Basics of Biology of Aging for MSU Non-biologists

Khokhlov A.N.

The author’s point of view is presented, according to which the cholesterol theory of aging and death, which was formulated back in the 1980s by Yu.M. Lopukhin et al., is now often misunderstood by many biologists and physicians. It is emphasized that this theory prioritized the structural role of cholesterol in the plasma membrane of the cell rather than the well-known role of cholesterol in metabolic processes. It was assumed that an increase in the microviscosity of the cell membrane due to the accumulation of cholesterol in it leads to a deterioration in the transfer of external signals and nutrients to the cell as well as to a decrease in the activity of membrane enzymes. This, according to the authors, triggers the occurrence of “senile” changes in cells, tissues, and organs and then in the entire multicellular organism, which ultimately leads to an increase in the probability of its death, that is, to aging. At the same time, the content of cholesterol (mainly cholesterol of low-density lipoproteins, which is considered especially “bad”) in serum was assigned only a minor role, because, according to the authors of the concept, its increase does not necessary lead to adverse changes in the body. This article briefly discusses the evolution of ideas about the possible role of cholesterol in aging and the development of various age-related pathologies, mainly cardiovascular diseases and mental disorders. Experimental data on the effect of liposomes containing both normal and oxidized cholesterol on the proliferative activity of cultured cells are analyzed. The data of longitudinal studies are considered, suggesting that, paradoxically, it is people with elevated levels of cholesterol in the blood plasma who live to an advanced age. At the same time, the consumption of large amounts of cholesterol-rich food may not affect the health of some patients. It is noted that a number of studies indicate the absence of a causal relationship between the level of cholesterol in serum and the occurrence of atherosclerotic changes. It is emphasized that cholesterol is a very important compound for our body, without which cell proliferation and, as a result, normal regeneration are impossible. It is noted that there is a continuously increasing amount of data on the negative role of statins in the formation of age-related pathologies, especially in people of older age groups. It is concluded that, at present, it becomes obvious that there is a need for a significant reappraisal (using the systems approach) of ideas about the possible role of cholesterol in aging and in the development of atherosclerosis and other age-related diseases as well as dementias caused by various factors, including Alzheimer’s disease.

Morgunova G.V., Khokhlov A.N.

Despite the great interest of scientists in the question of what cell aging is and the long history of its study, there are still many contradictions in this area. They arise because several different approaches to modeling aging in vitro have been developed. As a result, even different terms arose: cell senescence and cell aging. There are not only differences between models for studying aging at the cellular level; they also have common features. Moreover, it is now becoming apparent that some models complement others. This is evidenced, in particular, by the fact that biomarkers used in one model are suitable for use in another model (aging-associated β-galactosidase, lipofuscin, etc.). The approaches to studying cellular aging developed unevenly, and currently studies on this topic are experiencing another rise due to the prospects for the use of senolytics (drugs that selectively eliminate “senescent” cells) to increase the lifespan of multicellular organisms. This review considers the pros and cons of various models for studying aging on cultured cells of various nature.

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., Morgunova G.V., Klebanov A.A.

Aging organisms die out in accordance with the “Gompertz law,” i.e., the probability of their death increases with age. Survival curve construction is the main tool for gerontologists to study aging and test antiaging drugs. The analysis of survival curves includes obtaining some indices characterizing aging of the population, for example, the average and maximum lifespan, the mortality rate, and the aging rate. Testing geroprotectors can be correctly performed only by obtaining such curves. The dying out of stationary cell populations—bacteria, yeast, and mammalian cell cultures—also occurs in accordance with the Gompertz equation. In this regard, it is reasonable to use the construction of survival curves and their analysis to study the “aging” of non-subcultured cell cultures and testing anti-aging drugs on them. We used this approach in our experiments, due to which we were able to detect the positive anti-aging effect of the Quinton Marine Plasma on stationary phase aging culture of Chinese hamster cells.

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.

Gasparotto J., Girardi C.S., Somensi N., Ribeiro C.T., Moreira J.C., Michels M., Sonai B., Rocha M., Steckert A.V., Barichello T., Quevedo J., Dal-Pizzol F., Gelain D.P.

Patients recovering from sepsis have higher rates of CNS morbidities associated with long-lasting impairment of cognitive functions, including neurodegenerative diseases. However, the molecular etiology of these sepsis-induced impairments is unclear. Here, we investigated the role of the receptor for advanced glycation end products (RAGE) in neuroinflammation, neurodegeneration-associated changes, and cognitive dysfunction arising after sepsis recovery. Adult Wistar rats underwent cecal ligation and perforation (CLP), and serum and brain (hippocampus and prefrontal cortex) samples were obtained at days 1, 15, and 30 after the CLP. We examined these samples for systemic and brain inflammation; amyloid-β peptide (Aβ) and Ser-202–phosphorylated Tau (p-TauSer-202) levels; and RAGE, RAGE ligands, and RAGE intracellular signaling. Serum markers associated with the acute proinflammatory phase of sepsis (TNFα, IL-1β, and IL-6) rapidly increased and then progressively decreased during the 30-day period post-CLP, concomitant with a progressive increase in RAGE ligands (S100B, Nɛ-[carboxymethyl]lysine, HSP70, and HMGB1). In the brain, levels of RAGE and Toll-like receptor 4, glial fibrillary acidic protein and neuronal nitric-oxide synthase, and Aβ and p-TauSer-202 also increased during that time. Of note, intracerebral injection of RAGE antibody into the hippocampus at days 15, 17, and 19 post-CLP reduced Aβ and p-TauSer-202 accumulation, Akt/mechanistic target of rapamycin signaling, levels of ionized calcium-binding adapter molecule 1 and glial fibrillary acidic protein, and behavioral deficits associated with cognitive decline. These results indicate that brain RAGE is an essential factor in the pathogenesis of neurological disorders following acute systemic inflammation.

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.

According to our conception, the aging process is caused by cell proliferation restriction-induced accumulation of various macromolecular defects (mainly DNA damage) in cells of a mature organism or in a cell population. In the case of cell cultures, the proliferation restriction is related to so-called contact inhibition and to the Hayflick's limit, while in the case of multicellular organisms, it is related to the appearance, in the process of differentiation, of organs and tissues consisting of postmitotic and very slowly dividing cells. It is assumed that the proliferation of intact cells prevents accumulation of various errors in a cell population. However, the continuous propagation of all the cells in a multicellular organism is absolutely incompatible with its normal functioning. Thus, the program of development, when it generates postmitotic or slowly dividing cells, automatically leads also to the onset of the aging process (mortality increase with age). Therefore, any additional special program for aging simply becomes unnecessary. This, however, doesn't reject, for some organisms, the reasonability of programmed death, which makes possible the elimination of harmful, from the species point of view, individuals. It is also very important to emphasize that increase or decrease of an organism's lifespan under the effects of various external factors is not always necessarily related to modification of the aging process, though the experimental results in the field are usually interpreted in just this way. I called the experimental-gerontological models similar to the Hayflick's model "correlative", since they are based on some correlations only and not related necessarily to the gist of the aging phenomenon. So, for the Hayflick's model, it is the relationship between population doubling level and donor age, between population doubling potential and species lifespan, between some cell changes in vivo and in vitro, and so forth. If the rationale of the "Hayflick phenomenon" is used, we can't explain why we age. Nevertheless, many authors virtually put a sign of equality between aging in vitro and aging in vivo, which generates conclusions that are of quite doubtful accuracy. A classic illustration of this is the telomere concept of aging. Originally, the principle of shortening end-segments of DNA (telomeres) during each cell division was formulated at the beginning of seventies by the Russian scientist Aleksey Olovnikov and used by him to explain the limited "proliferative" lifespan in vitro of normal cells. Subsequently, the existence of this phenomenon was confirmed by the results of many research reports, the culmination of which was a publication in which the authors demonstrated the possibility of increasing the proliferative potential of normal cells by introducing the enzyme telomerase to them, thus restoring the lost telomere segments. At the moment it looks like the telomere shortening contributes to aging in vitro only, but not to aging in vivo because an organism never realizes the full proliferative potential of its cells. Besides, the most "responsive to aging" are the organs and tissues consisting of postmitotic cells, for which the concept of proliferative potential loses any meaning in practical terms. We developed another "correlative" model--a model for testing of geroprotectors and geropromoters--the "cell kinetics model." It is based on the well-known correlation between the "age" of cultured cells (age of their donor) and their saturation density. The model allowed us to perform preliminary testing of a lot of different compounds and factors that are interesting from a gerontological point of view, but it revealed no information about the real mechanisms of aging. However, the second model we use in our studies--the "stationary phase aging" model--obviously, is a "gist" model. It is based on the assumption that in the cells of stationary cultures various intracellular changes similar to those of an aging organism can be observed. The proliferation restriction in the case is provided, as a rule, just by contact inhibition. Many experimental results confirming this assumption were obtained. "Age-related" changes that are well known from organismal studies were shown to really occur in our experimental stationary cell culture model. Besides, such experiments can be carried out on nearly any type of cells from various biological species. Thus, the evolutionary approach to analysis of the data is provided. Moreover, the changes in the stationary cell cultures become detectable very soon--as a rule, in 2 to 3 weeks after beginning the experiment. All this allows us to suppose that the "stationary phase aging" model should provide a very effective approach to testing of different substances and their cocktails on their activities in terms of accelerating or retarding aging--of course, if their effect is realized on the cell level only.

Wei L., Li Y., He J., Khokhlov A.N.

Approaches to teaching the cell biology of aging (cytogerontology), within the appropriate agreements by scientists of the Biological Faculty of Moscow State University and at the Department of Life Science and Engineering of Harbin Institute of Technology (China), are described. The authors draw attention to certain differences in teaching biology between the two institutions and emphasize the significance of a system approach to teaching cytogerontology. This approach makes it absolutely necessary to introduce the course on the basics of biology of aging. It is concluded that full perception of the data from modern molecular cell cytogerontological research, by the students from both institutions, is impossible without understanding the fundamental notions and definitions used in both theoretical and experimental gerontology.

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.

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.

Khokhlov A.N., Morgunova G.V.

The authors try to consider the current situation with experimental gerontological research aimed at finding means to slow down aging and prolong life (geroprotectors/anti-aging drugs). The importance of correct defining aging as a set of age-related changes in the body, leading to an increase in the probability of its death, is emphasized, as well as the importance of attention to which lifespan (average, maximum, median, species-specific) changes under the influence of geroprotectors. It is noted that prolonging the life of non-aging animals with an exponential survival curve can hardly be considered a geroprotective effect. It is emphasized that the rate of aging and lifespan are not always strictly related, so the impact on one of these two indicators does not necessarily affect the second. The methodological aspects of works devoted to the study of the influence of diet restriction/modification on aging and lifespan are analyzed. The need for correct selection of a control group of animals in experimental gerontological studies is noted. According to the authors, such animals should not have any pathologies or genetic defects in order to exclude the possible influence of the studied factors on such abnormalities, and not on the aging process per se. In this regard, the authors do not consider antibiotics or drugs/vaccines against COVID-19 to be geroprotective, although the former led to the most significant increase of average lifespan in the 20th century, and the latter offset its decline in recent years caused by this infection. It is noted that the current situation in experimental gerontology makes it very desirable to have a thorough discussion on the question of what can still be considered geroprotectors/anti-aging drugs. It is proposed to organize such a discussion in the journal Advances in Gerontology.

Khokhlov A.N.

The history of research into the fundamental mechanisms of the pathogenesis of Alzheimer’s disease (AD) is briefly reviewed. Concepts in which a decisive role in the development of this disease was attributed to aluminum or free radicals are analyzed. The lack of reliable data to date to support these concepts is highlighted. The author’s point of view is stated, according to which almost all the results indicating the advisability of using antioxidants (as well as other potential drugs for AD) for the prevention and treatment of AD were obtained in model animals with certain pathologies (for example, with severe oxidative stress), which contribute to the formation of symptoms similar to those of AD in humans. In this regard, parallels with experimental gerontological work aimed at studying the effect of calorie-restricted nutrition on aging and life span are drawn. It is noted that these studies also used animals that were either not completely normal or were in unfavorable conditions. According to the author, the lack of serious success in the development of effective geroprotectors or drugs for the prevention/treatment of AD is due to the ignorance by most specialists of the principles of classical gerontology, in particular, the definitions of aging and age-related diseases, as well as correct approaches to the selection of control objects for their studies. It is emphasized that humans, unfortunately, cannot use the freshwater hydra method to combat aging and age-related diseases. Under certain conditions, it continuously renews all cells (including nerve cells) of its body and thereby ensures its “immortality.” In humans, replacing “old” neurons can lead to loss of personality/individuality, and “repairing” these cells seems impossible today. In this regard, the author considers it advisable to conduct studies of the aging of postmitotic cells in experiments on stationary cell cultures, which can accelerate, in particular, elucidation of the mechanisms of accumulation of beta-amyloid and senile pigments, such as lipofuscin, in neurons. The need to conduct clinical studies of AD as complementary to experimental work is noted, although the former are much more expensive and time-consuming. Only confirmation in human studies of the effectiveness of drugs developed in experiments on model animals will allow them to be recommended for clinical use.

Khokhlov A.N.

Editor-in-Chief of Advances in Gerontology describes the current editorial policy and the strategy of future development of the renewed journal which since 2023 is published as a separate independent edition. It is emphasized that now priority is given to publications devoted to (1) the fundamental mechanisms which may determine the increase in the probability of death of living organisms, including humans, with age, and to (2) identifying various factors of both chemical and physical nature that could potentially help to slow down the aging process. In addition, papers included in Volume 13, Issue 1 are shortly reviewed.