The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. The ability to modulate the life or death of a cell is recognized for its immense therapeutic potential. Therefore, research continues to focus on the elucidation and analysis of the cell cycle machinery and signaling pathways that control cell cycle arrest and apoptosis. To that end, the field of apoptosis research has been moving forward at an alarmingly rapid rate. Although many of the key apoptotic proteins have been identified, the molecular mechanisms of action or inaction of these proteins remain to be elucidated. The goal of this review is to provide a general overview of current knowledge on the process of apoptosis including morphology, biochemistry, the role of apoptosis in health and disease, detection methods, as well as a discussion of potential alternative forms of apoptosis.

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Apoptosis: A Review of Programmed Cell Death

A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.

https://science.sciencemag.org/content/sci/281/5381/1309.full.pdf

Mitochondria and apoptosis

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2−•). Even though O2−• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments. We also discuss various physiological and pathological scenarios resulting from an increased steady state concentration of mitochondrial oxidants.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-7793.2003.00335.x

Mitochondrial formation of reactive oxygen species.

Beckman, Kenneth B., and Bruce N. Ames. The Free Radical Theory of Aging Matures. Physiol. Rev. 78: 547–581, 1998. — The free radical theory of aging, conceived in 1956, has turned 40 and is rapidl...

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The Free Radical Theory of Aging Matures

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

Mitochondrial Membrane Permeabilization in Cell Death

Both physiological cell death (apoptosis) and, in some cases, accidental cell death (necrosis) involve a two-step process. At a first level, numerous physiological and some pathological stimuli trigger an increase in mitochondrial membrane permeability. The mitochondria release apoptogenic factors through the outer membrane and dissipate the electrochemical gradient of the inner membrane. Mitochondrial permeability transition (PT) involves a dynamic multiprotein complex formed in the contact site between the inner and outer mitochondrial membranes. The PT complex can function as a sensor for stress and damage, as well as for certain signals connected to receptors. Inhibition of PT by pharmacological intervention on mitochondrial structures or mitochondrial expression of the apoptosis-inhibitory oncoprotein Bcl-2 prevents cell death, suggesting that PT is a rate-limiting event of the death process. At a second level, the consequences of mitochondrial dysfunction (collapse of the mitochondrial inner transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins) entails a bioenergetic catastrophe culminating in the disruption of plasma membrane integrity (necrosis) and/or the activation of specific apoptogenic proteases (caspases) by mitochondrial proteins that leak into the cytosol (cytochrome c, apoptosis-inducing factor) with secondary endonuclease activation (apoptosis). The relative rate of these two processes (bioenergetic catastrophe versus protease and endonuclease activation) determines whether a cell will undergo primary necrosis or apoptosis. The acquisition of the biochemical and ultrastructural features of apoptosis critically relies on the liberation of apoptogenic proteases or protease activators from mitochondria. The fact that mitochondrial events control cell death has major implications for the development of cytoprotective and cytotoxic drugs.

The mitochondrial death/life regulator in apoptosis and necrosis

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.

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

Both physiological cell death (apoptosis) and at least some cases of accidental cell death (necrosis) involve a two-step-process. At first level, numerous physiological or pathological stimuli can trigger mitochondrial permeability transition which constitutes a rate-limiting event and initiates the common phase of the death process. Mitochondrial permeability transition (PT) involves the formation of proteaceous, regulated pores, probably by apposition of inner and outer mitochondrial membrane proteins which cooperate to form the mitochondrial PT pore complex. Inhibition of PT by pharmacological intervention on mitochondrial structures or mitochondrial expression of the apoptosis-inhibitory oncoprotein Bcl-2 thus can prevent cell death. At a second level, the consequences of mitochondrial dysfunction (collapse of the mitochondrial transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins) can entail a biogenetic catastrophe culminating in the disruption of plasma membrane integrity (necrosis) and/or the activation and action of apoptogenic proteases with secondary endonuclease activation and consequent oligonucleosomal DNA fragmentation (apoptosis). The acquisition of the biochemical and ultrastructural features of apoptosis critically relies on the liberation of apoptogenic proteases or protease activators from the mitochondrial intermembrane space. This scenario applies to very different models of cell death. The notion that mitochondrial events control cell death has major implications for the development of death-inhibitory drugs.

Mitochondrial Implication in Accidental and Programmed Cell Death: Apoptosis and Necrosis

According to current understanding, several metabolic alterations form part of the common phase of the apoptosis process. Such alterations include a disruption of the mitochondrial transmembrane potential (delta psi(m)), a depletion of nonoxidized glutathione (GSH) levels, an increase in the production of reactive oxygen species (ROS), and an elevation in cytosolic free Ca2+ levels. Using a cytofluorometric approach, we have determined each of these parameters at the single cell level in thymocytes or T cell hybridoma cells undergoing apoptosis. Regardless of the apoptosis induction protocol (glucocorticoids, DNA damage, Fas cross-linking, or CD3epsilon cross-linking), cells manifest a near-to-simultaneous delta psi(m) dissipation and GSH depletion early during the apoptotic process. None of the protocols for apoptosis inhibition (antioxidants, delta psi(m) stabilization, Bcl-2 hyperexpression, or inhibition of IL-1-converting enzyme) allowed for the dissociation of delta psi(m) disruption and GSH depletion, indicating that both parameters are closely associated with each other. At a later stage of the apoptotic process, cells manifest a near-simultaneous increase in ROS production and intracellular Ca2+ levels. Whereas the thapsigargin- or ionophore-induced elevation of calcium levels has no immediate consequence on delta psi(m') cellular redox potentials, or ROS production, pro-oxidants and menadione, an inducer of mitochondrial superoxide anion generation, cause a rapid (15 min) Ca2+ elevation. Together, these data suggest a two-step model of the common phase of apoptosis. After an initial delta psi(m) dissipation linked to GSH depletion (step 1), cells hyperproduce ROS with an associated disruption of Ca2+ homeostasis (step 2).

Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2897%2900623-6

Bruno Dallaporta

Papers

The mitochondrial death/life regulator in apoptosis and necrosis

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

Mitochondrial Implication in Accidental and Programmed Cell Death: Apoptosis and Necrosis

Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis.

Nitric oxide induces apoptosis via triggering mitochondrial permeability transition