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.     

Cyanide enhancement of dopamine-induced apoptosis in mesencephalic cells involves mitochondrial dysfunction and oxidative stress

Dopamine (DA)-induced neurotoxicity is potentiated when cellular metabolism is compromised. Since cyanide is a neurotoxin that produces mitochondrial dysfunction and stimulates intracellular generation of reactive oxygen species (ROS), KCN was used to study DA-induced apoptosis in primary cultured mesencephalon cells. Treatment of neurons with DA (300 microM) for 24h produced apoptosis as determined by TUNEL staining, DNA fragmentation and increased caspase activity. Pretreatment with KCN (100 microM) 30min prior to DA increased the number of cells undergoing apoptosis. When added to the cells alone, this concentration of KCN did not induce apoptosis. DA stimulated intracellular generation of ROS, and treatment with KCN enhanced ROS generation. Treatment of cells with glutathione or uric acid (antioxidants/scavengers) attenuated both the increase in ROS generation and the apoptosis, demonstrating that ROS are initiators of the cytotoxicity. Studies on the sequence of events mediating the response showed that DA-induced depolarization of the mitochondrial membrane was dependent on ROS generation and KCN enhanced this action of DA. Following changes in mitochondrial membrane potential, cytochrome c was released from mitochondria, leading to caspase activation and eventually cell death. These results demonstrate that oxidative stress and mitochondrial dysfunction are initiators of DA-induced apoptosis. Subsequent cytochrome c release activates the caspase effector component of apoptosis. Cyanide potentiates the neurotoxicity of DA by enhancing the generation of ROS and impairing mitochondrial function.     

Induction of cytochrome c-mediated apoptosis by amyloid beta 25-35 requires functional mitochondria

Accumulating data suggest a central role for mitochondria and oxidative stress in neurodegenerative apoptosis. We previously demonstrated that amyloid-beta peptide 25-35 (Abeta 25-35) toxicity in cultured cells is mediated by its effects on functioning mitochondria. In this study, we further explored the hypothesis that Abeta 25-35 might induce apoptotic cell death by altering mitochondrial physiology. Mitochondria in Ntera2 (NT2 rho+) human teratocarcinoma cells exposed to either staurosporine (STS) or Abeta 25-35 were found to release cytochrome c, with subsequent activation of caspases 9 and 3. However, NT2 cells depleted of mitochondrial DNA (rho0 cells), which maintain a normal mitochondrial membrane potential (Deltapsi(m)) despite the absence of a functional electron transport chain (ETC), demonstrated cytochrome c release and caspase activation only with STS. We further observed increased reactive oxygen species (ROS) production and decreased reduced glutathione (GSH) levels in rho+ and rho0 cells treated with STS, but only in rho+ cells treated with Abeta 25-35. We conclude that under in vitro conditions, Abeta can induce oxidative stress and apoptosis only when a functional mitochondrial ETC is present.     

Apoptosis-related changes in skeletal muscles of patients with mitochondrial diseases

Much interest has recently been shown in apoptosis-mediated roles in the pathophysiology of mitochondrial diseases, because mitochondrial defects are implicated in a wide variety of degenerative diseases. We investigated whether apoptotic events occurred in skeletal muscles of patients with mitochondrial diseases, including chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayer syndrome (KSS), and mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). In a immunohistochemical study, stainings for 8-hydroxy-deoxyguanosine (8-OH-dG), 4-hydroxy-nonenal (4-HNE), Mn-SOD, Bcl-2, cytochrome c, DNase I and Bcl-x L showed a pronounced granular distribution in the cytochrome c oxidase (COX)-negative ragged-red fibers (RRFs). On the other hand, the signals for Bax, p53, Fas and caspase 3 were not obviously increased in RRFs. In situ labeling of DNA breaks demonstrated preferential signals not only in myonuclei but also in subsarcolemmal regions of RRFs, indicating that mitochondrial as well as myonuclear DNA is fragmented in RRFs. An immunoblotting study demonstrated that cytochrome c was increased in the cytosol of diseased muscles and that DNase I was increased in mitochondria, compared to that of normal muscles. No difference was observed between protein bands at 20 kDa corresponding to caspase 3 in diseased and normal muscles. These findings demonstrate that these mitochondrial diseases harbor unique apoptosis-related changes that differ from caspase 3-dependent apoptosis. It is thought that these changes are induced by superoxide overproduction and cytochrome c release resulting from an inherent mitochondrial defect and that the events are associated with DNase I activation.     

Mild hypothermia attenuates cytochrome c release but does not alter Bcl-2 expression or caspase activation after experimental stroke.

Mild hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not well known. The authors previously found that hypothermia reduces the density of apoptotic cells, but it is not certain whether temperature alters associated biochemical events. Mitochondrial release of cytochrome c has recently been shown to be a key trigger in caspase activation and apoptosis via the intrinsic pathway. Using a model of transient focal cerebral ischemia, the authors determined whether mild hypothermia altered expression of Bcl-2 family proteins, mitochondrial release of cytochrome c, and caspase activation. Mild hypothermia significantly decreased the amount of cytochrome c release 5 hours after the onset of ischemia, but mitochondrial translocation of Bax was not observed until 24 hours. Mild hypothermia did not alter Bcl-2 and Bax expression, and caspase activation was not observed. The present study provides the first evidence that intraischemic mild hypothermia attenuates the release of cytochrome c in the brain, but does not appear to affect other biochemical aspects of the intrinsic apoptotic pathway. They conclude that necrotic processes may have been interrupted to prevent cytochrome c release, and that the ameliorative effect of mild hypothermia may be a result of maintaining mitochondrial integrity. Furthermore, the authors show it is unlikely that mild hypothermia alters the intrinsic apoptotic pathway.     

Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice.

Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. We have reported that cytosolic redistribution of cytochrome c in vivo occurred after transient focal cerebral ischemia (FCI) in rats and preceded the peak of DNA fragmentation. Although the involvement of reactive oxygen species in the cytosolic redistribution of cytochrome c in vitro has been suggested, the detailed mechanism by which cytochrome c release is mediated in vivo has not yet been established. Also, the role of mitochondrial oxidative stress in cytochrome c release is unknown. These issues can be addressed using knock-out mutants that are deficient in the level of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD). In this study we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and heterozygous knock-outs of the Mn-SOD gene (Sod2 -/+) after permanent FCI, in which apoptosis is assumed to participate. Cytosolic cytochrome c was detected as early as 1 hr after ischemia, and correspondingly, mitochondrial cytochrome c showed a significant reduction 2 hr after ischemia (p < 0.01). Cytosolic accumulation of cytochrome c was significantly higher in Sod2 -/+ mice compared with wild-type animals (p < 0.05). N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone (z-VAD.FMK), a nonselective caspase inhibitor, did not affect cytochrome c release after ischemia. A significant amount of DNA laddering was detected 24 hr after ischemia and increased in Sod2 -/+ mice. These data suggest that Mn-SOD blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after permanent FCI.     

Protein synthesis-dependent but Bcl-2-independent cytochrome C release in zinc depletion-induced neuronal apoptosis

Previously, we reported that chelation of intracellular zinc with N, N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-induced macromolecule synthesis-dependent apoptosis of cultured cortical neurons. According to the current theory of apoptosis, release of mitochondrial cytochrome C into the cytosol is required for caspase activation. In the present study, we examined whether cytochrome C release is dependent on macromolecule synthesis. Exposure of cortical cultures to 2 microM TPEN for 24 hr induced apoptosis as previously described. Fluorescence immunocytochemical staining as well as immunoblots of cell extracts revealed the release of cytochrome C into the cytosol 18-20 hr after the exposure onset. The cytochrome C release was completely blocked by the addition of cycloheximide or actinomycin D. Addition of the caspase inhibitor zVAD-fmk did not attenuate the cytochrome C release, whereas it blocked TPEN-induced apoptosis. Because Bcl-2 has been shown to block cytochrome C release potently, we exposed human neuroblastoma cells (SH-SY5Y) to TPEN. Whereas Bcl-2 overexpression completely blocked both cytochrome C release and apoptosis induced by staurosporine, it attenuated neither induced by TPEN. The present results suggest that, in neurons, macromolecule synthesis inhibitors act upstream of cytochrome C release to block apoptosis and that, in addition to the classical Bcl-2 sensitive pathway, there may exist a Bcl-2-insensitive pathway for cytochrome C release.     

Effects of EGb 761 Ginkgo biloba extract on mitochondrial function and oxidative stress

As major sources of reactive oxygen species (ROS), mitochondrial structures are exposed to high concentrations of ROS and may therefore be particularly susceptible to oxidative damage. Mitochondrial damage could play a pivotal role in the cell death decision. A decrease in mitochondrial energy charge and redox state, loss of transmembrane potential (depolarization), mitochondrial respiratory chain impairment, and release of substances such as calcium and cytochrome c all contribute to apoptosis. These mitochondrial abnormalities may constitute a part of the spectrum of chronic oxidative stress in Alzheimer's disease. Accumulation of amyloid beta (Abeta) in form of senile plaques is also thought to play a central role in the pathogenesis of Alzheimer's disease mediated by oxidative stress. In addition, increasing evidence shows that Abeta generates free radicals in vitro, which mediate the toxicity of this peptide. In our study, PC12 cells were used to examine the protective features of EGb 761(definition see editorial) on mitochondria stressed with hydrogen peroxide and antimycin, an inhibitor of complex III. In addition, we investigated the efficacy of EGb 761 in Abeta-induced MTT reduction in PC12 cells. Moreover, we examined the effects of EGb 761 on ROS levels and ROS-induced apoptosis in lymphocytes from aged mice after in vivo administration. Here, we will report that EGb 761 was able to protect mitochondria from the attack of hydrogen peroxide, antimycin and Abeta. Furthermore, EGb 761 reduced ROS levels and ROS-induced apoptosis in lymphocytes from aged mice treated orally with EGb 761 for 2 weeks. Our data further emphasize neuroprotective properties of EGb 761, such as protection against Abeta-toxicity, and antiapoptotic properties, which are probably due to its preventive effects on mitochondria.     

Mitochondrial involvement in nitric oxide-induced cellular death in cortical neurons in culture

Nitric oxide (NO) is an unstable molecule with physiological and pathological properties. In brain, NO acts as a modulator of neurotransmission as well as a protector against neuronal death from several death stimuli. However, beside this protector effect, high NO concentrations produce neuronal death by a mechanism in which the caspase pathway is implicated. In this work, we demonstrate that in cortical neurons the NO toxicity is mediated by mitochondrial dysfunction. SNAP, an NO donor, induces apoptosis in these cells because it 1) increases the p53 and 2) induces cytochrome c release and activation of caspase-9 and caspase-3. SNAP also induces necrosis, through 1) breakdown of the mitochondrial membrane potential, 2) ATP decrease, 3) ROS formation, and 4) LDH and ATP release, indicative of oxidative stress and death by necrosis. To sum up, in cortical neurons, high NO concentrations produced cellular death by both an apoptotic and a necrotic mechanism in which the mitochondria are implicated.     

Mitochondrial pathway is involved in hydrogen-peroxide-induced apoptotic cell death of oligodendrocytes

Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to oxidative stress and respond by the onset of programmed cell death (PCD). To further unravel the molecular events underlying their enhanced susceptibility, we have investigated whether mitochondrial damage occurs during oxidative stress-induced PCD in cultured rat brain oligodendrocytes. Mitochondria are considered as a central control point of apoptosis, and mitochondrial dysfunction has been linked to neurodegenerative disease. Upon a number of stimuli through the release of cytochrome c, they coordinate caspase activation, causing morphological and biochemical changes associated with PCD. Oxidative stress was exerted by the application of hydrogen peroxide. The data show that hydrogen peroxide-induced apoptosis in oligodendrocytes involves mitochondrial damage and cytochrome c release and is accompanied by the activation of the death-related caspases 3 and 9. Concomitantly, the activation and nuclear translocation of extracellular signal regulated kinases ERK1,2 are observed, which have been implicated to participate in the regulation of cell death and survival. DNA fragmentation could not be attenuated by the ERK1,2 inhibitor PD 98059, indicating that the ERK1,2- pathway in oligodendrocytes may be involved in the initial survival response after exposure to stressful stimuli.     

Release of mitochondrial Opa1 following oxidative stress in HT22 cells

Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.     

Acute ammonia neurotoxicity in vivo involves increase in cytoplasmic protein P53 without alterations in other markers of apoptosis

Acute intoxication with large ammonia doses leads to activation of NMDA receptors in the brain, resulting in oxidative stress and disturbance of mitochondrial function. Altered mitochondrial function is a crucial step in some mechanisms of cellular apoptosis. This study assesses whether ammonia intoxication in vivo leads to induction of apoptotic markers such as permeability transition pore (PTP) formation, caspase-3, and caspase-9 activation, changes in p53 protein, or cytochrome c release. Acute ammonia intoxication did not affect caspase-9 or caspase-3 activities. The mitochondrial membrane potential also remained unaltered in non-synaptic brain mitochondria after injection of ammonia, indicating that ammonia did not induce PTP formation in brain in vivo. The nuclear level of p53 did not change, whereas its cytoplasmic level increased approximately two-fold. In agreement with the theory that translocation of the p53 from cytosol to nuclei is an essential step for induction of apoptosis we did not find apoptotic nuclei in brain of rats injected with ammonia. This supports the idea that ammonia neurotoxicity does not involve apoptosis and points to impaired p53 transfer from cytoplasm to nuclei as a possible preventer of apoptosis. We did not find any release of cytochrome c from mitochondria to cytosol after ammonia injection. Cytochrome c content was significantly reduced (30%) in brain mitochondria from rats injected with ammonia. This decrease may contribute to the reduced state 3 respiration, decreased respiratory control index, and disturbances in the mitochondrial electron transport chain in brain mitochondria from rats injected with ammonia.     

Rasagiline prevents apoptosis induced by PK11195, a ligand of the outer membrane translocator protein (18 kDa), in SH-SY5Y cells through suppression of cytochrome c release from mitochondria

Rasagiline protects neuronal cells from cell death caused by various lines of insults. Its neuroprotective function is due to suppression of mitochondrial apoptosis signaling and induction of neuroprotective genes, including Bcl-2 and neurotrophic factors. Rasagiline inhibits the mitochondrial membrane permeabilization, an initial stage in apoptosis, but the mechanism has been elusive. In this paper, it was investigated how rasagiline regulates mitochondrial death cascade in apoptosis induced in SH-SY5Y cells by PK11195, a ligand of the outer membrane translocator protein of 18 kDa. Rasagiline prevented release of cytochrome c (Cyt-c), and the following caspase 3 activation, ATP depletion and apoptosis, but did not inhibit the mitochondrial membrane potential collapse, in contrast to Bcl-2 overexpression. Rasagiline stabilized the mitochondrial contact site and suppressed Cyt-c release into cytoplasm, which should be the critical point for the regulation of apoptosis. Monoamine oxidase was not associated with anti-apoptotic activity of rasagiline in PK11195-induced apoptosis.     

Delayed mitochondrial dysfunction in excitotoxic neuron death: cytochrome c release and a secondary increase in superoxide production.

An increased production of superoxide has been shown to mediate glutamate-induced neuron death. We monitored intracellular superoxide production of hippocampal neurons during and after exposure to the glutamate receptor agonist NMDA (300 microm). During a 30 min NMDA exposure, intracellular superoxide production increased significantly and remained elevated for several hours after wash-out of NMDA. After a 5 min exposure, superoxide production remained elevated for 10 min, but then rapidly returned to baseline. Mitochondrial membrane potential also recovered after wash-out of NMDA. However, recovery of mitochondria was transient and followed by delayed mitochondrial depolarization, loss of cytochrome c, and a secondary rise in superoxide production 4-8 hr after NMDA exposure. Treatment with a superoxide dismutase mimetic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibited by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relationship between cytochrome c release and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (rho(-) cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochrome c, caspase activation, and cell death in control and rho(-) cells. However, a delayed increase in superoxide production was only observed in control cells. Our data suggest that the delayed superoxide production in excitotoxicity and apoptosis occurs secondary to a defect in mitochondrial electron transport and that mitochondrial cytochrome c release occurs upstream of this defect.     

Impaired mitochondrial energy metabolism and neuronal apoptotic cell death after chronic dichlorvos (OP) exposure in rat brain

The present study elucidates a possible mechanism by which chronic organophosphate exposure (dichlorvos 6 mg/kg bw, s.c. for 12 weeks) causes neuronal degeneration. Mitochondria, as a primary site of cellular energy generation and oxygen consumption represent itself a likely target for organophosphate poisoning. Therefore, the objective of the current study was planned with an aim to investigate the effect of chronic dichlorvos exposure on mitochondrial calcium uptake, oxidative stress generation and its implication in the induction of neuronal apoptosis in rodent model. Mitochondrial preparation from dichlorvos (DDVP) treated rat brain demonstrated significant increase in mitochondrial Ca(2+) uptake (644.2 nmol/mg protein). Our results indicated decreased mitochondrial electron transfer activities of cytochrome oxidase (complex IV) along with altered mitochondrial complex I, and complex II activity, which might have resulted from elevated mitochondrial calcium uptake. The alterations in the mitochondrial calcium uptake and mitochondrial electron transfer enzyme activities in turn might have caused an increase in malondialdehyde, protein carbonyl and 8-hydroxydeoxyguanosine formation as a result of enhanced lipid peroxidation, and as well as protein and mtDNA oxidation. All this could have been because of enhanced oxidative stress, decreased GSH levels and also decreased Mn-SOD activity in the mitochondria isolated from dichlorvos treated rat brain. Thus, chronic organophosphate exposure has the potential to disrupt cellular antioxidant defense system which in turn triggers the release of cytochrome c from mitochondria to cytosol as well as caspase-3 activation in dichlorvos treated rat brain as revealed by immunoblotting experiments. Low-level long-term organophosphate exposure finally resulted in oligonucleosomal DNA fragmentation, a hallmark of apoptosis. These studies provide an evidence of impaired mitochondrial bioenergetics and apoptotic neuronal degeneration after chronic low-level exposure to dichlorvos.     

Lithium inhibits Abeta-induced stress in endoplasmic reticulum of rabbit hippocampus but does not prevent oxidative damage and tau phosphorylation

The goal of this study was to assess the in vivo effect of Abeta on apoptosis pathways involving the endoplasmic reticulum and mitochondria, and its relationship to the induction of tau phosphorylation and DNA oxidative damage. In rabbits treated intracisternally with aggregated Abeta(1-42), clear evidence of endoplasmic reticulum stress was observed by the activation of caspase-12 and cleavage of caspase-3 in the endoplasmic reticulum. Mitochondrial injury was evident from the release of cytochrome c into the cytosol and the induction of oxidized mitochondrial DNA. Tau phosphorylation and nuclear translocation of NF-kappaB and GSK-3beta were also observed. Treatment with lithium, an inhibitor of GSK-3beta, inhibited caspase activation but did not prevent mitochondrial DNA damage or tau hyperphosphorylation, suggesting that the translocation of GSK-3beta may represent an upstream event that leads to caspase activation but is unrelated to tau hyperphosphorylation or mitochondrial DNA oxidative damage. We propose that treatment by lithium alone is not sufficient to protect against the multiple adverse effects of Abeta, and the use of agents that prevent oxidative DNA damage and tau hyperphosphorylation, together with lithium, may provide better protection from the neurotoxic effect of Abeta.     

Regional expression of Bcl-2 mRNA and mitochondrial cytochrome c release after experimental brain injury in the rat

Regional levels of anti-apoptotic Bcl-2 mRNA and the cytosolic cytochrome c protein were measured after lateral fluid percussion (FP) brain injury in rats. Levels of Bcl-2 mRNA were significantly decreased in the injured left cortex (IC) and ipsilateral hippocampus (IH), but not in the contralateral right cortex (CC) and hippocampus (CH) after brain injury. Levels of Bcl-2 mRNA were significantly decreased as early as 2 h and stayed decreased as long as 48 h in the IC and IH after injury. Levels of the cytosolic cytochrome c protein were significantly increased in the IC and IH, but not in the CC and CH after brain injury. Levels of cytosolic cytochrome c were significantly increased in the IC at 30 min, 48 and 72 h, and in the IH at 2 h and as long as 72 h after injury. The increase of cytosolic cytochrome c suggests that the mitochondrial release of cytochrome is increased in the IC and IH after lateral FP brain injury. These data show that the reduction of anti-apoptotic Bcl-2 and increases of mitochondrial release of cytochrome c protein occur only in the IC and IH, regions which have been observed to undergo apoptosis and neuronal cell loss after lateral FP brain injury. Therefore, it is likely that the reduction of Bcl-2 and the increased cytochrome c protein in the cytosol contribute to the observed apoptosis and neuronal cell death in the IC and IH after lateral FP brain injury in rats.     

Protective function of taurine in glutamate-induced apoptosis in cultured neurons

Previously, we showed that taurine protects neurons against glutamate-induced excitotoxicity by inhibiting the glutamate-induced increase of [Ca2+](i). In this study, we report that taurine prevents glutamate-induced chromosomal condensation, indicating that taurine inhibits glutamate-induced apoptosis. We found that Bcl-2 was down-regulated while Bax was up-regulated by glutamate treatment, and these changes were prevented in the presence of taurine. We have also shown that taurine inhibits glutamate-induced activation of calpain. Furthermore, calpastatin, a specific calpain inhibitor, also prevented glutamate-induced cell death. Here we propose the mechanisms underlying glutamate-induced apoptosis and taurine's inhibition of glutamate-induced apoptosis to be as follows: glutamate stimulation induces [Ca2+](i) elevation, which in turn activates calpain; activation of calpain leads to a reduction of Bcl-2:Bax ratios; with decreased Bcl-2:Bax ratios Bax homodimers form, Bax homodimerization, and translocation to the mitochondria result in the release of cytochrome c; released cytochrome c in turn activates a downstream caspase cascade leading to apoptosis. The antiapoptotic function of taurine is due to its inhibition of glutamate-induced membrane depolarization.     

Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra

Focal ischemia by middle cerebral artery occlusion (MCAO) results in necrosis at the infarct core and activation of complex signal pathways for cell death and cell survival in the penumbra. Recent studies have shown activation of the extrinsic and intrinsic pathways of caspase-mediated cell death, as well as activation of the caspase-independent signaling pathway of apoptosis in several paradigms of focal cerebral ischemia by transient MCAO to adult rats and mice. The extrinsic pathway (cell-death receptor pathway) is initiated by activation of the Fas receptor after binding to the Fas ligand (Fas-L); increased Fas and Fas-L expression has been shown following focal ischemia. Moreover, focal ischemia is greatly reduced in mice expressing mutated (nonfunctional) Fas. Increased expression of caspase-1, -3, -8, and -9, and of cleaved caspase-8, has been observed in the penumbra. Activation of the intrinsic (mitochondrial) pathway following focal ischemia is triggered by Bax translocation to and competition with Bcl-2 and other members of the Bcl-2 family in the mitochondria membrane that is followed by cytochrome c release to the cytosol. Bcl-2 over-expression reduces infarct size. Cytochrome c binds to Apaf-1 and dATP and recruits and cleaves pro-caspase-9 in the apoptosome. Both caspase-8 and caspase-9 activate caspase-3, among other caspases, which in turn cleave several crucial substrates, including the DNA-repairing enzyme poly(ADP-ribose) polymerase (PARP), into fragments of 89 and 28 kDa. Inhibition of caspase-3 reduces the infarct size, further supporting caspase-3 activation following transient MCAO. In addition, caspase-8 cleaves Bid, the truncated form of which has the capacity to translocate to the mitochondria and induce cytochrome c release. The volume of brain infarct is greatly reduced in Bid-deficient mice, thus indicating activation of the mitochondrial pathway by cell-death receptors following focal ischemia. Recent studies have shown the mitochondrial release of other factors; Smac/DIABLO (Smac: second mitochondrial activator of caspases: DIABLO: direct IAP binding protein with low pI) binds to and neutralizes the effects of the X-linked inhibitor of apoptosis (XIAP). Finally, apoptosis-inducing factor (AIF) translocates to the mitochondria and the nucleus following focal ischemia and produces peripheral chromatin condensation and large-scale DNA strands, thus leading to the caspase-independent cell death pathway of apoptosis. Delineation of the pro-apoptotic and pro-survival signals in the penumbra may not only increase understanding of the process but also help to rationalize strategies geared to reducing brain damage targeted at the periphery of the infarct core.