Mitochondrial glutathione: Features,regulation and role in disease

Background

Mitochondria are the powerhouse of mammalian cells and the main source of reactive oxygen species (ROS) associated with oxygen consumption. In addition, they also play a strategic role in controlling the fate of cells through regulation of death pathways. Mitochondrial ROS production fulfills a signaling role through regulation of redox pathways, but also contributes to mitochondrial damage in a number of pathological states.

Scope of review

Mitochondria are exposed to the constant generation of oxidant species, and yet the organelle remains functional due to the existence of an armamentarium of antioxidant defense systems aimed to repair oxidative damage, of which mitochondrial glutathione (mGSH) is of particular relevance. Thus, the aim of the review is to cover the regulation of mGSH and its role in disease.

Major conclusions

Cumulating evidence over recent years has demonstrated the essential role for mGSH in mitochondrial physiology and disease. Despite its high concentration in the mitochondrial matrix, mitochondria lack the enzymes to synthesize GSH de novo, so that mGSH originates from cytosolic GSH via transport through specific mitochondrial carriers, which exhibit sensitivity to membrane dynamics. Depletion of mGSH sensitizes cells to stimuli leading to oxidative stress such as TNF, hypoxia or amyloid β-peptide, thereby contributing to disease pathogenesis.

General significance

Understanding the regulation of mGSH may provide novel insights to disease pathogenesis and toxicity and the opportunity to design therapeutic targets of intervention in cell death susceptibility and disease. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Glutathione and modulation of cell apoptosis

Apoptosis is a highly organized form of cell death that is important for tissue homeostasis, organ development and senescence. To date, the extrinsic (death receptor mediated) and intrinsic (mitochondria derived) apoptotic pathways have been characterized in mammalian cells. Reduced glutathione, is the most prevalent cellular thiol that plays an essential role in preserving a reduced intracellular environment. glutathione protection of cellular macromolecules like deoxyribose nucleic acid proteins and lipids against oxidizing, environmental and cytotoxic agents, underscores its central anti-apoptotic function. Reactive oxygen and nitrogen species can oxidize cellular glutathione or induce its extracellular export leading to the loss of intracellular redox homeostasis and activation of the apoptotic signaling cascade. Recent evidence uncovered a novel role for glutathione involvement in apoptotic signaling pathways wherein post-translational S-glutathiolation of protein redox active cysteines is implicated in the potentiation of apoptosis. In the present review we focus on the key aspects of glutathione redox mechanisms associated with apoptotic signaling that includes: (a) changes in cellular glutathione redox homeostasis through glutathione oxidation or GSH transport in relation to the initiation or propagation of the apoptotic cascade, and (b) evidence for S-glutathiolation in protein modulation and apoptotic initiation.     

Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion

Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts and lead to heart disease. At present, the source of these cardiac ROS or the subcellular site of cardiac GSH loss [i.e., cytosolic (cGSH) or mitochondrial (mGSH) GSH] has not been completely elucidated. With the use of rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced diabetic rat heart, the mitochondrial origin of ROS was established. Furthermore, mitochondrial damage, as evidenced by loss of membrane potential, increases in oxidative stress, and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and -3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, L-buthionine-sulfoximine (BSO; 10 mmol/kg ip), which blocks GSH synthesis, or diethyl maleate (DEM; 4 mmol/kg ip), which inactivates preformed GSH, was administered in diabetic rats for 4 days after STZ administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were coadministered in diabetic rats. In this setting, mGSH was undetectable and cardiac apoptosis was further aggravated compared with the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification of mGSH in diabetic rat hearts and prevented apoptosis. Our data suggest for the first time that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.     

Critical role of mitochondrial glutathione in the survival of hepatocytes during hypoxia

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.     

Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties,permeabilization and cell death

Mitochondria are known to actively regulate cell death with the final phenotype of demise being determined by the metabolic and energetic status of the cell. Mitochondrial membrane permeabilization (MMP) is a critical event in cell death, as it regulates the degree of mitochondrial dysfunction and the release of intermembrane proteins that function in the activation and assembly of caspases. In addition to the crucial role of proapoptotic members of the Bcl-2 family, the lipid composition of the mitochondrial membranes is increasingly recognized to modulate MMP and hence cell death. The unphysiological accumulation of cholesterol in mitochondrial membranes regulates their physical properties, facilitating or impairing MMP during Bax and death ligand-induced cell death depending on the level of mitochondrial GSH (mGSH), which in turn regulates the oxidation status of cardiolipin. Cholesterol-mediated mGSH depletion stimulates TNF-induced reactive oxygen species and subsequent cardiolipin peroxidation, which destabilizes the lipid bilayer and potentiates Bax-induced membrane permeabilization. These data suggest that the balance of mitochondrial cholesterol to peroxidized cardiolipin regulates mitochondrial membrane properties and permeabilization, emerging as a rheostat in cell death.     

Targeting the mitochondrial pathway to induce apoptosis/necrosis through ROS by a newly developed Schiff's base to overcome MDR in cancer

Multidrug resistance (MDR) in cancer, a major obstacle to successful application of cancer chemotherapy, is often characterized by over-expression of multidrug resistance-related proteins such as MRP1, P-gp or elevated glutathione (GSH) level. Efflux of drugs by functional P-gp, MRP1 and elevated GSH level can confer resistance to apoptosis induced by a range of different stimuli. Therefore, it is necessary to develop new cell death inducers with relatively lower toxicity toward non-malignant cells that can overcome MDR by induction of apoptotic or non-apoptotic cell death pathways. Herein we report the synthesis and spectroscopic characterization of a GSH depleting, redox active Schiff’s base, viz., potassium-N-(2-hydroxy-3-methoxy-benzaldehyde)-alaninate (PHMBA). Cytotoxic potential of PHMBA has been studied in doxorubicin-resistant and -sensitive T lymphoblastic leukemia cells and Ehrlich ascites carcinoma (EAC) cells. PHMBA kills both the cell types irrespective of their drug-resistance phenotype following apoptotic/necrotic pathways. Moreover, PHMBA-induced cell death is associated with oxidative stress mediated mitochondrial pathway as the H₂O₂ inhibitor PEG-Catalase abrogated PHMBA-induced apoptosis/necrosis. PHMBA induces anti-tumor activity in both doxorubicin-sensitive and -resistant EAC-tumor-bearing Swiss albino mice. The non-toxicity of PHMBA was also confirmed through cytotoxicity studies on normal cell lines like PBMC, NIH3T3 and Chang Liver. To summarise, our data provide compelling rationale for future clinical use of this redox active Schiff’s base in treatment of cancer patients irrespective of their drug-resistance status.     

Mitochondria,cholesterol and amyloid β peptide: a dangerous trio in Alzheimer disease

The molecular mechanisms of Alzheimer’s disease (AD) are not fully understood. Extensive evidence from experimental models has involved the overgeneration and accumulation of toxic amyloid β peptides (Aβ) in the onset and progression of the disease. The amyloidogenic processing of amyloid precursor protein into pathogenic Aβ fragments is thought to occur in specific domains of the plasma membrane and favored by cholesterol enrichment. Intracellular Aβ accumulation is known to induce oxidative stress, predominantly via mitochondria targeting of toxic Aβ. Recent evidence using mouse models of cholesterol loading has demonstrated that the specific mitochondrial cholesterol pool sensitizes neurons to Aβ-induced oxidant cell death and caspase-independent apoptosis due to selective mitochondrial GSH (mGSH) depletion induced by cholesterol-mediated perturbation of mitochondrial membrane dynamics. mGSH replenishment by permeable precursors such as glutathione ethyl ester protected against Aβ-mediated neurotoxicity and inflammation. Thus, these novel data expand the pathogenic role of cholesterol in AD indicating that in addition to fostering Aβ generation, mitochondrial cholesterol determines Aβ neurotoxicity via mGSH regulation.     

Mitochondrial effectors in caspase-independent cell death

Activation of caspases is recognized as a key element in the apoptotic process. However, new evidence is drawing attention to the emergent role of cell death pathways where caspases are not involved. Recent advances in the molecular understanding of these new ways to die, called caspase-independent, have revealed that mitochondria play an important role via the release of proapoptotic proteins. The purpose of this review is to integrate, from a biological and structural point of view, the most recent advances in the knowledge of the main mitochondrial proapoptotic proteins involved in this cell death cascade. The origin of programmed cell death is discussed through these strongly conserved effectors.     

Glutamine potentiates TNF-alpha-induced tumor cytotoxicity

L-glutamine (Gln) sensitizes tumor cells to tumor necrosis factor (TNF)-alpha-induced cytotoxicity. The type and mechanism of cell death induced by TNF-alpha was studied in Ehrlich ascites tumor (EAT)-bearing mice fed a Gln-enriched diet (GED; where 30% of the total dietary nitrogen was from Gln). A high rate of Gln oxidation promotes a selective depletion of mitochondrial glutathione (mtGSH) content to approximately 58% of the level found in tumor mitochondria of mice fed a nutritionally complete elemental diet (standard diet, SD). The mechanism of mtGSH depletion involves a glutamate-induced inhibition of GSH transport from the cytosol into mitochondria. The increase in reactive oxygen intermediates (ROIs) production induced by TNF-alpha further depletes mtGSH to approximately 35% of control values, which associates with a decrease in the mitochondrial transmembrane potential (MMP), and elicits mitochondrial membrane permeabilization and release of cytochrome c. Mitochondrial membrane permeabilization was also found in intact tumor cells cultured with a Gln-enriched medium under conditions of buthionine sulfoximine (BSO)-induced selective GSH synthesis inhibition. Enforced expression of the bcl-2 gene in tumor cells could not avoid the glutamine- and TNF-alpha-induced cell death under conditions of mtGSH depletion. However, addition of GSH ester, which delivers free intracellular GSH and increases mtGSH levels, preserved cell viability. These findings show that glutamine oxidation and TNF-alpha, by causing a change in the glutathione redox status within tumor mitochondria, activates the molecular mechanism of apoptotic cell death.     

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation.

Glycosphingolipids, including gangliosides, are emerging as signaling intermediates of extracellular stimuli. Because mitochondria play a key role in the orchestration of death signals, we assessed the interaction of GD3 ganglioside (GD3) with mitochondria and the subsequent cascade of events that culminate in cell death. In vitro studies with isolated mitochondria from rat liver demonstrate that GD3 elicited a burst of peroxide production within 15-30 min, which preceded the opening of the mitochondrial permeability transition, followed by cytochrome c (cyt c) release. These effects were mimicked by lactosylceramide and N-acetyl-sphingosine but not by sphinganine or sphingosine and were prevented by cyclosporin A and butylated hydroxytoluene (BHT). Reconstitution of mitochondria pre-exposed to GD3 with cytosol from rat liver in a cell-free system resulted in the proteolytic processing of procaspase 3 and subsequent caspase 3 activation. Intact hepatocytes or U937 cells selectively depleted of glutathione in mitochondria by 3-hydroxyl-4-pentenoate (HP) with the sparing of cytosol reduced glutathione (GSH) were sensitized to GD3, manifested as an apoptotic death. Inhibition of caspase 3 prevented the apoptotic phenotype of HP-treated cells caused by GD3 without affecting cell survival; in contrast, BHT fully protected HP-treated cells to GD3 treatment. Treatment of cells with tumor necrosis factor increased the level of GD3, whereas blockers of mitochondrial respiration at complex I and II protected sensitized cells to GD3 treatment. Thus, the effect of GD3 as a lipid death effector is determined by its interaction with mitochondria leading to oxidant-dependent caspase activation. Mitochondrial glutathione plays a key role in controlling cell survival through modulation of the oxidative stress induced by glycosphingolipids.     

Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals

Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.     

Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis

Nitric oxide, generated by endogenous nitric oxide synthases or nitric oxide donors, can promote or prevent apoptosis induced by diverse pro-apoptotic stimuli in cell culture models. Both mitochondrial-dependent and -independent apoptotic signaling pathways mediate this dichotomous cellular response to nitric oxide. The molecular mechanisms behind these effects are complex and involve a number of nitrogen oxide-related species that are more reactive than nitric oxide itself. The local cellular environment plays a dynamic role in determining the nature and concentration of these species. Important components of the microenvironment include: the cellular redox state, glutathione, transition metals and the presence of other oxygen- and nitrogen-centered radicals. In particular, redox-sensitive nitrosating species are favorably generated under physiological conditions and capable of modifying multiple cell signaling pathways through reversible S-nitrosation reactions. Cytochrome c release from mitochondria is an important mechanism for the activation of caspase-3 and the initiation of cell death in response to 'intrinsic' pro-apoptotic stimuli, including oxidative and nitrosative stress. In turn, caspases and mitogen associated protein kinases may modulate cytochrome c release through their effects on the Bcl-2 family of proteins. This review will focus on (i) the importance of the cellular environment in determining the fate of nitric oxide and (ii) the ability of S-nitrosation to regulate mitochondrial-dependent apoptosis at the level of mitochondrial bioenergetics, cytochrome c release, caspases, mitogen associated protein kinases, and the Bcl-2 family of proteins.     

The role of mitochondria in apoptosis

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca2+ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize Ca2+ signaling. Massive accumulation of Ca2+ in the mitochondria leads to apoptosis. The Ca2+ dynamics of ER and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis. The number and morphology of mitochondria are precisely controlled through mitochondrial fusion and fission process by numerous mitochondria-shaping proteins. Mitochondrial fission accompanies apoptotic cell death and appears to be important for progression of the apoptotic pathway. Here, we highlight and discuss the role of mitochondrial calcium handling and mitochondrial fusion and fission machinery in apoptosis.     

The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet

Mitochondria are 'life-essential' organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a 'loss of function' resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.     

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.     

A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death

In ischemia-reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries.