Overcome aging. Part I.

Olga Volkova, "Biomolecule" 

Aging is a complex, multifactorial and almost universal process. And most importantly – not very clear yet. It is even less clear, but the non-aging of living organisms is very encouraging. In this article we will talk about animals for which the insignificant (negligible) mode is shown or only assumed aging. As an example of the analysis of this phenomenon in a specific group of organisms, let's consider testing popular theories of aging on sea urchins, touch on other centenarians and evaluate the prospects of working with such animals. And for dessert, let's "try" a couple of specific approaches to achieving negligible aging in humans. In the second (separate) part of the article, we will get acquainted with the molecular features of long-lived rodents and analyze the mechanisms of their resistance to cancer.

He also took care of the development of criteria for classifying organisms as negligibly (extremely slowly) aging: the rate of their mortality should not increase with age and fertility, physiological capabilities and resistance to diseases should not noticeably decrease [1]. But for biogerontological research, any animals with an early plateau on the mortality curve with an abnormally long life for their weight and taxon are considered valuable. In the case of mammals, an equally valuable feature is the absence of typical senile ailments for the class.

The "officially" confirmed list is negligible (insignificant) there are no aging animals. The respected thematic resource AnAge has included eight organisms in this category so far, but the number of candidate species is constantly growing - in fact, purposefully collecting information on them began only at the end of the last century. 

Negligible aging does not imply immortality at all (modular creatures like sponges and hydroids can only take a swing at it). Such organisms, firstly, are "suddenly mortal" (like all others – from diseases, injuries, changes in conditions), and secondly, in old age, specific factors can act on them, often associated with their continuous growth. Giant turtles become cramped shell, too heavy bivalve mollusk Arctica islandica problems with movement doom to starvation, huge fish lose to nimble small relatives in the competition for food, the Asian elephant loses the last set of teeth, the whale fails vision... Nevertheless, animals with negligible (delayed) aging are not characterized by an exponential increase in mortality rates with age.

Examples of animals with negligible (delayed) aging: 

animal name*, maximum life expectancy (years)

The phenomenon of negligible aging has arisen in evolution many times – independently in different systematic categories: there are plants and representatives of different classes of animals among the slightly aging ones (Fig. 1), and even within the same family, some species have developed this trait, while others have not. It may seem strange, but the assessment of the maximum life expectancy and aging regime in a particular animal is extremely difficult. It is a great success if some individuals were helpfully marked, for example, a couple of centuries ago - and they live and live to this day. It takes decades to observe potential centenarians in protected conditions; it is usually difficult to judge the age of such animals by their appearance. Marine life – some mollusks and echinoderms – is easier to work with: their age is determined by the annual growth zones of solid structures (shells and shells), and confirmed by radiocarbon analysis.

P isunok 1. The range of life expectancy and types of aging of organisms. It is obvious that the MPJ – the maximum life expectancy – in organisms differs by orders of magnitude, and the regime of negligible aging (horizontal scale) has arisen repeatedly in evolution – independently in phylogenetically distant organisms. Rapid aging is typical, for example, for yeast Saccharomyces cerevisiae (live 2-4 days with asexual reproduction), nematodes Caenorhabditis elegans (30 days), flies Drosophila melanogaster (60 days), Pacific salmon (3-6 years), plants Phyllostachys bambusoides, P. nigra f. henonis (bamboo species, MPJ – 120 years) and Puya raimondii (it is a relative of pineapple, living up to 150 years). Gradual aging is observed in the mouse Mus musculus (MPJ – 4.2 years) and human (122.4 years). Negligible aging is endowed with: ray-finned fish (Aleutian sea bass, Atlantic largemouth, deep-sea sunfish, lake sturgeon), living for more than 130 years; turtles (some of them are >150 years old), bivalve mollusk Arctica islandica (the authors of the drawing are guided by a 220-year-old MPJ, but later discovered a 507-year-old specimen), intermountain spinous pine (about 5000 years old) and many other organisms. Figure from [1], adapted.It is believed that it is more likely to "stumble" on a negligibly aging animal in extreme, but protected from enemies, niches:

stress resistance seems to be a common feature of centenarians [2], and high life expectancy contributes to the selection of the determinants of longevity. The approximate life expectancy of individuals of a particular species is predicted by size (mass) animal bodies. In general, the doubling of the species body weight in terrestrial mammals is accompanied by a 16 percent prolongation of life. Of course, species with negligible or delayed aging (and humans, by the way) these mathematical expectations do not meet. It is customary to refer to long-lived species as organisms that live two or more times longer than it is "prescribed" by their mass. More often, indeed, large animals age slowly, but the developed brain is the determining factor here. Portuguese scientist Juan Pedro de Magallanes and his colleagues have shown that the life expectancy of vertebrates directly correlates with the age of puberty and vice versa – with the rate of postnatal development (in mammals, but not in birds). But the metabolic rate (adjusted for phylogeny and body weight) is not related to the lifespan of birds and placental mammals [3].

The wider the spectrum of long-lived organisms discovered and studied, the clearer the prospects for interventions in the human aging process. The schemes of inter- and intracellular signaling, the systems for maintaining the stability of the genome and proteome, and in general the cellular structure of all animals are similar, and the difference in their lifespan reaches several orders of magnitude (Fig. 1). Hence, the solution may lie in the peculiarities of the regulation of gene expression and their polymorphisms. Therefore, animals with delayed aging, having short-lived relatives and phylogenetically close to humans are especially valuable as gerontological models. In this sense, the naked digger became a real gift – a mammal, if not with negligible, then with greatly delayed aging, living at least 6-7 times longer than mice and rats (we will discuss the features of this unique rodent separately.)

Even before the digger, the attention of biogerontologists was attracted by marine non–colonial inhabitants - bivalves and sea urchins. These are commercial animals, and hedgehogs are also "experimental" developmental biology from the century before last, so a lot of information has accumulated about their lifestyle, growth and reproduction. Evolutionarily, they are much closer to humans than worms or flies, and the mollusk Min (1499-2006) – a representative of the species Arctica islandica – is generally the current champion of longevity among animals. At the same time, life expectancy varies greatly in these groups of marine invertebrates [4], which makes them excellent objects for comparative analysis of life support mechanisms.

However, biogerontologists often resort to "running through" the biological features of a negligibly aging organism according to the most popular theories – this orders the structure of comparative analysis, gives at least some initial guidelines, and most importantly, tests the theories of aging themselves for viability. This is how Andrea Bodnar built her analytical review on the basics of the longevity of sea urchins [5].

shortening of telomeres leads to aging of the cell (through replicative aging or genomic instability) and the organism as a whole. On the contrary, the stabilization of telomeres due to their regular completion by telomerase grants cells immortality [6]. Human aging is accompanied by a reduction in telomere length, telomere dysfunction accelerates aging in mice and humans, and activation of telomerase delays aging in mice with a deficiency of this enzyme.

The biology of telomeres was studied in short-, medium- and long-lived species of sea urchins: Lytechinus variegatus (life expectancy about 4 years), Echinometra lucunter (40 years) and Strongylocentrotus franciscanus (100-200 years). In none of the species did the telomere length decrease with age, which is most likely due to the constant activity of telomerase. Moreover, longevity in sea urchins is not related to telomere length at all, on the contrary: L. variegatus has the longest terminal repeats.

Conclusion: there is no replicative aging in these animals, therefore it makes no sense to look for the origins of the neglect of their aging in the biology of telomeres. However, one point is interesting: with lifelong telomerase activity, echinoderms rarely suffer from neoplasia (cancer). This means that the mechanisms of repair and (or) getting rid of the "traitor cells" characteristic of them should certainly be sought.

reactive oxygen species cause the accumulation of damage to cellular structures, play a key role in aging and determining life expectancy. Oxidative stress is the result of an imbalance between the production of reactive oxygen species (ROS) – in mitochondria and not only – and the prevention/elimination of damage due to the work of antioxidant systems and mechanisms of repair or disposal of damaged biomolecules [7-9]. Although a number of studies have shown an increase in the level of oxidative damage in the tissues of many organisms with age, it is not yet clear whether this is the cause or the effect of aging.

A comparison of three species of sea urchins with different lifespans did not reveal an age-dependent change in their overall level of oxidative damage. This can be explained by the lifelong stable functioning of antioxidant systems and proteasomes in these organisms. However, over the years, the content of lipofuscin - a fluorescent yellow-brown "aging pigment" - increases in their tissues. It is assumed that during intensive oxidation of proteins, proteolysis systems cannot cope with their removal, damaged proteins are "crosslinked" with each other and other molecules (including in the composition of organelle membranes) – a lipofuscin conglomerate is formed, in which, in addition to defective proteins, oxidized lipids, sugars and transition metals are "preserved". The latter – especially iron – form a surface capable of oxidation-reduction, making lipofuscin granules strong oxidizers (and according to some data, stimulators of apoptosis). Moreover, it is suspected that lipofuscin competitively inhibits the activity of proteasomes [10]. All this closes the pathological circle.

In humans, lipofuscin usually accumulates in lysosomes, and it cannot be "digested" or cured by exocytosis. Postmitotic cells such as neurons are particularly affected by this. In sea urchins, lipofuscin accumulates not inside, but outside the cells. Perhaps the export of cytotoxic "junk" reduces the level of oxidative damage in cells and allows proteasomes to work stably at any age. However, the question remains open, does lipofuscin interfere with tissues in general? The authors of the work note that in the short-lived L.variegatus, the level of oxidative damage and the content of lipofuscin were higher than in long–lived relatives - that is, there seems to be a link between oxidation and aging. However, the studied species of sea urchins live in completely different climatic conditions, which may explain the differences in ROS production.

Conclusion: the relationship of oxidative stress with life expectancy in sea urchins is not obvious and requires confirmation (another sample is needed for a correct comparison). Unlike humans and classical model animals, antioxidant systems and proteasomes that utilize oxidized proteins work stably throughout life in three species of echinoderms and without much interspecific difference. The organizers of the study believe that it makes sense to understand why L.variegatus produces more ROS (since other levels of protection against oxidative stress are "not broken").

It has been repeatedly shown that in model animals and humans, the expression of many genes changes with age (this has already been discussed on the example of J. Zan [11], see the article "Senile vagaries of nature: why people stop aging, and mice do not have time to live" [12]). Cause-and-effect relationships, again, are not obvious, and complex changes in the intensity of signaling along one or another pathways during aging are not completely clear.

In 2012, some age-dependent (and mostly tissue-specific) changes in the transcription profile were found in the sea urchin S. purpuratus [13]. Common to the three studied tissues was an increase in the activity of Notch signaling pathway genes over the years, and for two tissues – a decrease in the activity of the Wnt1 gene (a little later, additional, receptor-dependent suppression of Wnt signaling was shown). A specific balance between these signaling pathways, which are important for the development of the organism, can determine the ability to moderate regeneration in mature S. purpuratus and contribute to their constant growth and high life expectancy (more than 50 years). The ability to regenerate does not depend on age in mature sea urchins with different lifespans (S. franciscanus, S. purpuratus, and L. variegatus) and can be provided either by maintaining a decent pool of stem cells (which has not yet been confirmed), or by dedifferentiating specialized cells into stem cells.

Described by J. Zan as a marker of aging common to different branches of the phylogenetic tree of animals – a decrease in the expression of ETC genes – does not work in S. purpuratus (and this is logical for species with negligible aging): the activity of genes associated with energy production is stable in him, and even grows in the nervous tissue. The expression of some proteasome degradation genes supporting protein homeostasis is also increased.

Conclusion: age-dependent changes in gene expression in S. purpuratus have been identified, but their comparison with transcription profiles of other echinoderms is necessary.

The general conclusion is that the ability to regenerate tissues for life without allowing hyperproliferation leading to cancer is the most important feature of echinoderms, which deserves the closest attention of gerontologists [5].

A. Bodnar believes that the negligibly aging sea urchins (S.franciscanus) and bivalves (A.islandica, Panopea abrupta) will become excellent model animals, especially if they choose a decent "background" – to identify the closest relatives living in similar conditions: this will eliminate the "noise" when comparing [4].

The main hopes are pinned on systematic studies comparing cellular processes in different age cohorts of closely related species aging rapidly vs. negligibly. Genome matching analytics is already available for some groups. More often, we have to be content with the results obtained on small samples and therefore contradictory.

In addition to the analysis of echinoderms, A. Bodnar's team tried to systematize the available data on bivalves, which include the long-lived record holder A.islandica [4]. It turned out that in this species, after puberty, antioxidant protection works stably throughout life, while in some short-lived species it gradually weakens, mitochondrial function suffers, ROS is formed more, the proportion of oxidized proteins increases. On the other hand, the excellent condition of mitochondria and proteins in long–lived mollusks contrasts with the accumulation of peroxidation products of membrane lipids (this may be a common property of cold water organisms associated with the fatty acid composition of their membranes) - and this does not prevent them from setting age records. Another interesting point is that the "senile pigment" lipofuscin still accumulates in A.islandica, but only in certain areas of the body, which minimizes its effect on tissue functions. A later study showed that long-lived mollusks are extremely resistant to the action of not only oxidizing agents, but also genotoxic substances with different mechanisms of DNA damage [14]. Such "multistress tolerance" may be a common property of negligibly aging organisms.

Nothing is yet clear about the size and rate of telomere shortening in bivalve mollusks – except that telomerase does not "turn off" in them – and differences in susceptibility to cancer are still associated with polymorphism of the p53 protein gene. Oddly enough, representatives of some species have not recorded a single case of oncopathology, while their closest relatives often die from one of two types of cancer – and die at any age, there is no link between the incidence and aging. It is clear that researchers will pay special attention to the anticancer mechanisms of immune species.

It's even more difficult with other centenarians. The obvious protection from predators in adulthood in large turtles, their low metabolic rate and time–stretched development, the ability to survive for a long time in adverse natural conditions with a shortage of food resources - all this is the tip of the iceberg. The molecular basis has yet to be established. Interestingly, the pronounced telomerase activity in somatic cells is probably characteristic of many, if not all reptiles and amphibians. This can be said even more definitely about fish. This means that cold–blooded people use other mechanisms of protection against cancer than replicative aging, and their telomerase honestly works for growth and regeneration - after all, many species grow until death, while preserving the ability to divide most somatic cells.

Birds and mammals are of particular interest to gerontology, and there are already small advances in the disclosure of their molecular secrets.

Brandt's moth. This insectivorous bat has the most impressive "gap" between body weight and life expectancy among mammals. The genome and transcriptomes of the moth were sequenced in 2013 (by the way, the "read" animals were Russian, and the project coordinator was Vadim Gladyshev). In addition to the expected lifestyle adaptations (echolocation and hibernation), her DNA also revealed features that are currently quite expected for a long-lived person: mutations of growth hormone receptor genes (GHR) and insulin-like growth factor 1 (IGF1R). Previously, GHR dysfunction was shown to be associated with increased resistance to diabetes and cancer in mice and humans, and knockout of the IGF1 receptor daf-2 gene in nematodes was shown to prolong the life of the Caenorhabditis elegans worm. Weak sensitivity to growth hormone leads to low insulin secretion and suppression of "senile" signaling, which should contribute to resistance to diabetes, cancer and aging. But do not forget that growth hormone also performs a lot of useful functions. The project participants believe that it is the changes in metabolism caused by mutations of the GH/IGF1 node and seasonal hibernation, coupled with a low rate of reproduction and a protected habitat, that allow these small bats to live up to almost half a century [15]. However, in a large comparative analysis of the genes of these receptors in different species of bats and rodents, it was not possible to unambiguously link GHR/IGF1R variations with the longevity of bats and diggers. [16]. It is possible that the peculiarities of the evolution of their mitochondrial DNA contribute to the longevity of some bats, which can determine the relatively low production of ROS and resistance to their mutagenic action of mtDNA itself [17].

Birds. The life expectancy of birds varies greatly, but they all surprise not only gerontologists, but also endocrinologists. Like waking bats, birds have a very high metabolic rate (and body temperature), and they live much longer than terrestrial animals with similar dimensions and a moderate "pace of life". It is clear that both groups are better protected from external threats (some birds are also due to high intelligence and sociality), but they should also have internal mechanisms of longevity. Interestingly, the large-scale production of ROS, which accompanies intensive exchange, does not harm birds in any way. This is partly explained, for example, by the special properties of their mitochondrial proteins and DNA and/or the low content of unsaturated fatty acids in cell membranes (the impossibility of large-scale peroxide oxidation of membrane lipids).

But the most surprising thing is that the concentration of glucose in the blood plasma of birds reaches 17 mmol / l. Such an indicator could bring a person to a comatose state, and birds do not have a hint of diabetic symptoms. And although much more needs to be confirmed, the enviable resistance of avian tissues to chronic hyperglycemia is explained by the probable absence of a receptor for glycation end products (RAGE). The binding of these products to RAGE in the vascular endothelium changes the work of signaling systems, activating pro-inflammatory genes and causing rearrangements of the basement membrane with subsequent accumulation of fluid in the tissues. It is assumed that RAGE signaling is involved in the development of not only complications of type II diabetes, but also atherosclerosis, Alzheimer's disease and some types of cancer.

Harmful receptor ligands are actively formed in the blood against a background of high sugar content by non-enzymatic glycation of proteins (Maillard reaction), and the carbonyl group of sugar interacts with amino acids. There is an opinion that birds not only do not have a RAGE receptor, but also the end products of glycation themselves are formed less. It has been shown that their blood contains much more taurine and other free amino acids, wonderful "traps" of reactive carbonyl groups, compared to mammals. In addition, birds produce less of the glycating agent methylglyoxal, which can also interact directly with nerve endings, participating in the development of symptoms of diabetic neuropathy. Some biologists believe that methylglyoxal may be a key mediator in the development of diabetes complications, respectively, and it is possible to combat the latter by suppressing the production of methylglyoxal or purifying the blood from it [18].

Bowhead whale. The findings on the sequencing of the genome and transcriptomes of this potential mammalian longevity champion were published by the de Magalhaens group in 2015. The authors paid special attention to the possible causes of the whale's resistance to age-dependent diseases, including cancer (with an animal weight of up to 100 tons, and hence the most powerful cell proliferation, the mechanisms of cancer control should be simply outstanding!). Comparison with the closest, but not too long-lived relative showed that of the proteins associated with aging and oncogenesis, HDAC1 and HDAC2 histone deacetylases and repair helicase XPB, or ERCC3 (excision repair cross-completion group 3) are the most peculiar in the Bowhead whale. It is known that without the last protein, mice age and die faster. Histone deacetylases are epigenetic regulators involved in the change of chromatin structure, regulation of transcription and cell division; their activity is associated with the longevity of fruit flies.

Biogerontologists were also interested in duplications of genes associated with translation and ubiquitin-proteasome system, mitosis and stress response, and especially duplications with mutations in new copies of PCNA (proliferating cell nuclear antigen) and LAMTOR1 (late endosomal/lysosomal adapter, MAPK and MTOR activator 1) genes. The product of the first gene, a DNA polymerase cofactor, participates in DNA replication and repair, and the second gene participates in the regulation of TOR signaling. The loss of carboxypeptidase A – CPA2 and CPA3 genes can also contribute to the resistance of whales to cancer: CPA polymorphism is associated with an increased risk of prostate cancer in humans. [19].

A number of organisms hardly age or age extremely slowly – even compared to closely related species. The probability of their death does not depend on age: the mortality curve can reach a plateau after puberty. The molecular mechanisms of aging are evolutionarily ancient, which means that the establishment of ways to transfer it to the negligible mode in phylogenetically different long-lived model animals will give a chance for a radical prolongation of active human life and a shift of the cherished plateau to an early age.

The path is unlikely to be simple: it is difficult to find a way to reprogram systems configured for millions of years that are not fraught with serious side effects – this is eloquently evidenced by a number of failed clinical trials of medicines with billions of investments [20]. Therefore, it will be necessary to measure "seven times" on animals: to evaluate changes in key molecular and biochemical processes in different age categories of organisms with negligible aging, to compare their transcriptomes and proteomes with those of the closest short–lived relatives, to identify points of possible interventions, to confirm them by genetic modification of short-lived or traditional model animals - will sensitivity to age-related diseases or aging mode, will there be extremely undesirable effects?

Most likely, at first it will be possible to transfer to a person the results of the fight against certain diseases (metabolic syndrome, diabetes, autoimmune and neurodegenerative pathologies, cancer, atherosclerosis), and only then to take a swing at "normal" aging – at least medically [21]. Nevertheless, encouraging results are already coming from laboratories working with both classical model organisms [22, 23] and aging negligibly. An optimistic part of gerontologists believes that with proper investments in technologies that slow down aging, we can become the last generation with a relatively short life, or even the first with a significantly extended one.

Protection from negative external factors seems to have played a major role in this: antibiotics, vaccinations, availability of resources, protection from enemies and climatic stresses ... (It is clear that not everyone is so lucky, but we are talking about averaged processes.) This could also affect the nature of our evolution. There is an opinion that earlier signs were selected from a person that were useful for experiencing a kind of catastrophic event that happened, and not for a long individual life. But now, having created special conditions for ourselves, we may have already set foot on another evolutionary path – the path of an ageless species [2].

But it's too long to wait for mercy from nature, while you can only rely on your own technology. Major modern projects aimed at correcting aging and related pathologies are based on different, sometimes diametrically opposed ideas.

The developer of the new concept of aging, Michael Rose (Fig. 2, right), has been managing the evolution of laboratory animals since 1977 to this day – but with the help of hundreds of colleagues [24]. This evolution is called experimental. It is completely controlled and allows you to quickly and arbitrarily prolong or shorten the life of members of experimental populations under different conditions, evaluate the genetic and physiological mechanisms by which nature "turns on" or "turns off" aging (makes it negligible), track the side effects of selection on a specific trait. Rose's team managed to double their life expectancy and improve their health indicators by postponing the start of reproduction in a series of generations of fruit flies. These insects were called Methuselah flies. Methuselah's mice are next in line. In the end, according to the pioneers of the direction, the results of experimental evolution should create a solid foundation for engineering interventions in human aging [25].

Methuselah's mice, by the way, are well paid by the founder of the Methuselah Foundation and the M-Prize, Aubrey de Grey (Fig. 2, left). To receive the award, long-lived mice can be bred by breeding and genetic engineering, and you can rejuvenate already adult individuals or even just work worthily in the field of life extension. The prize has been awarded four times already. In particular, Professor Andrzej Bartke was awarded for the record extension of mouse life in 2003. His mouse with the unpretentious and very working nickname GHR-KO 11C (from the already familiar Growth Hormone Receptor) did not last just a week before its fifth anniversary. The knockout of the growth hormone receptor gene allowed her (or rather, him: the male turned out to be the record holder) to live 1819 days [26]. The individual turned out to be dwarfed, with a reduced level of IGF-1, insulin and glucose in the blood and increased resistance to oxidative stress [27]. Such a typical long-lived phenotype!

Aubrey de Grey is the author of the strategy for achieving negligible aging by engineering methods (SENS, Strategies for Engineered Negligible Senescence) – rather ambiguous and doubtful about its speedy implementation and, in general, as extravagant as its author. He believes that the existing level of knowledge about the mechanisms of aging (accumulation of damage) and age-dependent diseases is sufficient for the development of human anti-aging techniques. There is no need to wait for a full understanding of the causes of aging, and in general it is not worth interfering with multiple and intertwined mechanisms – the result is unpredictable. Therefore, it is necessary to focus directly on the regular repair of damage – but before they cause irreparable damage to health. Moreover, according to di Gray, only seven classes of "problems" require correction: 

04.02.2015