Brian Herman

THE MITOCHONDRIAL PERMEABILITY TRANSITION IN CELL DEATH : A COMMON MECHANISM IN NECROSIS, APOPTOSIS AND AUTOPHAGY

Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reyes syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reyes syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.

The TGF-α precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction

The 50 amino acid form of TGF-alpha is cleaved from a conserved integral membrane glycoprotein by a protease that, in many tumor cells, appears to be limiting. To test whether the membrane-bound precursor has biological activity in the absence of processing, we introduced amino acid substitutions at the proteolytic cleavage sites. BHK cells transfected with expression vectors containing these altered sequences do not secrete detectable levels of mature TGF-alpha into the medium, but express high levels of proTGF-alpha at the cell surface. Coincubation of these BHK cells with A431 cells demonstrates that membrane-bound proTGF-alpha may bind to EGF receptors on the surface of contiguous cells, induce receptor autophosphorylation, and thereby produce a rapid rise in A431 intracellular calcium levels. Thus, proTGF-alpha can be biologically active in the absence of processing, a fact that may have implications for the integral membrane precursors of related growth factors.

Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death.

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca2+ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca2+, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-α.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.

Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes.

To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to <1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained >90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na+-free buffer at pH 7.4, pHi did not rise and cell viability remained >80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5-1 μM) at pH 7.4, the MPT was prevented and cell viability remained >80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30-40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to < 1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained > 90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na(+)-free buffer at pH 7.4, pHi did not rise and cell viability remained > 80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5-1 microM) at pH 7.4, the MPT was prevented and cell viability remained > 80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30-40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.

Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis

BACKGROUND The caspase-mediated proteolysis of many cellular proteins is a critical event during programmed cell death or apoptosis. It is important to determine which caspases are activated in mammalian cells, and where and when activation occurs, upon receipt of specific death stimuli. Such information would be useful in the design of strategies to regulate the activation of caspases during apoptosis. RESULTS We developed two novel fluorescent substrates that were specifically cleaved by caspase-1 or caspase-3. For in vitro studies, four-amino-acid recognition sequences, YVAD for caspase-1 and DEVD for caspase-3, were introduced between blue fluorescent protein (BFP) and green fluorescent protein (GFP), expressed in bacteria and purified. For in vivo studies, YVAD and DEVD were introduced between cyan fluorescent protein and yellow fluorescent protein, and expression was monitored in live mammalian cells. The proximity between fluorophores was determined using fluorescence resonance energy transfer. Purified substrates were cleaved following exposure to purified caspase-1 and caspase-3. In Cos-7 cells, caspase-1 and caspase-3 substrates were cleaved upon induction of apoptosis with staurosporine, a protein-kinase inhibitor, whereas caspase-3 but not caspase-1 substrate was cleaved upon treatment of cells with the DNA-damaging agent mitomycin c. CONCLUSIONS These substrates allow the spatial activation of specific members of the caspase family to be deciphered during the initiation and execution phase of programmed cell death, and allow activation of specific caspases to be monitored both in vivo and in vitro. This technology is also likely to be useful for high-throughput screening of reagents that modulate caspase activity.

Ageing and apoptosis

Ageing is accompanied by a general decline of physiological function, especially at later stages, and significant increases in the incidence of cancer and other degenerative diseases. It has recently been hypothesized that alterations in apoptosis may contribute to these age-associated changes. However, whether there is a role for apoptosis in the ageing process and how ageing may modify the regulatory machinery of apoptosis remains obscure. Although the literature addressing these issues is scarce, research in this area is gaining momentum. Molecules involved in apoptosis signaling in mammals have been found to regulate ageing in organisms such as Caenorhabditis elegans and Drosophila melanogaster. Caloric restriction studies in a wide variety of organisms, ranging from yeast to mammals, suggest the conserved nature of the ageing regulatory systems. It seems very likely that signals that regulate ageing will impact apoptosis and the extent of apoptosis may then impact ageing. However, to date, there has been no direct evidence supporting the existence of such cross-communication between ageing and apoptosis in mammalian system. Here we review progress in the field.

Transgenic Mice Overexpressing Glutathione Peroxidase 4 Are Protected against Oxidative Stress-induced Apoptosis

Glutathione peroxidase 4 (Gpx4) is uniquely involved in the detoxification of oxidative damage to membrane lipids. Our previous studies showed that Gpx4 is essential for mouse survival and that Gpx4 deficiency makes cells vulnerable to oxidative injury. In the present study, we generated two lines of transgenic mice overexpressing Gpx4 (Tg(GPX4) mice) using a genomic clone containing the human GPX4 gene. Both lines of Tg-(GPX4) mice, Tg5 and Tg6, had elevated levels of Gpx4 (mRNA and protein) in all tissues investigated, and overexpression of Gpx4 did not cause alterations in activities of glutathione peroxidase 1, catalase, Cu/Zn superoxide dismutase, and manganese superoxide dismutase. The human GPX4 transgene rescued the lethal phenotype of null mutation of the mouse Gpx4 gene, indicating that the transgene can replace the essential role of mouse Gpx4 in mouse development. Cell death induced by t-butylhydroperoxide and diquat was significantly less in murine embryonic fibroblasts from Tg(GPX4) mice compared with wild type mice. Liver damage and lipid peroxidation induced by diquat were reduced significantly in Tg(GPX4) mice. In addition, diquat-induced apoptosis was decreased in Tg(GPX4) mice, as evidenced by attenuated caspase-3 activation and reduced cytochrome c release from mitochondria. These data demonstrate that Gpx4 plays a role in vivo in the mechanism of apoptosis induced by oxidative stress that most likely occurs through oxidative damage to mitochondrial phospholipids such as cardiolipin.

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm).

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH(m)). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.

Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability.

Using low‐light digitized video microscopy, the onset, progression, and reversibility of anoxic injury were assessed in single hepatocytes isolated from fasted rats. Cell‐surface bleb formation occurred in three stages over 1‐3 h after anoxia. Stage I was characterized by formation of numerous small blebs. In stage II, small blebs enlarged by coalescence and fusion to form a few large terminal blebs. Near the end of stage II, cells began to swell rapidly, ending with the apparent breakdown of one of the terminal blebs. Breakdown of the bleb membrane initiated stage III of injury and was coincident with a rapid increase of nonspecific permeability to organic cationic and anionic molecules. On reoxygenation, stages I and II were fully reversible, and plasma membrane blebs were resorbed completely within 6 min of reoxygenation without loss of viability. Stage III, however, was not reversible, and no morphological changes occurred on reoxygenation. The results indicate that onset of cell death owing to anoxia is a rapid event initiated by a sudden increase of nonspecific plasma membrane permeability caused by rupture of a terminal bleb. Anoxic injury is reversible until this event occurs.— Herman, B.; Nieminen, A.‐L.; Gores, G. J.; Lemasters, J. J. Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability. FASEB J. 2: 146‐151; 1988.

Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals☆

The importance of mitochondrial ATP formation and extracellular acidosis was evaluated in hepatocyte suspensions after different toxic treatments. Acidotic pH was protective against cell killing from all toxic treatments examined except for pronase, a toxic protease. Fructose, a substrate for glycolytic ATP formation, provided good protection against toxicity from cyanide, oligomycin, t-butyl hydroperoxide, menadione and cystamine. Protection by fructose against CCCP, gramicidin and Br-A23187 required oligomycin. This indicated that these ionophores were causing cytotoxicity by uncoupling oxidative phosphorylation. Fructose provided little protection against pronase and HgCl2, the latter compound being a potent inhibitor of glycolysis. In conclusion, disruption of mitochondrial ATP formation was a common event contributing to the toxicity of chemical oxidants and ionophores. Acidotic pH was generally protective under these conditions of impaired ATP generation.