Biochemistry (Moscow) (v.82, #12)

(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.
Keywords: phenoptosis; aging program; mitochondria; evolution; Heterocephalus glaber

Programmed aging theories contend that evolved biological mechanisms purposely limit internally determined lifespans in mammals and are ultimately responsible for most instances of highly age-related diseases and conditions. Until recently, the existence of programmed aging mechanisms was considered theoretically impossible because it directly conflicted with Darwin’s survival-of-the-fittest evolutionary mechanics concept as widely taught and generally understood. However, subsequent discoveries, especially in genetics, have exposed issues with some details of Darwin’s theory that affect the mechanics of the evolution process and strongly suggest that programmed aging mechanisms in humans and other mammals can and did evolve, and more generally, that a trait that benefits a population can evolve even if, like senescence, it is adverse to individual members of the population. Evolvability theories contend that organisms can possess evolved design characteristics (traits) that affect their ability to evolve, and further, that a trait that increases a population’s ability to evolve (increases evolvability) can be acquired and retained even if it is adverse in traditional individual fitness terms. Programmed aging theories based on evolvability contend that internally limiting lifespan in a species-specific manner creates an evolvability advantage that results in the evolution and retention of senescence. This issue is critical to medical research because the different theories lead to dramatically different concepts regarding the nature of biological mechanisms behind highly age-related diseases and conditions.
Keywords: aging theory; senescence; medicine; gerontology; evolutionary mechanics theories

Programmed (adaptive) aging refers to the idea that mammals, including humans and other complex organisms, have evolved mechanisms that purposely cause or allow senescence or otherwise internally limit their lifespans in order to obtain an evolutionary advantage. Until recently, programmed aging had been thought to be theoretically impossible because of the mechanics of the evolution process. However, there is now substantial theoretical and empirical support for the existence of programmed aging in mammals. Therefore, a comprehensive approach to medical research on aging and age-related diseases must consider programmed aging mechanisms and the detailed nature of such mechanisms is of major importance. Theories of externally regulated programmed aging suggest that in mammals and other complex organisms, genetically specified senescence mechanisms detect local or temporary external conditions that affect the optimal lifespan for a species population and can adjust the lifespans of individual members in response. This article describes why lifespan regulation in response to external conditions adds to the evolutionary advantage produced by programmed aging and why a specific externally regulated programmed aging mechanism provides the best match to empirical evidence on mammal senescence.
Keywords: senescence; evolution; gerontology; health policy; medical research; evolvability

Phenoptosis is a phenomenon that is genetically programmed and favored by natural selection, and that determines death or increased risk of death (fitness reduction) for the individual that manifests it. Aging, here defined as agerelated progressive mortality increase in the wild, if programmed and favored by natural selection, falls within the definition of phenoptosis. Sexual reproduction (sex), as for the involved individuals determines fitness reduction and, in some species, even certain death, also falls within the definition of phenoptosis. In this review, sex and aging are analyzed as phenoptotic phenomena, and the similarities between them are investigated. In particular, from a theoretical standpoint, the genes that cause and regulate these phenomena: (i) require analyses that consider both individual and supra-individual selection because they are harmful in terms of individual selection, but advantageous (that is, favored by natural selection) in particular conditions of supra-individual selection; (ii) determine a higher velocity of and greater opportunities for evolution and, therefore, greater evolutionary potential (evolvability); (iii) are advantageous under ecological conditions of K-selection and with finite populations; (iv) are disadvantageous (that is, not favored by natural selection) under ecological conditions of r-selection and with unlimited populations; (v) are not advantageous in all ecological conditions and, so, species that reproduce asexually or species that do not age are predicted and exist.
Keywords: aging; sex; phenoptosis; supra-individual selection; K-selection; r-selection

An experimental design is proposed for high-throughput testing of combined interventions that might increase life expectancy in rodents. There is a growing backlog of promising treatments that have never been tested in mammals, and known treatments have not been tested in combination. The dose-response curve is often nonlinear, as are the interactions among different therapies. Herein are proposed two experimental designs optimized for detecting high-value combinations. In Part I, numerical simulation is used to explore a protocol for testing different dosages of a single intervention. With reasonable and general biological assumptions about the dose-response curve, information is maximized when each animal receives a different dosage. In Part II, numerical simulation is used to explore a protocol for testing interactions among many combinations of treatments, once their individual dosages have been established. Combinations of three are identified as a sweet spot for statistics. To conserve resources, the protocol is designed to identify those outliers that lead to life extension greater than 50%, but not to offer detailed survival curves for any treatments. Every combination of three treatments from a universe of 15 total treatments is represented, with just three mice replicating each combination. Stepwise regression is used to infer information about the effects of individual treatments and all their pairwise interactions. Results are not quite as robust as for the dosage protocol in Part I, but if there is a combination that extends lifespan by more than 50%, it will be detected with 80% certainty. These two screening protocols offer the possibility of expediting the identification of treatment combinations that are most likely to have the largest effect, while controlling costs overall.
Keywords: lifespan; life extension; combined treatments; high-throughput testing; rodents

On the cause and mechanism of phenoptosis by R. F. Walker (1462-1479).
Based upon evolvability theory, phenotypes like aging that offer no apparent individual benefit may evolve nonetheless. Pursuant to that concept, the evolution of a hypothetical, genome-based aging program called phenoptosis was proposed. However, while aging may facilitate evolvability, it need not result from a program specifically selected for that purpose. Instead, it is possible that the potential for aging is conserved within the genome as a part of a beneficial program that orchestrates and integrates developmental transformation of the soma from conception to maturation. Because somatic remodeling is inherently unstable, its continued non-programmatic expression beyond young adulthood (developmental inertia) erodes internal order, initiating and exacerbating aging. Thus, aging may result paradoxically from post-maturational expression of the same programmatic function for somatic transformation that previously provided individual benefit. It did so by ensuring acquisition of reproductive competence, post-reproductive development of parents to nurture offspring and thereby, to guarantee species survival. In an attempt to identify genes capable of controlling developmental inertia, we sequenced DNA from a series of subjects displaying extreme neoteny, i.e. retention of youthful characteristics during adulthood. We hoped to identify mutations associated with delayed development and to compare each subject’s biological and chronological ages. De novo mutations of coding-genes were found in all the subjects, but they could not be definitively identified as a cause of developmental delay. Nonetheless, genetic and epigenetic studies of neotenic subject’s DNA are on-going. We are attempting to determine if phenoptosis specifically evolved to cause aging, or rather if it exists as a cryptic component of the developmental program that expresses its lethal potential serendipitously and only after individual benefit is realized.
Keywords: developmental inertia; temporal order; neoteny; homeodynamics; somatic transformation; DNA sequencing

Coefficient of variation of lifespan across the tree of life: Is it a signature of programmed aging? by G. A. Shilovsky; T. S. Putyatina; V. V. Ashapkin; O. S. Luchkina; A. V. Markov (1480-1492).
Measurements of variation are of great importance for studying the stability of pathological phenomena and processes. For the biology of aging, it is very important not only to determine average mortality, but also to study its stability in time and the size of fluctuations that are indicated by the variation coefficient of lifespan (CVLS). It is believed that a relatively small (∼20%) value of CVLS in humans, comparable to the coefficients of variation of other events programmed in ontogenesis (for example, menarche and menopause), indicates a relatively rigid determinism (N. S. Gavrilova et al. (2012) Biochemistry (Moscow), 77, 754-760). To assess the prevalence of this phenomenon, we studied the magnitude of CVLS, as well as the coefficients of skewness and kurtosis in diverse representatives of the animal kingdom using data provided by the Institute for Demographic Research (O. R. Jones et al. (2014) Nature, 505, 169-173). We found that, unlike humans and laboratory animals, in most examined species the values of CVLS are rather high, indicating heterogeneity of the lifespan in the cohorts studied. This is probably due to the large influence of background mortality, as well as the non-monotonicity of total mortality in the wild, especially at the earliest ages. One way to account for this influence is to “truncate” the data (removing the earliest and latest ages from consideration). To reveal the effect of this procedure, we proposed a new indicator, the stability coefficient of mortality dynamics, which indicates how quickly CVLS is reduced to values that characterize a relatively homogeneous population (33%) when the data are “truncated”. Such indicators facilitate the use of the parameters of survival curves for analysis of the effects of geroprotectors, lifestyle, and other factors on lifespan, and for the quantification of relative contributions of genetic and environmental factors to the dynamics of aging in human and animal populations, including those living in the wild.
Keywords: aging; lifespan; mortality rate; survival curves; skewness; coefficient of variation

Mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyl)decyltriphenylphosphonium bromide) inhibits mast cell degranulation in vivo and in vitro by M. A. Chelombitko; O. A. Averina; T. V. Vasilyeva; O. Yu. Pletiushkina; E. N. Popova; A. V. Fedorov; B. V. Chernyak; V. S. Shishkina; O. P. Ilinskaya (1493-1503).
The therapeutic effect of mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decyltriphenylphosphonium bromide (SkQ1) in experimental models of acute inflammation and wound repair has been shown earlier. It was suggested that the antiinflammatory activity of SkQ1 is related to its ability to suppress inflammatory activation of the vascular endothelium and neutrophil migration into tissues. Here, we demonstrated that SkQ1 inhibits activation of mast cells (MCs) followed by their degranulation and histamine release in vivo and in vitro. Intraperitoneal injections of SkQ1 in the mouse air-pouch model reduced the number of leukocytes in the air-pouch cavity and significantly decreased the histamine content in it, as well as suppressing MC degranulation in the air-pouch tissue. The direct effect of SkQ1 on MCs was studied in vitro in the rat basophilic leukemia RBL-2H3 cell line. SkQ1 inhibited induced degranulation of RBL-2H3 cells. These results suggest that mitochondrial reactive oxygen species are involved in the activation of MCs. It is known that MCs play a crucial role in regulation of vascular permeability by secreting histamine. Suppression of MC degranulation by SkQ1 might be a significant factor in the antiinflammatory activity of this mitochondria-targeted antioxidant.
Keywords: inflammation; mast cell; degranulation; histamine; mitochondria-targeted antioxidant

Spontaneous and experimentally induced pathologies in the naked mole rat (Heterocephalus glaber) by V. N. Manskikh; O. A. Averina; A. I. Nikiforova (1504-1512).
The naked mole rat (Heterocephalus glaber, Rüppell, 1842) is a unique eusocial rodent with unusually long lifespan. Therefore, the study of spontaneous and experimentally induced pathologies in these animals is one of the most important tasks of gerontology. Various infections, noninfectious pathologies (including age-dependent changes), and tumors have been described in the naked mole rat. The most frequent pathologies are traumas (bite wounds), purulent and septic complications of traumatic injuries, renal tubular calcinosis, chronic progressive nephropathy, hepatic hemosiderosis, testicular interstitial cell hyperplasia, calcinosis cutis, cardiomyopathy, and dysbiosis-related infectious lesions of the digestive system. However, the summarized data on pathology (including tumor incidence) and on the causes of mortality are insufficient. There are only few publications about the results of experiments where pathologies were induced in the naked mole rat. All these problems could be subjects for promising future studies without which adequate studies on mechanisms providing the long lifespan of the naked mole rat are impossible, as well as the elucidation of causes of tumor resistance of this species.
Keywords: aging and age-related diseases; naked mole rat; resistance of animals to tumors; spontaneous pathologies; laboratory animals

Effects of mitochondrial antioxidant SkQ1 on biochemical and behavioral parameters in a Parkinsonism model in mice by V. V. Pavshintsev; L. S. Podshivalova; O. Y. Frolova; M. V. Belopolskaya; O. A. Averina; E. A. Kushnir; N. V. Marmiy; M. L. Lovat (1513-1520).
According to one hypothesis, Parkinson’s disease pathogenesis is largely caused by dopamine catabolism that is catalyzed on mitochondrial membranes by monoamine oxidase. Reactive oxygen species are formed as a byproduct of these reactions, which can lead to mitochondrial damage followed by cell degeneration and death. In this study, we investigated the effects of administration of the mitochondrial antioxidant SkQ1 on biochemical, immunohistochemical, and behavioral parameters in a Parkinson-like condition caused by protoxin MPTP injections in C57BL/6 mice. SkQ1 administration increased dopamine quantity and decreased signs of sensory-motor deficiency as well as destruction of dopaminergic neurons in the substantia nigra and ventral tegmental area in mice with the Parkinson-like condition.
Keywords: Parkinson’s disease; MPTP; mitochondrial antioxidants; SkQ1; sensorimotor deficiency; dopaminergic neurons

Y-box-binding protein 1 stimulates abasic site cleavage by E. E. Alemasova; K. N. Naumenko; N. A. Moor; O. I. Lavrik (1521-1528).
Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions. The first step in the AP site repair involves the magnesium-dependent enzyme AP endonuclease 1 (APE1) that catalyzes hydrolytic cleavage of the DNA phosphodiester bond at the 5′ side of the AP site, thereby generating a single-strand DNA break flanked by the 3′-OH and 5′-deoxyribose phosphate (dRP) groups. Increased APE1 activity in cancer cells might correlate with tumor chemoresistance to DNA-damaging treatment. It has been previously shown that the multifunctional oncoprotein Y-box-binding protein 1 (YB-1) interacts with APE1 and inhibits APE1-catalyzed hydrolysis of AP sites in single-stranded DNAs. In this work, we demonstrated that YB-1 stabilizes the APE1 complex with double-stranded DNAs containing the AP sites and stimulates cleavage of these AP sites at low magnesium concentrations.
Keywords: Y-box-binding protein 1; AP endonuclease 1; base excision repair; apurinic/apyrimidinic site

Heterologous expression and isolation of influenza A virus nuclear export protein NEP by A. O. Golovko; O. N. Koroleva; V. L. Drutsa (1529-1537).
Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N-or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.
Keywords: influenza A virus; nuclear export protein (NEP); affinity chromatography; protein aggregation

Metagenomics of bolidophyceae in plankton and ice of the White Sea by T. A. Belevich; L. V. Ilyash; I. A. Milyutina; M. D. Logacheva; A. V. Troitsky (1538-1548).
The molecular diversity of poorly studied algae of Bolidophyceae class was first estimated by Illumina sequencing of V4 region of 18S rRNA gene in ice, under-ice water and summer water of the subarctic White Sea. We used two clustering thresholds–93 and 97%–and revealed 31 phylotypes of Bolidophyceae. Triparma pacifica and Т. strigata were identified to species level. The association of individual phylotypes to certain biotopes (ice or plankton) and stages of seasonal succession (under ice or summer plankton) has been established. Some phylotypes are found in different biotopes and over a wide temperature range. Due to changing their genetic composition, Bolidophyceae are a constant component of the photoautotrophic plankton and ice communities.
Keywords: metagenomic analysis; picophytoplankton; White Sea; Bolidophyceae; Triparma pacifica ; Triparma strigata

Bacterial therapy and mitochondrial therapy by V. A. Popkov; E. Y. Plotnikov; D. N. Silachev; L. D. Zorova; I. B. Pevzner; S. S. Jankauskas; S. D. Zorov; N. V. Andrianova; V. A. Babenko; D. B. Zorov (1549-1556).
Current methods for treatment of cellular and organ pathologies are extremely diverse and constantly evolving, going beyond the use of drugs, based on chemical interaction with biological targets to normalize the functions of the system. Because pharmacological approaches are often untenable, recent strategies in the therapy of different pathological conditions are of particular interest through introducing into the organism of some living system or its components, in particular, bacteria or isolated subcellular structures such as mitochondria. This review describes the most interesting and original examples of therapy using bacteria and mitochondria, which in perspective can dramatically change our views on the principles for the treatment of many diseases. Thus, we analyze such therapeutic effects from the perspective of the similarities between mitochondria and bacteria as the evolutionary ancestors of mitochondria.
Keywords: bacteria; mitochondria; microbiota; pathology; transplantation; treatment