Senile vagaries of nature

Olga Volkova, "Biomolecule" 

Birth, growth, aging, death – we perceive this sequence of events as absolutely natural and inevitable for at least all animals. But are their paths parallel to the end point, and do everyone reach it? These questions have always interested philosophers and naturalists, but it has become possible to solve them on a scientific platform only recently: firstly, biological methodology has tightened up, secondly, long-term observations of animals in protected conditions (laboratories, zoos) and lost natural corners have begun to yield results, and finally, a public request for the extension of active life sounded louder. Let's look at how our ideas about aging have changed, how some theories try to explain aging and its neglect, and why it is so necessary for a person to "disassemble" ageless animals to molecules.

Aging is usually defined as a natural process of gradual disruption and loss of important body functions, especially the ability to reproduce and regenerate. As a result, the ability to adapt to environmental conditions, resist predators and diseases decreases. However, research in recent years has cast doubt on the universality of this law: it seems that it does not apply to a number of living organisms (in more detail, this phenomenon, called negligible or insignificant aging, and its happy "owners" will be considered in a separate article), and not everything is so unambiguous with human aging. Actually, there was no complete unanimity about the causes, "goals" and mechanisms of aging before, and there is no now: there are many theories of aging and approaches to its study at different levels – evolutionary, population, organismic, cellular, molecular [1]. Some of them consider aging as a programmed, predetermined process, "intentionally" (from the point of view of evolutionary expediency) working to clear space for new generations – that is, evolutionary plasticine – others – as a quasi-program (imitation of a program that works "without malicious intent"), others – as a confluence of unfavorable circumstances, a failure in the operation of protective systems, the accumulation of all kinds of damage.

That is, it corresponds to the generally recognized "law of mortality" of B. Gompertz, who for almost two centuries claimed that the probability of a multiple organism (and this is the majority of animals) to die increases exponentially over the years. Individual reports of a slowdown in the mortality rate of Europeans in old age were attributed to health successes or a decrease in mortality from non-permanent causes such as wars and epidemics [2].

But in 1992, two studies at once recorded a plateau at a late age on the mortality curve of Ceratitis and Drosophila flies, which could not be suspected of caring for elderly brethren. Later, a slowdown in mortality rates up to the plateau was found in other aging organisms: yeast, nematodes, wasps – and confirmed in humans. The rate of aging during this period, apparently, is also negligible. Thus, at the end of the XX century, the conditionality of Gompertz's law appeared: apparently, it is unconditionally valid only for middle-aged animals. During the newborn period, there is a peak on the mortality curve (due to immaturity of immune mechanisms, cannibalism and maternal negligence), and at a late age - a plateau (the probability of not living to the next year is high, but it is no longer growing and even decreases slightly). Of course, Gompertz could not even imagine any plateaus, because a large sample of 100-year–old people is needed to fix them - a condition impossible for the beginning of the XIX century due to high mortality from accidental causes – infections, conflicts, etc.

What has changed in the understanding of aging since 1992? A simple concept (the gradual accumulation of physiological defects) was replaced by a more complex one and therefore not accepted by everyone. Apparently, the phenomenon of late inhibition of aging is not a population artifact, but a pattern that manifests itself at the individual level (Fig. 1). There is no scientific evidence that any physiological deterioration associated with aging steadily progresses until death. The luminary of the evolutionary biology of aging, Michael Rose, and his numerous like–minded people believe that aging is not necessarily a cumulative and indomitable process of simple physiological wear and tear, but a multifaceted phenomenon in which physiological changes – as a particular manifestation of it - are directed and restrained by the dictate of adaptation (hence, natural selection). Within the framework of the evolutionary approach, aging is an adaptation disorder in the first half of maturity. After the completion of a gradual decrease in the power of natural selection (which is understandably active during the reproductive period), aging stops, adaptive capabilities stabilize, albeit at a lower level. Selection is indifferent to the genetic effects manifested in old age, since they did not affect the well-being of the population throughout its evolutionary history [2].

Some of them may work to maintain life and productivity at a later age "accidentally" – because of age-independent benefits. The role here can be played by alleles that are "beneficial" at the reproductive age and therefore fixed by selection, but according to the principle of multiple action positively affect survival in the post-productive period (protagonist pleiotropy, according to de Grey) [3]. But antagonistic pleiotropy (M. Rose's term) can contribute to aging – the effects of the work of genes that provide advantages during the breeding season, but harmful after – for the individual, but not the species. There are few confirmed examples of such a kind of reckoning for past success: in bacteria it is sigma factor σ70, in eukaryotes it is telomerase. Traditionally, they included the tumor suppressor protein p53, but recently data have accumulated on the benefits of p53 signaling at any age (see below, "Longevity of short–lived"). On the other hand, the whole theory of quasi–programmed aging - TOR-centric (from the name of the kinase FK506 binding protein 12–rapamycin associated protein 1, FRAP1) - is based on the positive role of TOR signaling during the development of the organism and its reverse role in the future: when the body components no longer need to grow, intracellular hypersignalization in response to food intake (insulin and amino acid reception) leads to aging and organ damage. The effect of antagonistic pleiotropy is especially clearly visible here for men [4]. However, other population-genetic mechanisms have been proposed to explain aging.

In the light of a new concept (or, more precisely, a new understanding) that considers aging as a complex set of adaptations, the physiology of an elderly organism should decently differ from the physiology of an aging organism. This, of course, introduces difficulties in the design of aging studies, but it is unproductive to neglect the peculiarity of "late life" responsible for the plateau on the mortality curve. After all, the understanding of the mechanisms that form this plateau in humans is around 100 years old, and in some animals at a young age (Fig. 2), opens up prospects for shifting the point of aging inhibition during the period of life when a person is in optimal condition*. There is an opinion that with the probability of death characteristic of a ten-year-old person, we could live up to 1000 years [5].

Until the age of 100, she rode a bicycle, fenced and played tennis, indulging in a couple of cigarettes a day and a kilogram of good chocolate a week. In some interviews, she attributed her longevity to the fact that she was a parasite all her life and did pleasant things – playing the piano and painting. (And it's true, after all – the daily washing of nerves by the boss at an unloved job hardly prolongs life ...)

However, nature occasionally "throws up" phenomena that are more difficult to explain, independently, without the efforts of scientists, shifting the regime of neglect of aging to an early age. Unfortunately, nature has not finalized the strategy yet... The case of Brooke Megan Greenberg, known as "the girl who doesn't age," is particularly revealing. Throughout her short (20-year-old) life, she suffered from severe manifestations of asynchronous slowing down of the development of body systems: the only thing that somehow corresponded to her chronological age was the indicators of the "telomeres-telomerase" system. At the age of 16, the condition of her teeth (milk!) and bones corresponded to the age of 8-10 years, the development of brain structures, endocrine functions and anthropometric indicators corresponded to infancy (Fig. 3, right), and elements of the respiratory and digestive systems could not develop smoothly at all. Brooke never learned to talk or swallow food. Anyway, her family claims that they got a lot of joy from communicating with the girl, and biologists had a unique opportunity to establish the mechanisms of aging of ordinary people by understanding the causes of Brooke's non-aging [6]. Thorough studies of her genome have not revealed any known "profile" mutations, and the disease has so far remained "syndrome X". No less than the possibility of clarifying the eternal question of the existence of the aging program (as an integral part of the development program), scientists were intrigued by the episode of the sudden appearance and spontaneous disappearance of a brain tumor in a girl.

Obviously, some research can be continued after Brooke's death in 2013 due to the defective work of the bronchi. Moreover, at the University of California they work with seven more girls (one of them, however, is already 25) with "syndrome X" of different "purity". It has already been established, in particular, that the epigenetic age of their blood is quite consistent with the chronological age (the level of DNA methylation is still considered the most reliable age marker). Perhaps this is not typical for all their tissues, but at least one is still aging [7].

Initially, each of them claimed to be universal, but now they got along well within the framework of one coherent concept – the network. The complex of damages accumulating over the years includes: free radical oxidation of biomolecules, mutations of chromosomal and mitochondrial DNA, epigenetic disorders, malfunctions in the work of chaperones and proteasomes, and in general products of "noise" (inaccuracies) of all biological processes – from gene expression to enzymatic reactions [8]. Thus, the damage is a by–product of the normal vital activity of the cell, but their spectrum and number in an individual are not predetermined. Although all the described damages are applicable to any cell, the intensity of their accumulation can vary depending on the type of tissue and change over time, and the accumulation of some defects can trigger a cascade of others (Fig. 4).

But the tendency to accumulate is just less significant damage, to prevent and "repair" which, in a particular biological species, nature may consider an excessively expensive undertaking – at least according to the theory of disposable catfish. The latter, in its modernized form, complements the concept of damage accumulation quite well, although it is far from being accepted unconditionally, in particular, by supporters of the TOR-centric theory of aging [9] – despite the fact that even there we are talking about payback, but for success in youth. Since the body's resources are limited, they need to be reasonably distributed between the functions of maintaining and repairing the body, growth, reproduction, etc. It is reasonable – from the point of view of the survival of the population in a specific ecological niche: if most wild mice die in the first year of life from hypothermia (and nature is satisfied with this situation), then why should they invest in the development of expensive, but useless for 90% of the population mechanisms of protection against cancer, and not in reproduction? The body's protection from climatic shocks, hunger and predators, on the contrary, works to maintain the body and increase life expectancy – counting on repeated stable reproduction in the future. And indeed, there are many centenarians among flying, shell-like (shell-like), underground and relatively large-brained animals. Thus, ecological and economic expediency can determine the choice between strategies for species survival and strategies for combating damage accumulation, and consequently aging (Fig. 5).

The availability of food is the most important variable in nature. Short-term periods of fasting should cause a redistribution of resources from reproduction to body maintenance. And it is not for nothing that insulin signaling has become one of the central mechanisms of regulating life expectancy. The plasticity of the regulation of the life span of individuals within the same species is illustrated by the example of different destinies of summer and winter bees. Even more interesting are the variations in the biography of one individual: in conditions of crowding (lack of resources), C. elegans nematodes form dower larvae - stress–resistant forms that can live 10 times longer (until better times) than an ordinary worm. The switch of development modes is the "environment detector" – the daf-2 gene of the insulin-like growth factor 1 receptor (IGF-1), acting through an intermediary of FOXO proteins – Daf16, a regulator of hundreds of genes associated with the functions of maintaining the body: response to stress, protein metabolism, resistance to microbes, etc. [10]. By the way, in adult worms, the activity of the daf-2 gene, on the contrary, promotes aging.

Harvard University professor V. Gladyshev believes that evolutionarily, the first strategy to combat moderate damage was simple dilution by cell division: like other cellular contents, damage is distributed between two daughter cells, which reduces the load on each of them. This strategy was developed by prokaryotes, stem cells are used, and cancer cells have also returned to it. The emergence of multicellular postmitotic cells in the process of evolution required a redistribution of investments in the development of systems for the prevention and elimination of damage, of which, however, there are an inexhaustible multitude. The price of precision control against all is unaffordable, constant dilution by division in differentiated cells is impossible – systems of strict control of division and suppression of tumors preserve the architecture of tissues and protect against cancer (the most famous "controller" is p53). Apoptosis is an ideal solution for severely damaged cells, and especially for stem cells endowed with the greatest responsibility of actively renewing tissues. But less traumatized, but "unreliable" cells are cheaper to "arrest" – to deny the possibility of division: temporarily, until the damage is repaired, or forever, condemning them to gradual transformation into deeply senescent cells [8]. The latter are real masters of "SUSPense" (SASP, senescence–associated secretory phenotype): they secrete substances that destroy the extracellular matrix (proteases), attract immunocytes (cytokines) and cause neighboring cells to divide or also age (growth factors), and all this in an altruistic attempt to withdraw with the help of the immune system and release a place for healthy cells. But the immune system is also not free from age–related dysfunction, the result is a pro-inflammatory microenvironment, a violation of tissue architecture and the risk of oncogenesis. (The ambiguity of the role of cellular aging in the process of development, maintenance and aging of the body, as well as the issues of cleaning the body of old cells are discussed in detail and clearly in the review [11].)

Thus, at the cellular level in each tissue, the issues of expediency of large–scale cell protection are constantly being resolved, and for those already damaged – survival (then the risk of cancer), aging and death [10, 12]. The accumulation of cellular damage (cellular aging) entails immune changes (for example, in the profile of cytokines and growth factors) with the development of chronic inflammation, which stimulates the accumulation of damage – the vicious circle closes (Fig. 4) [13].

Malignant tumor cells get rid of expensive cycle blocking options, which are also directly or indirectly "broken" – otherwise they would not allow transformation – and return to primitive dilution by division. DNA mutations in cancer cells lead to a distortion of metabolism and a more intense accumulation of damage, and in order to survive, cells must divide faster and faster. Not surprisingly, cancer is closely related to aging. Although aging is sometimes considered as a kind of payment for turning off the mechanisms leading to malignancy [12], cancer is essentially a disease of aging: the damage that accumulates with age is removed by protective mechanisms (apoptosis, immune system, etc.), until the violation (aging) of them will not allow some cells to rebuild metabolism and get out ofunder control [8].

But if overload by damage is considered the cause (or one of the reasons) aging, then life expectancy can be regulated by metabolic reprogramming – interventions that change the rate of damage accumulation, and most importantly, their profile. It is the landscape of damage (and its dynamics) that can explain paradoxical natural examples of longevity against a background of pronounced oxidative stress – for example, in birds. No specific type of damage reflects the full picture, and interventions that prolong the life of model animals often increase the production of reactive oxygen species (ROS), but by changing the damage profile, they activate protection mechanisms against oxidative stress and other malfunctions. Often such a phenomenon is interpreted as hormesis – stimulation of resistance with small doses of stressors [8].

Calorie rating systems can be deceived, for example, by suppressing signaling pathways that control cell growth (TOR and insulin/IGF-1), then the death of animals is postponed without starvation, although aging does not switch to negligible mode.

As for positive regulators, in a recent article by A. Moskalev's team [14] one can find an impressive list of alleged "longevity genes" of drosophila having human orthologs, and critical remarks about the staging of experiments in which the role of these genes was determined. Drosophila, in particular, benefits from the overexpression of many genes of metabolic regulation pathways (IGF-1R, PI3K, PKB, AMPK, TOR) and stress response (FOXO, HDAC, p53). Here it should be borne in mind that overexpression of some genes of the pathway may lead not to increased signaling along it, but to suppression, and it is blocking the transmission of information along some paths (the same TOR, for example) capable of prolonging life. The activity of the supposed "longevity genes" is aimed at preventing or repairing damage caused by physiological and environmental factors, and as a result – at increasing the resistance of cells to stress. Moreover, the work of stress response systems (Sir2, FOXO, JNK) can turn off life-shortening metabolic genes and affect other mechanisms that determine longevity: reparative, epigenetic, "cleaning" (neutralization of toxins and free radicals, proteolysis, autophagy).

A group of biologists from Columbia University (New York) emphasizes the importance of the normal functioning of transcription factors FOXO and p53 for prolonging life, although according to traditional ideas, cell cycle arrest and apoptosis provoked by them lead to cellular aging and the formation of senile phenotype (pathologies associated with the termination of stem cell division and tissue degeneration) [15]. But facts are a stubborn thing. A decrease in p53 activity in aging mice correlates with an increase in the incidence of tumors and a decrease in life expectancy in general. Conversely, an increased amount of this protein (and another cell cycle controller, ARF Ink4a, which we will discuss in an article about naked and not very diggers) protects against cancer – which in itself prolongs the life of mice – and oxidative damage, postpones aging. The ability of this protein to situationally suppress TOR signaling may play an important role in this effect of p53 on life expectancy (Fig. 6). TOR kinase (or mTOR, "target of rapamycin in mammals") integrates the overlying signaling branches (insulin, growth factors, amino acids), "senses" the level of nutrients, oxygen and the energy in the cell, directs the exchange along the anabolic path. TOR hyperactivity is associated with the development of type II diabetes, obesity, cancer, depression; the immunosuppressant rapamycin inhibits TOR signaling. Factor p53 can fight not only the work of TOR, but also its consequences by increasing the expression of antioxidant genes necessary for the capture of ROS – by-products of intensive metabolism. As one of the main targets for anti–aging drugs – geroprotectors – the authors of the article [15] consider the antagonist of FOXO and p53 and at the same time the "accomplice" of TOR - ubiquitin ligase Mdm2.

Another link of the scheme shown in Figure 6, PI3K kinase, is delicately suppressed by the American gerontologist Robert Schmuckler, who created the famous long–lived worms, Fig. In the group of nematodes C. elegans, by an order of magnitude (!) surviving relatives, Rice "turned off" the age-1 gene (aka - daf-23), encoding just PI3K [16]. The effect of such a knockout is associated with the suppression of signaling along the insulin/IGF-1/FOXO pathway, which merges with PI3K/AKT/mTOR at the "PI3K" point, by Rice and scientists who previously doubled the life span of nematodes by disabling the daf-2 gene (homologue of human insulin and IGF receptor genes). Insulin/IGF-1 reception is essentially one of the "peripheries" of the system that regulates metabolism and stress response (catabolism/anabolism balance, growth, division, apoptosis, etc.) depending on food and other conditions. By the way, PI3K hyperactivity provokes the development of oncological diseases, because inhibitors of this enzyme are already being created. A detailed description of the discovery of the insulin/IGF-1/FOXO pathway and the life-prolonging C. elegans mutations can be found in the article of a direct participant in the events, Cynthia Kenyon [17].

For example, the glycolysis enzyme GAPDH, in addition to energy metabolism, participates in DNA repair and regulation of apoptosis. The malate dehydrogenase (men) gene is involved in the regulation of energy, lipid, carbohydrate metabolism, ROS levels, activation of protection systems against oxidative stress. The effects of this gene, by the way, fall well under the description of hormesis: overexpression of men in drosophila larvae leads to oxidative stress, but shifts the metabolic balance towards catabolism and activates a number of protective mechanisms that overcome stress at a late age and postpone death [14].

An equally important problem is the transfer of results from laboratory animals, usually small and with a short life, to humans. Although the mechanisms of aging have been formed in yeast, and many regulatory pathways that control the lifespan of model animals are conservative, we know that the determinants of longevity were not selected during the evolution of these species. Human defense mechanisms are much more effective. Moreover, experiments with each model suggest serious methodological limitations: knockouts are possible in some organisms, hyperexpression in others, etc. Nevertheless, these animals gave us basic knowledge about the life and death of cells and directed the biogerontological search in the right direction. For example, studies comparing the dynamics of gene expression associated with life expectancy in humans and laboratory animals are interesting. And here it is important to find the connection of expression not only with aging, but also with its transition to the phase of neglect: we remember that in old age, many small "martyrs of science" have a mortality curve that also reaches a plateau.

Yes, and the tissues age in different ways... Research teams rarely, but still resort to large-scale comparisons of transcriptomes of people of different ages and classical model organisms.

Interesting data were presented in 2006 by J. Zan and co-authors [18]. Comparing the age dynamics of gene expression in human muscles, kidneys and brain, they identified six genetic pathways that change activity over the years: the expression of four of them increased (genes of extracellular matrix components, complement activation factors, components of cytosolic ribosomes and genes associated with cell growth), and two decreased (genes of components of mitochondrial electron transport chains (ETC) and transporters of chloride ions). A comparison of this "age signature" of a person with data on mice and fruit flies showed that only one pathway changes activity during life in all three organisms, and in the same direction and pace – the pathway encoding proteins of ETC.

However, this fact does not mean at all that a decrease in the production of ETC proteins is a universal cause of aging. The fact is that in another popular model animal, the worm C. elegans, blocking the synthesis of components of the ETC decently prolongs life. Therefore, the authors of the work believe that a decrease in the activity of this genetic pathway, on the contrary, may contribute to human longevity or be a manifestation of antagonistic pleiotropy. And most importantly, it can be considered as one of the common markers of aging for animals. Another very important conclusion (based not only on the results of this work): the age-dependent expression pattern of almost all human genes is species-specific, that is, radically different from that of worms, flies and mice. This is one of the reasons why short-lived organisms cannot be considered ideal models for studying the mechanisms of "normal" aging. However, awareness of this fact in itself would have done little. If not...

If at the beginning of the XXI century they had not paid attention to the hairless rat-like inhabitants of the dungeon with an unprecedented life expectancy for rodents who do not suffer from cancer, atherosclerosis and much more. And most importantly, the cherished plateau on the curve of their mortality began already in their youth (Fig. 2). What is not a worthy object for analyzing the mechanisms of longevity and negligible aging? And in addition to this small mammal, called the naked digger, there are already a lot of centenarians with delayed aging (or without it at all) in different classes and kingdoms. But they will be discussed in a separate article.

There are hundreds of hypotheses and theories explaining the causes of aging and its mechanisms, and so far the scientific community has not chosen the most plausible of them. Integrating the ideas of some reputable biogerontologists about the molecular mechanisms of aging, we can assume the following sequence of events: the accumulation of heterogeneous damage to cellular structures and certain patterns of signaling systems lead to blocking cell division or their death (which protects the body from cancer until the protective mechanisms themselves are damaged), disruption of the ability of tissues to regenerate and trigger inflammatory processes, as a result, it further accelerates the accumulation of damage and aging. The schemes of systems regulating cellular metabolism and response to stress, DNA repair and disposal of damaged structures are confusing and intersect at many points. The task facing biogerontologists is to establish in working with long–lived animals the most promising points of influence on the mechanisms that determine life expectancy. Well, then check everything on classical models, at the same time confirming or refuting some of the hypotheses of aging.

29.01.2015