Mitochondrion-targeted antioxidant SkQ1 prevents rapid animal death caused by highly diverse shocks.

The response to stress involves the activation of pathways leading either to protection from the stress origin, eventually resulting in development of stress resistance, or activation of the rapid death of the organism. Here we hypothesize that mitochondrial reactive oxygen species (mtROS) play a key role in stress-induced programmed death of the organism, which we called "phenoptosis" in 1997. We demonstrate that the synthetic mitochondria-targeted antioxidant SkQ1 (which specifically abolishes mtROS) prevents rapid death of mice caused by four mechanistically very different shocks: (a) bacterial lipopolysaccharide (LPS) shock, (b) shock in response to intravenous mitochondrial injection, (c) cold shock, and (d) toxic shock caused by the penetrating cation C12TPP. Importantly, under all these stresses mortality was associated with a strong elevation of the levels of pro-inflammatory cytokines and administration of SkQ1 was able to switch off the cytokine storms. Since the main effect of SkQ1 is the neutralization of mtROS, this study provides evidence for the role of mtROS in the activation of innate immune responses mediating stress-induced death of the organism. We propose that SkQ1 may be used clinically to support patients in critical conditions, such as septic shock, extensive trauma, cooling, and severe infection by bacteria or viruses.

Biological Diversity and Remodeling of Cardiolipin in Oxidative Stress and Age-Related Pathologies.

Age-related dysfunctions are accompanied by impairments in the mitochondrial morphology, activity of signaling pathway, and protein interactions. Cardiolipin is one of the most important phospholipids that maintains the curvature of the cristae and facilitates assembly and interaction of complexes and supercomplexes of the mitochondrial respiratory chain. The fatty acid composition of cardiolipin influences the biophysical properties of the membrane and, therefore, is crucial for the mitochondrial bioenergetics. The presence of unsaturated fatty acids in cardiolipin is the reason of its susceptibility to oxidative damage. Damaged cardiolipin undergoes remodeling by phospholipases, acyltransferases, and transacylases, creating a highly specific fatty acyl profile for each tissue. In this review, we discuss the variability of cardiolipin fatty acid composition in various species and different tissues of the same species, both in the norm and at various pathologies (e.g., age-related diseases, oxidative and traumatic stresses, knockouts/knockdowns of enzymes of the cardiolipin synthesis pathway). Progressive pathologies, including age-related ones, are accompanied by cardiolipin depletion and decrease in the efficiency of its remodeling, as well as the activation of an alternative way of pathological remodeling, which causes replacement of cardiolipin fatty acids with polyunsaturated ones (e.g., arachidonic or docosahexaenoic acids). Drugs or special diet can contribute to the partial restoration of the cardiolipin acyl profile to the one rich in fatty acids characteristic of an intact organ or tissue, thereby correcting the consequences of pathological or insufficient cardiolipin remodeling. In this regard, an urgent task of biomedicine is to study the mechanism of action of mitochondria-targeted antioxidants effective in the treatment of age-related pathologies and capable of accumulating not only in vitro, but also in vivo in the cardiolipin-enriched membrane fragments.

SkBQ - prooxidant addressed to mitochondria.

Oxidative stress and mitochondrial dysfunction are the key links in the chain of development of pathologies associated with the violation of cellular energy metabolism. Development of mitochondria-addressed compounds highly specific for chemical processes is one of the most promising ways to develop approaches to the treatment of inherited and age-related diseases with mitochondrial etiology. Correlation of structure and chemical activity of the test compounds from a class of lipophilic cations revealed the key role of substituents in the aromatic ring of 1,4-benzoquinones in the manifestation of high antioxidant properties. In this work, it is shown that a synthesized benzoquinone derivative conjugated in position 6 with membrane-penetrating cation of decyltriphenylphosphonium and with substituents at position 2, 3, and 5 (SkBQ) has much lower antioxidant and significantly higher prooxidant activity in comparison with similar derivatives of plasto- and toluquinone SkQ1 and SkQT1 in experiments on isolated mitochondria. At the same time, SkBQ, like SkQ1 and SkQT1, can be reduced by the respiratory chain in the center i of complex III and decrease the mitochondrial membrane potential. In cell cultures of human fibroblasts, it was revealed that SkBQ does not protect cells from apoptosis induced by hydrogen peroxide. Under the same conditions, SkQ1 and SkQT1 exhibit a powerful protective effect. Thus, SkBQ can be seen as a mitochondria-addressed prooxidant. The possibility of using SkBQ as an anticancer drug for the treatment of cancers such as prostate cancer whose cells are sensitive to mitochondrial reactive oxygen species is discussed.

Mitochondrial-targeted plastoquinone derivatives. Effect on senescence and acute age-related pathologies.

Plastoquinone, a very effective electron carrier and antioxidant of chloroplasts, was conjugated with decyltriphenylphosphonium to obtain a cation easily penetrating through membranes. This cation, called SkQ1, is specifically targeted to mitochondria by electrophoresis in the electric field formed by the mitochondrial respiratory chain. The respiratory chain also regenerates reduced SkQ1H(2) from its oxidized form that appears as a result of the antioxidant activity of SkQ1H(2). SkQ1H(2) prevents oxidation of cardiolipin, a mitochondrial phospholipid that is especially sensitive to attack by reactive oxygen species (ROS). In cell cultures, SkQ1 and its analog plastoquinonyl decylrhodamine 19 (SkQR1) arrest H(2)O(2)-induced apoptosis. When tested in vivo, SkQs (i) prolong the lifespan of fungi, crustaceans, insects, fish, and mice, (ii) suppress appearance of a large number of traits typical for age-related senescence (cataract, retinopathies, achromotrichia, osteoporosis, lordokyphosis, decline of the immune system, myeloid shift of blood cells, activation of apoptosis, induction of ß-galactosidase, phosphorylation of H2AX histones, etc.) and (iii) lower tissue damage and save the lives of young animals after treatments resulting in kidney ischemia, rhabdomyolysis, heart attack, arrhythmia, and stroke. We suggest that the SkQs reduce mitochondrial ROS and, as a consequence, inhibit mitochondria-mediated apoptosis, an obligatory step of execution of programs responsible for both senescence and fast "biochemical suicide" of an organism after a severe metabolic crisis.

Molecular mechanisms of transformation of SkQ mitotropic quinones and the search for new approaches to creation of selective free radical traps.

Features of the mechanism of action of positively charged benzoquinone derivatives (SkQ), which are the analogs of coenzyme Q (I), plastoquinone (II), and tocopherol (III), are discussed. It is usually considered that the main target of these compounds is mitochondria, where they accumulate due to the positive charge of the molecule. In the present work, it is shown with model systems that the reduced forms of compounds (I-III) under certain conditions can transform into electrically neutral cyclic zwitterions, which theoretically can escape from the matrix of energized mitochondria against the concentration gradient. A weak uncoupling effect of molecules I-III has been found on mitochondria. Its existence is in agreement with the abovementioned transformation of positively charged hydroquinones of type Ia-IIIa into electrically neutral molecules. The data obtained with model systems suggest that the target of SkQ hydroquinones as free radical traps may be not only mitochondria but also biochemical systems of the cytoplasm. Due to the presence of a large number of reactive oxygen species (ROS)-dependent signal systems in a cell, the functioning of cytoplasmic systems might be disturbed under the action of antioxidants. The problem of selective effect of antioxidants is discussed in detail in the present work, and a functional diagram of selective decrease of the "background level" of ROS based on differences in the intensity of background and "signal" ROS fluxes is considered.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 5. SkQ1 prolongs lifespan and prevents development of traits of senescence.

Very low (nano- and subnanomolar) concentrations of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney.

Reoxygenation following ischemia causes tissue oxidative stress. We studied the role of oxidative stress caused by kidney ischemia/reperfusion (I/R) on the mitochondria of renal tissue slices. I/R caused the mitochondria to be swollen, fragmented, and have lower membrane potential. The mitochondria generated more reactive oxygen species (ROS) and nitric oxide (NO) in situ as measured by fluorescence of ROS- and NO-sensitive probes. Infusion of lithium ion, an inhibitor of glycogen kinase synthase-3, caused phosphorylation of its Ser-9 and restored the membrane potential and decreased ROS production of the mitochondrial fraction. Ischemic kidney and hypoxic rat preconditioning improved mitochondrial membrane potential and lowered ROS production caused by subsequent I/R similar to lithium ion infusion. Preconditioning normalized NO production in mitochondria as well. The drop in the mitochondrial membrane potential was prevented by NO synthase inhibition, demonstrating a strong contribution of NO to changes in mitochondrial energy metabolism during the I/R transition. Mitochondria in the I/R-stressed kidney contained less cytochrome c and more pro-apoptotic Bax, consistent with apoptotic degradation.

On the nature of obligate intracellular symbiosis of rickettsiae--Rickettsia prowazekii cells import mitochondrial porin.

Mitochondrial porin was identified in Rickettsia prowazekii by Western blot analysis of whole cells and membrane fractions with monoclonal antibody against porin VDAC 1 of animal mitochondria. Using the BLAST server, no protein sequences homologous to mitochondrial porin were found among the rickettsial genomes. Rickettsiae also do not contain their own porin. The protein imported by rickettsiae is weakly extracted by nonionic detergents and, like porin in mitochondria, is insensitive to proteinase K in whole cells. Immunocytochemical analysis showed that it localizes to the outer membrane of the bacterial cells. These data support an earlier suggestion about import by rickettsiae of indispensable proteins from cytoplasm of the host cell as a molecular basis of obligate intracellular parasitism. They are also consistent with the hypothesis invoking a transfer of genes specifying surface proteins from the last common ancestor of rickettsiae and mitochondria to the host genome, and preservation by rickettsiae of the primitive ability to import these proteins.

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal.

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.

Uncoupling effect of lauryl sulfate on mitochondria can be mediated by release of bound endogenous fatty acids.

The mechanism of uncoupling by lauryl sulfate (LS) has been studied. The very fact that uncoupling by low concentration of LS (a strong acid) resembles very much that by fatty acids (weak acids) was used as an argument against the fatty acid cycling scheme of uncoupling where protonated fatty acids operate as a protonophore. We have found that rat liver and heart muscle mitochondria can be uncoupled by low (70 microM) LS concentration in a fashion completely arrested by the ATP/ADP antiporter inhibitor carboxyatractylate (CAtr). On the other hand, uncoupling by two-fold higher LS concentration is not sensitive to CAtr. Addition of oleate desensitizes mitochondria to low LS so that addition of bovine serum albumin becomes necessary to recouple mitochondria. The data are accounted for assuming that low LS releases endogenous fatty acids from some mitochondrial depots, and these fatty acids are responsible for uncoupling. As to high LS, it causes a nonspecific (CAtr-insensitive) damage to the mitochondrial membrane.

Reactive oxygen and nitrogen species: friends or foes?

Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS) and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as "electric cables", as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive "ROS-induced ROS release".

Cyclosporin A-sensitive cytochrome c release and activation of external pathway of NADH oxidation in liver mitochondria due to pore opening by acidification of phosphate-containing incubation medium.

Acidification of a high phosphate incubation medium from pH 7.4 to 6.5 promotes increase in rates of succinate oxidation and exogenous NADH oxidation via external (rotenone-and myxothiazol-resistant) pathway by factors 2 and 2.3 respectively. Cyclosporin A prevents these effects. To measure the cytochrome c release, mitochondrial cytochrome c concentration was calculated from absorption spectrum of alpha-band of cytochromes c + c1. The cytochrome c release is shown to be equal to 27 +/- 4%, 40 +/- 12%, 70 +/- 5% at pH 7.4, 7.0, 6.5, respectively, the last value being reduced by cyclosporin A to 10 +/- 3%. Immunoblot method gives the similar results. It is concluded that acidification of the high phosphate medium induces release of a large part of the cytochrome c pool from liver mitochondria due to opening the Ca(2+)-dependent cyclosporin A-sensitive permeability transition pore and subsequent high amplitude swelling.

Oligomeric C-terminal truncated Bax preferentially releases cytochrome c but not adenylate kinase from mitochondria, outer membrane vesicles and proteoliposomes.

UNLABELLED: The mechanism by which the proapoptotic protein Bax releases cytochrome c from mitochondria is not fully understood. The present work approaches this problem using C-terminal truncated oligomeric Bax (BaxDeltaC). Micromolar concentrations of BaxDeltaC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. BaxDeltaC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, BaxDeltaC was ineffective in releasing cytochrome c when outer membrane vesicles were obtained in the presence of glycerol, conditions under which the number of contact sites was drastically reduced. BaxDeltaC did not liberate encapsulated cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles reconstituted with porin. However, when the hexokinase-porin-adenine nucleotide translocase complex from brain mitochondria was reconstituted in vesicles, BaxDeltaC released internal cytochrome c but not adenylate kinase. In all these systems, only a small portion of total cytochrome c present in either mitochondria or vesicles could be liberated by BaxDeltaC. BaxDeltaC also increased the accessibility of external cytochrome c to either oxidation by complex IV or reduction by complex III in intact liver and heart mitochondria. CONCLUSIONS: (1) BaxDeltaC selectively releases cytochrome c and enables a bidirectional movement of cytochrome c across the outer mitochondrial membrane. (2) A multiprotein complex that resembles the mitochondrial contact sites is a prerequisite for BaxDeltaC action. (3) A limited pool of cytochrome c becomes the first target for BaxDeltaC.

Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.

Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein.

Proteinaceous complexes from mitochondrial contact sites.

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.

Folding and refolding of thermolabile and thermostable bacterial luciferases: the role of DnaKJ heat-shock proteins.

Bacterial luciferases are highly suitable test substrates for the analysis of refolding of misfolded proteins, as they are structurally labile and loose activity at 42 degrees C. Heat-denatured thermolabile Vibrio fischeri luciferase and thermostable Photorhabdus luminescens luciferase were used as substrates. We found that their reactivation requires the activity of the DnaK chaperone system. The DnaKJ chaperones were dispensable in vivo for de novo folding at 30 degrees C of the luciferase, but essential for refolding after a heat-shock. The rate and yield of DnaKJ refolding of the P. luminescens thermostable luciferase were to a marked degree lower as compared with the V. fischeri thermolabile luciferase. The refolding activity of the DnaKJ chaperones was examined at various temperatures. Between 30 and 37 degrees C, the refolding rates of the V. fischeri luciferase decreased and the reaction reached a complete arrest at temperatures above 40 degrees C. The rate of DnaKJ-mediated refolding of the thermostable luciferase at first increased between 30 and 37 degrees C and then decreased at the range of 37-44 degrees C. We observed that the rate of DnaKJ-mediated refolding of the heat-denatured P. luminescens thermostable luciferase, but not of the thermolabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47 degrees C) temperature. The efficiency and reversibility of protein refolding arrest during and after heat-shock strongly depended on the stability of the DnaKJ-denatured luciferase complex. It is supposed that the thermostable luciferase is released during the heat-shock, whereas the thermolabile luciferase remained bound to the chaperone.