The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

In several different cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(m) that is not inhibited by the broad spectrum caspase inhibitor z-VAD.fmk, although z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(m) disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamide-triggered delta psi(m) collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-purified PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X(L) proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.     

The central role of the mitochondrial megachannel in apoptosis: evidence obtained with intact cells, isolated mitochondria, and purified protein complexes

The mitochondrial megachannel (also called permeability transition pore) is a polyprotein complex formed in the contact site between the inner and the outer mitochondrial membranes and participates in the regulation of mitochondrial membrane permeability. We have obtained three independent lines of evidence suggesting the implication of the mitochondrial megachannel in apoptosis. First, in intact cells, apoptosis is accompanied by an early dissipation of the mitochondrial transmembrane potential (delta psi m). In several models of apoptosis, specific agents inhibiting the mitochondrial megachannels prevent this delta psi m dissipation and simultaneously abolish the manifestations of caspase- and endonuclease activation, indicating that megachannel opening is a critical event of the apoptotic process. Second, mitochondria are rate-limiting for caspase and nuclease activation in several cell-free systems of apoptosis. Isolated mitochondria release apoptogenic factors capable of activating pro-caspases or endonucleases upon opening of the mitochondrial megachannel in vitro. Third, opening of the purified megachannel reconstituted into liposomes is inhibited by recombinant Bcl-2 or Bcl-XL, two apoptosis-inhibitory proteins which also prevent megachannel opening in cells and isolated mitochondria. This indicates that the megachannel is under the direct regulatory control of anti-apoptotic members of the Bcl-2 family. Altogether, our results suggest that megachannel opening is sufficient and (mostly) necessary for triggering apoptosis.     

Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins

Bcl-2 is the prototype of a class of oncogenes which regulates apoptosis. Bcl-2-related gene products with either death-promoting and death-inhibitory activity are critically involved in numerous disease states and thus constitute prime targets for therapeutic interventions. The relative amount of death agonists and antagonists from the Bcl-2 family constitutes a regulatory rheostat whose function is determined, at least in part, by selective protein-protein interactions. Bcl-2 and its homologs insert into intracellular membranes including mitochondria, the endoplasmatic reticulum and the nuclear envelope. Many of the molecular genetic, ultrastructural, crystallographic and functional studies suggest that Bcl-2-related molecules exert their apoptosis-regulatory effects via regulating mitochondrial alterations preceding the activation of apoptogenic proteases and nucleases. Via a direct effect on mitochondrial membranes, Bcl-2 prevents all hallmarks of the early stage of apoptosis including disruption of the inner mitochondrial transmembrane potential and the release of apoptogenic protease activators from mitochondria. The mitochondrial permeability transition (PT) pore, also called mitochondrial megachannel or multiple conductance channel, is a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes, exactly at the same localization at which Bax, Bcl-2, and Bcl-XL are particularly abundant. The PT pore participates in the regulation of matrix Ca2+, pH, deltapsim, and volume and functions as a Ca2+-, voltage-, pH-, and redox-gated channel with several levels of conductance and little if any ion selectivity. Experiments involving the purified PT pore complex indicate that Bax, Bcl-2, and Bcl-XL exert at least part of their apoptosis-regulatory function by facilitating (Bax) or inhibiting (Bcl-2, Bcl-XL) PT pore opening. These findings clarify the principal (but not exclusive) mechanism of Bcl-2-mediated cytoprotection.     

Imaging the permeability pore transition in single mitochondria

In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.     

Release of cytochrome c from liver mitochondria during permeability transition

The mitochondrial permeability transition (PT) follows opening of megachannels in the inner membrane and may be part of a programmed death pathway. Recently a role for cytochrome c in programmed cell death has been proposed, although its relationship to PT has not been been determined. We studied the release of cytochrome c from liver mitochondria undergoing PT. Well-coupled mitochondria treated with 5 mM atractyloside (ATR) or 100 microM calcium chloride were found to undergo PT and release cytochrome c into the incubation buffer within 5 minutes. Control mitochondria and mitochondria treated with the uncoupler FCCP did not undergo PT or release cytochrome c at 5 or 15 minutes. PT induced by ATR could be prevented by pretreatment with 10 microM cyclosporin A. Mitochondria incubated with ATR or calcium caused a 20-30% decrease in electron transfer rate via cytochrome c and cytochrome c oxidase. We conclude that cytochrome c release is an early event during mitochondrial PT, and is sufficient to decrease electron transfer through the terminal electron transport components of the mitochondrion.     

Comparison of Mg2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system

Mitochondrial cytochrome c (cyt c) has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Through interaction with apoptotic protease activating factors (Apaf), cyt c can initiate the activation cascade of caspases once it is released into the cytosol. The loss of a component of the mitochondrial electron transport chain also triggers the generation of superoxide. Although cyt c can be released independent of the mitochondrial permeability transition (MPT), the accompanying cellular redox change can trigger the MPT. Since another apoptotic protease, AIF, is released by MPT, the two separate pathways provide redundancy that ensures effective execution of the cell death program. Anti-apoptotic Bcl-2 family proteins function as gatekeepers to prevent the release of both cyt c and AIF. In spite of their stabilization effect on the mitochondrial outer membrane, Bcl-2 proteins may also be involved in the direct binding of Apaf molecules as regulatory elements further downstream from the mitochondrial apoptotic signals.     

The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition

Contact sites between the outer and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase-porin-ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through porin was regulated by its interaction with a specific antiporter, ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT-pore-like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT-pore-like properties concerning Ca2+ sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin was not observed.     

Differential use of immunoglobulin light chain genes and B lymphocyte expansion at sites of disease in rheumatoid arthritis (RA) compared with circulating B lymphocytes

Patients infected with HIV-1 often exhibit cognitive deficits that are related to progressive neuronal degeneration and cell death. The protein Tat, which is released from HIV-1-infected cells, was recently shown to be toxic toward cultured neurons. We now report that Tat induces apoptosis in cultured embryonic rat hippocampal neurons. Tat induced caspase activation, and the caspase inhibitor zVAD-fmk prevented Tat-induced neuronal death. Tat induced a progressive elevation of cytoplasmic-free calcium levels, which was followed by mitochondrial calcium uptake and generation of mitochondrial-reactive oxygen species (ROS). The intracellular calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red protected neurons against Tat-induced apoptosis. zVAD-fmk suppressed Tat-induced increases of cytoplasmic calcium levels and mitochondrial ROS accumulation, indicating roles for caspases in the perturbed calcium homeostasis and oxidative stress induced by Tat. An inhibitor of nitric oxide synthase, and the peroxynitrite scavenger uric acid, protected neurons against Tat-induced apoptosis, indicating requirements for nitric oxide production and peroxynitrite formation in the cell death process. Finally, Tat caused a delayed and progressive mitochondrial membrane depolarization, and cyclosporin A prevented Tat-induced apoptosis, suggesting an important role for mitochondrial membrane permeability transition in Tat-induced apoptosis. Collectively, our data demonstrate that Tat can induce neuronal apoptosis by a mechanism involving disruption of calcium homeostasis, caspase activation, and mitochondrial calcium uptake and ROS accumulation. Agents that interupt this apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in AIDS patients.     

The mitochondrial permeability transition

This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.     

Calcium in ischemic cell death

BACKGROUND: This review article deals with the role of calcium in ischemic cell death. A calcium-related mechanism was proposed more than two decades ago to explain cell necrosis incurred in cardiac ischemia and muscular dystrophy. In fact, an excitotoxic hypothesis was advanced to explain the acetylcholine-related death of muscle end plates. A similar hypothesis was proposed to explain selective neuronal damage in the brain in ischemia, hypoglycemic coma, and status epilepticus. SUMMARY OF REVIEW: The original concepts encompass the hypothesis that cell damage in ischemia-reperfusion is due to enhanced activity of phospholipases and proteases, leading to release of free fatty acids and their breakdown products and to degradation of cytoskeletal proteins. It is equally clear that a coupling exists between influx of calcium into cells and their production of reactive oxygen species, such as .O2, H2O2, and .OH. Recent results have underscored the role of calcium in ischemic cell death. A coupling has been demonstrated among glutamate release, calcium influx, and enhanced production of reactive metabolites such as .O2-, .OH, and nitric oxide. It has become equally clear that the combination of .O2- and nitric oxide can yield peroxynitrate, a metabolite with potentially devastating effects. The mitochondria have again come into the focus of interest. This is because certain conditions, notably mitochondrial calcium accumulation and oxidative stress, can trigger the assembly (opening) of a high-conductance pore in the inner mitochondrial membrane. The mitochondrial permeability transition (MPT) pore leads to a collapse of the electrochemical potential for H+, thereby arresting ATP production and triggering production of reactive oxygen species. The occurrence of an MPT in vivo is suggested by the dramatic anti-ischemic effect of cyclosporin A, a virtually specific blocker of the MPT in vitro in transient forebrain ischemia. However, cyclosporin A has limited effect on the cell damage incurred as a result of 2 hours of focal cerebral ischemia, suggesting that factors other than MPT play a role. It is discussed whether this could reflect the operation of phospholipase A2 activity and degradation of the lipid skeleton of the inner mitochondrial membrane. CONCLUSIONS: Calcium is one of the triggers involved in ischemic cell death, whatever the mechanism.     

Hormonal factors in the development of differences in strength between boys and girls during adolescence: a longitudinal study

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinson's disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP+) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca2+ and Pi, MPP+ induced a permeability transition in both liver and brain mitochondria. MPP+ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP(+)-induced swelling and release of cytochrome c. The MPP(+)-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP(+)-induced pore opening and cytochrome c release were blocked by CsA, the Ca2+ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP+ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca2+ and cytochrome c known to be involved in apoptosis.     

Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3

Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.     

Molecular characterization of mitochondrial apoptosis-inducing factor

Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.     

A caspase-activated factor (CAF) induces mitochondrial membrane depolarization and cytochrome c release by a nonproteolytic mechanism

It is well established that apoptosis is accompanied by activation of procaspases and by mitochondrial changes, such as decrease in mitochondrial transmembrane potential (DeltaPsim) and release of cytochrome c. We analyzed the causal relationship between activated caspases and these mitochondrial phenomena. Purified recombinant caspase-1, -11, -3, -6, -7, and -8 were incubated with mitochondria in the presence or absence of additional cellular components, after which DeltaPsim was determined. At lower caspase concentrations, only caspase-8 was able to activate a cytosolic factor, termed caspase-activated factor (CAF), which resulted in decrease in DeltaPsim and release of cytochrome c. Both CAF-mediated activities could not be blocked by protease inhibitors, including oligopeptide caspase inhibitors. CAF-induced cytochrome c release, but not decrease of DeltaPsim, was blocked in mitochondria from cells overexpressing Bcl-2. CAF is apparently involved in decrease of DeltaPsim and release of cytochrome c, whereas Bcl-2 only prevents the latter. Hence, CAF may form the link between death domain receptor-dependent activation of procaspase-8 and the mitochondrial events studied.     

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.     

Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid

Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochrome c to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria.     

Bax directly induces release of cytochrome c from isolated mitochondria

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-XL protein abrogated Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria.     

Calcium-induced activation of the mitochondrial permeability transition in hippocampal neurons

The mitochondrial permeability transition (mPT) has been implicated in both excitotoxic and apoptotic neuronal cell death, despite the fact that it has not been previously identified in neurons. To study the mPT in hippocampal neurons, cultures were loaded with the mitochondrial dye JC-1 and observed with confocal and conventional microscopy. After pretreatment with 4Br-A23187 and subsequent calcium addition, the initially rodlike mitochondria increased in diameter until mitochondria became rounded in appearance. Morphological changes reversed when calcium was removed by EGTA. When neurons were loaded with both fura-2-AM and rhodamine 123, calcium loading produced an increase in cytosolic calcium, mitochondrial depolarization, and similar alterations in mitochondrial morphology. Smaller calcium challenges produced calcium cycling, delaying morphological changes until after secondary depolarization and calcium release to the cytosol. In neurons exposed to glutamate, confocal observation of JC-1 fluorescence revealed comparable changes in mitochondrial morphology that were prevented when barium was substituted for calcium, or following pretreatment with the mPT inhibitor, cyclosporin A. These experiments establish conditions in which the mPT could be observed in situ in neurons in response to calcium loading. In addition, the timing of changes suggested that induction of the permeability transition in situ represents a sequence of multiple events that may reflect the multiple open conformations of the mPT pore.     

Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis

Release of cytochrome c is important in many forms of apoptosis. Recent studies of CD95 (Fas/APO-1)-induced apoptosis have implicated caspase-8 cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family, in this release. We now demonstrate that both receptor-induced (CD95 and tumor necrosis factor) and chemical-induced apoptosis result in a similar time-dependent activation of caspases-3, -7, -8, and -9 in Jurkat T cells and human leukemic U937 cells. In receptor-mediated apoptosis, the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD. FMK), inhibits apoptosis prior to commitment to cell death by inhibiting the upstream activator caspase-8, cleavage of Bid, release of mitochondrial cytochrome c, processing of effector caspases, loss of mitochondrial membrane potential, and externalization of phosphatidylserine. However, Z-VAD.FMK inhibits chemical-induced apoptosis at a stage after commitment to cell death by inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases. Cleavage of Bid but not release of cytochrome c is blocked by Z-VAD.FMK demonstrating that in chemical-induced apoptosis cytochrome c release is caspase-independent and is not mediated by activation of Bid. We propose that caspases form an integral part of the cell death-inducing mechanism in receptor-mediated apoptosis, whereas in chemical-induced apoptosis they act solely as executioners of apoptosis.