Aging: telomeres + mitochondria + stem cells + …

The results of the study of hereditary human diseases and their mouse models provided scientists with evidence that the development of aging processes is facilitated by a complex violation of the mechanisms that support the stability of the genome, signaling DNA damage and regulating metabolism. It is believed that the main stimulators of the deterioration of the functional state of tissue stem cells and mitochondria, which reduces the ability of tissues to recover, are the shortening of telomeres and the deterioration of their ability to stabilize chromosomes, as well as the associated activation of the p53 gene, which occurs as the body ages. However, for a detailed study of the role of various factors in the processes of aging and the development of age-related diseases, an experimental model is needed that simulates the interaction of telomeres, stem cells and mitochondria with key molecules, ensuring the maintenance of genome stability, the normal functioning of metabolism and the ability of stem cells to unlimited division and differentiation.

The achievements of medical science over the past century have almost doubled the life expectancy of the population of developed countries. According to statistical estimates, this longevity boom will lead to the fact that by 2025 the population of people over the age of 60 will reach 1.2 billion. To maintain the health and well-being of this growing stratum of the population, a detailed understanding of the molecular mechanisms and biological processes underlying aging and associated diseases is necessary.

Progress in the study of aging mechanisms is hampered by a number of factors, including the variety of options for the onset of tissue deterioration, the complexity and diversity of its phenotypic manifestations, as well as the lack of adequate biomarkers that allow quantifying the "degree" of aging at the molecular and tissue levels. The situation is further complicated by the influence of many external and internal factors on the rate of aging of mammals, such as diet, synthesis of reactive oxygen species (ROS) and telomere shortening. The study of the aging processes of various model organisms (from yeast to primates) revealed a number of important metabolic mechanisms, including those controlled by phosphatidylinositol-3-OH-kinase (PI(3)K), sirtuins and other key metabolic regulators. Mechanisms for maintaining genome stability, the level of genotoxic stress caused by the destruction of telomeres or the formation of an excessive amount of ROS, activation of mechanisms for suppressing tumor growth mediated by proteins p53 and p16, the number and functional state of mitochondria, as well as the balance of the extracellular environment and cytokine profile also play important roles in the aging processes. However, how the interaction of these mechanisms provides acceleration or deceleration of the aging process is largely unclear today.

Recently, there is increasing evidence that telomeres and mechanisms mediated by the p53 gene signaling chromosome damage play a central role in age-related tissue degeneration and a decrease in stem cell reserves through their apoptosis or premature biological aging. The participation of these mechanisms in the processes of functional aging of actively dividing tissues is obvious, whereas their negative effect on low-proliferating, but also rapidly aging tissues (such as the heart and brain) is quite difficult to explain. This article presents a theoretical model demonstrating the mechanisms by which telomere degradation and p53 activation contribute to the disruption of stem cell and mitochondrial functions.

The phenotype of agingA universal manifestation of aging is the deterioration of the ability of organs to maintain a basic level of homeostasis and adequately respond to stress.

Many tissues in middle age are characterized by a gradual moderate deterioration of functions, which significantly accelerates in later years and becomes especially evident in conditions that force the body to overcome strong stress effects by triggering physiological or regenerative processes. Apparently, at the anatomical and physiological levels, the reduced ability of tissues to self-renewal and inadequate regenerative reactions are closely interrelated with classic age-related problems: muscle atrophy, anemia, weakened immunity and slow wound healing.

Due to the total deterioration of the functional state of tissues, old age is the main risk factor for the development of chronic diseases. So, in the USA, about 50% of people over the age of 65 develop diseases of the cardiovascular system, 35% - joint diseases, 15% – type 2 diabetes and 10% – lung diseases. Old age is also a major risk factor for cancer. Thus, in general, diseases of the elderly are the main cause of poor health of people and consume most of the resources of the health system. From this point of view, it is obvious that there is an urgent need to study the nature of aging and develop methods to combat the consequences of deterioration of the physiological state of the body.

Tissue stem cells and age-related diseasesThe human body has an amazing ability to renew itself, provided by stem cells of somatic tissues.

Due to the fact that in an aging organism, especially in actively dividing tissues, violations of regenerative reactions and differentiation of stem cells localized in them are observed (Fig. 1), these cells attract the close attention of researchers. In addition, stem cells of aging tissues are at increased risk of malignancy, which also negatively affects the health of the elderly.

Figure 1. Deterioration of the functional state of aging hematopoietic cellsDespite the fact that aging hematopoietic cells may have an increased ability to self-renew, they are characterized by a reduced functional regenerative ability, which is especially pronounced under stress.

The differentiation program of such cells is usually disrupted, which leads to a decrease in the population of lymphoid progenitor cells, while maintaining the normal number of myeloid progenitor cells. Ultimately, the result of such a differentiation program is a low level of mature T- and B-lymphocytes and an increased level of granulocytes and macrophages in the blood of elderly people. The number of red blood cells and platelets in this case, as a rule, does not suffer. Possible causes of this phenomenon include age-related changes in the composition of the stroma and the cytokine profile of the bone marrow.

Tissues of different types show different levels of proliferative activity and regenerative potential. Stem cells of actively renewing tissues generate a huge number of specialized progenitor cells, which makes it possible to maintain the functionality of these tissues throughout the life of the organism. Identification of stem cells in tissues with low proliferative or regenerative potential, such as the heart and brain, turned out to be more difficult, but their presence in the tissues of most of these organs has been proven. The hypothesis that maintaining an adequate population of tissue stem cells is necessary to preserve the functions of aging organs is confirmed by the results of experiments on transgenic mice with genetic defects that negatively affect the state of stem cells. These models provide some information, but the question of whether quantitative and qualitative degradation of stem cell populations contributes to the deterioration of the health of older people and whether it underlies at least some aspects of aging remains open. In this regard, the analysis of the state of well-described stem cells of three types of tissues with different regenerative profiles, namely hematopoietic, nervous and muscular, is of particular value.

Hematopoiesis systemThe functions of hematopoietic stem cells fade away as the body ages under the influence of cellular factors and microenvironment factors (niches).

This leads to a decrease in the effectiveness of innate immunity (a decrease in the activity of natural killers, the ability of macrophages and neutrophils to phagocytosis and the development of pro-inflammatory status) and an increase in the population of myeloid cells, accompanied by mild or moderate normocytic anemia (not accompanied by a change in the size of erythrocytes). A retrospective analysis of the results of bone marrow transplantation demonstrated the existence of a statistically significant pronounced negative relationship between the age of the donor and the probability of survival of the recipient. These observations fully confirmed the results of experiments on aging mice, in which there was a gradual decrease in the population of functionally competent hematopoietic stem cells and a shift in their differentiation towards myeloid growth.

Nervous systemIn the adult brain (at least in humans and mice), the formation of new neurons continues from neural stem cells capable of migration, localized in the subventricular zone and subgranular zone of the dentate gyrus of the hippocampus.

The results of the work on mice demonstrated a decrease in the activity of neurogenesis in the aging brain, accompanied by a number of functional consequences, including a violation of olfactory function due to a decrease in the number of nerve stem cells of the subventricular zone, providing olfactory epithelium renewal.

The muscular systemThe regenerative potential of skeletal muscles also decreases with age, which is manifested by a decrease in the number of muscle fibers and their replacement with fibrous tissue.

This is based on a decrease in the number of satellite cells (muscle tissue stem cells) and their ability to proliferate and differentiate. The age-related changes in satellite cells are mainly due to the "aging" of the microenvironment, since with parabiosis (surgical connection of the circulatory systems) of two mice, old and young, the effect of the microenvironment of young muscle tissue completely restores the functional abilities of the satellite cells of old muscles.

Molecular mechanisms of agingAnalysis of the state of stem cells of these three types of tissues revealed the role of a number of molecular mechanisms in the aging process.

In the first place is the signaling mechanism mediated by the enzyme phosphatidylinositol-3-OH-kinase, which affects both the rate of aging processes and life expectancy. Violations of the functions of the FOXO family proteins, which are important components of this mechanism, lead to a decrease in the number of tissue stem cells induced by active oxygen forms. Similarly, deletion of the gene encoding the protein TSC1, which suppresses the activity of the mammalian rapamycin target (mTOR), increases the concentration of ROS and profoundly disrupts the functions of hematopoietic stem cells and their mobilization from the bone marrow.

In second place are the mechanisms of DNA repair, the violation of which also negatively affects the state of tissue stem cell populations. Interestingly, various violations of these mechanisms lead to different consequences. For example, defects in excision repair mechanisms (DNA repair by removing nucleotides) lead to the development of progeroid syndromes (premature aging), while violations of the mechanisms of mismatch repair (correction of nucleotide pairing errors) do not accelerate aging, but increase the risk of cancer. DNA damage that disrupts telomere functions also accelerates aging, causing global tissue atrophy and depletion of stem cell reserves in all tested tissues. The decrease in the number of stem cells in this case occurs mainly as a result of p53-mediated cessation of proliferation, premature biological aging and/or apoptosis.

The third place is occupied by molecules of cell death mechanisms, such as tumor suppressor p16, whose increased expression in cells of aging tissues of mice and humans (including hematopoietic and neural stem cells, as well as beta cells of the pancreas) indicates its participation in aging processes.

Finally, mitochondria play a critical role in ensuring the stability of stem cell populations. Mitochondrial disorders lead to an increase in the level of reactive oxygen species, which not only damage cytoplasmic proteins and accelerate the shortening of telomeres, but also trigger a number of molecular mechanisms that disrupt the functioning of stem cells and cause their premature biological aging and death.

In general, experiments on animal models and the study of human tissues have shown that aging is associated with a decrease in the ability of stem cells of various organs to proliferate and differentiate, which does not always affect their number. These changes occur simultaneously with a deterioration in the functioning of organs, a violation of the physiological reactions of the body to stressful effects and an increase in the likelihood of developing diseases. Apparently, a large number of genetic factors and mechanisms affecting the state of stem cells and mitochondria are involved in the aging process.

Telomeres and agingThe end sections of chromosomes – telomeres – are nucleoprotein structures that ensure the stability of chromosomes.

Early experiments on human fibroblasts showed that in order to ensure the ability of cells to divide, it is necessary to restore the length of telomeres carried out by the telomerase enzymatic complex, shortening with each cell division. In culture, after a certain number of divisions, the telomeres of cells reach a critical value (the Hayflick limit), which leads to their biological aging and loss of the ability to divide. In case of resumption of cell division, further degradation of telomeres occurs, resulting in numerous chromosome breakdowns (mainly mergers of their end sections) and cell malignancy.

In further experiments, scientists managed to stabilize the length of fibroblast telomeres and endow cells with the ability to divide indefinitely without the risk of malignancy. To do this, it was sufficient to increase the expression of the catalytic TERT telomerase subunit. The results of these and other experiments on human cell lines and transgenic mice prompted scientists to study the role of telomeres in the aging process and the development of various age-related diseases.

Population studies have demonstrated a correlation between the short telomere length of peripheral blood leukocytes with a high mortality rate of people over the age of 60. A recent large-scale study did not reveal such a relationship, but showed a pronounced positive correlation between the length of telomeres and the duration of a "healthy life" of people. At the same time, recent work involving centenarians and their families has revealed a positive correlation between telomere length and longevity, as well as good health in old age.

Studies have shown that women aged 20-50 years, subject to severe psychological stress, have the shortest telomeres and the lowest levels of telomerase activity in peripheral blood leukocytes, and also demonstrate high levels of oxidative stress. This fact is of great interest, since it is known that individuals living under chronic stress are characterized by shorter life expectancy and early development of age-related diseases. The reason for the rapid shortening of telomeres in this case may be the activation of the autonomic nervous and neuroendocrine systems, followed by the release of glucocorticoid hormones that stimulate the production of ROS.

The results of studying the chromosome features of patients with hereditary degenerative diseases characterized by premature aging of the body, including autosomal dominant congenital dyskeratosis (Zinsser-Engman-Cole syndrome), adult progeria (Werner syndrome) and ataxia-telangiectasia (Louis-Bar syndrome), also indicate an important role belonging to telomeres in the aging process. There is also evidence of the role of telomeres in the development of acquired degenerative diseases. The most illustrative example is cirrhosis of the liver, in which a significant increase in the rate of renewal of hepatocytes is accompanied by progressive shortening of telomeres. As a result, the cells lose their ability to divide and die, which leads to the development of liver failure.

In general, the results of studying a wide range of degenerative human diseases indicate that telomere shortening is a key factor triggering the development of degenerative diseases, increasing the risk of cancer and reducing life expectancy. Based on this, the assessment of various aspects of the state of telomeres can help in predicting the course of various diseases and provide new opportunities for preventive and therapeutic interventions, including the temporary activation of endogenous telomerase.

Mice with the telomerase gene knocked outInitially, the participation of telomeres in the processes of aging and the development of degenerative diseases and cancer was studied in transgenic mice that do not have genes of one of the telomerase subunits, Terc or Tert.

In the first generation (G1), such mice had telomeres of normal length (Fig. 2, above) and at a young age looked almost normal, but as they aged, the symptoms of tissue degeneration developed in them a little faster than in ordinary animals. The telomeres of the second (G2), third (G3) and subsequent generations, which appeared as a result of crossing such mice, were shortened with each generation. Such animals were more likely to have chromosomal abnormalities (Fig. 2, at the bottom, the arrow points to the place of chromosome fusion). 

Figure 2. Chromosomes of transgenic mice with defective telomeraseThe result was a short life expectancy of animals, general weakness of the body, low fertility and tissue atrophy, accompanied by organ dysfunction.

Total tissue atrophy (both actively proliferating and postmitotic) caused the presence in mice of a whole complex of symptoms typical for the elderly, including anemia, leukopenia, kyphosis, osteoporosis, cardiomyopathy and moderate glucose tolerance. The severity of these manifestations correlated with the degree of chromosome dysfunction in successive generations of mice.

The results of a number of studies indicate that telomere dysfunction leads to a decrease in the number and functionality of populations of tissue stem cells and progenitor cells in various tissues, which has a particularly strong effect on the condition of actively proliferating organs. The molecular mechanisms mediating the relationship between telomere disorders and cellular reactions leading to stem cell degeneration are largely unclear today, but there is convincing evidence that the p53 gene plays an important role in this.

P53 tumor growth suppressor gene and genome stability maintenanceThe study of the genetic characteristics of patients with impaired DNA repair mechanisms and experiments on genetically modified mice have demonstrated the exceptional importance of genome integrity in the processes of aging and the development of degenerative diseases.

The important role of the signaling mechanism mediated by the p53 gene and triggered by DNA damage and the subsequent development of degenerative symptoms of aging is obvious based on the almost complete disappearance of such symptoms with the deletion of this gene.

Maintaining genome stabilityHuman genetic diseases characterized by defects in the mechanisms of maintaining the stability of the genome are often associated with premature aging.

In transgenic mice with similar mutations, there is an increase in the amount of DNA damage and a rapid extinction of stem cell populations. Experiments on mice have also shown that forced hyperactivation of signaling mechanisms, usually triggered by DNA damage, also accelerates the degradation of stem cells under normal conditions. Moreover, a number of proteins involved in DNA repair processes, including RAD50, KU70, ATM and WRN, are necessary to maintain telomere stability. Separately, it is worth highlighting the SIRT6 protein, which belongs to the family of sirtuins associated with telomeres. Deletion of the gene of this protein in mice causes telomere dysfunction, accompanied by chromosome fusion. This accelerates the biological aging of cells, causes the development of typical manifestations of aging of the body and shortens the life span of animals to three weeks.

P53 tumor growth suppressor geneThe "defender of the genome" p53 gene is the main cellular stress sensor.

Its expression is activated by DNA damage resulting from telomere dysfunction and exposure to various adverse factors, including reactive oxygen species, hypoxia and oncogen activation. The consequences of triggering the p53-mediated mechanism depend on the degree of gene activation and are manifested by the cessation of cell growth and division, followed by damage repair or premature biological aging of cells and/or their apoptosis. This theory is supported by the results of experiments on transgenic mice, including the fact that the deletion of the p53 gene in mice with critically short telomere length significantly reduces the level of apoptosis and stimulates cell proliferation in many tissues of these animals. In addition, the restoration of p53 functions in mice with defective variants of this gene improves the functioning of various organs, including testicles, intestines, skin and hematopoietic organs. These and other experimental data indicate the existence of a mechanism controlled by the p53 gene in stem cells of various tissues that responds to a critical increase in the level of DNA damage.

Despite the fact that the removal of the p53 gene has a positive effect on the functioning of the stem cells of mice with short telomeres, it does not increase the life expectancy of these animals that die prematurely from malignant diseases. This fact confirms the importance of the p53 gene for maintaining the integrity of the stem cell genome.

Additional evidence for the role of p53 gene activation in aging was obtained by studying two genetically modified mouse lines with hyperactive variants of this gene. Such mice are resistant to malignant diseases, but they age much faster than normal animals. As the tissues of such animals age, the number of cells in the biological aging phase increases. In addition, the hematopoietic cell transplants obtained from them have a reduced ability to engraft and restore the recipient's hematopoiesis. These data are on a par with the results of experiments on numerous mouse models of progeroid syndromes: the removal or dysfunction of the p53 gene in such mice protected them from the symptoms of premature aging. At the same time, transgenic mice with one additional copy of the normal p53 gene had a normal or increased (by a maximum of 16%) life expectancy, while they were characterized by a later appearance of signs of aging, apparently due to low levels of ROS and damage to proteins and lipids caused by them. The complexity of the mechanisms mediated by p53 is also evidenced by the fact that mice with a hypomorphic (poorly expressed) version of the Mdm2 gene, which is the main inhibitor of p53 activity, do not show obvious signs of premature aging under the condition of increased activity of the p53 gene. Finally, it should be noted that inactivation of the p53 gene does not ensure the viability of mice with exceptionally severe telomere dysfunction and pronounced chromosome instability.

Despite the fact that deletion of the p53 gene prevents the development of many symptoms of aging at the cellular and organizational levels, the positive effect of its inactivation on the state of stem cells is negated by the associated increased risk of malignant diseases. Therefore, only a detailed study of the mechanisms of the p53 gene-mediated processes will allow us to develop effective methods to combat aging and prevent malignant diseases.

Genotoxic model of agingAt first glance, genotoxic stress, mainly caused by defective telomeres, is least suitable for the role of the main cause of functional exhaustion of poorly proliferating organs, such as the heart and liver, progressing with age.

However, in later generations of mutant mice with defective telomerase, pronounced cardiomyopathy is observed. Therefore, to study the processes occurring in the heart and other organs with an active metabolism, it is advisable to use a model in which a violation of the mitochondria triggers a cascade of genotoxic damage, which in turn causes activation of the p53 gene, an increase in the concentration of reactive oxygen species and, accordingly, a new round of violations of mitochondrial functions and DNA damage. This downward spiral clearly explains the nature of the increase and aggravation of the symptoms of aging observed in the final stages of life.

The cellular reactions that develop when the p53 gene is activated depend on the concentration of ROS. Under conditions of moderate oxidative stress, activation of p53 predominantly induces the expression of genes that have an antioxidant effect, resulting in a temporary cessation of cell division and restoration of DNA damage. At the same time, at a high concentration of ROS, activation of p53 triggers the work of pro-oxidative genes, which leads to premature biological aging of cells and their apoptosis and/or disruption of mitochondria, leading to tissue atrophy and the extinction of organ functions.

In the proposed model of aging, which is based on genotoxic stress (Fig. 3), the central "axis", represented by telomeres and the p53 gene, integrates well with almost all important of the known genetic elements of the aging process. Firstly, it explains the premature aging of transgenic mice with both defective telomeres and hyperactive variants of the p53 gene. Secondly, it shows the relationship between the absence of SIRT1 or SIRT6 proteins suppressing the activity of the p53 gene and premature aging. Thirdly, it explains the relationship between mitochondria and key factors of aging, such as the proteins PGC-1α, PGC-1β, FOXO and BMI1. Mice lacking one of the genes encoding these proteins are characterized by mitochondrial dysfunction and rapid tissue degeneration.

Figure 3. Model of interaction between DNA damage, p53 gene activation and mitochondrial disordersIn this model, genotoxic stress caused by telomere degeneration, disruption of DNA repair mechanisms, exposure to ultraviolet and ionizing radiation, chemical compounds, ROS and other mechanisms activates p53 and induces the termination of cell division, their premature biological aging and apoptosis.

It is possible that p53 directly or indirectly disrupts the mitochondria, which triggers a continuous cycle of DNA damage, activation of p53, disruption of mitochondria and an increase in the level of ROS, causing additional DNA damage. Disorders of mitochondria negatively affect the functionality of various organs by reducing the amount of ATP produced by them and changes in metabolism. The model also reflects the interaction between p53 and a low-calorie diet, sirtuins and other mechanisms and factors involved in the aging process.

The proposed model of aging emphasizes the need for a more complete understanding of the factors affecting the degradation of telomeres, the functions of genes protecting cells from ROS-induced damage, signals controlling the activity of p53, and mechanisms ensuring the stability of mitochondrial reserves and functions. It is also necessary to find out how the differential activation of these various mechanisms is carried out in the aging process and how they are interfaced with each other. Decoding this complex biological system will allow identifying biomarkers of aging and developing effective therapeutic strategies aimed at rejuvenation of both actively proliferating and postmitotic tissues of the elderly.

Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of Nature: Ergün Sahin & Ronald A. DePinho, Linking functional decline of telomeres, mitochondria and stem cells during aging.

18.05.2010