Teaching the cell biology of aging at the Harbin Institute of Technology and Moscow State University

Khokhlov A.N.

The history of gerontological experiments on cell cultures is reviewed. Cytogerontological studies and aging theories by Weismann, Carrel, Hayflick, and the author are compared. It is emphasized that the basic notion of aging mechanisms was deeply revised several times within the 20th century. It is concluded that at present the aging of multicellular organisms cannot be satisfactorily explained with the help of cytogerontological studie’s data. Experiments on cell cultures need to be combined with fundamental gerontological studies, including survival curve analysis for humans or experimental animals.

Khokhlov A.N.

According to author’s concept, the aging process is caused by cell proliferation restriction-induced accumulation of various macromolecular defects (mainly DNA damage) in cells of an organism or cell population. In case of cell cultures, this proliferation restriction is related to either so-called contact inhibition or Hayflick’s limit, and in case of multicellular organisms, to the appearance, in the process of differentiation, of organs and tissues consisting of postmitotic or very slowly dividing cells. Cell proliferation is absolutely incompatible with normal functioning of a macroorganism. Thus, the development program automatically leads to a situation inducing the aging process (martaliry rates increase with age). Therefore, any special program of aging simply becomes senseless. This, however, does not reject for some organisms the reasonability of programmed death, which makes possible the elimination of harmful, from the species’s point of view, individuals. It is also very important to understand that increase or decrease of organism’s life span under the action of various factors are not necessarily related to a modification of the aging process, even though the experimental results in this field are interpreted just in this way.

Khokhlov A.N.

For the most part, research in the area of cytogerontology, i.e., investigation of the mechanisms of aging in the experiments on cultured cells, is carried out using the “Hayflick's model”. More than forty years have passed since the appearance of that model, and during this period of time, very much data were obtained on its basis. These data contributed significantly to our knowledge of the behavior of both animal and human cultured cells. Specifically, we already know of the mechanisms underlying the aging in vitro. On the other hand, in my opinion, little has changed in our knowledge of the aging of the whole organism. In all likelihood, this can be explained by that the Hayflick's model is, like many others used in the experimental gerontology, correlative, i.e. based on a number of detected correlations. In the case of Hayflick's model, these are correlations between the mitotic potential of cells (cell population doubling potential) and some “gerontological” parameters and indices: species life-span, donor age, evidence of progeroid syndromes, etc., as well as various changes of normal (diploid) cells during long-term cultivation and during aging of the organism. It is, however, well known that very frequently a good correlation has nothing to do with the essence (gist) of the phenomenon. For example, we do know that the amount of gray hair correlates quite well with the age of an individual but is in no way related to the mechanisms of his/her aging and probability of death. In this case, the absence of cause-effect relationships is evident, which are, at the same time, indispensable for the development of gist models. These models, as distinct from the correlative ones, are based on a certain concept of aging. In the case of Hayflick's model, such a concept is absent: we cannot explain, using the “Hayflick's limit,” why our organism ages. This conclusion was convincingly confirmed by the discovery of telomere mechanism which determines the aging of cellsin vitro. That discovery initiated the appearance of theories attempting to explain the process of aging in vivo also on its basis. However, it has become clear that the mechanisms of aging of the entire organism, located, apparently, in its postmitotic cells, such as neurons or cardiomyocytes, cannot be explained in the framework of this approach. Hence, we believe that it is essential to develop “gist” models of aging using cultured cells. The mechanisms of cell aging in such models should be similar to the mechanisms of cell aging in the entire organism. Our “stationary phase aging” model could be one of such models, which is based on the assumption of the leading role of cell proliferation restriction in the processes of aging. We assume that the accumulation of “senile” damage is caused by the restriction of cell proliferation either due to the formation of differentiated cell populations during development (in vivo) or to the existence of saturation density phenomenon (in vitro). Cell proliferation changes themselves do not induce aging, they only lead to the accumulation of macromolecular defects, which, in turn, lead to the deterioration of tissues, organs, and, eventually, of the entire organism, increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of various types of damage identical to the damage arising in senescing multicellular organism. And, finally, it is essential to determine how the cell is dying and what the death of the cell is. These definitions will help to draw real parallels between the “genuine” aging of cells (i.e., increasing probability of their death with “age”) and the aging of multicellular organisms.

Zhang D., Lai M.C., Constable I.J., Rakoczy P.E.

This work aims to investigate the effect of compromised lysosomal enzyme activity on the accumulation of photoreceptor-derived debris in the retinal pigment epithelial (RPE) cells and to examine if this accelerated debris accumulation can induce retinal abnormalities similar to those observed in aged individuals. A mutated, enzymatically inactive form of cathepsin D (CatD), generated by site-directed mutagenesis was used to produce stable cell lines and transgenic mice. There was a strong increase in enzymatically inactive CatD protein production in the mutated CatD DNA transfected D407 cells (D407MCD). The presence of the inactive CatD has been linked to an impairment in bovine rod outer segment(BROS) digestion and was confirmed by astatistically significant increase of undigested residual BROS in the medium ofD407MCD when compared to the control vector-transfected D407 cells (t-test,P ≤ 0.016, P ≤ 0.003) or untransfectedD407 cells (t-test, P ≤ 0.008,P ≤ 0.003). The impairment was also confirmed in vivo by demonstration of BROS-derived debris accumulation in the RPE cell layer of transgenic mice. These results demonstrated that the mutated and inactive CatD form could lead to impairment of photo receptorouter segments (POS) proteolysis. It is proposed that this initial impairment of POS proteolysis may result in the accumulation of CatD-opsin-like complexes in the pigment epithelium, which further compromises RPE cell functions and thus causes the changes observed in aging humans.

Cristofalo V.J., Allen R.G., Pignolo R.J., Martin B.G., Beck J.C.

Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared "healthy," according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.

Hayflick L.

The finite in vitro lifetime of cultured normal cells is interpreted to be aging at the cellular level. In addition to the inverse relationship between donor age and population doubling potential (PDP), a number of biochemical and physiological increments and decrements occur prior to the cessation of cell division. The reconstruction of replicating normal human cells from the nuclei of "young" cells and the cytoplasm of "old" cells (and the reverse) suggests that the nucleus governs PDP. Several morphological changes were found to occur in late phase III cells held for up to one year in culture. Autoradiography studies show that (1) a cell population may be composed of several subpopulations, each of which is at a different stage in its life history and (2) lipid synthesis is affected much less as cells age than is DNA, RNA and protein synthesis. Changes occurring in the genetic program of individual cells seem to be the most tenable hypothesis to explain fundamental causes of aging.

Khokhlov A.N.

The history of the creation of the course of lectures “Basics of the Biology of Aging” at the School of Biology of Lomonosov Moscow State University, as well as at the Department of Life Science and Engineering of Harbin Institute of Technology is brie y described. In the process of teaching this course, the author got the impression that its main provisions may also be of interest to students of non-biological specialties, who have recently been quite often involved in the work on the implementation of gerontological grants. This is largely determined, apparently, by the signi cantly increased funding for this kind of research in recent years. In turn, this is a consequence of the fact that the average life span of people in developed countries has increased dramatically over the past decades. However, the maximum life span has not changed much (it is now the same as it was thousands of years ago, it is just that the chances of living to the age of a centenarian have become much greater). If earlier people often died at an early age from various diseases not related to age (mainly infectious diseases), now, due to signi cant advances in medicine, most people live to old age. As a result, death “from aging” is becoming more common. At the same time, many people have a very vague idea of what aging is, what are its mechanisms and how to ght it. In this regard, in 2022, an interschool elective course of lectures was organized at MSU for students of any departments of the university, except for the School of Biology itself. It is called “Basics of the biology of aging, or everything you wanted to know about aging (but were afraid to ask).” The material of the lectures was specially adapted for students who are not biologists by their main specialty. The main emphasis in the course, consisting of 12 lectures, is made on the fundamental de nitions and methodical/methodological approaches used in gerontology. The article lists the questions submitted for the students’ test and brie y analyzes its results.

Khokhlov A.N.

The history of the creation of the course of lectures “Basics of the Biology of Aging” at the School of Biology of Moscow State University, as well as at the Department of Life Science and Engineering of Harbin Institute of Technology, is briefly described. In the process of teaching this course, the author got the impression that its main provisions may also be of interest to students of non-biological specialties who have recently been quite often involved in the work on the implementation of gerontological grants. This is largely determined, apparently, by the significantly increased funding for this kind of research in recent years. In turn, this is a consequence of the fact that the average life span of people in developed countries has increased dramatically over the past decades. However, the maximum life span has not changed much (it is now the same as it was thousands of years ago, it is just that the chances of living to the age of a centenarian have become much greater). If earlier people often died at an early age from various diseases not related to age (mainly infectious diseases), now, due to significant advances in medicine, most people live to old age. As a result, death “from aging” is becoming more common. At the same time, many people have a very vague idea of what aging is, what are its mechanisms and how to fight it. In this regard, in 2022, an interschool elective course of lectures was organized at MSU for students of any schools of the university, except for the School of Biology itself. It is called “Basics of the Biology of Aging or Everything You Wanted to Know about Aging (but Were Afraid to Ask).” The material of the lectures was specially adapted for students who are not biologists by their main specialty. The main emphasis in the course, consisting of 12 lectures, is made on the fundamental definitions and methodical/methodological approaches used in gerontology. The article lists the questions submitted for the students’ test and briefly analyzes its results.

Khokhlov A.N.

The author’s view on the current state of gerontological research is presented. He believes that the widespread departure from the principles of classical gerontology, formulated back in the 20th century, has not been reflected in the works in the field of biology of aging (both theoretical and experimental) in the best way. The neglect of the fundamental principles of gerontological research has led to the fact that, in most works, the classical definition of aging as a set of age-related changes in practically healthy individuals leading to an increase in the rate of mortality is ignored. The emphasis is on assessing the average and maximum life span of the studied organisms, even if they are ageless. Extending the lifetime of such objects cannot be considered a modification of the rate of their aging. It is emphasized that special attention is now being paid to molecular age-related changes, which some gerontologists consider aging, although this is just its possible mechanism or consequence. However, geroprotectors are very often studied exactly by assessing the modification of the rate of such age-related changes. At the same time, as classical gerontology rightly believes, the principles of which the author urges to adhere to, without taking the survival curves of the control and experimental cohorts, it is impossible to draw a correct conclusion about whether the studied compound is a geroprotector. At the same time, an approach to the formation of such cohorts, including an assessment of the minimum required number of organisms in them, as well as the “quality” of their health, is very important. Several gerontological articles that have been published in the most highly ranked scientific journals and, therefore, have attracted much attention of relevant specialists are considered. This attention was expressed, among other things, in the high citation rate of these works, although they were performed with significant violations of the principles of classical gerontology, which were subsequently identified by other researchers. It is also emphasized that, at present, the rating of a scientific journal for many gerontological readers has become much more important than the correctness of the results and ideas presented in the article. A list of methodological problems is given, which, according to the author, not only complicate the situation with modern gerontological research but also make tangible progress in this area practically unattainable.

Khokhlov A.N., Morgunova G.V.

Over the past decades, the approaches to writing and formatting scientific articles, as well as to choosing editions by scientists for publishing the results of their research (both experimental and theoretical) have changed dramatically. Today, the majority of specialists pay much attention to formal ratings of scientific journals, since it is they that mainly determine how great the chances of the scientists published in them are to get grants for their research. And at the present stage, it is practically impossible to engage in not only applied, but also basic research without serious funding. In particular, this has become especially important for biologists and biomedical specialists working in a wide variety of fields, because they usually use expensive equipment, reagents, and experimental animals in their work. In this regard, any scientists working in the field of Life Sciences must be able to choose the appropriate journals for their publications on the basis of the scientometric indicators of the editions. No less important is the problem of formatting/designing scientific articles, because high ranked journals reject a significant percentage of manuscripts that do not meet the requirements not only after peer-reviewing but also before it (in the “rapid rejection” mode). We consider it necessary to introduce appropriate courses of lectures into the curricula of students of biological and biomedical specialties. A list of issues that are proposed to be touched in such lectures is considered, including the basics of scientometrics, work on lists of references, search for possible borrowings in manuscripts, requirements for illustrations, compliance with ethical standards, determining whether a scientific edition is a “predatory” one, peer-reviewing scientific articles, their correct structuring, etc.

Khokhlov A.N., Klebanov A.A., Morgunova G.V.

Recently, a large number of papers have appeared that describe the successful use of various biologically active compounds (short peptides, mitochondrial antioxidants, antidiabetic biguanides, mimetics of dietary restriction, autophagy modulators, etc.) as geroprotectors. However, in our opinion, in most cases, the positive results of such studies are determined by a “successful” selection of control objects. Animals with certain abnormalities are often used for this purpose, so that any favorable effect on the corresponding pathological processes leads to an increase in their lifespan. In addition, control animals can be normal (i.e., wildtype) but placed under certain extreme conditions that can be overcome just by using certain biologically active compounds. Thus, in this case, the treatment of pathologies rather than the effect on fundamental processes of aging is observed. There is a point of view that the results of Clive McCay’s well-known experiments, which have significantly prolonged the life of rats by limiting caloric intake, were determined by the facts that, firstly, the control animals fed ad libitum (which is absolutely untypical for animals in the wild) and, secondly, Fisher-344 rats, which were used in these experiments, are short-lived. The above considerations, apparently, also apply to the gerontological experiments on cultured cells. In particular, we sometimes hear remarks from our colleagues regarding the model of “stationary phase aging” of cell cultures, which is used in our laboratory, due to the fact that most of the experiments are performed on transformed rather than normal cells. However, this approach seems to us quite justified, because the phenomenon of “stationary phase”/chronological aging is common to a wide variety of cells, including bacteria, yeasts, cyanobacteria, mycoplasmas, as well as animal and plant cells. Cells with an unlimited mitotic potential do not change either from experiment to experiment or during long-term cultivation both with and without subcultivation (within the framework of the stationary phase aging model), which cannot be said of the normal diploid fibroblasts, whose telomeres are shortened with each division. In the period from seeding to entering the stationary phase of growth, the cells divide up to ten times! We believe that, to search for effective geroprotectors that affect the fundamental mechanisms of aging, it is necessary to perform studies on “maximally healthy” animals or on “maximally stable” model systems.

Shilovsky G.A., Shram S.I., Morgunova G.V., Khokhlov A.N.

It is well known that the number of dividing cells in an organism decreases with age. The average rate of cell division in tissues and organs of a mature organism sharply decreases, which is probably a trigger for accumulation of damage leading to disturbance of genome integrity. This can be a cause for the development of many age-related diseases and appearance of phenotypic and physiological signs of aging. In this connection, the protein poly(ADP-ribosyl)ation system, which is activated in response to appearance of various DNA damage, attracts great interest. This review summarizes and analyzes data on changes in the poly(ADP-ribosyl)ation system during development and aging in vivo and in vitro, and due to restriction of cell proliferation. Special attention is given to methodological aspects of determination of activity of poly(ADP-ribose) polymerases (PARPs). Analysis of relevant publications and our own data has led us to the conclusion that PARP activity upon the addition of free DNA ends (in this review referred to as stimulated PARP activity) is steadily decreasing with age. However, the dynamics of PARP activity measured without additional activation of the enzyme (in this review referred to as unstimulated activity) does not have such a clear trend: in many studies, the presented differences are statistically non-significant, although it is well known that the number of unrepaired DNA lesions steadily increases with aging. Apparently, the cell has additional regulatory systems that limit its own capability of reacting to DNA damage. Special attention is given to the influence of the cell proliferative status on PARP activity. We have systematized and analyzed data on changes in PARP activity during development and aging of an organism, as well as data on differences in the dynamics of this activity in the presence/absence of additional stimulation and on cellular processes that are associated with activation of these enzymes. Moreover, data obtained in different models of cellular aging are compared.

Khokhlov A.N., Morgunova G.V., Ryndina T.S., Coll F.

The effect of isotonic Quinton Marine Plasma (QMP) solution on the growth and “stationary phase aging” (accumulation of “age”-related changes in cultured cells during cell proliferation slowing down within a single passage and subsequent “aging” in the stationary phase of growth) of transformed Chinese hamster cells was studied. No positive effects of QMP on the studied viability indexes of the cultured cells were found in any of the experiments. It is assumed that QMP, like many other potential anti-aging agents the authors studied recently (2,4-dinitrophenol in concentrations that provide mild uncoupling, the essential oil of oregano, hydrated C60-fullerene, etc.), can demonstrate its beneficial effect only at the level of the whole organism, triggering neurohumoral mechanisms that are not present in cytological model systems.

Khokhlov A.N.

The long history of ideas about the most famous “immortal” (non-aging) organism, freshwater hydra, is shortly reviewed. Over the years this polyp has attracted the attention of naturalists interested in problems of aging and longevity. In recent years, this interest has abruptly increased with the accent on fine mechanisms providing an almost complete lack of aging in hydra. It is emphasized that hydra immortality is based on indefinite self-renewal capacity of its stem cells. It is this fact that allows the polyp to continuously replace the “outworn” cells of the organism, keeping all its characteristics unchanged for an almost unlimited time. It is concluded that the applicability of the data obtained in gerontological experiments on hydra to human being is, unfortunately, very limited because normal functioning of many important organs and tissues in highly developed organisms is determined by the presence of postmitotic cells (neurons, cardiomyocytes, etc.), which actually cannot be replaced.

Kirpichnikov M.P., Khokhlov A.N.

The paper considers the history of how the scientific journal Moscow University Biological Sciences Bulletin (MUBSB) evolved during the last 7 years. It is the English edition of the Russian scientific peer-reviewed journal of the School of Biology of Lomonosov Moscow State University MSU Vestnik (Herald). Series 16. Biology. MUBSB is published by Allerton Press, a member of the Nauka/Interperiodica International Academic Publishing Company since 2007. The rapid progress of MUBSB in recent years is apparently due to the journal having been distributed since 2007 by the internationally renowned Springer publishing consortium that places electronic versions of all articles on its website, which has, to all appearances, led to a manifold increase in the number of journal subscribers. As a result, the number of downloads of MUBSB papers from the publishing company website also raised by an order of magnitude from 2007 to 2013. The growing popularity of the journal is noted to have lead to its inclusion in a number of international databases, and this, in turn, has increased its attractiveness for a large number of authors, including Russian nonmembers of Moscow State University, as well as scientists from research institutes and universities of other countries. The main features of the spectrum of the papers published in MUBSB are briefly considered.

Khokhlov A.N.

Today, gerontologists usually employ certain molecular or cellular biomarkers of aging to evaluate the effects of various interventions in this process, since this approach is much more time-efficient than the construction of survival curves. However, arguments for the expediency of using such biomarkers are often based on the results of studies on what is called cell/cellular senescence. Unfortunately, the usage of this term has recently evolved so that it has largely lost its initial meaning, which is that normal cultured cells are subject to replicative senescence (according to the Hayflick phenomenon) and undergo changes similar to those in the cells of an aging organism. Most of recent studies in this field deal with the induction of relevant changes in cultured (usually transformed) cells by various DNA-damaging factors. Such an approach is important for defining the strategy of cancer control but, yet again, leads away from the study of actual mechanisms of organismal aging. Moreover, there are grounds to consider that biomarkers of aging identified in these studies (in particular, senescence-associated beta-galactosidase activity, the most popular among them) are basically linked to cell proliferative status. At the organismal level, this status is generally determined by the program of development and differentiation of tissues and organs, which in a definitive state are composed of postmitotic or very slowly propagating cells. Therefore, it appears that canceling the aging program will not cause any significant changes in the age-dependent dynamics of the above biomarkers. This conclusion brings us back to the necessity of constructing the survival curves for test groups of animals or humans as the only reliable (though expensive and time-inefficient) approach to evaluating the efficiency of means to modify the aging process.

Khokhlov A.N., Klebanov A.A., Karmushakov A.F., Shilovsky G.A., Nasonov M.M., Morgunova G.V.

We believe that cytogerontological models, such as the Hayflick model, though very useful for experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our “stationary phase aging” model appears to be a “gist model,” since it is based on the hypothesis that the main cause of both various “age-related” changes in stationary cell cultures and similar changes in the cells of aging multicellular organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, mycoplasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the Gompertz law, which describes exponential increase of the death probability with time. Therefore, the “stationary phase aging” model may prove effective in testing of various geroprotectors (anti-aging factors) and geropromoters (pro-aging factors) in cytogerontological experiments. It should be emphasized, however, that even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this complies with ethical principles of human subject research).

Khokhlov A.N.

There is a standpoint according to which the suppression of the ability of cells in a multicellular organism to proliferate, taking place during aging, as well as the corresponding decline in the regenerative capacities of tissues and organs, is caused by the specialized mechanisms having emerged in the evolution that decrease the risk of malignant transformation and, thereby, provide for protection against cancer. At the same time, various macromolecular defects start to accumulate in senescent cells of the body, which, on the contrary, elevate the probability for malignant transformation of these cells. Thus, according to the mentioned concept, the restriction of cell proliferation is a double-edged sword, which, on the one hand, decreases the probability for malignant tumor development in young age and, on the other hand, limits the lifespan due to accumulation of “spoiled” cells in old age. However, it remains unclear why normal human cells placed under in vitro conditions and thus having no mentioned “anticancer” barriers, which function at the body level only, NEVER undergo spontaneous malignant transformation. In addition, it is unclear how the freshwater hydra escapes both aging and cancer, as it under certain conditions contains no postmitotic and senescent cells at all and under these conditions (excluding the need for sexual reproduction) can live almost indefinitely, possessing a tremendous regenerative potential (a new organism can emerge from even 1/100 part of the old one). Presumably, the restriction of cell proliferation in an aging multicellular organism is not the result of a certain special program. Apparently, there is no program of aging at all, the aging being a “byproduct” of the program of development, whose implementation in higher organisms necessarily requires emergence of cell populations with a very low and even zero proliferative activity, which actually determines the limited ability of the corresponding organs and tissues to regenerate. On the other hand, the populations of highly differentiated cells incapable or poorly capable of reproduction (e.g., neurons, cardiomyocytes, and hepatocytes) are the particular factor that determines the normal functioning of higher animals and humans. Even regeneration of such organs with the help of stem cells may interfere with the necessary links in elaborate systems. The reductionism (“everything is determined by adverse changes in individual cells”), which has recently become widespread in experimental gerontological research, has brought about several model systems for studying the aging mechanisms in isolated cells (Hayflick phenomenon, stationary phase aging model, cellular kinetic model for testing of geroprotectors and geropromoters, etc.). However, it currently seems that data obtained using such models are inappropriate for an automatic extrapolation to the situation in the whole body. Presumably, impairments in regulatory processes functioning at the neurohumoral level are the major players in the mechanisms underlying aging of multicellular organisms rather than a mere accumulation of macromolecular damage in individual cells. It cannot be excluded that a disturbance of such regulation is the particular reason for the abnormal INCREASE in proliferation intensity of some cell populations that are frequently observed in old age and that lead to senile acromegaly and development of numerous benign tumors. It looks like the quality of CONTROL over cells, organs, and tissues becomes poorer with age rather than the quality of the cells themselves, which leads to an increase in the death rate.