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.

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to th...

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.

No abstract

A unique phenomenon of mitochondria-targeted protonophores is described.ItconsistsinatransmembraneH þ -conductingfattyacidcycling mediated by penetrating cations such as 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C 12 TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (ΔpH) to a membrane potential (Δψ) of the Nernstian value (about 60 mV Δψ at ΔpH ¼ 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C 12 TPP, but not cetyltrimethylammonium, potentiatedfattyacid-induced(i)uncouplingofrespirationandphosphorylation, and (ii) inhibition of H 2 O 2 formation. In intact yeast cells, C 12 TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanidephenylhydrazone)stimulatedrespiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.mild uncoupling | membrane | Mitochondria-targeted uncoupler | penetrating ion | antioxidant S ome decrease in mitochondrial membrane potential (Δψ) in the resting state may be favorable in treating obesity and hypothyroidism as well as in preventing senescence and certain types of cancer [for reviews, see refs. 1, 2]. In the first two cases, Δψ lowering stimulates respiratory metabolism. As to senescence and cancer, such an effect seems to be related to a decrease in production of reactive oxygen species (ROS) in mitochondria. ROS, in turn, were assumed to mediate senescence and some steps of cancerogenesis (2, 3). As was shown in our group (4), there is a very steep dependence of mitochondrial ROS formation on Δψ. Small (10-15%) lowering of Δψ resulted in ten-fold decrease in the ROS production rate (4). In isolated mitochondria, this can be achieved by adding a low concentration of a protonophorous uncoupler (4-6). This approach, called "mild uncoupling" (4, 6), was recently used by Padalko (7) and by Kowaltowski and coworkers (8) to prolong the lifespan of Drosophila and mice, respectively. However, long-term treatment of animals with uncouplers results in toxic side effects (9).In this paper, we put forward an alternative approach based on the use of synthetic cations that easily penetrate through biological membranes. Penetrating ions were suggested by our group to reveal electric potential difference across mitochondrial membrane (9, 10). In tetraphenylphosphonium (TPP), a typical representative of such ions, the positive charge is strongly displaced over four phenyl residues. As a result, water dipoles cannot be held by t...

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.

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 β-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.

Genes coding for small peptides have been frequently misannotated as long noncoding RNA (lncRNA) genes. Here we have demonstrated that one such transcript is translated into a 56-amino-acid-long peptide conserved in chordates, corroborating the work published while this manuscript was under review. The Mtln peptide could be detected in mitochondria of mouse cell lines and tissues. In line with its mitochondrial localization, lack of the Mtln decreases the activity of mitochondrial respiratory chain complex I. Unlike the integral components and assembly factors of NADH:ubiquinone oxidoreductase, Mtln does not alter its enzymatic activity directly. Interaction of Mtln with NADH-dependent cytochrome b5 reductase stimulates complex I functioning most likely by providing a favorable lipid composition of the membrane. Study of Mtln illuminates the importance of small peptides, whose genes might frequently be misannotated as lncRNAs, for the control of vitally important cellular processes.