A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro.

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase-3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl-xL, cyclosporin A and z-VAD.fmk. Moreover, caspase-3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl-xL- and cyclosporin A-inhibitable manner. These results suggest that caspase-3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.     

In vivo application of mitochondrial pore inhibitors blocks the induction of apoptosis in axotomized neonatal facial motoneurons

Axotomy induces apoptosis in motoneurons of neonatal rodents. To identify the key players in motoneuron apoptosis, we assessed the progression of apoptosis at 4 h intervals following facial motoneuron axotomy. The mitochondrial release of cytochrome c, caspase-3 activation and nuclear condensation were first observed in the motoneuron cell bodies 16 h postaxotomy. In vivo application of inhibitors of the mitochondrial permeability transition pore, Bongkrekic acid and cyclosporin A prevented cytochrome c release as well as caspase-3 activation and attenuated motoneuron apoptosis. Similarly, in vivo application of RU360, an inhibitor of the mitochondrial calcium uniporter, also protected axotomized motoneurons from apoptosis. Taken together, our results show that cytochrome c release and subsequent caspase-3 activation are critical events that precipitate the apoptotic death of axotomized neonatal motoneurons in vivo. In addition, these results provide evidence that application of mitochondrial pore inhibitors in vivo can block the induction of apoptosis following motoneuron axotomy.     

Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.     

The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release,caspase activation,and mitochondrial dysfunction

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.     

Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.     

ROS-dependent caspase-9 activation in hypoxic cell death

Mitochondria are known to play a fundamental role in apoptosis by releasing apoptogenic molecules such as cytochrome c into the cytoplasm, thereby sequentially activating initiator caspase-9. However, the mechanisms of cytochrome c release or caspase-9 activation in response to hypoxia are unclear. In this report, we show that caspase-9 is activated by reactive oxygen species (ROS) without involvement of cytochrome c release in hypoxic injury. In addition, activated caspase-9 induces permeability transition (PT)-independent cytochrome c release, suggesting that caspase-9 may disrupt mitochondrial diffusion limit of cytochrome c and serve to amplify further release of cytochrome c.     

Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria

This study tested the hypothesis that mitochondrial precursor targeting peptides can elicit the release of cytochrome c from both liver and brain mitochondria by a mechanism distinct from that mediated by the classical, Ca2+-activated permeability transition pore. Human cytochrome oxidase subunit IV signal peptide (hCOXIV1-22) at concentrations from 15 to 100 microM induced swelling, a decrease in membrane potential, and cytochrome c release in both types of mitochondria. Although cyclosporin A and bongkrekic acid were without effect, dibucaine, propanolol, dextran, and the uncoupler FCCP were each able to inhibit signal peptide-induced swelling and cytochrome c release. Adenylate kinase was coreleased with cytochrome c, arguing against a signal peptide-induced cytochrome c-specific pathway of efflux across the outer membrane. Taken together, the data indicate that a human mitochondrial signal peptide can evoke the release of cytochrome c from both liver and brain mitochondria by a unique permeability transition that differs in several characteristics from the classical mitochondrial permeability transition.     

Microcin J25 induces the opening of the mitochondrial transition pore and cytochrome c release through superoxide generation

Microcin J25, an antimicrobial lasso-structure peptide, induces the opening of mitochondrial permeability transition pores and the subsequent loss of cytochrome c. The microcin J25 effect is mediated by the stimulation of superoxide anion overproduction. An increased uptake of calcium is also involved in this process. Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclosporin A and Mn(2+), allowed us to establish a time sequence of events starting with the binding of microcin J25, followed by superoxide anion overproduction, opening of mitochondrial permeability transition pores, mitochondrial swelling and the concomitant leakage of cytochrome c.     

Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A

Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms. In this study, the mitochondria-dependent cell death pathway was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochondria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifically inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells. These morphological and biochemical observations correlated with decreased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important linked events in hyperoxia-induced lung injury and can be efficiently blocked by cyclosporin A.     

Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells

Cholesterol enrichment of rat liver mitochondria (CHM) impairs atractyloside-induced mitochondrial permeability transition (MPT) due to decreased membrane fluidity. In this study we addressed the effect of cholesterol enrichment on MPT induced by reactive oxygen species (ROS). Superoxide anion generated by xanthine plus xanthine oxidase triggered mitochondrial swelling and cytochrome c release in CHM, which was prevented by butylated hydroxytoluene, an anti-voltage-dependent anion channel antibody, or cyclosporin A. Furthermore, hydrogen peroxide generated by the combination of ganglioside GD3 and mitochondrial GSH depletion elicited mitochondrial swelling and release of cytochrome c, Smac/Diablo and apoptosis-inducing factor in control mitochondria and CHM. Thus, ROS induce MPT and apoptosome activation regardless of decreased mitochondrial membrane dynamics due to cholesterol enrichment.     

Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.

The overexpression of Bax kills cells by a mechanism that depends on induction of the mitochondrial permeability transition (MPT) (Pastorino, J. G., Chen, S.-T., Tafani, M., Snyder, J. W., and Farber, J. L. (1998) J. Biol. Chem. 273, 7770-7775). In the present study, purified, recombinant Bax opened the mitochondrial permeability transition pore (PTP). Depending on its concentration, Bax had two distinct effects. At a concentration of 125 nM, Bax caused the release of the intermembranous proteins cytochrome c and adenylate kinase and the release from the matrix of sequestered calcein, effects prevented by the inhibitor of the PTP cyclosporin A (CSA). At this concentration of Bax, there was no detectable mitochondrial swelling or depolarization. These effects of low Bax concentrations are interpreted as the consequence of transient, non-synchronous activation of the PTP followed by a prompt recovery of mitochondrial integrity. By contrast, Bax concentrations between 250 nM and 1 microM caused a sustained opening of the PTP with consequent persistent mitochondrial swelling and deenergization (the MPT). CSA prevented the MPT induced by Bax. Increasing concentrations of calcium caused a greater proportion of the mitochondria to undergo the MPT in the presence of Bax. Importantly, two known mediators of apoptosis, ceramide and GD3 ganglioside, potentiated the induction by Bax of the MPT. The data imply that Bax mediates the opening of the mitochondrial PTP with the resultant release of cytochrome c from the intermembranous space.     

Apop-1, a novel protein inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis

In the intrinsic pathway of apoptosis, mitochondria play a crucial role by releasing cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c release through Bax/Bak-dependent channels in mitochondria has been well documented. In contrast, cyclophilin D (CypD), an important component of permeability transition pore-dependent protein release, remains largely undefined, and no apoptogenic proteins that act specifically in a CypD-dependent manner have been reported to date. Here, we describe a novel and evolutionarily conserved protein, apoptogenic protein (Apop). Mouse Apop-1 expression induces apoptotic death by releasing cytochrome c from mitochondria into the cytosolic space followed by activation of caspase-9 and -3. Apop-1-induced apoptosis is not blocked by Bcl-2 or Bcl-xL, inhibitors of Bax/Bak-dependent channels, whereas it is completely blocked by cyclosporin A, an inhibitor of permeability transition pore. Cells lacking CypD were resistant to Apop-induced apoptosis. Moreover, inhibition of Apop expression prevented the cell death induced by apoptosis-inducing substances. Our findings, thus, indicate that the expression of Apop-1 induces apoptosis though CypD-dependent pathway and that Apop-1 plays roles in cell death under physiological conditions.     

Critical role of mitochondrial damage in determining outcome of macrophage infection with Mycobacterium tuberculosis

Human macrophages (Mphi) respond to Mycobacterium tuberculosis (Mtb) infection by undergoing apoptosis, a cornerstone of effective antimycobacterial host defense. Virulent mycobacteria override this reaction by inducing necrosis leading to uncontrolled Mtb replication. Accordingly, Mphi death induced by inoculation with Mtb had the characteristics of apoptosis and necrosis and correlated with moderate increase of mitochondrial permeability transition (MPT), mitochondrial cytochrome c release, and caspase-9 and -3 activation. We hypothesized that changes in intramitochondrial Ca(2+) concentration ([Ca(2+)](m)) determine whether Mphi undergo either apoptosis or necrosis. Therefore, we induced mechanism(s) leading to predominant apoptosis or necrosis by modulating [Ca(2+)](m) and examined their physiological consequences. Adding calcium ionophore A23187 to Mphi inoculated with Mtb further increased calcium flux into the cells which is thought to lead to increased [Ca(2+)](m), blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome c release, lowered caspase activation, and accompanied effective antimycobacterial activity. In contrast, Mphi infected with Mtb in presence of the mitochondrial calcium uniporter inhibitor ruthenium red showed increased mitochondrial swelling and cytochrome c release and decreased MPT and antimycobacterial activity. Thus, in Mtb-infected Mphi, high levels of mitochondrial membrane integrity, low levels of caspase activation, and diminished mitochondrial cytochrome c release are hallmarks of apoptosis and effective antimycobacterial activity. In contrast, breakdown of mitochondrial membrane integrity and increased caspase activation are characteristic of necrosis and uncontrolled Mtb replication.     

Targeted gene inactivation of calpain-1 suppresses cortical degeneration due to traumatic brain injury and neuronal apoptosis induced by oxidative stress

Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role.     

Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.     

Group B Streptococcus induces macrophage apoptosis by calpain activation

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.     

Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes

Previous studies have shown that microcystin-LR (MLR), a specific hepatotoxin, induces onset of mitochondrial permeability transition (MPT) and apoptosis in cultured rat hepatocytes. Here we attempted to investigate the downstream events after the onset of MPT in MLR-treated hepatocytes. Various mitochondrial electron transport chain (ETC) inhibitors effectively prevented the onset of MPT, suggesting that the mitochondrial ETC plays an important role in MLR-induced MPT. MLR also induced mitochondrial cytochrome c release, which can be prevented by a specific MPT inhibitor (cyclosporin A, CsA), and by various ETC inhibitors. Interestingly, the release of cytochrome c did not activate caspase-9 and -3, the main caspases involved in apoptosis. Instead, MLR activated calpain in rat hepatocytes, probably through the increase of intracellular Ca(2+) released from mitochondria. Both ALLN and ALLM, two calpain inhibitors, significantly blocked MLR-induced calpain activation and subsequent cell death. CsA also prevented MLR-induced calpain activation and cell death, suggesting that the activation of calpain may be a post-mitochondrial event. These data demonstrate for the first time that calpain rather than caspases plays an important role in MLR-induced apoptosis.     

Cellular characterization of leukotoxin diol-induced mitochondrial dysfunction

Leukotoxin, a cytochrome P450-derived epoxide of linoleic acid, has been implicated as a causative factor in acute respiratory distress syndrome. Conversion of this fatty acid epoxide to leukotoxin diol by epoxide hydrolase has been hypothesized as the critical activation step in leukotoxin-induced cellular toxicity. In both human and insect cells, we observed that leukotoxin diol causes acute cellular toxicity and that cyclosporin A, an inhibitor of the mitochondrial permeability transition, ameliorates leukotoxin diol-associated toxicity. To evaluate mitochondria as a target of leukotoxin diol, multiple aspects of mitochondrial integrity were evaluated in both cell- and organelle-based assays. Leukotoxin diol specifically activated the mitochondrial permeability transition, resulting in release of cytochrome c and subsequent cell death. Pretreatment with cyclosporin A inhibited these effects and, furthermore, limited in vivo toxicity. While the mechanisms underlying leukotoxin-mediated toxicity remain to be fully elucidated, the observation that leukotoxin diol disrupts mitochondrial function specifically through activation of the mitochondrial permeability transition suggests at least one mechanism through which leukotoxin diol may exert its activity in physiological contexts.     

Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.     

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.