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The authors examined the ultrastructure of mitochondrial apparatus of skeletal muscles of naked mole rats (Heterocephalus glaber) from the age of 6 months to 11 years. The obtained results have demonstrated that the mitochondria in skeletal muscles of naked mole rats aged below 5 years is not well-developed and represented by few separate small mitochondria. Mitochondrial reticulum is absent. Starting from the age of 5 years, a powerful mitochondrial structure is developed. By the age of 11 years, it become obvious that the mitochondrial apparatus formed differs from that in the skeletal muscle of adult rats and mice, but resembles that of cardiomyocytes of rats or naked mole rats cardiomyocytes. From the age of 6 months to 11 years, percentage area of mitochondria in the skeletal muscle of naked mole rat is increasing by five times. The growth of mitochondria is mainly driven by increased number of organelles. Such significant growth of mitochondria is not associated with any abnormal changes in mitochondrial ultrastructure. We suppose that specific structure of mitochondrial apparatus developed in the skeletal muscle of naked mole rats by the age of 11 years is necessary for continual skeletal muscle activity of these small mammals burrowing very long holes in stony earth, resembling continual activity of heart muscle. In any case, ontogenesis of naked mole rat skeletal muscles is much slower than of rats and mice (one more example of neoteny).

This review discusses genetic and molecular pathways that link circadian timing with metabolism, resulting in the emergence of positive and negative regulatory feedback loops. The Nrf2 pathway is believed to be a component of the anti-aging program responsible for the healthspan and longevity. Nrf2 enables stress adaptation by activating cell antioxidant defense and other metabolic processes via control of expression of over 200 target genes in response to various types of stress. The GSK3 system represents a “regulating valve” that controls fine oscillations in the Nrf2 level, unlike Keap1, which prevents significant changes in the Nrf2 content in the absence of oxidative stress and which is inactivated by the oxidative stress. Furthermore, GSK3 modifies core circadian clock proteins (Bmal1, Clock, Per, Cry, and Rev-erbα). Phosphorylation by GSK3 leads to the inactivation and degradation of circadian rhythm-activating proteins (Bmal1 and Clock) and vice versa to the activation and nuclear translocation of proteins suppressing circadian rhythms (Per and Rev-erbα) with the exception of Cry protein, which is likely to be implicated in the fine tuning of biological clock. Functionally, GSK3 appears to be one of the hubs in the cross-regulation of circadian rhythms and antioxidant defense. Here, we present the data on the crosstalk between the most powerful cell antioxidant mechanism, the Nrf2 system, and the biorhythm-regulating system in mammals, including the impact of GSK3 overexpression and knockout on the Nrf2 signaling. Understanding the interactions between the regulatory cascades linking homeostasis maintenance and cell response to oxidative stress will help in elucidating molecular mechanisms that underlie aging and longevity.

Homo sapiens and naked mole rats (Heterocephalus glaber) are vivid examples of social mammals that differ from their relatives in particular by an increased lifespan and a large number of neotenic features. An important fact for biogerontology is that the mortality rate of H. glaber (a maximal lifespan of more than 32 years, which is very large for such a small rodent) negligibly grows with age. The same is true for modern people in developed countries below the age of 60. It is important that the juvenilization of traits that separate humans from chimpanzees evolved over thousands of generations and millions of years. Rapid advances in technology resulted in a sharp increase in the life expectancy of human beings during the past 100 years. Currently, the human life expectancy has exceeded 80 years in developed countries. It cannot be excluded that the potential for increasing life expectancy by an improvement in living conditions will be exhausted after a certain period of time. New types of geroprotectors should be developed that protect not only from chronic phenoptosis gradual poisoning of the body with reactive oxygen species (ROS) but also from acute phenoptosis, where strong increase in the level of ROS immediately kills an already aged individual. Geroprotectors might be another anti-aging strategy along with neoteny (a natural physiological phenomenon) and technical progress.

Significance The mitochondria, organelles that produce the largest amounts of ATP and reactive oxygen species (mROS) in living cells, are equipped with a universal mechanism that can completely prevent mROS production. This mechanism consists of mild depolarization of the inner mitochondrial membrane to decrease the membrane potential to a level sufficient to form ATP but insufficient to generate mROS. In short-lived mice, aging is accompanied by inactivation of the mild depolarization mechanism, resulting in chronic poisoning of the organism with mROS. However, mild depolarization still functions for many years in long-lived naked mole rats and bats., 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.

As inhabitants of soda lakes, Thioalkalivibrio versutus are halo- and alkaliphilic bacteria that have previously been shown to respire with the first demonstrated Na+-translocating cytochrome-c oxidase (CO). The enzyme generates a sodium-motive force (Δs) as high as −270 mV across the bacterial plasma membrane. However, in these bacteria, operation of the possible Δs consumers has not been proven. We obtained motile cells and used them to study the supposed Na+ energetic cycle in these bacteria. The resulting motility was activated in the presence of the protonophore 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), in line with the same effect on cell respiration, and was fully blocked by amiloride—an inhibitor of Na+-motive flagella. In immotile starving bacteria, ascorbate triggered CO-mediated respiration and motility, both showing the same dependence on sodium concentration. We concluded that, in T. versutus, Na+-translocating CO and Na+-motive flagella operate in the Na+ energetic cycle mode. Our research may shed light on the energetic reason for how these bacteria are confined to a narrow chemocline zone and thrive in the extreme conditions of soda lakes.

As inhabitants of soda lakes

The data on the neoteny (prolongation of youth and retardation of aging) in naked mole rat (Heterocephalus glaber) and Homo sapiens are summarized. Fifty-eight neotenic traits have been described by now in the naked mole rat at the organismal, tissue, cellular, and metabolism levels. Among them, there are traits that increase the lifespan, including mild depolarization of mitochondria that prevents generation by these organelles of reactive oxygen species known to strongly promote aging. Mild mitochondrial depolarization disappears with age in short-lived mammals (mouse Mus musculus) much faster than in long-lived mammals (e.g., naked mole rats and bats). The development of neoteny in naked mole rats has been due to the social organization. These animals live in subterranean colonies, where sexual reproduction is monopolized by the queen and one or several males who are defended and provided with nutrition by numerous subordinates. Humans have achieved a gradual increase in the lifespan first due to neoteny, and then to the technical progress, which can be observed by comparing the lifespan curves of chimpanzees, hunter-gatherers of the Paraguayan Ache tribe, and residents of Sweden from the XVII century to the present day. Significantly different rates of neoteny and technical progress make it possible to discriminate between the contributions of these two longevity mechanisms.

The protective effect of SkQR1, a mitochondria-targeted antioxidant, was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region from 1 to 7 days after the injury. TBI caused a reliable disruption of the functions of the limbs contralateral to injury focus. The intravenous single injection of SkQR1 (250 nmol/kg) but not C12R1 (a SkQR1 homologue devoid of the antioxidant group) 30 min after TBI reduced the impairment of the motor functions of the limbs. A statistically significant improvement in limb function in animals was shown using 3 different tests: limb-placing test, beam-walking test and grip strength test. A pronounced therapeutic effect appeared on the 1th day and lasted until the end of the experiment - the 7th day after TBI. Histopathological examination showed that in the group of animals that did not receive SkQR1 in the marginal layer of the lesion there was a marked increase in astroglial expression, infiltration with segmented neutrophils, and poor survivability of neurons compared with animals treated with SkQR1. The obtained results demonstrate that the single use of plastoquinone-containing mitochondria-targeted antioxidant SkQR1 at the early stages of development of traumatic brain damage can reduce TBI-related disruptions of limb functions, and that mechanisms of the brain damage after trauma are dependent on the production of mitochondrial reactive oxygen species.

Homo sapiens and naked mole rats (Heterocephalus glaber) are vivid examples of social mammals that differ from their relatives in particular by an increased lifespan and a large number of neotenic features. An important fact for biogerontology is that the mortality rate of H. glaber (a maximal lifespan of more than 32 years, which is very large for such a small rodent) negligibly grows with age. The same is true for modern people in developed countries below the age of 60. It is important that the juvenilization of traits that separate humans from chimpanzees evolved over thousands of generations and millions of years. Rapid advances in technology resulted in a sharp increase in the life expectancy of human beings during the past 100 years. Currently, the human life expectancy has exceeded 80 years in developed countries. It cannot be excluded that the potential for increasing life expectancy by an improvement in living conditions will be exhausted after a certain period of time. New types of geroprotectors should be developed that protect not only from chronic phenoptosis gradual poisoning of the body with reactive oxygen species (ROS) but also from acute phenoptosis, where strong increase in the level of ROS immediately kills an already aged individual. Geroprotectors might be another anti-aging strategy along with neoteny (a natural physiological phenomenon) and technical progress.

Electron microscopic study of cardiomyocytes taken from healthy Wistar and OXYS rats and naked mole rats (Heterocephalus glaber) revealed mitochondria in nuclei that lacked part of the nuclear envelope. The direct interaction of mitochondria with nucleoplasm is shown. The statistical analysis of the occurrence of mitochondria in cardiomyocyte nuclei showed that the percentage of nuclei with mitochondria was roughly around 1%, and did not show age and species dependency. Confocal microscopy of normal rat cardiac myocytes revealed a branched mitochondrial network in the vicinity of nuclei with an organization different than that of interfibrillar mitochondria. This mitochondrial network was energetically functional because it carried the membrane potential that responded by oscillatory mode after photodynamic challenge. We suggest that the presence of functional mitochondria in the nucleus is not only a consequence of certain pathologies but rather represents a normal biological phenomenon involved in mitochondrial/nuclear interactions.

(i) In 2015-2017 we compared possible reasons for longevity of two mammalian highly social species, i.e. naked mole rats and humans. We proposed that in both cases longevity is a result of neoteny, prolongation of youth by deceleration of late ontogeny (Skulachev, V. P. (2015) Abst. 11th Conf. on Mitochondrial Physiology (MiP2015), Lucni Bouda, Czech Republic, pp. 64-66; Skulachev, V. P., Holtze, S., Vyssokikh, M. Y., Bakeeva, L. E., Skulachev, M. V., Markov, A. V., Hildebrandt, T. B., and Sadovnichii, V. A. (2017) Physiol. Rev., 97, 699-720). Both naked mole rats and humans strongly decreased the pressure of natural selection, although in two different manners. Naked mole rats preferred an "aristocratic" pathway when reproduction (and, hence, involvement in evolution) is monopolized by the queen and her several husbands. Huge number of subordinates who have no right to take part in reproduction and hence in evolution serves the small queen's family. Humans used an alternative, "democratic" pathway, namely technical progress facilitating adaptation to the changing environmental conditions. This pathway is open to all humankind. (ii) As a result, aging as a mechanism increasing evolvability by means of facilitation of natural selection became unnecessary for naked mole rats and humans due to strong attenuation of this selection. This is apparently why aging became a counterproductive atavism for these two species and was strongly shifted to late ages. This shift is direct evidence of the hypothesis that aging is programmed, being the last step of late ontogeny. (iii) Further deceleration of aging for humans by means of neoteny is unrealistic since the development of neoteny probably takes million years. (iv) However, if biological aging is a program, an alternative and much simpler way to avoid it seems possible. We mean inhibition of an essential step of this program. (v) At present, the most probable scheme of the aging program assumes that it is a mechanism of slow poisoning of an organism by reactive oxygen species produced by mitochondria. If this is the case, a mitochondria-targeted antioxidant might be an inhibitor of the aging program. During the last 12 years, such an antioxidant, namely SkQ1, was synthesized and studied in detail in our group. It consists of plastoquinone and decyltriphenylphosphonium (a penetrating cation responsible for electrophoretic accumulation of SkQ1 in mitochondria). It was shown that long-term treatment with SkQ1 increased the lifespan of plants, fungi, invertebrates, fish, and mammals. Moreover, SkQ1 is effective in the therapy of various age-related diseases. It was also shown that a single SkQ1 injection could save life in certain models of sudden death of animals. (vi) A tentative scheme is proposed considering aging as a process of chronic phenoptosis, which eventually results in initiation of acute phenoptosis and death. This scheme also suggests that under certain conditions chronic phenoptosis can be neutralized by an anti-aging program that is activated by food restriction regarded by an organism as a signal of starvation. As for acute phenoptosis, it is apparently controlled by receptors responsible for measuring key parameters of homeostasis. The first experimental indications have been already obtained indicating that both chronic and acute phenoptosis are suppressed by SkQ1.

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 1) a specific social structure where only the “queen” and her “husband(s)” are involved in reproduction (naked mole rats) or 2) 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.

Oxidative stress is widely recognized as an important factor in the delayed wound healing in diabetes. However, the role of mitochondrial reactive oxygen species in this process is unknown. It was assumed that mitochondrial reactive oxygen species are involved in many wound-healing processes in both diabetic humans and animals. We have applied the mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) to explore the role of mitochondrial reactive oxygen species in the wound healing of genetically diabetic mice. Healing of full-thickness excisional dermal wounds in diabetic C57BL/KsJ-db−/db− mice was significantly enhanced after long-term (12 weeks) administration of SkQ1. SkQ1 accelerated wound closure and stimulated epithelization, granulation tissue formation, and vascularization. On the 7th day after wounding, SkQ1 treatment increased the number of α-smooth muscle actin-positive cells (myofibroblasts), reduced the number of neutrophils, and increased macrophage infiltration. SkQ1 lowered lipid peroxidation level but did not change the level of the circulatory IL-6 and TNF. SkQ1 pretreatment also stimulated cell migration in a scratch-wound assay in vitro under hyperglycemic condition. Thus, a mitochondria-targeted antioxidant normalized both inflammatory and regenerative phases of wound healing in diabetic mice. Our results pointed to nearly all the major steps of wound healing as the target of excessive mitochondrial reactive oxygen species production in type II diabetes.

Author congratulates David Gems and co-authors on a brilliant discovery — direct proof of acute phenoptosis in the nematode — but argues that the authors underappreciate the significance of their work by suggesting that phenoptosis is a rare natural phenomenon not typically observed in mammals.

Social insects with identical genotype that form castes with radically different lifespans are a promising model system for studying the mechanisms underlying longevity. The main direction of progressive evolution of social insects, in particular, ants, is the development of the social way of life inextricably linked with the increase in the colony size. Only in a large colony, it is possible to have a developed polyethism, create large food reserves, and actively regulate the nest microclimate. The lifespan of ants hugely varies among genetically similar queens, workers (unproductive females), and males. The main advantage of studies on insects is the determinism of ontogenetic processes, with a single genome leading to completely different lifespans in different castes. This high degree of determinacy is precisely the reason why some researchers (incorrectly) call a colony of ants the "superorganism", emphasizing the fact that during the development, depending on the community needs, ants can switch their ontogenetic programs, which influences their social roles, ability to learn (i.e., the brain [mushroom-like body] plasticity), and, respectively, the spectrum of tasks performed by a given individual. It has been shown that in many types of food behavior, older ants surpass young ones in both performing the tasks and transferring the experience. The balance between the need to reduce the "cost" of non-breeding individuals (short lifespan and small size of workers) and the benefit from experienced long-lived workers possessing useful skills (large size and "non-aging") apparently determines the differences in the lifespan and aging rate of workers in different species of ants. A large spectrum of rigidly determined ontogenetic trajectories in different castes with identical genomes and the possibility of comparison between "evolutionarily advanced" and "primitive" subfamilies (e.g., Formicinae and Ponerinae) make ants an attractive object in the studies of both normal aging and effects of anti-aging drugs.

In this study, the ultrastructure of mitochondria in cardiomyocytes of naked mole rats (Heterocephalus glaber) aged from 6 months to 11 years was examined. Mitochondria in cardiomyocytes of naked mole rats have a specific ultrastructure that is different from those in cardiomyocytes of other mammalian species studied to date. In contrast to mitochondria of other mammalian cardiomyocytes, where the internal space is completely filled by tightly packed parallel rows of cristae, mitochondria in cardiomyocytes of naked mole rats have a chaotic pattern of cristae organization with wave-like contours. Gradual formation of mitochondrial ultrastructure occurs in naked mole rats for many years. Two mitochondrial populations are developed to the age of 5 years. In addition to the main population, there are some large organelles which exceed normal sizes by two to three times. Most cristae in these mitochondria are assembled into small groups, which form the curved and ring-like structures. The appearance of some specific structural changes (i.e. bundles of parallel cristae) is observed in the mitochondrial population of naked mole rat after 11 years of age. However, these bundles are very rare and of sporadic nature. Morphometric analysis has shown that the superficial density of the inner mitochondrial membrane is similar in all examined age groups of naked mole rats: 21.1 at 6 months, 23.21 at 3 years, 23.55 at 5 years, and 20.8 at 11 years. This level is almost two times lower than in other animals studied (mice and rats). The data demonstrate that pathological changes in mitochondrial apparatus are not present in naked mole rats at least until the age of 11 years. The mitochondrial apparatus corresponds to the phenotype in young animals, thus being another neotenic feature in naked mole rats.

Accumulation of various types of lesions in the course of aging increases an organism's vulnerability and results in a monotonous elevation of mortality rate, irrespective of the position of a species on the evolutionary tree. Stroustrup et al. (Nature, 530, 103-107) [1] showed in 2016 that in the nematode Caenorhabditis elegans, longevity-altering factors (e.g. oxidative stress, temperature, or diet) do not change the shape of the survival curve, but either stretch or shrink it along the time axis, which the authors attributed to the existence of an "aging program". Modification of the accelerated failure time model by Stroustrup et al. uses temporal scaling as a basic approach for distinguishing between quantitative and qualitative changes in aging dynamics. Thus we analyzed data on the effects of various longevity-increasing genetic manipulations in flies, worms, and mice and used several models to choose a theory that would best fit the experimental results. The possibility to identify the moment of switch from a mortality-governing pathway to some other pathways might be useful for testing geroprotective drugs. In this work, we discuss this and other aspects of temporal scaling.

The mitochondrial genome provides not only respiratory chain function, but it also ensures the impact of mitochondria on nearly all crucial metabolic processes. It is well known that mitochondria regulate aging and lifespan. However, until now there were no direct experimental data concerning the influence of various mitochondrial DNA variants on lifespan of animals with identical nuclear genome. In a recent paper of J. A. Enríquez and coworkers (Latorre-Pellicer, A., et al. (2016) Nature, 535, 561-565), it was shown that mice carrying nuclear DNA from one strain and mitochondrial DNA from another had longer median lifespan and retarded development of various aging traits. This review critically analyzes that paper and considers some aspects of the crosstalk between the nuclear and mitochondrial genomes. We also discuss new perspectives of gerontology in the light of the discovery made by Enríquez's group.

// Galina Shagieva 1 , Lidiya Domnina 1 , Olga Makarevich 2 , Boris Chernyak 3 , Vladimir Skulachev 3, 4 , Vera Dugina 1 1 Department of Mathematical Methods in Biology, Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 2 Faculty of Basic Medicine, Lomonosov Moscow State University, Moscow, Russia 3 Department of Bioenergetics, Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 4 Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia Correspondence to: Galina Shagieva, email: gshagieva@genebee.msu.su Keywords: epithelial-to-mesenchymal transition, mitochondrial reactive oxygen species, ERK1/2, cervical cancer cells, SkQ1 Received: July 22, 2016 Accepted: November 14, 2016 Published: November 25, 2016 ABSTRACT In the course of cancer progression, epithelial cells often acquire morphological and functional characteristics of mesenchymal cells, a process known as epithelial-to-mesenchymal transition (EMT). EMT provides epithelial cells with migratory, invasive, and stem cell capabilities. Reactive oxygen species produced by mitochondria (mtROS) could be of special importance for pro-tumorigenic signaling and EMT. In our study, we used mitochondria-targeted antioxidant SkQ1 to lower the mtROS level and analyze their role in the regulation of the actin cytoskeleton, adhesion junctions, and signaling pathways critical for tumorigenesis of cervical carcinomas. A decrease in mtROS was found to induce formation of β-cytoplasmic actin stress fibers and circumferential rings in cervical cancer SiHa and Ca-Ski cells. It was accompanied by an upregulation of E-cadherin in SiHa cells and a downregulation of N-cadherin in Ca-Ski cells. In SiHa cells, an increase in E-cadherin expression was accompanied by a reduction of Snail, E-cadherin negative regulator. A stimulation of mtROS by epidermal growth factor (EGF) caused a Snail upregulation in SiHa cells that could be downregulated by SkQ1. SkQ1 caused a decrease in activation of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in SiHa and Ca-Ski. EGF produced an opposite effect. Incubation with SkQ1 suppressed EGF-induced p-ERK1/2 upregulation in SiHa, but not in Ca-Ski cells. Thus, we showed that scavenging of mtROS by SkQ1 initiated reversal of EMT and suppressed proliferation of cervical cancer cells.

A membrane-penetrating cation, dodecyltriphenylphosphonium (C12TPP), facilitates the recycling of fatty acids in the artificial lipid membrane and mitochondria. C12TPP can dissipate mitochondrial membrane potential and may affect total energy expenditure and body weight in animals and humans. We investigated the metabolic effects of C12TPP in isolated brown-fat mitochondria, brown adipocyte cultures and mice in vivo. Experimental approaches included the measurement of oxygen consumption, carbon dioxide production, western blotting, magnetic resonance imaging and bomb calorimetry. In mice, C12TPP (50 μmol per (day•kg body weight)) in the drinking water significantly reduced body weight (12%, P

An increase in the production of reactive oxygen species (ROS) in mitochondria due to targeted delivery of redox active compounds may be useful in studies of modulation of cell functions by mitochondrial ROS. Recently, the mitochondria-targeted derivative of menadione (MitoK3) was synthesized. However, MitoK3 did not induce mitochondrial ROS production and lipid peroxidation while exerting significant cytotoxic action. Here we synthesized 1,4-naphthoquinone conjugated with alkyltriphenylphosphonium (SkQN) as a prototype of mitochondria-targeted prooxidant, and its redox properties, interactions with isolated mitochondria, yeast cells and various human cell lines were investigated. According to electrochemical measurements, SkQN was more active redox agent and, due to the absence of methyl group in the naphthoquinone ring, more reactive as electrophile than MitoK3. SkQN (but not MitoK3) stimulated hydrogen peroxide production in isolated mitochondria. At low concentrations, SkQN stimulated state 4 respiration in mitochondria, decreased membrane potential, and blocked ATP synthesis, being more efficient uncoupler of oxidative phosphorylation than MitoK3. In yeast cells, SkQN decreased cell viability and induced oxidative stress and mitochondrial fragmentation. SkQN killed various tumor cells much more efficiently than MitoK3. Since many tumors are characterized by increased oxidative stress, the use of new mitochondria-targeted prooxidants may be a promising strategy for anticancer therapy.

Neonatal hypoxia&ndash, ischemia is one of the main causes of mortality and disability of newborns. To study the mechanisms of neonatal brain cell damage, we used a model of neonatal hypoxia&ndash, ischemia in seven-day-old rats, by annealing of the common carotid artery with subsequent hypoxia of 8% oxygen. We demonstrate that neonatal hypoxia&ndash, ischemia causes mitochondrial dysfunction associated with high production of reactive oxygen species, which leads to oxidative stress. Targeted delivery of antioxidants to the mitochondria can be an effective therapeutic approach to treat the deleterious effects of brain hypoxia&ndash, ischemia. We explored the neuroprotective properties of the mitochondria-targeted antioxidant SkQR1, which is the conjugate of a plant plastoquinone and a penetrating cation, rhodamine 19. Being introduced before or immediately after hypoxia&ndash, ischemia, SkQR1 affords neuroprotection as judged by the diminished brain damage and recovery of long-term neurological functions. Using vital sections of the brain, SkQR1 has been shown to reduce the development of oxidative stress. Thus, the mitochondrial-targeted antioxidant derived from plant plastoquinone can effectively protect the brain of newborns both in pre-ischemic and post-stroke conditions, making it a promising candidate for further clinical studies.

Mitochondrial dysfunction plays a crucial role in the macroautophagy/autophagy cascade. In a recently published study Sun et al. described the induction of autophagy by the membranophilic triphenylphosphonium (TPP)-based cation 10-(6'-ubiquinonyl) decyltriphenylphosphonium (MitoQ) in HepG2 cells (Sun C, et al. "MitoQ regulates autophagy by inducing a pseudo-mitochondrial membrane potential [PMMP]", Autophagy 2017, 13:730-738.). Sun et al. suggested that MitoQ adsorbed to the inner mitochondrial membrane with its cationic moiety remaining in the intermembrane space, adding a large number of positive charges and establishing a "pseudo-mitochondrial membrane potential," which blocked the ATP synthase. Here we argue that the suggested mechanism for generation of the "pseudo-mitochondrial membrane potential" is physically implausible and contradicts earlier findings on the electrophoretic displacements of membranophilic cations within and through phospholipid membranes. We provide evidence that TPP-cations dissipated the mitochondrial membrane potential in HepG2 cells and that the induction of autophagy in carcinoma cells by TPP-cations correlated with the uncoupling of oxidative phosphorylation. The mild uncoupling of oxidative phosphorylation by various mitochondria-targeted penetrating cations may contribute to their reported therapeutic effects via inducing both autophagy and mitochondria-selective mitophagy.

Mitochondria-targeted cationic plastoquinone derivatives (SkQs, e.g. SkQ1, SkQR1) and their analogs lacking plastoquinol moiety (C12TPP, C12R1) can pass through bilayer phospholipid membrane. Their cationic forms are accumulated in isolated mitochondria or in mitochondria of living cells, driven by membrane potential. These compounds were extensively tested in model lipid membranes, isolated mitochondria and in living human cells in culture. It was found that SkQs are antioxidants that quench reactive oxygen species (ROS) in mitochondria, and mild uncouplers that facilitate transmembrane proton transport by fatty acids. Both properties result in efficient prevention of oxidative stress and protection of mitochondria and cells from damage by ROS, making SkQs a promising drug candidate against pathologies caused by excess mitochondrial ROS generation. Recent discovery of SkQ1 antibacterial activity at concentrations not toxic to human cells opens a perspective for development of new antibiotics. In this chapter, we summarize recent in vitro experiments with mitochondria-targeted plastoquinones.

Mitochondria are important regulators of tumour growth and progression due to their specific role in cancer metabolism and modulation of apoptotic pathways. In this paper we describe that mitochondria-targeted antioxidant SkQ1 designed as a conjugate of decyl-triphenylphosphonium cation (TPP+) with plastoquinone, suppressed the growth of fibrosarcoma HT1080 and rhabdomyosarcoma RD tumour cells in culture and tumour growth of RD in xenograft nude mouse model. Under the same conditions, no detrimental effect of SkQ1 on cell growth of primary human subcutaneous fibroblasts was observed. The tumour growth suppression was shown to be a result of the antioxidant action of low nanomolar concentrations of SkQ1. We have revealed significant prolongation of mitosis induced by SkQ1 in both tumour cell cultures. Prolonged mitosis and apoptosis could be responsible for growth suppression after SkQ1 treatment in RD cells. Growth suppression in HT1080 cells was accompanied by the delay of telophase and cytokinesis, followed by multinuclear cells formation. The effects of SkQ1 on the cell cycle were proved to be at least partially mediated by inactivation of Aurora family kinases. Abbreviations: TPP+: Triphenylphosphonium cation; ROS: Reactive oxygen species; mtROS: Mitochondrial reactive oxygen species; NAC: N-acetyl-L-cysteine; DCFH-DA: Dichlorodihydrofluorescein diacetate; APC: Anaphase promoting complex; ABPs: Actin-binding proteins; DMEM: Dulbecco’s modified Eagle media; SDS: sodium dodecyl sulfate; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Alzheimer's disease is characterized by progressive memory loss and cognitive decline accompanied by degeneration of neuronal synapses, massive loss of neurons in the brain, eventually resulting in complete degradation of personality and death. Currently, the cause of the disease is not fully understood, but it is believed that the person's age is the major risk factor for development of Alzheimer's disease. People who have survived after cerebral stroke or traumatic brain injury have substantially increased risk of developing Alzheimer's disease. Social exclusion, low social activity, physical inactivity, poor mental performance, and low level of education are among risk factors for development of this neurodegenerative disease, which is consistent with the concept of phenoptosis (Skulachev, V. P., et al. (1999) Biochemistry (Moscow), 64, 1418-1426; Skulachev, M. V., and Skulachev, V. P. (2014) Biochemistry (Moscow), 79, 977-993) stating that rate of aging is related to psychological and social aspects in human behavior. Here we assumed that Alzheimer's disease might be considered as an exacerbation of senile phenoptosis. If so, then development of this disease could be slowed using mitochondria-targeted antioxidants due to the accumulated data demonstrating a link between mitochondrial dysfunction and oxidative stress both with normal aging and Alzheimer's disease.

Accumulation of various types of unrepaired damage of the genome because of increasing production of reactive oxygen species and decreasing efficiency of the antioxidant defense system and repair systems can cause age-related diseases and emergence of phenotypic signs of senescence. This should lead to increasing vulnerability and to mortality monotonously increasing with age independently of the position of the species on the evolutionary tree. In this light, the survival, mortality, and fertility curves for 45 animal and plant species and one alga published by the Max Planck Institute for Demographic Research (Germany/Denmark) are of special interest (Jones, O. R., et al. (2014) Nature, 505, 169-173). We divided all species treated in that study into four groups according to the ratio of mortality at the terminal age (which corresponds to 5% survival) and average mortality during the entire studied period. For animals of group IV (long-lived and senescent), including humans, the Jones method makes it possible to trace mortality during the entire life cycle. The same applies to short-lived animals (e.g. nematodes or the tundra vole), whether they display the Gompertz type of senescence or not. However, in long-lived species with a less pronounced increase in mortality with age (e.g. the freshwater crocodile, hermit crab, or Scots pine), as well as in animals of average lifespan that reach the terminal age earlier than they could have enough time to become senescent, the Jones method is capable of characterizing only a small part of the life cycle and does not allow judging how senescence manifests itself at late stages of the life cycle. Thus, it is known that old trees display signs of biological senescence rather clearly; although Jones et al. consider them non-senescent organisms because less than 5% of sexually mature individuals survive to display the first manifestations of these characters. We have concluded that the classification proposed by Jones et al. makes it possible to approximately divide animals and plants only by their levels of the Gompertz type of senescence (i.e. actuarial senescence), whereas susceptibility to biological senescence can be estimated only when principally different models are applied.

During the last decade, several pieces of convincing evidence were published indicating that aging of living organisms is programmed, being a particular case of programmed death of organism (phenoptosis). Among them, the following observations can be mentioned. (1) Species were described that show negligible aging. In mammals, the naked mole rat is the most impressive example. This is a rodent of mouse size living at least 10-fold longer than a mouse and having fecundity higher than a mouse and no agerelated diseases. (2) In some species with high aging rate, genes responsible for active organization of aging by poisoning of the organism with endogenous metabolites have been identified. (3) In women, standard deviations divided by the mean are the same for age of menarche (an event controlled by the ontogenetic program) and for age of menopause (an aging-related event). (4) Inhibitors of programmed cell death (apoptosis and necrosis) retard and in certain cases even reverse the development of age-dependent pathologies. (5) In aging species, the rate of aging is regulated by the individual which responds by changes in this rate to changes in the environmental conditions. In this review, we consider point (5) in detail. Data are summarized suggesting that inhibition of aging rate by moderate food restriction can be explained assuming that such restriction is perceived by the organism as a signal of future starvation. In response to this dramatic signal, the organism switches off such an optional program as aging, mobilizing in such a way additional reserves for survival. A similar explanation is postulated for geroprotective effects of heavy muscle work, a lowering or a rise in the external temperature, small amounts of metabolic poisons (hormesis), low doses of radiation, and other deleterious events. On the contrary, sometimes certain positive signals can prolong life by inhibiting the aging program in individuals who are useful for the community (e.g., geroprotective psychological factors). Similarly, dangerous individuals can be eliminated by programmed death due to operation of progeric psychological factors. The interplay of all these signals results in the final decision of the organism concerning its aging – to accelerate or to decelerate this process. Thus, paradoxically, such an originally counterproductive program as aging appears to be useful for the individual since this program can be switched off by the individual for a certain period of time, an action that thereby increases its resources in crucial periods of life.

Radioprotection appeared to be an important problem of today due to atom energetic development and utilization of radiation material in the industry, science and medicine. It has been shown that mitochondrial targeted antioxidant SkQR1 could attenuate radiation injury of human erythroleukemia K562 cells. Pretreatment with SkQR1 before irradiation decreased DNA double strand breaks formation, diminished the number of chromosomal aberrations and suppressed delayed ROS production. Prevention of oxidative stress and normalization of mitochondrial function by mitochondria-targeted antioxidants may be a potential therapeutic strategy not only against immediate consequences of radiation, but, either against its late consequences such as genomic instability. SkQR1 did not protect against radiation-induced damage the K562 subline with high level of multidrug resistance (MDR) due to SkQR1 extrusion with Pgp 170 MDR pump. We suggest that mitochondria-targeted antioxidants might be used for selective protection of normal cells against radiation-induced damage without interference with radiotherapy of MDR-positive tumors.

Glaucoma is the main cause of irreversible blindness worldwide. This disease is characterized by apoptosis of retinal ganglion cells (RGC) and visual field loss that seems to be related to elevated intraocular pressure (IOP). Several lines of evidences have implicated the crucial role of mitochondrial dysfunction in the pathogenesis of glaucoma. Increased mitochondrial oxidative stress in RGC may underlie or contribute to susceptibility of RGC to apoptosis. In our work we (i) designed a rabbit model of chronic, moderately elevated IOP for studying glaucoma and (ii) demonstrated efficacy of mitochondria-targeted antioxidant SkQ1 as a tool to reverse several traits of experimental glaucoma induced by a series of injections of hydroxypropylmethylcellulose (HPMC) to the anterior chamber of the rabbit eye. It is shown that 6 months instillations of drops of 0.2.5-5 microM solution of SkQ1 normalize IOP and eye hydrodynamics and abolish an increase in lens thickness that accompanies glaucoma.

We show here that mitochondria-targeted antioxidant composed of plastoquinone conjugated through hydrocarbon linker with cationic rhodamine 19 (SkQR1) protected against nuclear DNA damage induced by gamma radiation in K562 erythroleukemia cells. We also demonstrate that SkQR1 prevented the early (1 h postirradiation) accumulation of phosphorylated histone H2AX (γ-H2AX) an indicator of DNA double-strand break formation, as well as the radiation-induced increase in chromosomal aberrations. These data suggested that nuclear DNA damage induced by gamma radiation may be mediated by mitochondrial reactive oxygen species (ROS) production. We show that SkQR1 suppressed delayed accumulation of ROS 32 h after irradiation probably by inhibiting mitochondrial ROS-induced ROS release mechanisms. This suggests that mitochondria-targeted antioxidants may protect cells from the late consequences of radiation exposure related to delayed oxidative stress. We have previously reported that SkQRl is the substrate of multidrug resistance pump P-glycoproten (Pgp 170) and selectively protects Pgp 170-negative cells against oxidative stress. In line with this finding, we demonstrate here that SkQR1 did not protect Pgp170-positive K562 subline against DNA damage induced by gamma radiation. The selective radioprotection of normal Pgp 170-negative cells by mitochondria-targeted antioxidants could be a promising strategy to increase the efficiency of radiotherapy for multidrug-resistant tumors.

Background Multiple sclerosis (MS) is one of the most widespread chronic neurological diseases that manifests itself by progressive demyelination in the central nervous system. The study of MS pathogenesis begins with the onset of the relapsing-remitting phase of the disease, which becomes apparent due to microglia activation, neuroinflammation and demyelination/ remyelination in the white matter. The following progressive phase is accompanied by severe neurological symptoms when demyelination and neurodegeneration are spread to both gray and white matter. In this review, we discuss a possible role of mitochondrial reactive oxygen species (mtROS) in MS pathogenesis, mechanisms of mtROS generation and effects of some mitochondria-targeted antioxidants as potential components of MS therapy. Results In the early phase of MS, mtROS stimulate NLRP3 inflammasomes, which is critical for the formation of local inflammatory lesions. Later, mtROS contribute to blood-brain barrier disruption induced by mediators of inflammation, followed by infiltration of leukocytes. ROS generated by leukocytes and activated microglia promote mitochondrial dysfunction and oligodendrocyte cell death. In the progressive phase, neurodegeneration also depends on excessive mtROS generation. Currently, only a few immunomodulatory drugs are approved for treatment of MS. These drugs mainly reduce the number of relapses but do not stop MS progression. Certain dietary and synthetic antioxidants have demonstrated encouraging results in animal models of MS but were ineffective in the completed clinical trials. Conclusion Novel mitochondria-targeted antioxidants could be promising components of combined programs for MS therapy considering that they can be applied at extremely low doses and concurrently demonstrate anti-inflammatory and neuroprotective activities.

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per μm2 of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles–from 0.21 ± 0.01 to 0.29 ± 0.03 μm2. Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.

Initially regarded solely as “power plants of the living cell”, mitochondria are now also acknowledged as key regulators of cell survival, death, and ageing. Mitochondrial malfunction correlates with aging in a broad variety of species, including yeast, invertebrates and mammals. An important consequence of the mitochondrial malfunction is an increase in the level of reactive oxygen species (ROS). Excessive level of mitochondrial ROS (mROS) results in imbalance of regulatory mitochondrial pathways, oxidative stress, and cell death, which triggers several age-related pathologies. Mitochondria-targeted rechargeable antioxidants (e.g. SkQ1, SkQR, MitoQ) are promising candidates for development of therapeutic drugs capable of suppressing mitochondria-initiated oxidative stress and thereby promoting healthy aging and longevity. In this review, we focus on the studies of mitochondria-targeted rechargeable antioxidants in animal models, with a special focus on experiments with Drosophila melanogaster.

Mitochondrial-derived reactive oxygen species have been deemed an important contributor in sepsis pathogenesis. We investigated whether two mitochondria-targeted antioxidants (mtAOX; SkQ1 and MitoTEMPO) improved long-term outcome, lessened inflammation, and improved organ homeostasis in polymicrobial murine sepsis. 3-month-old female CD-1 mice (n=90) underwent cecal ligation and puncture (CLP) and received SkQ1 (5 nmol/kg), MitoTEMPO (50 nmol/kg), or vehicle 5 times post-CLP. Separately, 52 SkQ1-treated CLP mice were sacrificed at 24 h and 48 h for additional endpoints. Neither MitoTEMPO nor SkQ1 exerted any protracted survival benefit. Conversely, SkQ1 exacerbated 28-day mortality by 29%. CLP induced release of 10 circulating cytokines, increased urea, ALT, and LDH, and decreased glucose but irrespectively of treatment. Similar occurred for CLP-induced lymphopenia/neutrophilia and the NO blood release. At 48 h post-CLP, dying mice had approximately 100-fold more CFUs in the spleen than survivors, but this was not SkQ1 related. At 48 h, macrophage and granulocyte counts increased in the peritoneal lavage but irrespectively of SkQ1. Similarly, hepatic mitophagy was not altered by SkQ1 at 24 h. The absence of survival benefit of mtAOX may be due to the extended treatment and/or a relatively moderate-risk-of-death CLP cohort. Long-term effect of mtAOX in abdominal sepsis appears different to sepsis/inflammation models arising from other body compartments.

This review summarizes the latest data on biochemistry and physiology of living organisms. These data suggest that aging, i.e. coordinated age-dependent weakening of many vital functions leading to gradual increase in the probability of dying, is not common to all organisms. Some species have been described whose probability of death does not depend on age or even decreases with age, this being accompanied by constant or increasing fertility. In the case of the naked mole rat (a non-aging mammal), a mechanism has been identified that protects this animal from cancer and the most common age-related diseases. The high molecular weight polysaccharide hyaluronan, a linear polymer composed of multiple repeated disaccharide of glucuronic acid and glucosamine, plays the key role in this mechanism. Hyaluronan is accumulated in the intercellular spaces in the organs and tissues of the naked mole rat. This polysaccharide provides early contact inhibition of cell division (anti-cancer effect). In addition, hyaluronan prevents the development of certain types of apoptosis, in particular, those induced by reactive oxygen species (ROS) (geroprotective effect preventing ROS-induced decrease in cellularity in the organs and tissues of aging organisms). Extraordinary longevity of the naked mole rat (over 30 years, which is long for a rodent the size of a mouse) is connected to its eusocial lifestyle, when only the "queen" and its few "husbands" breed, while the huge army of non-breeding "subordinates" provide the "royal family" with protection from predators, food, and construction and maintenance of an underground labyrinth size of a football field. This way of life removes the pressure of natural selection from the "family" and makes aging - the program that is counterproductive for the individual but increases "evolvability" of its offspring - unnecessary. The example of the naked mole rat demonstrates the optional character of the aging program for the organism. Many facts indicating that aging can be regulated by an organism provide another argument in favor of optionality of aging. Cases have been described when aging as a program useful for the evolution of offspring but counterproductive for the parental individual slows under conditions that threaten the very existence of the individual. These conditions include food restriction (the threat of death from starvation), heavy muscular work, decrease or increase in the environmental temperature, small amounts of poisons (including ROS; here we speak about the paradoxical geroprotective effect of the low doses of prooxidants that inhibit apoptosis). On the other hand, aging can be inhibited (and maybe even cancelled) artificially. This can be done by turning off the genes encoding the proteins participating in the aging program, such as FAT10, p66shc, and some others. In addition, the gene of the antioxidant enzyme catalase can be addressed into mitochondria, where it will split mitochondrial hydrogen peroxide, the level of which increases with age. However, today the simplest way to slow down the aging program is the use of mitochondria-targeted low molecular weight antioxidant compounds of plastoquinonyl decyltriphenylphosphonium-type (SkQ1), which prolong the life of animals, plants, and fungi and inhibit the development of many age-related diseases and symptoms.

A comparative electron-microscopic study of ultrastructure of mitochondria in skeletal muscles of the 3- and 24-month-old Wistar and OXYS rats revealed age-dependent changes in both general organization of the mitochondrial reticulum and ultrastructure of mitochondria. The most pronounced ultrastructure changes were detected in the OXYS rats suffering from permanent oxidative stress. In the OXYS rats, significant changes in mitochondrial ultrastructure were detected already at the age of 3 months. Among them, there were the appearance of megamitochondria and reduction of cristae resulting in formation of cristae-free regions inside mitochondria. In the 24-month-old OXYS rats, mitochondrial reticulum was completely destroyed. In the isotropic region of muscle fiber, only small solitary mitochondria were present. There appeared regions of lysed myofibrils as well as vast regions filled with autophagosomes. A mitochondrial antioxidant SkQ1 (given to rats with food daily in the dose of 250 nmol/kg of body weight for 5 months beginning from the age of 19 months) prevented development of age-dependent destructive changes in both the Wistar and OXYS rats.

Induced and frequently unwanted alterations in the mitochondrial structure and functions are a key component of the pathological cascade in many kidney pathologies, including those associated with acute damage. One of the principal pathogenic elements causing mitochondrial dysfunction in Acute Kidney Injury (AKI) is oxidative stress. After ischemia and nephrotoxic action of drugs, sepsis and systemic inflammation are the most frequent causes of AKI. As the kidney suffers from oxidative stress during sepsis, one of the most promising approaches to alleviate such damaging consequences is the use of antioxidants. Considering administration of lipopolysaccharide (LPS) as a model of sepsis, we demonstrate that the mitochondria of neonatal renal tissue are severely affected by LPS-induced AKI, with pathological ultrastructural changes observed in both the mitochondria of the renal tubular epithelium and the vascular endothelium. Upon mitochondrial damage, we evaluated the effect of the mitochondria-targeted antioxidant plastoquinol decylrhodamine 19 (SkQR1) on the development of acute renal failure in newborn rats associated with systemic inflammation induced by the administration of LPS. We found that SkQR1 administration 3 h before LPS led to decreased urinal expression of the AKI marker neutrophil gelatinase-associated lipocalin 2 (NGAL), in addition to a decrease in urea and creatinine levels in the blood. Additionally, an observed impairment of proliferative activity in the neonatal kidney caused by LPS treatment was also prevented by the treatment of rat pups with SkQR1. Thus, one of the key events for renal tissue damage in neonatal sepsis is an alteration in the structure and function of the mitochondria and the mitochondria-targeted antioxidant SkQR1 is an effective nephroprotective agent, which protects the neonatal kidney from sepsis-induced AKI.