Six Functions of Respiration: Isn't It Time to Take Control over ROS Production in Mitochondria, and Aging Along with It?

Cellular respiration is associated with at least six distinct but intertwined biological functions. (1) biosynthesis of ATP from ADP and inorganic phosphate, (2) consumption of respiratory substrates, (3) support of membrane transport, (4) conversion of respiratory energy to heat, (5) removal of oxygen to prevent oxidative damage, and (6) generation of reactive oxygen species (ROS) as signaling molecules. Here we focus on function #6, which helps the organism control its mitochondria. The ROS bursts typically occur when the mitochondrial membrane potential (MMP) becomes too high, e.g., due to mitochondrial malfunction, leading to cardiolipin (CL) oxidation. Depending on the intensity of CL damage, specific programs for the elimination of damaged mitochondria (mitophagy), whole cells (apoptosis), or organisms (phenoptosis) can be activated. In particular, we consider those mechanisms that suppress ROS generation by enabling ATP synthesis at low MMP levels. We discuss evidence that the mild depolarization mechanism of direct ATP/ADP exchange across mammalian inner and outer mitochondrial membranes weakens with age. We review recent data showing that by protecting CL from oxidation, mitochondria-targeted antioxidants decrease lethality in response to many potentially deadly shock insults. Thus, targeting ROS- and CL-dependent pathways may prevent acute mortality and, hopefully, slow aging.

Extrusion of mitochondria: Garbage clearance or cell-cell communication signals?

Mitochondria are dynamic organelles that regulate various intracellular signaling pathways, including the mechanisms of programmed cell death, differentiation, inflammation, and so on. Mitochondria may be extruded as membrane enveloped or as free organelles during developmental processes, inflammatory activation, and in the process of "garbage clearance" of damaged mitochondria in postmitotic cells. Extracellular mitochondria can be engulfed by immune and nonimmune cells and trigger intracellular signaling leading to an inflammatory response. At the same time, it was reported that the release of extracellular vesicles containing mitochondria from mesenchymal stem cells contributes to their therapeutic anti-inflammatory effects. Numerous studies claim that engulfed mitochondria improve cellular bioenergetics, but this assumption requires further investigation. This review aims at a critical discussion of the mechanisms of mitochondrial extrusion in mammals, the reception of mitochondrial components, and the responses of recipient cells to extracellular mitochondria.

Innate Immunity and Phenoptosis.

The hypothesis is proposed that activation of innate immunity is the primary mechanism of phenoptosis (programmed death of an organism). In support of the hypothesis, we discuss (i) the data on active release of signaling molecules from the cell producing excessive inflammation; (ii) the data on contribution of mitochondrial production of reactive oxygen species to immune response.

Innate Immunity as an Executor of the Programmed Death of Individual Organisms for the Benefit of the Entire Population.

In humans, over-activation of innate immunity in response to viral or bacterial infections often causes severe illness and death. Furthermore, similar mechanisms related to innate immunity can cause pathogenesis and death in sepsis, massive trauma (including surgery and burns), ischemia/reperfusion, some toxic lesions, and viral infections including COVID-19. Based on the reviewed observations, we suggest that such severe outcomes may be manifestations of a controlled suicidal strategy protecting the entire population from the spread of pathogens and from dangerous pathologies rather than an aberrant hyperstimulation of defense responses. We argue that innate immunity may be involved in the implementation of an altruistic programmed death of an organism aimed at increasing the well-being of the whole community. We discuss possible ways to suppress this atavistic program by interfering with innate immunity and suggest that combating this program should be a major goal of future medicine.

Mitochondria-Targeted Antioxidants and Uncouplers of Oxidative Phosphorylation in Treatment of the Systemic Inflammatory Response Syndrome (SIRS).

Systemic inflammatory response syndrome (SIRS) development is accompanied by mitochondrial dysfunction and excessive ROS production. Mitochondrial dysfunctions also occur in many SIRS-related diseases and may be critical for their pathogenesis; therefore, a use of mitochondria-targeted drugs is a promising trend in SIRS research and therapy. Here, we review recent studies concerning the application of the mitochondria-targeted antioxidants and uncouplers of oxidative phosphorylation in animal models of SIRS and related diseases. We propose that a new class of uncouplers of oxidative phosphorylation, lipophilic cations could be a base for a new generation of drugs for SIRS treatment. J. Cell. Physiol. 232: 904-912, 2017. © 2016 Wiley Periodicals, Inc.

Low concentration of uncouplers of oxidative phosphorylation decreases the TNF-induced endothelial permeability and lethality in mice.

Mitochondrial dysfunctions occur in many diseases linked to the systemic inflammatory response syndrome (SIRS). Mild uncoupling of oxidative phosphorylation is known to rescue model animals from pathologies related to mitochondrial dysfunctions and overproduction of reactive oxygen species (ROS). To study the potential of SIRS therapy by uncoupling, we tested protonophore dinitrophenol (DNP) and a free fatty acid (FFA) anion carrier, lipophilic cation dodecyltriphenylphosphonium (C12TPP) in mice and in vitro models of SIRS. DNP and C12TPP prevented the body temperature drop and lethality in mice injected with high doses of a SIRS inducer, tumor necrosis factor (TNF). The mitochondria-targeted antioxidant plastoquinonyl decyltriphenylphosphonium (SkQ1) which also catalyzes FFA-dependent uncoupling revealed similar protective effects and downregulated expression of the NFκB-regulated genes (VCAM1, ICAM1, MCP1, and IL-6) involved in the inflammatory response of endothelium in aortas of the TNF-treated mice. In vitro mild uncoupling rescued from TNF-induced endothelial permeability, disassembly of cell contacts and VE-cadherin cleavage by the matrix metalloprotease 9 (ММР9). The uncouplers prevented TNF-induced expression of MMP9 via inhibition of NFκB signaling. Water-soluble antioxidant Trolox also prevented TNF-induced activation and permeability of endothelium in vitro via inhibition of NFκB signaling, suggesting that the protective action of the uncouplers is linked to their antioxidant potential.

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector.

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C4R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C12R1). Surprisingly, C12R1 increased H(+) conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.

The Role Played by Autophagy in FcεRI-Dependent Activation of Mast Cells.

The significant role of mast cells in the development of allergic and inflammatory diseases is well-established. Among the various mechanisms of mast cell activation, the interaction of antigens/allergens with IgE and the subsequent binding of this complex to the high-affinity IgE receptor FcεRI stand out as the most studied and fundamental pathways. This activation process leads to the rapid exocytosis of granules containing preformed mediators, followed by the production of newly synthesized mediators, including a diverse array of cytokines, chemokines, arachidonic acid metabolites, and more. While conventional approaches to allergy control primarily focus on allergen avoidance and the use of antihistamines (despite their associated side effects), there is increasing interest in exploring novel methods to modulate mast cell activity in modern medicine. Recent evidence suggests a role for autophagy in mast cell activation, offering potential avenues for utilizing low-molecular-weight autophagy regulators in the treatment of allergic diseases. More specifically, mitochondria, which play an important role in the regulation of autophagy as well as mast cell activation, emerge as promising targets for drug development. This review examines the existing literature regarding the involvement of the molecular machinery associated with autophagy in FcεRI-dependent mast cell activation.

Exogenous Iron Induces Mitochondrial Lipid Peroxidation, Lipofuscin Accumulation, and Ferroptosis in H9c2 Cardiomyocytes.

Lipid peroxidation plays an important role in various pathologies and aging, at least partially mediated by ferroptosis. The role of mitochondrial lipid peroxidation during ferroptosis remains poorly understood. We show that supplementation of exogenous iron in the form of ferric ammonium citrate at submillimolar doses induces production of reactive oxygen species (ROS) and lipid peroxidation in mitochondria that precede ferroptosis in H9c2 cardiomyocytes. The mitochondria-targeted antioxidant SkQ1 and the redox mediator methylene blue, which inhibits the production of ROS in complex I of the mitochondrial electron transport chain, prevent both mitochondrial lipid peroxidation and ferroptosis. SkQ1 and methylene blue also prevented accumulation of lipofuscin observed after 24 h incubation of cardiomyocytes with ferric ammonium citrate. Using isolated cardiac mitochondria as an in vitro ferroptosis model, it was shown that rotenone (complex I inhibitor) in the presence of ferrous iron stimulates lipid peroxidation and lipofuscin accumulation. Our data indicate that ROS generated in complex I stimulate mitochondrial lipid peroxidation, lipofuscin accumulation, and ferroptosis induced by exogenous iron.

Mitochondrial fragmentation in early differentiation of human MB135 myoblasts: Role of mitochondrial ROS production in the absence of depolarization.

AIMS: Study of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis. MAIN METHODS: Mitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria. KEY FINDINGS: In immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation. SIGNIFICANCE: Mitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.

Redox processes are major regulators of leukotriene synthesis in neutrophils exposed to bacteria Salmonella typhimurium; the way to manipulate neutrophil swarming.

Neutrophils play a primary role in protecting our body from pathogens. When confronted with invading bacteria, neutrophils begin to produce leukotriene B4, a potent chemoattractant that, in cooperation with the primary bacterial chemoattractant fMLP, stimulates the formation of swarms of neutrophils surrounding pathogens. Here we describe a complex redox regulation that either stimulates or inhibits fMLP-induced leukotriene synthesis in an experimental model of neutrophils interacting with Salmonella typhimurium. The scavenging of mitochondrial reactive oxygen species by mitochondria-targeted antioxidants MitoQ and SkQ1, as well as inhibition of their production by mitochondrial inhibitors, inhibit the synthesis of leukotrienes regardless of the cessation of oxidative phosphorylation. On the contrary, antioxidants N-acetylcysteine and sodium hydrosulfide promoting reductive shift in the reversible thiol-disulfide system stimulate the synthesis of leukotrienes. Diamide that oxidizes glutathione at high concentrations inhibits leukotriene synthesis, and the glutathione precursor S-adenosyl-L-methionine prevents this inhibition. Diamide-dependent inhibition is also prevented by diphenyleneiodonium, presumably through inhibition of NADPH oxidase and NADPH accumulation. Thus, during bacterial infection, maintaining the reduced state of glutathione in neutrophils plays a decisive role in the synthesis of leukotriene B4. Suppression of excess leukotriene synthesis is an effective strategy for treating various inflammatory pathologies. Our data suggest that the use of mitochondria-targeted antioxidants may be promising for this purpose, whereas known thiol-based antioxidants, such as N-acetylcysteine, may dangerously stimulate leukotriene synthesis by neutrophils during severe pathogenic infection.

Pyrimidine biosynthesis links mitochondrial respiration to the p53 pathway.

While many functions of the p53 tumor suppressor affect mitochondrial processes, the role of altered mitochondrial physiology in a modulation of p53 response remains unclear. As mitochondrial respiration is affected in many pathologic conditions such as hypoxia and intoxications, the impaired electron transport chain could emit additional p53-inducing signals and thereby contribute to tissue damage. Here we show that a shutdown of mitochondrial respiration per se does not trigger p53 response, because inhibitors acting in the proximal and distal segments of the respiratory chain do not activate p53. However, strong p53 response is induced specifically after an inhibition of the mitochondrial cytochrome bc1 (the electron transport chain complex III). The p53 response is triggered by the deficiency in pyrimidines that is developed due to a suppression of the functionally coupled mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). In epithelial carcinoma cells the activation of p53 in response to mitochondrial electron transport chain complex III inhibitors does not require phosphorylation of p53 at Serine 15 or up-regulation of p14(ARF). Instead, our data suggest a contribution of NQO1 and NQO2 in stabilization of p53 in the nuclei. The results establish the deficiency in pyrimidine biosynthesis as the cause of p53 response in the cells with impaired mitochondrial respiration.

Current perspectives of mitochondria-targeted antioxidants in cancer prevention and treatment.

Oxidative stress nearly always accompanies all stages of cancer development. At the early stages, antioxidants may help to reduce reactive oxygen species (ROS) production and exhibit anticarcinogenic effects. In the later stages, ROS involvement becomes more complex. On the one hand, ROS are necessary for cancer progression and epithelial-mesenchymal transition. On the other hand, antioxidants may promote cancer cell survival and may increase metastatic frequency. The role of mitochondrial ROS in cancer development remains largely unknown. This paper reviews experimental data on the effects of both endogenous and exogenous antioxidants on cancerogenesis focusing on the development and application of mitochondria-targeted antioxidants. We also discuss the prospects for antioxidant cancer therapy, focusing on the use of mitochondria-targeted antioxidants.

Mitochondrial Lipid Peroxidation Is Responsible for Ferroptosis.

Ferroptosis induced by erastin (an inhibitor of cystine transport) and butionine sulfoximine (an inhibitor of glutathione biosynthesis) was prevented by the mitochondria-targeted antioxidants SkQ1 and MitoTEMPO. These effects correlate with the prevention of mitochondrial lipid peroxidation, which precedes cell death. Methylene blue, a redox agent that inhibits the production of reactive oxygen species (ROS) in complex I of the mitochondrial electron transport chain, also inhibits ferroptosis and mitochondrial lipid peroxidation. Activation of ROS production in complex I with rotenone in the presence of ferrous iron stimulates lipid peroxidation in isolated mitochondria, while ROS produced by complex III are ineffective. SkQ1 and methylene blue inhibit lipid peroxidation. We suggest that ROS formed in complex I promote mitochondrial lipid peroxidation and ferroptosis.

Role of Mitochondria in the Regulation of Effector Functions of Granulocytes.

Granulocytes (neutrophils, eosinophils, and basophils) are the most abundant circulating cells in the innate immune system. Circulating granulocytes, primarily neutrophils, can cross the endothelial barrier and activate various effector mechanisms to combat invasive pathogens. Eosinophils and basophils also play an important role in allergic reactions and antiparasitic defense. Granulocytes also regulate the immune response, wound healing, and tissue repair by releasing of various cytokines and lipid mediators. The effector mechanisms of granulocytes include the production of reactive oxygen species (ROS), degranulation, phagocytosis, and the formation of DNA-containing extracellular traps. Although all granulocytes are primarily glycolytic and have only a small number of mitochondria, a growing body of evidence suggests that mitochondria are involved in all effector functions as well as in the production of cytokines and lipid mediators and in apoptosis. It has been shown that the production of mitochondrial ROS controls signaling pathways that mediate the activation of granulocytes by various stimuli. In this review, we will briefly discuss the data on the role of mitochondria in the regulation of effector and other functions of granulocytes.

The critical role of mitochondrial lipid peroxidation in ferroptosis: insights from recent studies.

Ferroptosis is a regulated form of necrotic cell death reliant on iron-catalyzed lipid peroxidation. Although the precise involvement of mitochondria in ferroptosis remains incompletely elucidated, recent research indicates that mitochondrial oxidative events wield a pivotal influence in this mechanism. This article centers on the most recent discoveries, spotlighting the significance of mitochondrial lipid peroxidation in the occurrence of ferroptosis. Modern investigative tools, such as mitochondria-specific dyes responsive to lipid peroxidation and antioxidants targeting mitochondria, have been employed to delve into this phenomenon. The authors' recent empirical evidence demonstrates that mitochondrial lipid peroxidation, quantified using the innovative fluorescent ratiometric probe MitoCLox, takes place prior to the onset of ferroptotic cell death. The mitochondria-targeted antioxidant SkQ1 hinders mitochondrial lipid peroxidation and thwarts ferroptosis, all while leaving unaffected the buildup of reactive oxygen species within the cytoplasm, an antecedent to mitochondrial lipid peroxidation. Similarly, the redox agent methylene blue, impeding the genesis of reactive oxygen species in complex I of the electron transport chain, also imparts a comparable protective effect. These findings collectively imply that reactive oxygen species originating from complex I might hold particular significance in fomenting mitochondrial lipid peroxidation, a pivotal trigger of ferroptosis.

Mitochondrial Oxidative Stress and Mitophagy Activation Contribute to TNF-Dependent Impairment of Myogenesis.

Many muscular pathologies are associated with oxidative stress and elevated levels of the tumor necrosis factor (TNF) that cause muscle protein catabolism and impair myogenesis. Myogenesis defects caused by TNF are mediated in part by reactive oxygen species (ROS), including those produced by mitochondria (mitoROS), but the mechanism of their pathological action is not fully understood. We hypothesized that mitoROS act by triggering and enhancing mitophagy, an important tool for remodelling the mitochondrial reticulum during myogenesis. We used three recently developed probes-MitoTracker Orange CM-H2TMRos, mito-QC, and MitoCLox-to study myogenesis in human myoblasts. Induction of myogenesis resulted in a significant increase in mitoROS generation and phospholipid peroxidation in the inner mitochondrial membrane, as well as mitophagy enhancement. Treatment of myoblasts with TNF 24 h before induction of myogenesis resulted in a significant decrease in the myoblast fusion index and myosin heavy chain (MYH2) synthesis. TNF increased the levels of mitoROS, phospholipid peroxidation in the inner mitochondrial membrane and mitophagy at an early stage of differentiation. Trolox and SkQ1 antioxidants partially restored TNF-impaired myogenesis. The general autophagy inducers rapamycin and AICAR, which also stimulate mitophagy, completely blocked myogenesis. The autophagy suppression by the ULK1 inhibitor SBI-0206965 partially restored myogenesis impaired by TNF. Thus, suppression of myogenesis by TNF is associated with a mitoROS-dependent increase in general autophagy and mitophagy.

Mitochondria-targeted antioxidant SkQ1 inhibits leukotriene synthesis in human neutrophils.

Leukotrienes are among the most potent mediators of inflammation, and inhibition of their biosynthesis, is becoming increasingly important in the treatment of many pathologies. In this work, we demonstrated that preincubation of human neutrophils with the mitochondria targeted antioxidant SkQ1 (100 nM) strongly inhibits leukotriene synthesis induced by three different stimuli: the Ca2+ ionophore A23187, the chemotactic formyl-peptide fMLP in combination with cytocholasin B, and opsonized zymosan. The SkQ1 analogue lacking the antioxidant quinone moiety (C12TPP) was ineffective, suggesting that mitochondrial production of reactive oxygen species (ROS) is critical for activating of leukotriene synthesis in human neutrophils. The uncoupler of oxidative phosphorylation FCCP also inhibits leukotriene synthesis, indicating that a high membrane potential is a prerequisite for stimulating leukotriene synthesis in neutrophils. Our data show that activation of mitogen-activated protein kinases p38 and ERK1/2, which is important for leukotriene synthesis in neutrophils is a target for SkQ1: 1) the selective p38 inhibitor SB203580 inhibited fMLP-induced leukotriene synthesis, while the ERK1/2 activation inhibitor U0126 suppressed leukotriene synthesis induced by any of the three stimuli; 2) SkQ1 effectively prevents p38 and ERK1/2 activation (accumulation of phosphorylated forms) induced by all three stimuli. This is the first study pointing to the involvement of mitochondrial reactive oxygen species in the activation of leukotriene synthesis in human neutrophils. The use of mitochondria-targeted antioxidants can be considered as a promising strategy for inhibiting leukotriene synthesis and treating various inflammatory pathologies.

Mitochondria-Targeted Antioxidant SkQ1 Prevents the Development of Experimental Colitis in Mice and Impairment of the Barrier Function of the Intestinal Epithelium.

Mitochondria-targeted antioxidants have become promising candidates for the therapy of various pathologies. The mitochondria-targeted antioxidant SkQ1, which is a derivative of plastoquinone, has been successfully used in preclinical studies for the treatment of cardiovascular and renal diseases, and has demonstrated anti-inflammatory activity in a number of inflammatory disease models. The present work aimed to investigate the therapeutic potential of SkQ1 and C12TPP, the analog of SkQ1 lacking the antioxidant quinone moiety, in the prevention of sodium dextran sulfate (DSS) experimental colitis and impairment of the barrier function of the intestinal epithelium in mice. DSS-treated animals exhibited weight loss, bloody stool, dysfunction of the intestinal epithelium barrier (which was observed using FITC-dextran permeability), reduced colon length, and histopathological changes in the colon mucosa. SkQ1 prevented the development of clinical and histological changes in DSS-treated mice. SkQ1 also reduced mRNA expression of pro-inflammatory molecules TNF, IL-6, IL-1ß, and ICAM-1 in the proximal colon compared with DSS-treated animals. SkQ1 prevented DSS-induced tight junction disassembly in Caco-2 cells. Pretreatment of mice by C12TPP did not protect against DSS-induced colitis. Furthermore, C12TPP did not prevent DSS-induced tight junction disassembly in Caco-2 cells. Our results suggest that SkQ1 may be a promising therapeutic agent for the treatment of inflammatory bowel diseases, in particular ulcerative colitis.

Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs).

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273-1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663-668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH2 interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ- being reduced back to SkQH2 by heme bH of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C9 or C13 of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (capital ES, Cyrillic12TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since capital ES, Cyrillic12TPP is specifically accumulated in mitochondria and increases the H+ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by capital ES, Cyrillic12TPP.