. The bcl-2 family member protein Bax is a potent proapoptotic molecule that, on translocation from cytosol to mitochondria, triggers the activation of terminal caspases by increasing mitochondrial membrane permeability and resulting in the release of apoptosis-promoting factors, including cytochrome c. In the present study, the role of intracellular Bax translocation in ischemic brain injury was investigated in a rat model of transient focal ischemia (30 minutes) and reperfusion (1 to 72 hours). Immunochemical studies revealed that transient ischemia induced a rapid translocation of Bax from cytosol to mitochondria in caudate neurons, with a temporal profile and regional distribution coinciding with the mitochondrial release of cytochrome c and caspase-9. Further, in postischemic caudate putamen in vivo and in isolated brain mitochondria in vitro, the authors found enhanced heterodimerization between Bax and the mitochondrial membrane permeabilization-related proteins adenine nucleotide translocator (ANT) and voltage-dependent anion channel. The ANT inhibitor bongkrekic acid prevented Bax and ANT interactions and inhibited Bax-triggered caspase-9 release from isolated brain mitochondria in vitro. Bongkrekic acid also offered significant neuroprotection against ischemia-induced caspase-3 and caspase-9 activation and cell death in the brain. These results strongly suggest that the Bax-mediated mitochondrial apoptotic signaling pathway may play an important role in ischemic neuronal injury.