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...

Abstract. An hypothesis considering mitochondria as intracellular power-transmitting protonic cables was tested in human fibroblasts where mitochondria are thin and long and in rat cardiomyocytes where they show cluster organization. Mitochondria in the cell were specifically stained with fluorescent-penetrating cation ethylrhodamine, which electrophoretically accumulates in the mitochondrial matrix.A 40-1xm-long mitochondrial filament of fibroblast was illuminated by a very narrow (~0.5 ~tm) laser beam to induce local damage of the mitochondrial membranes. Such a treatment was found to induce quenching of the ethylrhodamine fluorescence in the entire filament. According to the electron microscope examination, the laser-treated filament retained its continuity after the laser illumination. Other mitochondrial filaments (some of which were localized at a distance <10 ~tm from the laser-treated one) remained fluorescent. In a cell where mitochondrial filaments seemed to be united in a network, laser illumination of one filament resulted in fluorescence quenching in the whole network, whereas fluorescence of small mitochondria not connected with the network was unaffected.The illumination of cardiomyocyte was found to result in the fluorescence quenching not only in a laserilluminated mitochondrion but also in a large cluster of organeUes composed of many mitochondria. Electron microscopy showed that all the mitochondria in the cluster change from the orthodox to the condensed state. It was also found that mitochondria in the cluster are connected to one another with specific junctions. If a mitochondrion did not form junctions with a quenched cluster, its fluorescence was not decreased even when this mitochondrion was localized close to an illuminated one. The size of the mitochondrial cluster may be as long as 50 lam. The cluster is formed by branched chains of contacting mitochondria, which may be defined as Streptio mitochondriale. In the cardiomyocyte there are several mitochondrial clusters or, alternatively, the quenched cluster is a result of decomposition of a supercluster uniting all the mitochondria of the cell. Cluster organization of mitochondria could also be revealed when a single mitochondrion was punctured in situ with a microcapillary. The obtained data are in agreement with the idea that mitochondrial junctions are H ÷ permeable so that, within the cluster, A~ may be transmitted from one mitochondrion to another.The above results are consistent with the assumption that mitochondrial filaments or networks represent a united electrical system. (Possible functions of extended mitochondrial systems are discussed.) M EMBRANE structures containing energy-releasing and energy-consuming enzymes are defined as coupling membranes. If within one membrane there are a AIiH-generating respiratory chain and an H ÷-ATP synthase, respiration and phosphorylation can be coupled via A~H (19). It is obvious that both A~ and ApH constituents of AIiH, once they are formed across it, immediately spread along the membran...

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Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age-induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 microM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

It has been suggested that highly social mammals, such as naked mole rats and humans, are long-lived due to neoteny (the prolongation of youth). In both species, aging cannot operate as a mechanism facilitating natural selection because the pressure of this selection is strongly reduced due to ) a specific social structure where only the "queen" and her "husband(s)" are involved in reproduction (naked mole rats) or) substituting fast technological progress for slow biological evolution (humans). Lists of numerous traits of youth that do not disappear with age in naked mole rats and humans are presented and discussed. A high resistance of naked mole rats to cancer, diabetes, cardiovascular and brain diseases, and many infections explains why their mortality rate is very low and almost age-independent and why their lifespan is more than 30 years, versus 3 years in mice. In young humans, curves of mortality versus age start at extremely low values. However, in the elderly, human mortality strongly increases. High mortality rates in other primates are observed at much younger ages than in humans. The inhibition of the aging process in humans by specific drugs seems to be a promising approach to prolong our healthspan. This might be a way to retard aging, which is already partially accomplished via the natural physiological phenomenon neoteny.

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