Is mPTP the gatekeeper for necrosis, apoptosis, or both?

Permeabilization of the mitochondrial membranes is a crucial step in apoptosis and necrosis. This phenomenon allows the release of mitochondrial death factors, which trigger or facilitate different signaling cascades ultimately causing the execution of the cell. The mitochondrial permeability transition pore (mPTP) has long been known as one of the main regulators of mitochondria during cell death. mPTP opening can lead to matrix swelling, subsequent rupture of the outer membrane, and a nonspecific release of intermembrane space proteins into the cytosol. While mPTP was purportedly associated with early apoptosis, recent observations suggest that mitochondrial permeabilization mediated by mPTP is generally more closely linked to events of late apoptosis and necrosis. Mechanisms of mitochondrial membrane permeabilization during cell death, involving three different mitochondrial channels, have been postulated. These include the mPTP in the inner membrane, and the mitochondrial apoptosis-induced channel (MAC) and voltage-dependent anion-selective channel (VDAC) in the outer membrane. New developments on mPTP structure and function, and the involvement of mPTP, MAC, and VDAC in permeabilization of mitochondrial membranes during cell death are explored. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosis-induced channel (MAC) is the mitochondrial apoptosis-induced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets.     

Isolated mouse liver mitochondria are devoid of glucokinase

Glucokinase is a hexokinase isoform with low affinity for glucose that has previously been identified as a cytosolic enzyme. A recent report claims that glucokinase physically associates with liver mitochondria to form a multi-protein complex that may be physiologically important in apoptotic signaling [N.N. Danial, C.F. Gramm, L. Scorrano, C.Y. Zhang, S. Krauss, A.M. Ranger, S.R. Datta, M.E. Greenberg, L.J. Licklider, B.B. Lowell, S.P. Gygi, S.J. Korsmeyer, Nature 424 (2003) 952-956]. Here, we re-examined the association of glucokinase with isolated mouse liver mitochondria. When glucokinase activity was measured by coupled enzyme assay, robust activity was present in whole liver homogenates and their 9500 g supernatants (cytosol), but activity in the purified mitochondrial fraction was below detection (<0.2% of homogenate). Furthermore, addition of 45 mM glucose in the presence of ATP did not increase mitochondrial respiration, indicating the absence of ADP formation by glucokinase or any other hexokinase isoform. Immunoblots of liver homogenates and cytosol revealed strong glucokinase bands, but no immunoreactivity was detected in mitochondria. In conclusion, mouse liver mitochondria lack measurable glucokinase. Thus, functional linkage of glucokinase to mitochondrial metabolism and apoptotic signaling is unlikely to be mediated by the physical association of glucokinase with mitochondria.     

A tale of two mitochondrial channels,MAC and PTP,in apoptosis

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.     

Very long chain ceramides interfere with C16-ceramide-induced channel formation: A plausible mechanism for regulating the initiation of intrinsic apoptosis

Mitochondria mediate both cell survival and death. The intrinsic apoptotic pathway is initiated by the permeabilization of the mitochondrial outer membrane to pro-apoptotic inter-membrane space (IMS) proteins. Many pathways cause the egress of IMS proteins. Of particular interest is the ability of ceramide to self-assemble into dynamic water-filled channels. The formation of ceramide channels is regulated extensively by Bcl-2 family proteins and dihydroceramide. Here, we show that the chain length of biologically active ceramides serves as an important regulatory factor. Ceramides are synthesized by a family of six mammalian ceramide synthases (CerS) each of which produces a subset of ceramides that differ in their fatty acyl chain length. Various ceramides permeabilize mitochondria differentially. Interestingly, the presence of very long chain ceramides reduces the potency of C₁₆-mediated mitochondrial permeabilization indicating that the intercalation of the lipids in the dynamic channel has a destabilizing effect, reminiscent of dihydroceramide inhibition of ceramide channel formation (Stiban et al., 2006). Moreover, mitochondria isolated from cells overexpressing the ceramide synthase responsible for the production of C₁₆-ceramide (CerS5) are permeabilized faster upon the exogenous addition of C₁₆-ceramide whereas they are resistant to permeabilization with added C₂₄-ceramide. On the other hand mitochondria isolated from CerS2-overexpressing cells show the opposite pattern, indicating that the product of CerS2 inhibits C₁₆-channel formation ex vivo and vice versa. This interplay between different ceramide metabolic enzymes and their products adds a new dimension to the complexity of mitochondrial-mediated apoptosis, and emphasizes its role as a key regulatory step that commits cells to life or death.     

Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel

Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.     

Alteration of mitochondrial function and cell sensitization to death

Stimulation of cell death is a powerful instrument in the organism’s struggle with cancer. Apoptosis represents one mode of cell death. However, in a variety of tumor cells proapoptotic mechanisms are downregulated, or not properly activated, whereas antiapoptotic mechanisms are upregulated. Mitochondria are known as key players in the regulation of apoptotic pathways. Specifically, permeabilization of the mitochondrial outer membrane and subsequent release of proapoptotic proteins from the intermembrane space are viewed as decisive events in the initiation and/or execution of apoptosis. Disruption of mitochondrial functions by anticancer drugs, which induce oxidative stress, inhibit mitochondrial respiration, or uncouple oxidative phosphorylation, can sensitize mitochondria in these cells and facilitate outer membrane permeabilization.     

Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1

The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC) inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.     

Mitochondrial membrane permeabilization upon interaction with lysozyme fibrillation products: Role of mitochondrial heterogeneity

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders, although the relative degree of this functional impairment and the mechanism of selective vulnerability in different regions of the brain related to various neurological diseases are not completely understood. In a recent study, we reported on brain mitochondrial membrane permeabilization upon interaction with hen egg white lysozyme (HEWL) protofibrils, and came to the conclusion that mitochondrial heterogeneity could offer an explanation for some of our observations. Accordingly, the first part of the present investigation was devoted on studies involving interaction of HEWL fibrillation products with mitochondria isolated from various areas of the brain, known to be affected in a number of well-characterized neurodegenerative conditions. This was followed by looking at heart and liver mitochondria. Membrane permeabilization was investigated by monitoring release of mitochondrial enzymes. Mitochondria isolated from cortex and hippocampus showed greater sensitivities than those prepared from substantia nigra. Results clearly demonstrate heterogeneity in brain mitochondria together with a higher resistance to permeabilization in these organelles in comparison with those isolated from liver and heart. Calcium and spermine were found to be more effective in preventing permeabilization in brain mitochondria, as compared to liver and heart. The structure-function aspects and physiological significance of the observations in relation to differences in composition, biophysical nature and morphological properties of mitochondria are discussed. It is argued that studies on heterogeneity in cellular membranes, the primary targets of toxic protofibrils, may provide important insights into mechanism of toxicity, with clinical and pathological manifestations.     

Glibenclamide interferes with mitochondrial bioenergetics by inducing changes on membrane ion permeability

The interference of glibenclamide, an antidiabetic sulfonylurea, with mitochondrial bioenergetics was assessed on mitochondrial ion fluxes (H+, K+, and Cl-) by passive osmotic swelling of rat liver mitochondria in K-acetate, KNO3, and KCl media, by O2 consumption, and by mitochondrial transmembrane potential (Deltapsi). Glibenclamide did not permeabilize the inner mitochondrial membrane to H+, but induced permeabilization to Cl- by opening the inner mitochondrial anion channel (IMAC). Cl- influx induced by glibenclamide facilitates K+ entry into mitochondria, thus promoting a net Cl-/K+ cotransport, Deltapsi dissipation, and stimulation of state 4 respiration rate. It was concluded that glibenclamide interferes with mitochondrial bioenergetics of rat liver by permeabilizing the inner mitochondrial membrane to Cl- and promoting a net Cl-/K+ cotransport inside mitochondria, without significant changes on membrane permeabilization to H+.     

Mitochondrial regulation of apoptotic cell death

Mitochondria play a decisive role in the regulation of both apoptotic and necrotic cell death. Permeabilization of the outer mitochondrial membrane and subsequent release of intermembrane space proteins are important features of both models of cell death. The mechanisms by which these proteins are released depend presumably on cell type and the nature of stimuli. Of the mechanisms involved, mitochondrial permeability transition appears to be associated mainly with necrosis, whereas the release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins. However, there is increasing evidence for interaction and co-operation between these two mechanisms. The multiple mechanisms of mitochondrial permeabilization may explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal approximately 4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.     

Adriamycin as a probe for the transversal distribution of cardiolipin in the inner mitochondrial membrane

The ability of adriamycin to complex cardiolipin was used to determine the distribution of cardiolipin across the inner membrane of rat liver and heart mitochondria. In both mitochondrial types, about 57 +/- 5% of the total cardiolipin was found to be located in the cytoplasmic face of the inner membrane. Mitochondria and mitoplasts were used to study the cytoplasmic face of the inner membrane, purified submitochondrial vesicles with inverted membrane orientation for the matrix face. The cardiolipin amount titrated by adriamycin in the latter was found to be complementary to the amount titrated in the cytoplasmic face. The adriamycin association constant determined for the first saturation level of mitochondria was in good agreement with the value published by Goormaghtigh et al. (Goormaghtigh, E., Chatelain, P., Caspers, J., and Ruysschaert, J. M. (1980) Biochim. Biophys. Acta 597, 1-14) for cardiolipin in artificial membranes. Two binding plateaus were observed when increasing amounts of adriamycin were added to mitochondria. The plateau at higher concentrations is conveniently explained by the penetration of adriamycin into mitochondria and the titration of cardiolipin in the matrix face. Scatchard plot analysis of the binding curves leading to the two plateaus produced almost identical association constants. The total amount of cardiolipin in mitochondria calculated from curves of this type corresponded to the total amount of cardiolipin determined by phosphate analysis of extracts, analyzed by thin layer chromatography.     

The mitochondrion in cell death control: certainties and incognita

Apoptosis research has recently experienced a change from a paradigm in which the nucleus determined the apoptotic process to a paradigm in which caspases and, more recently, mitochondria constitute the center of death control. Mitochondria undergo major changes in membrane integrity before classical signs of cell death become manifest. These changes concern both the inner and the outer mitochondrial membranes, leading to the dissipation of the inner transmembrane potential (DeltaPsi(m)) and/or the release of intermembrane proteins through the outer membrane. An ever-increasing number of endogenous, viral, or xenogeneic effectors directly act on mitochondria to trigger permeabilization. At least in some cases, this is achieved by a direct action on the permeability transition pore complex (PTPC), a multiprotein ensemble containing proteins from both mitochondrial membranes, which interact with pro- and antiapoptotic members of the Bcl-2 family. At present, it is elusive whether opening of the PTPC is the only physiological mechanism leading to mitochondrial membrane permeabilization. Proteins released from mitochondria during apoptosis include caspases (mainly caspases 2, 3, and 9), caspase activators (cytochrome c, hsp 10), as well as a caspase-independent death effector, AIF (apoptosis inducing factor). The functional hierarchy among these proteins and their actual impact on the decision between death and life is elusive.     

Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118-125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-x(L) and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-x L and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-x L act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis.     

Assessing mitochondrial outer membrane permeabilization during apoptosis

Mitochondria play a pivotal role in the regulation of apoptosis. An imbalance in apoptosis can lead to disease. Unscheduled apoptosis has been linked to neurodegeneration while inhibition of apoptosis can cause cancer. An early and key event during apoptosis is the release of factors from mitochondria. In apoptosis the mitochondrial outer membrane becomes permeable, leading to release of apoptogenic factors into the cytosol. One such factor, cytochrome c, is an electron carrier of the respiratory chain normally trapped within the mitochondrial intermembrane space. Many apoptotic studies investigate mitochondrial outer membrane permeabilization (MOMP) by monitoring the release of cytochrome c. Here, we describe three reliable techniques that detect cytochrome c release from mitochondria, through subcellular fractionation or immunocytochemistry and fluorescence microscopy, or isolated mitochondria and recombinant Bax and t-Bid proteins in vitro. These techniques will help to identify mechanisms and characterize factors regulating MOMP.     

Modulation of the mitochondrial megachannel by divalent cations and protons.

In patch-clamp experiments on rat liver mitoplasts, the 1.3 nanosiemens (in 150 mM KCl) mitochondrial megachannel was activated by Ca2+ and competitively inhibited by Mg2+, Mn2+, Ba2+, and Sr2+. Cyclosporin A, which inhibits the megachannel, also showed a competitive behavior versus Ca2+. The pore is regulated by pH in the physiological range; lower pH values cause its closure in a Ca(2+)-reversible manner. The modulating sites involved in these effects are located on the matrix side of the membrane. As illustrated in the companion paper (Bernardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabó, I., and Zoratti, M. (1992) J. Biol. Chem. 267, 2934-2939), the calcium-induced permeability transition of mitochondria is affected by these various agents in a similar manner. The results support the identification of the megachannel with the pore believed to be involved in the permeabilization process. The kinetic characteristics of the single channel events support the idea that the megachannel is composed of cooperating subunits.     

Multiple pathways of cytochrome c release from mitochondria in apoptosis

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating permeabilization of the outer mitochondrial membrane and the release of intermembrane space proteins remain controversial. Here, we discuss possible scenarios of the outer membrane permeabilization. The mechanisms by which the intermembrane space proteins are released from mitochondria depend presumably on cell type and on the nature of the apoptotic stimulus. The variety of mechanisms that can lead to outer membrane permeabilization might explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

Evidence against functionally significant aquaporin expression in mitochondria

Recent reports suggest the expression of aquaporin (AQP)-type water channels in mitochondria from liver (AQP8) (Calamita, G., Ferri, D., Gena, P., Liquori, G. E., Cavalier, A., Thomas, D., and Svelto, M. (2005) J. Biol. Chem. 280, 17149-17153) and brain (AQP9) (Amiry-Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., Rinvik, E., Torgner, I. A., and Ottersen, O. P. (2005) FASEB J. 19, 1459-1467), where they were speculated to be involved in metabolism, apoptosis, and Parkinson disease. Here, we systematically examined the functional consequence of AQP expression in mitochondria by measurement of water and glycerol permeabilities in mitochondrial membrane preparations from rat brain, liver, and kidney and from wild-type versus knock-out mice deficient in AQPs -1, -4, or -8. Osmotic water permeability, measured by stopped-flow light scattering, was similar in all mitochondrial preparations, with a permeability coefficient P(f) approximately 0.009 cm/s. Glycerol permeability was also similar ( approximately 5 x 10(-6) cm/s) in the various preparations. HgCl(2) slowed osmotic equilibration comparably in mitochondria from wild-type and AQP-deficient mice, although the slowing was explained by altered mitochondrial size rather than reduced P(f). Immunoblot analysis of mouse liver mitochondria failed to detect AQP8 expression, with liver homogenates from wild-type/AQP8 null mice as positive/negative controls. Our results provide evidence against functionally significant AQP expression in mitochondria, which is consistent with the high mitochondrial surface-to-volume ratio producing millisecond osmotic equilibration, even when intrinsic membrane water permeability is not high.