Aging and ways to prolong life: taking lessons from yeast
The last decade has significantly increased the level of understanding of aging processes and inspired people with optimism about the imminent appearance of ways to prolong life. A huge contribution to this progress was made by the ordinary baker's yeast Saccharomyces cerevisiae, the study of which clarified many questions concerning the mechanisms of aging of various organisms, including invertebrates and mammals. In addition, the mechanisms identified as a result of these studies have become targets of the most promising geroprotectors currently being developed – anti-aging drugs.Yeast is one of the most important model organisms used in the study of aging. The simplicity of working with yeast and their short life cycle made it possible to identify the molecular mechanisms of aging of these organisms and identify several dozen factors affecting their life expectancy. To date, it is unclear to what extent the data obtained are applicable to humans, however, despite the specificity of a number of aspects of yeast aging, many of the most important mechanisms have been preserved during evolution and are identified, at least in invertebrates and rodents.
Yeast has two aging models: chronological and replicative.
The chronological aging model is applied to postmitotic cells, whose life expectancy is estimated as the period between the moment a cell loses its ability to divide and its death. This model is usually applied to yeast cultivated in a liquid medium, which stops dividing immediately after depletion of available carbon reserves in the medium. The viability of cells in this case is assessed by their ability to resume division when placed in a fresh environment.
Replicative aging is a model of aging of cells actively dividing by mitosis, in which the life expectancy of the mother cell is determined by the number of daughter cells that budded before its entry into the phase of biological aging. In this case, daughter cells that are easily distinguishable in an optical microscope are removed from the culture to estimate the life span.
The main task of studying the aging process is to determine the nature of the damage that stimulates the aging process and the development of age-related diseases.
In the chronological model, an increase in the level of damage within non-dividing cells to a certain threshold value leads to the loss of cells' ability to resume division. At the same time, ethanol accumulates in the culture medium, which is converted into acetic acid that is toxic to cells. In the replicative model, the cause of aging is an asymmetric distribution of damage between the mother and daughter cells. In the process of cell division, extra-chromosomal ring DNA, oxidized cytoplasmic proteins and damaged mitochondria are removed from the daughter cell and moved to the mother cell. However, as a rule, the replicative lifespan of the mother cell ends not much later than the daughter cell stops dividing. This indicates the ability of a healthy maternal cell to cope with most of the age-associated molecular damage. Interestingly, when dividing very old cells, the division asymmetry is disrupted and daughter cells can inherit a significant part of the damage that causes their premature aging.
Despite a number of differences, there are several interesting parallels between the two types of yeast aging. For example, the availability of nutrients has a pronounced effect on both models of aging. In addition, chronological age reduces the replicative lifespan of a yeast cell, regardless of the number of daughter cells produced by it.
Chronological aging and toxic effects of metabolitesUsually, when studying replication aging, yeast is cultured in a synthetic medium containing 2% (0.5% or less when studying the effects of a low-calorie diet) glucose as a carbon source.
The cells convert glucose into ethanol, which gradually accumulates in the culture medium. When glucose reserves are depleted, they begin to break down ethanol through mitochondrial respiration. The reactive oxygen species (ROS) formed in this case damage proteins and mitochondria, accelerating the chronological aging of cells. This hypothesis is confirmed by data according to which the effects that increase the chronological lifespan of yeast simultaneously increase their resistance to oxidative stress.
The transition of cells to the use of ethanol as a secondary carbon source leads to the release of acetic and other organic acids into the culture medium and, accordingly, an increase in the acidity of the medium. Neutralization or removal of acid from the depleted environment, as well as the transfer of cells into water, increases their life expectancy, which indicates the influence of extracellular factors on this indicator. The transfer of postmitotic cells into water containing physiological concentrations of acetic acid (but not other acids) prevents an increase in life expectancy. Thus, acetic acid is a factor necessary and sufficient to accelerate the chronological aging of yeast cells. At the same time, according to existing data, it not only stimulates the synthesis of ROS, but also induces cell death similar to apoptosis. All this is comparable with observations according to which the addition of ethanol to the cultivation medium shortens the life of yeast.The identification of acetic acid as a factor limiting the chronological survival of yeast under standard conditions has called into question the feasibility of using this model to study human aging. However, if acetic acid is a factor that specifically accelerates the chronological aging of yeast, then the destructive effect of ROS and the cellular reactions developing at the same time may well be important for other organisms. In addition, a number of modifications, for example, suppression of the activity of the rapamycin target kinase complex (target of rapamycin, TOR) or deletion of the ribosomal S6 kinase gene, which increase chronological lifespan, also increase the replicative lifespan, as well as the lifespan of invertebrates and even mammals.
In the future, scientists should pay attention to the development of alternative conditions for the chronological aging model, which will allow identifying additional molecular factors affecting the survival of non-dividing yeast cells. The simplest approach is to use a buffer medium that neutralizes the toxic effect of acetic acid. Perhaps the removal of acetic acid as a factor limiting cell survival will increase the value of this model for studying the aging of multicellular eukaryotes.
Asymmetric distribution of damage in the process of replicative agingThe most described type of molecular damage associated with replicative aging is the accumulation of extra-chromosomal ring ribosomal DNA in maternal cells, which are a by-product of ribosomal DNA (rDNA) recombination.
It is known that proteins that modulate rDNA recombination and the formation of extra-chromosomal ring RDNAS also affect the replicative lifespan of yeast.
The mechanism by which extrachromosomal ring RDNAS cause biological aging of yeast cells is unknown. According to one theory, in aging cells, important replication or transcription factors come into physical contact with extra-chromosomal ring RDNAS, which deprives them of the ability to perform their normal functions. Another possible explanation is that the accumulation of DNA encoding ribosomal RNAs (rRNAs) shifts the balance between rRNAs and ribosomal proteins, which disrupts the functioning of chromosomes. Recent data suggest that an excess of ring extra-chromosomal RDNAS can reduce the replicative lifespan of aging cells by inducing rDNA instability. The researchers who received these data claim that the mechanism described by them is the main cause of biological aging of cells, but this hypothesis, like all others, requires convincing evidence.
In addition to extra-chromosomal ring RDNAS, damaged cytoplasmic proteins and mitochondria accumulate asymmetrically in the mother cell, which also contribute to its replicative aging. Segregation of damaged cytoplasmic proteins in the mother cell, carried out by the actin skeleton with the participation of histone deacetylase Sir2, increases the resistance of the daughter cell to oxidative stress. The mitochondrial ATPase component Atp2 is necessary for the preferential distribution of normally functioning mitochondria into the daughter cell. At the same time, damaged mitochondria, which reduce the stability of the genome and reduce the replicative lifespan, move to the mother cell.
Low-calorie diet and mechanisms of longevity preserved during evolutionA low-calorie diet is the most studied of the factors that slow down the aging of a wide range of organisms, ranging from yeast to mammals.
The traditional protocol for reducing the caloric content of the yeast diet is to reduce the glucose concentration in the culture medium from 2% to 0.5% or lower, which increases both the chronological and replicative lifespan of cells. Limiting the availability of amino acids also increases the replicative lifespan of yeast, but the mechanisms of this phenomenon have been studied very little. The literature also describes several genetic models of a low-calorie diet in which the yeast genome contains mutations that reduce the activity of kinases that perform the functions of recognizing nutrients, including protein kinase A, TOR and Sch9 (ribosomal S6 yeast kinase).
A decrease in glucose availability leads to a number of physiological shifts affecting aging processes, including the metabolic transition from fermentation to mitochondrial respiration, increased autophagy, suppression of information RNA translation and increased resistance to stress. Changing the nature of metabolism and eliminating damaged molecules through autophagy increases the chronological lifespan. The mechanisms by which a low-calorie diet increases replicative life expectancy are still the subject of controversy. According to existing hypotheses, activation of Sir2 and inhibition of TOR play a certain role in them.
SirtuinsSirtuin proteins belong to the family of NAD+-dependent protein deacetylases.
The significance of these proteins for aging processes was demonstrated by the results of experiments, according to which deletion of the Sir2 gene reduces, and overexpression increases, the replicative lifespan of yeast. The results of later studies have shown that the activation of orthologs of the Sir2 gene increases the life expectancy of nematodes, fruit flies and mice. Currently, a number of sirtuin activators are at the stages of development and testing on models of various mammalian diseases.
According to the generally accepted hypothesis, Sir2 contributes to an increase in the lifespan of yeast by suppressing the recombination of ribosomal DNA and, accordingly, the formation of extra-chromosomal ring RDNAS. However, this mechanism has no significance for multicellular organisms. This fact indicates that sirtuins perform other functions that ensure longevity that are not related to rDNA. One of these functions, apparently, is the participation of Sir2 in the asymmetric movement of damaged cytoplasmic proteins into the mother cell (Fig. 3).
The expression of Sir2 decreases in replicatively aging cells, which is accompanied by a decrease in the level of histones and their acetylation in the telomere region. Telomere dysfunction is considered to be the cause of cell aging, therefore, the participation of Sir2 in maintaining the stability of telomere chromatin is potentially a factor contributing to an increase in the replicative lifespan of yeast.
There is a fairly large amount of evidence that sirtuins mediate the positive effect of a low-calorie diet on the health and life expectancy of yeast, roundworms and mice. However, scientists have also identified several mechanisms independent of Sir2 that contribute to prolonging the life of yeast and nematodes in a low-calorie diet. At the same time, SIRT1 activity is a mandatory factor in prolonging the life of starving mice. In addition, there is a fairly large amount of evidence for the role of this protein in the regulation of mitochondrial function in conditions of nutrient deficiency. It is obvious that sirtuins play an important role in regulating the lifespan of the body, but their role in the mechanisms triggered by a low-calorie diet is not clear today.
TOR-mediated signaling mechanism and mRNA translationIn recent years, scientists have been paying much attention to the TOR-mediated signaling mechanism as a factor of longevity preserved in the process of evolution and a possible component of the processes triggered by a low-calorie diet.
The TOR kinases identified in a wide range of living species (from yeast to humans) register the levels of growth factors and nutrients in the body and accordingly regulate its growth, metabolism and resistance to stress. The decrease in TOR activity observed in a low-calorie diet prolongs the life of all model organisms.
Studies have shown that inhibition of rRNA recombination and translation of informational RNAs are key components of the TOR-mediated signaling mechanism that prolongs the life of yeast, invertebrates and, according to recent studies, mice (Fig. 3).
There is also a hypothesis according to which the activation of Sir2 occurs when the activity of the TOR-mediated signaling mechanism decreases. This assumption is based on the observation that TOR inhibition activates stress-responsive transcription factors Msn2 and Msn4, which, in turn, stimulate the expression of nicotinamidase Pnc1, which reduces intracellular nicotinamide levels, which can cause activation of Sir2. This hypothesis looks very interesting, but to date there is no convincing evidence of the existence of a relationship between the activity of TOR and Sir2.
New models of working with yeastBudding yeast Saccharomyces cerevisiae is the most popular model for studying the mechanisms of aging, but experiments with other types of yeast also bring valuable results.
The most studied of the alternative models is the dividing yeast Schizosaccharomyces pombe. The study of the mechanisms of chronological aging of this type of yeast, as well as factors affecting this process, including a low-calorie diet and mutations modulating glucose recognition mechanisms, has yielded results similar to those obtained in the study of budding yeast. Scientists have yet to find out whether the toxicity of acetic acid is the main cause of premature biological aging of S.pombe. If this is not the case, then this type of yeast can be a very informative model for studying additional processes that cause oxidative damage and disrupt the mitochondria during aging.
Unlike the division of S.cerevisiae, the division of S.pombe is morphologically symmetrical and leads to the formation of two practically indistinguishable sister cells. However, careful observation of many generations of cells allows morphologically differentiating aging maternal cells and assessing their replicative lifespan. As in S.cerevisiae, when S.pombe divides, as a result of the launch of the Sir2-dependent mechanism, damaged proteins accumulate in the mother cell.
Another interesting model is yeast of the Kluyveromyces lactis species, which breaks down glucose mainly through mitochondrial respiration, rather than alcoholic fermentation, as S.cerevisiae does. A low-calorie diet consisting in reducing the concentration of glucose in the culture medium does not affect the chronological life expectancy of this species. Thus, alternative evolutionary strategies chosen by S.pombe, K.lactis and other yeast species may well provide specialists with new data on the mechanisms of aging.
Directions of future work and prospectsThe results of studying the mechanisms of yeast aging cannot be underestimated.
Some of the most promising longevity factors and potential anti-aging drugs have been identified and characterized for the first time when working with yeast. The ability of rapamycin (rapamycin) and resveratrol (resveratrol) to slow down aging was first demonstrated in yeast. The first drug, unfortunately, is an immunosuppressor and is not suitable for general use, but the second is already undergoing clinical trials as a drug for the treatment of type 2 diabetes mellitus and possibly other age-related diseases. An important task of future research is to separate aspects of aging specific to yeast from mechanisms that have passed the test of evolution and have reached mammals in a little-modified form.
The study of the mechanisms of yeast aging does not lose relevance. Currently, this field continues to develop rapidly due to the emergence of new methods and technologies of systems biology, metabolomics and proteomics. New, constantly improving high-performance methods for assessing life expectancy in the near future will allow not only to study the influence of thousands of genetic variants on the aging process, but also to screen small molecules in order to find new drugs that prevent aging. The recently proposed method for estimating the replicative lifespan, which is based on the selective destruction of daughter cells, rather than micromanipulations carried out under a microscope, can significantly speed up and facilitate work with yeast cells.
The relationship between chronological and replicative lifespan is still one of the unexplored aspects of yeast aging. Despite the fact that the decrease in replicative lifespan caused by chronological aging of cells has been demonstrated for more than a decade, the underlying mechanisms of this phenomenon remain a mystery. Recently, scientists have demonstrated that in addition to reducing the replicative lifespan, chronological aging increases the level of genome instability and leads to the loss of morphological asymmetry of daughter and maternal cells when the last mitotic activity is resumed. Perhaps this phenomenon reflects an aspect of chronological aging masked by cell death induced by acetic acid. Hybrid models that combine chronological and replicative aging will allow us to study the aspects of cell aging in more detail.
A very important task of future research is to study in detail the mechanisms by which various types of molecular damage lead to biological aging of yeast cells. As described above, at least three types of damage (extra-chromosomal ring rDNA, damaged cytoplasmic proteins and defective mitochondria), asymmetrically inherited by the mother cell, accelerate the replicative aging of yeast. It is very important to find out how these damages block the cell's ability to divide, as well as to study the nature of their possible interactions with each other. A detailed study of these mechanisms that disrupt the functioning of aging yeast cells will help to understand the mechanisms of aging of mammalian and, ultimately, human cells.
Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of Nature: Lessons on longevity from budding yeast.
04.05.2010