A possible cooperation of SOD1 and cytochrome c in mitochondria-dependent apoptosis

A small amount of reactive oxygen species (ROS) is generated through aerobic respiration even under physiological conditions. Because ROS are known to have various deteriorating actions, the way cells could evade the effects of ROS in and around mitochondria would determine the fate of cells. We previously reported that Cu,Zn-superoxide dismutase (SOD1), a cytosolic enzyme, is also localized in mitochondria in various types of cells. Therefore, we undertook this study to elucidate the physiological significance of SOD1 localization in and around mitochondria. We analyzed the effects of various reagents that could modulate mitochondrial respiration, ROS metabolism, and subcellular localization of SOD1 and cytochrome c. Using rat liver mitochondria, we have shown that Ca2+, Fe2+, or long-chain fatty acids increased the mitochondrial generation of ROS and that the resulting ROS oxidized the critical thiol groups in adenine nucleotide translocase (ANT). The oxidation of ANT induced mitochondrial swelling followed by the release of SOD1 and cytochrome c. Although inhibitors of electron transport, such as rotenone, antimycin A, and KCN, also increased ROS generation, they failed to (i) oxidize the critical thiol groups in ANT, (ii) induce swelling, and (iii) release SOD1 and cytochrome c. These results suggest that the oxidation of ANT thiols and the opening of the membrane permeability transition pores induce the release of both SOD1 and cytochrome c. We demonstrated that the loss of SOD1 increases the susceptibility of mitochondria to oxidative stresses and that the simultaneous release of SOD1 enhances the vicious cycle of apoptotic reactions triggered by the released cytochrome c. Therefore, SOD1 must have important roles in protecting mitochondria from ROS-induced injury. Our data also suggest that SOD1 release parallels cytochrome c release under all conditions. We propose that intramembranously localized SOD1 is a third reagent (along with AIF) that will regulate apoptosis.     

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.     

Identification of a novel protein MICS1 that is involved in maintenance of mitochondrial morphology and apoptotic release of cytochrome c

Mitochondrial morphology dynamically changes in a balance of membrane fusion and fission in response to the environment, cell cycle, and apoptotic stimuli. Here, we report that a novel mitochondrial protein, MICS1, is involved in mitochondrial morphology in specific cristae structures and the apoptotic release of cytochrome c from the mitochondria. MICS1 is an inner membrane protein with a cleavable presequence and multiple transmembrane segments and belongs to the Bi-1 super family. MICS1 down-regulation causes mitochondrial fragmentation and cristae disorganization and stimulates the release of proapoptotic proteins. Expression of the anti-apoptotic protein Bcl-XL does not prevent morphological changes of mitochondria caused by MICS1 down-regulation, indicating that MICS1 plays a role in maintaining mitochondrial morphology separately from the function in apoptotic pathways. MICS1 overproduction induces mitochondrial aggregation and partially inhibits cytochrome c release during apoptosis, regardless of the occurrence of Bax targeting. MICS1 is cross-linked to cytochrome c without disrupting membrane integrity. Thus, MICS1 facilitates the tight association of cytochrome c with the inner membrane. Furthermore, under low-serum condition, the delay in apoptotic release of cytochrome c correlates with MICS1 up-regulation without significant changes in mitochondrial morphology, suggesting that MICS1 individually functions in mitochondrial morphology and cytochrome c release.     

Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.     

Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin.

To examine whether valinomycin induces a mitochondrial permeability transition (PT), we investigated its effects on mitochondrial functions under various conditions. The acceleration of mitochondrial respiration and swelling, induced by valinomycin, were found to be insensitive to inhibitors of the ordinary PT, indicating that valinomycin does not induce the ordinary PT. Results of experiments using mitochondria isolated from transgenic mice expressing human bcl-2 also supported this conclusion. Furthermore, evidence for induction of PT pores by valinomycin was not obtained by either electron microscopic analysis of mitochondrial configurations or by measurement of the permeability of the inner mitochondrial membrane by use of polyethylene glycol. However, valinomycin did induce a significant release of cytochrome c, and thus it may be a nice tool to study the processes of mitochondrial cytochrome c release.     

Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

Differential interactions of apo- and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria

Monomolecular layers of lipid extracts of microsomal, mitochondrial outer and inner membranes, and pure lipid species have been used to measure their interaction with apo- and holocytochrome c. Large differences were observed both with respect to the nature and the lipid specificity of the interaction. The initial electrostatic interaction of the hemefree precursor apocytochrome c with anionic phospholipids is followed by penetration of the protein in between the acyl chains. Apocytochrome c shows similar interactions for all anionic lipids tested. In strong contrast the holoprotein discriminates enormously between cardiolipin for which it has a high affinity and phosphatidylserine and phosphatidylinositol for which it has a much lower affinity. For these latter lipids the interaction with cytochrome c is primarily electrostatic. The cytochrome c-cardiolipin interaction shows several unique features which suggest the formation of a specific complex between the two molecules. These properties account for the preference in interaction of the apoprotein with the lipid extract of the outer mitochondrial membrane over that of the endoplasmic reticulum and the large preference of cytochrome c for the inner over that of the outer mitochondrial membrane lipid extract. Only apocytochrome c was able to induce close contacts between monolayers of the mitochondrial outer membrane lipids and vesicles of mitochondrial inner membrane lipids. Experiments with fragments of both protein and unfolding experiments with cytochrome c revealed that the differences in interaction between the two proteins are mainly due to differences in their tertiary structure and not the presence of the heme group itself. The initial unfolded structure of apocytochrome c is responsible for the high penetrative power of the protein and its ability to induce close membrane contact, whereas the folded structure of cytochrome c is responsible for the specific interaction with cardiolipin. The results are discussed in the light of the apocytochrome c import process in mitochondria and suggest that lipid-protein interactions contribute to targeting the precursor toward mitochondria and are important for its translocation across the outer mitochondrial membrane and the final localization of cytochrome c toward the outside of the inner mitochondrial membrane.     

Cholesterol impairs the adenine nucleotide translocator-mediated mitochondrial permeability transition through altered membrane fluidity

Mitochondrial permeability transition (MPT) has been proposed to play a key role in cell death. Downstream MPT events include the release of apoptogenic factors that sets in motion the mitochondrial apoptosome leading to caspase activation. The current work examined the regulation of MPT by membrane fluidity modulated upon cholesterol enrichment. Mitochondria enriched in cholesterol displayed increased microviscosity resulting in impaired MPT induced by atractyloside, a c-conformation stabilizing ligand of the adenine nucleotide translocator (ANT). This effect was dependent on the dose of cholesterol loaded and reversed upon the fluidization of mitochondria by the fatty acid derivative A2C. Mitoplasts derived from cholesterol-enriched mitochondria responded to atractyloside in a similar fashion as intact mitochondria, indicating that a significant amount of cholesterol is still found in the inner membrane. The effects of cholesterol on MPT induced by atractyloside were mirrored by the release of intermembrane proteins, cytochrome c, Smac/Diablo, and apoptosis inducing factor. However, cholesterol loading did not affect the uptake rate of adenine nucleotide hence dissociating the function of ANT as a MPT-mediated protein from its adenine nucleotide exchange function. Thus, these findings indicate that the ability of atractyloside to induce MPT via ANT requires an appropriate membrane fluidity range.     

Tributyltin causes cytochrome C release from isolated mitochondria by two discrete mechanisms

Using isolated liver mitochondria we show that low concentrations of TBT (0.5 microM) cause the release of mitochondrial cytochrome c, in the presence of Ca(2+). This is reflected in a rapid loss of membrane potential (DeltaPsi(m)), and a large-amplitude swelling characteristic of mitochondrial permeability transition (MPT). Despite this, the inclusion of cyclosporin A could not prevent the release of cytochrome c. Further, in the absence of Ca(2+), low concentrations of TBT (0.5 microM) resulted in a slow sub-maximal shift of DeltaPsi(m), not characteristic of MPT, which was still paralleled by a release of cytochrome c. Further experiments showed that the loss of DeltaPsi(m) in the absence of Ca(2+) was due to a combination of inhibition of respiration and a direct uncoupling effect on the respiratory chain. Under these conditions, rapid swelling of mitochondria could be demonstrated, due to chloride exchange over the inner mitochondrial membrane. Taken together these data suggest that TBT can induce the release of cytochrome c in intact cells by at least two mechanisms. The first and critical mechanism is initiated immediately the mitochondria sense the presence of TBT and involves a slow loss of DeltaPsi(m) and induction of swelling, which allows release of cytochrome c in a relatively non-specific manner and independently from a rise in [Ca(2+)](i). The second mechanism involves the induction of formal MPT as intracellular [Ca(2+)](i) increases. These data help to explain previous observations in intact lymphocytes demonstrating TBT-induced release of mitochondrial cytochrome c in the absence of a rise in [Ca(2+)](i) (Stridh, H., Gigliotti, D., Orrenius, S., and Cotgreave, I. A. (1999) Biochem. Biophys. Res. Commun. 266, 460-465).     

VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.     

The intra-mitochondrial cytochrome c distribution varies correlated to the formation of a complex between VDAC and the adenine nucleotide translocase: this affects Bax-dependent cytochrome c release

The mechanism of Bax-dependent cytochrome c release is still controversial and may also depend on the actual localisation of cytochrome C: (i) we studied the distribution of cytochrome c in sub-fractions of rat kidney mitochondria and found that 10-20% of the total cytochrome c was associated at the peripheral inner membrane and to some extent organised in the contact sites. (ii) Cytochrome c concentrations in the contact site fractions varied related to surface bound hexokinase activity. It decreased upon reduction of contact sites by glycerol or specific dissociation of the VDAC-ANT complexes by bongkrekate, whereas it increased upon induction of contacts by dextran or association of VDAC-ANT complexes by atractyloside. (iii) The outer membrane pore (VDAC) acquires high capacity for hexokinase binding by interacting with the ANT. Thus, surface-attached hexokinase protein indicated the frequency of VDAC-ANT complexes and the correlation between hexokinase activity and cytochrome c suggested association of the latter to the complexes. (iv) Substances affecting exclusively the structure of either hexokinase (glucose-6P) or cytochrome c (borate) led to a decrease only of the effected protein without changing the concentration of other contact site constituents. (v) Hexokinase was furthermore used as a tool to isolate the contact site forming complex of outer membrane VDAC and inner membrane ANT from Triton-dissolved membranes. Cytochrome c remained attached to the hexokinase VDAC-ANT complexes that were reconstituted in phospholipid vesicles. (vi) The vesicles were loaded with malate and BaxDeltaC released the endogenous cytochrome c from the reconstituted complexes without forming unspecific pores for malate. BaxDeltaC targeted a cytochrome c fraction associated at the VDAC-ANT complex. The cytochrome c organisation was dependent on the actual structure of VDAC and ANT. Thus, the BaxDeltaC effect was suppressed either by hexokinase utilising glucose and ATP or by bongkrekic acid both influencing the pore and ANT structure.     

Role of alpha-tocopherol in the regulation of mitochondrial permeability transition

We previously showed that Ca2+-induced cyclosporin A-sensitive membrane permeability transition (MPT) of mitochondria occurred with concomitant generation of reactive oxygen species (ROS) and release of cytochrome c (Free Rad. Res.38, 29-35, 2004). To elucidate the role of alpha-tocopherol in MPT, we investigated the effect of alpha-tocopherol on mitochondrial ROS generation, swelling and cytochrome c release induced by Ca2+ or hydroxyl radicals. Biochemical analysis revealed that alpha-tocopherol suppressed Ca2+-induced ROS generation and oxidation of critical thiol groups of mitochondrial adenine nucleotide translocase (ANT) but not swelling and cytochrome c release. Hydroxyl radicals also induced cyclosporin A-sensitive MPT of mitochondria. alpha-Tocopherol suppressed the hydroxyl radical-induced lipid peroxidation, swelling and cytochrome c release from mitochondria. These results indicate that alpha-tocopherol inhibits ROS generation, ANT oxidation, lipid peroxidation and the opening of MPT, thereby playing important roles in the prevention of oxidative cell death.     

Mechanisms of cytochrome c release by proapoptotic BCL-2 family members

A crucial amplificatory event in several apoptotic cascades is the nearly complete release of cytochrome c from mitochondria. Proteins of the BCL-2 family which include both anti- and proapoptotic members control this step. Here, we review the proposed mechanisms by which proapoptotic BCL-2 family members induce cytochrome c release. Data support a model in which the apoptotic pathway bifurcates following activation of a "BH3 only" family member. BH3 only molecules induce the activation of the multidomain proapoptotics BAX and BAK, resulting in the permeabilization of the outer mitochondrial membrane and the efflux of cytochrome c. This is coordinated with the activation of a distinct pathway characterized by profound changes of the inner mitochondrial membrane morphology and organization. This mitochondrial remodelling insures complete release of cytochrome c and the onset of mitochondrial dysfunction that is a typical feature of many apoptotic deaths.     

Cytochrome c dissociation and release from mitochondria by truncated Bid and ceramide

We have examined the effects of truncated Bid (tBid) and ceramide on mitochondrial membrane integrity and cytochrome c release, using mitochondria with intact outer membranes. While tBid permeabilizes the outer membrane and efficiently stimulates cytochrome c release, digitonin is unable to cause cytochrome c release in the absence of salt. Ceramides did not permeabilize the mitochondrial outer membrane, and stimulated cytochrome c release only in the presence of digitonin. Taken together, these observations support a model for cytochrome c release in which the first step is dissociation from the inner membrane followed by transit across the outer membrane.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Mitochondria and cell death

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.     

Oxidative stress underlies the mechanism for Ca(2+)-induced permeability transition of mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca₂₊ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca₂₊ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca₂₊ increased the generation of H₂O₂ by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca₂₊ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca₂₊-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca₂₊ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Heat stress prevents mitochondrial injury in ATP-depleted renal epithelial cells

The events that precipitate cell death and the stress proteins responsible for cytoprotection during ATP depletion remain elusive. We hypothesize that exposure to metabolic inhibitors damages mitochondria, allowing proapoptotic proteins to leak into the cytosol, and suggest that heat stress-induced hsp72 accumulation prevents mitochondrial membrane injury. To test these hypotheses, renal epithelial cells were transiently ATP depleted with sodium cyanide and 2-deoxy-D-glucose in the absence of medium dextrose. Recovery from ATP depletion was associated with the release into the cytosol of cytochrome c and apoptosis-inducing factor (AIF), proapoptotic proteins that localize to the intermitochondrial membrane space. Concomitant with mitochondrial cytochrome c leak, a seven- to eightfold increase in caspase 3 activity was observed. In controls, state III mitochondrial respiration was reduced by 30% after transient exposure to metabolic inhibitors. Prior heat stress preserved mitochondrial ATP production and significantly reduced both cytochrome c release and caspase 3 activation. Despite less cytochrome c release, prior heat stress increased binding between cytochrome c and hsp72. The present study demonstrates that mitochondrial injury accompanies exposure to metabolic inhibitors. By reducing outer mitochondrial membrane injury and by complexing with cytochrome c, hsp72 could inhibit caspase activation and subsequent apoptosis.     

Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?

Several mitochondrial proteins, such as cytochrome c, are directly involved in the pathway for caspase activation following induction of apoptosis. Release of mitochondrial cytochrome c early in apoptosis is rapid and almost complete. Microinjection of cytochrome c into resting cells induces apoptosis, but the amount needed approaches the total cellular content. These observations suggest that mitochondrial protein release is an all-or-nothing process inside the cell and not an amplifiable apoptotic signal. To test this hypothesis, laser micro-irradiation was used to rupture membranes of individual mitochondria within living rat neural cells. Laser micro-irradiation caused swelling, fragmentation, depolarization, and cytochrome c depletion in targeted mitochondria. These effects were explained by correlative electron microscopic analysis showing local rupture of outer and inner membranes at the site of irradiation. In all cases, there were no detectable changes in the structure, membrane potential, or cytochrome c content of neighboring, non-irradiated organelles. Furthermore, irradiation of up to 15% of the mitochondria in a cell did not induce apoptosis. The results from these laser micro-irradiation experiments prove that local release of mitochondrial proteins does not constitute an amplifiable apoptotic signal in resting neural cells.