Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.     

Zinc and calcium modulate mitochondrial redox state and morphofunctional integrity

Zinc and calcium have highly interwoven functions that are essential for cellular homeostasis. Here we first present a novel real-time flow cytometric technique to measure mitochondrial redox state and show it is modulated by zinc and calcium, individually and combined. We then assess the interactions of zinc and calcium on mitochondrial H₂O₂ production, membrane potential (ΔΨ_m), morphological status, oxidative phosphorylation (OXPHOS), complex I activity, and structural integrity. Whereas zinc at low doses and both cations at high doses individually and combined promoted H₂O₂ production, the two cations individually did not alter mitochondrial redox state. However, when combined at low and high doses the two cations synergistically suppressed and promoted, respectively, mitochondrial shift to a more oxidized state. Surprisingly, the antioxidants vitamin E and N-acetylcysteine showed pro-oxidant activity at low doses, whereas at high antioxidant doses NAC inhibited OXPHOS and dyscoupled mitochondria. Individually, zinc was more potent than calcium in inhibiting OXPHOS, whereas calcium more potently dissipated the ΔΨ_m and altered mitochondrial volume and ultrastructure. The two cations synergistically inhibited OXPHOS but antagonistically dissipated ΔΨ_m and altered mitochondrial volume and morphology. Overall, our study highlights the importance of zinc and calcium in mitochondrial redox regulation and functional integrity. Importantly, we uncovered previously unrecognized bidirectional interactions of zinc and calcium that reveal distinctive foci for modulating mitochondrial function in normal and disease states because they are potentially protective or damaging depending on conditions.     

Redox regulation of ER and mitochondrial Ca2+ signaling in cell survival and death

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca²⁺ signaling at the ER/mitochondrial interface – notably, inositol trisphosphate receptors (IP₃Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.     

Synthetic ceramides induce growth arrest or apoptosis by altering cellular redox status

Reactive oxygen species (ROS) and ceramide are each partly responsible for the signal transduction of a variety of extracellular agents. Furthermore, the application of synthetic, short-chain ceramides mimics the cellular responses to these extracellular agents. However, the significance of ROS involvement in ceramide signaling pathways is poorly understood. Here we describe that the cellular responses to C2-/C6-ceramide of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells are preceded by a rise in mitochondrial peroxide production. In Jurkat T-cells, this is associated with a large time- and dose-dependent loss of cellular glutathione. However, in U937 monocytes, glutathione loss is transient. Differences in the magnitude and kinetics of this alteration in cellular redox state associate with discrete outcomes, namely growth arrest or apoptosis.     

Role of Mitochondria in Human Aging

Mitochondria are the major intracellular source and target sites of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in animal and human cells. It has been demonstrated that mitochondrial respiratory function declines with age in various human tissues and that a defective respiratory chain results in enhanced production of ROS and free radicals in mitochondria. On the other hand, accumulating evidence now indicates that lipid peroxidation, protein modification and mitochondrial DNA (mtDNA) muutation are concurrently increased during aging. On the basis of these observations and the fact that the rate of cellular production of superoxide anions and hydrogen peroxide increases with age, it has recently been postulated that oxidative stress is a major contributory factor in the aging process. A causal relationship between oxidative modification and mutation of mtDNA, mitochondrial dysfunction and aging has emerged, although some details have remained unsolved. In this article, the role of mitochondria in the human aging process is reviewed on the basis of recent findings gathered from our and other laboratories.     

Blockade of SOCE protects HT22 cells from hydrogen peroxide-induced apoptosis

Oxidative stress is an established event in the pathology of neurobiological diseases. Previous studies indicated that store-operated Ca²⁺ entry (SOCE) has been involved in oxidative stress. The present study was carried out to investigate the effects of SOCE inhibition on neuronal oxidative stress injury induced by hydrogen peroxide (H₂O₂) in HT22 cells, a murine hippocampal neuronal model. H₂O₂ insult induced significant intracellular Ca²⁺ overload, mitochondrial dysfunction and cell viability decrease. Inhibition of SOCE by pharmacological inhibitor and STIM1 RNAi significantly alleviated intracellular Ca²⁺ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and eventually inhibited H₂O₂-induced cell apoptosis. These findings suggest that SOCE inhibition exhibited neuroprotection against oxidative stress induced by H₂O₂ and SOCE might be a useful therapeutic target in neurobiological disorders.     

Modulation of the matrix redox signaling by mitochondrial Ca~(2+)

Mitochondria sense,shape and integrate signals,and thus function as central players in cellular signal transduction. Ca2+ waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca2+ transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance,the molecular nature of the proteins involvedin mitochondrial Ca2+ transport has been revealed only recently. Mitochondrial Ca2+ promotes energy metabolism through the activation of matrix dehydrogenases and downstream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)+ ratio,but at the same time will increase reactive oxygen species(ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state,which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redoxsensitive sensors,real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca2+ combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca2+ and redox signals and their impact on cell function. In this review,we describe mitochondrial Ca2+ handling,focusing on a number of newly identified proteins involved in mitochondrial Ca2+ uptake and release. We further discuss our recent findings,revealing how mitochondrial Ca2+ influences the matrix redox state. As a result,mitochondrial Ca2+ is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease.     

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection.     

Mitochondrial ATP-sensitive K channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK_ATP). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. Isolated HTG liver mitochondria presented lower rates of H₂O₂ production, which were reversed by mitoK_ATP antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoK_ATP activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote mitoK_ATP activation. The mild uncoupling mediated by mitoK_ATP increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK_ATP as a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess.     

Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H₂O₂) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H₂O₂ derives from superoxide (O₂^˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O₂^˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O₂^˙̄ and H₂O₂ may provide feedback signals to modulate mitochondrial metabolism.     

Age-associated cellular relocation of Sod 1 as a self-defense is a futile mechanism to prevent vascular aging

Vascular aging is characterized by the presence of chronic oxidative stress. Although cytosolic Sod 1 has a key role in the detoxification of superoxide ((*)O(2)(-)), little is known about its importance in vascular aging. We found that inhibition of Sod 1 had no effect on (*)O2- generation. Furthermore, its expression decreased in an age-dependent manner. Interestingly, Sod 1 loses its membrane-association and is also lost from the caveolae with increasing age. Instead, a relocation of Sod 1 to the mitochondria takes place, presumably in an attempt to maintain mitochondrial integrity and to counter-balance age-associated oxidative stress. Unlike Sod 2, which is constitutively expressed in mitochondria to control (*)O2- radical fluxes, Sod 1 is not inactivated by peroxynitrite and is not nitrated as a function of age. These novel insights into oxidative stress-associated vascular aging and the understanding about how redox-systems are regulated in old age may identify new targets to ameliorate aging as the greatest cardiovascular risk factor.     

Mitochondria: Participation to infertility as source of energy and cause of senescence

Mitochondria is a powerhouse organelle involved in ATP synthesis, calcium signaling, reactive oxygen species (ROS) by oxidative stress production, cell cycle arrest via apoptosis and sex steroid hormones biosynthesis. Improvement of sperm parameters such as motility, capacitation, acrosome reaction, and oocyte interaction, involve regulation of ROS levels by the mitochondria. In human, the relation between the quantitative level of mitochondrial DNA (mtDNA), oocyte cytoplasm maturation and fertilization potential, is not clear. It has been hypothesized that oocytes without sufficient wild type mtDNA and therefore able to generate ATP, would not normally be ovulated. This is reflected in the low numbers of mtDNA observed in degenerate oocytes obtained through super ovulation protocols during assisted reproductive technology programs. Different theories place mitochondria in a central role of oxidative damage to cells and tissues related to infertility declining and aging. Mitochondria-dependent apoptosis seems to be responsible for the pre and post-natal decline in germ cells, embryo development, implantation failure, and miscarriages.     

Mechanisms of vanadate-induced cellular toxicity: role of cellular glutathione and NADPH

Increased production of reactive oxygen species (ROS) by the mitochondrion has been implicated in the pathogenesis of numerous liver diseases. However, the exact sites of ROS production within liver mitochondria and the electron transport chain are still uncertain. To determine the sites of ROS generation in liver mitochondria we evaluated the ability of a variety of mitochondrial respiratory inhibitors to alter the steady state levels of ROS generated within the intact hepatocyte and in isolated mitochondria. Treatment with myxothiazol alone at concentrations that significantly inhibit respiration dramatically increased the steady-state levels of ROS in hepatocytes. Similar results were also observed in isolated mitochondria oxidizing succinate. Coincubation with antimycin or rotenone had no effect on myxothiazol-induced ROS levels. Myxothiazol stimulation of ROS was mitochondrial in origin as demonstrated by the colocalization of MitoTracker Red and dichlorofluorescein staining using confocal microscopy. Furthermore, diphenyliodonium, an inhibitor that blocks electron flow through the flavin mononucleotide of mitochondrial complex I and other flavoenzymes, significantly attenuated the myxothiazol-induced increase in hepatocyte ROS levels. Together, these data suggest that in addition to the ubiquinone-cytochrome bc(1) complex of complex III, several of the flavin-containing enzymes or iron-sulfur centers within the mitochondrial electron transport chain should also be considered sites of superoxide generation in liver mitochondria.     

Mitochondria and arrhythmias

Mitochondria are essential to providing ATP, thereby satisfying the energy demand of the incessant electrical activity and contractile action of cardiac muscle. Emerging evidence indicates that mitochondrial dysfunction can adversely affect cardiac electrical functioning by impairing the intracellular ion homeostasis and membrane excitability through reduced ATP production and excessive reactive oxygen species (ROS) generation, resulting in increased propensity to cardiac arrhythmias. In this review, the molecular mechanisms linking mitochondrial dysfunction to cardiac arrhythmias are discussed with an emphasis on the impact of increased mitochondrial ROS on the cardiac ion channels and transporters that are critical to maintaining normal electromechanical functioning of the cardiomyocytes. The potential of using mitochondria-targeted antioxidants as a novel antiarrhythmia therapy is highlighted.     

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase,an enzyme essential for the alleviation of oxidative stress

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H₂O₂. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC_1 -172 and EcAhpC_1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC_1 -172 and EcAhpC_1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC–AhpF assembly has been demonstrated.     

Role of calcium and cyclophilin D in the regulation of mitochondrial permeabilization induced by glutathione depletion

The mitochondrial permeability transition (MPT) is a calcium and oxidative stress sensitive transition in the permeability of the mitochondrial inner membrane that plays a crucial role in cell death. However, the mechanism regulating the MPT remains controversial. To study the role of oxidative stress in the regulation of the MPT, we used diethyl maleate (DEM) to deplete glutathione (GSH) in human leukemic CEM cells. GSH depletion increased mitochondrial calcium and reactive oxygen species (ROS) levels in a co-dependent manner causing loss of mitochondrial membrane potential (deltapsi(m)) and cell death. These events were inhibited by the calcium chelator BAPTA-AM and the antioxidants N-acetylcysteine (NAC) and the triphenyl phosphonium-linked ubiquinone derivative MitoQ. In contrast, the MPT inhibitor cyclosporine A (CsA) and small interference RNA (siRNA) knockdown of cyclophilin D (Cyp-D) were not protective. These results indicate that mitochondrial permeabilization induced by GSH depletion is not regulated by the classical MPT.     

Mitochondria and Organismal Longevity

Mitochondria are essential for various biological processes including cellular energy production. The oxidative stress theory of aging proposes that mitochondria play key roles in aging by generating reactive oxygen species (ROS), which indiscriminately damage macromolecules and lead to an age-dependent decline in biological function. However, recent studies show that increased levels of ROS or inhibition of mitochondrial function can actually delay aging and increase lifespan. The aim of this review is to summarize recent findings regarding the role of mitochondria in organismal aging processes. We will discuss how mitochondria contribute to evolutionarily conserved longevity pathways, including mild inhibition of respiration, dietary restriction, and target of rapamycin (TOR) signaling.     

On the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid

Glycyrrhetinic acid (GE), the aglycone of glycyrrhizic acid, a triterpene glycoside which represents one of the main constituents of licorice root, induces an oxidative stress in liver mitochondria responsible for the induction of membrane permeability transition. In fact, GE, by interacting with the mitochondrial respiratory chain, generates hydrogen peroxide which in turn oxidizes critical thiol groups and endogenous pyridine nucleotides leading to the opening of the transition pore. Most likely the reactive group of GE is the carbonyl oxygen in C-11 which, by interacting mainly with a Fe/S centre of mitochondrial complex I, generates an oxygen-centered radical responsible for the pro-oxidant action.     

A spontaneous mutation in the nicotinamide nucleotide transhydrogenase gene of C57BL/6J mice results in mitochondrial redox abnormalities

NADPH is the reducing agent for mitochondrial H₂O₂ detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP⁺ ratio. This enzyme catalyzes the reduction of NADP⁺ at the expense of NADH oxidation and H⁺ reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-Nnt^MUT) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-Nnt^MUT mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-Nnt^W) and from B6J-Nnt^MUT mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-Nnt^MUT mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H₂O₂ release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca²⁺-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-Nnt^MUT mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-Nnt^W mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-Nnt^MUT mice in biomedical research should not be overlooked.