Senescence-associated β-galactosidase—A biomarker of aging, DNA damage, or cell proliferation restriction?

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.

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.

Alinkina E.S., Vorobyova A.K., Misharina T.A., Fatkullina L.D., Burlakova E.B., Khokhlov A.N.

In order to clarify possible cytological mechanisms that underlie the beneficial effects of carvacrol-bearing essential oils on health and mental abilities, we studied one of them (oregano essential oil) in experiments on transformed cultured Chinese hamster cells. Possible cytotoxic or mitogenic effects of the preparation at various concentrations were preliminarily estimated by analyzing the cell culture density after 4 days of cultivation. The preparation concentration in the growth medium (on carvacrol basis) varied from 1 × 10−15 up to 5 × 10−4 M (on carvacrol basis). As a result, two concentrations were selected for further experiments, including 2.5 × 10−5 M as the maximal absolutely non-toxic concentration and 2.5 × 10−4 M as the concentration at which the oregano essential oil decreased approximately 2-fold the final cell density of the grown culture. It was found that the preparation at 2.5 × 10−5 M had no effect on either the colony-forming ability of the cells or the saturation density of the culture (which is a marker of its “biological age”) or kinetics of its “stationary phase aging” (degradation of cultured cells in the stationary phase of growth, similar to age-related changes of the cells in aging organism). On the contrary, the oregano essential oil at 2.5 × 10−4 M abruptly diminished colony-forming ability of the cells and influenced as a “pro-aging” factor on the saturation density of the cell culture and kinetics of the cell death induced by “stationary phase aging.” Based on our own concept of aging and the data obtained, we assumed that detected increase in the life span of mice under the influence of the oregano essential oil could be determined by certain functional changes at the organismal level only, but is not associated with any geroprotective (anti-aging) activity of the preparation, which is manifested at the cellular level and improves the cell viability.

Sikora E., Arendt T., Bennett M., Narita M.

Cellular senescence as the state of permanent inhibition of cell proliferation is a tumour-suppressive mechanism. However, due to the associated secretory phenotype senescence can also contribute to cancer and possibly other age-related diseases, such as obesity, diabetes, atherosclerosis and Alzheimer's disease. There are two major mechanisms of cellular senescence; replicative senescence depends on telomere erosion or dysfunction whilst stress-induced premature senescence (SIPS) is telomere-independent and also includes oncogene-induced senescence (OIS). The senescence phenotype is characterised by altered cellular morphology, increased activity for senescence-associated-β-galactosidase (SA-β-GAL), increased formation of senescence-associated heterochromatin foci (SAHF) and promyelocytic leukemia protein nuclear bodies (PML NBs), permanent DNA damage, chromosomal instability and an inflammatory secretome. Some of these markers have been identified in cells from age-related pathologies. However, to improve our understanding of the contribution of cellular senescence to organismal ageing and age-related disease, it is imperative to define an unequivocal signature of cellular senescence that is functionally connected with normal and pathological ageing. Herein, we describe the processes leading to senescence, and the current biomarkers of cellular senescence, with particular emphasis on the causal role of DNA damage responses involved in the process. We highlight the gaps in our knowledge both of the processes leading to senescence, and the signature of cellular senescence both in vitro and in vivo. A well-defined set of senescence biomarkers for ageing and age-related disease would have a strong impact on the diagnosis, staging and predicted outcomes of age-related disease, providing the basis for a pharmacological intervention to postpone ageing and age-related disease.

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.

Harman D.

My short paper, ‘‘Origin and evolution of the free radical theory of aging: a brief personal history, 1954–2009’’ by myself was recently published in Biogerontology. In this paper I inadvertently failed to make clear the difference between the research fields of oxygen toxicity and aging. I indicated that Drs. Rebeca Gershman and Daniel Gilbert were reported to apparently equate ‘‘oxygen toxicity’’ with ‘‘aging.’’ Although free radicals are involved in both fields (see Harman, D., Ann. N.Y. Acad. Sci., 1067: 10–21, 2006; pages 16–17), time is required to manifest the complex product attributed to aging. Further, the research fields of aging and oxygen toxicity differ clinically. Oxygen toxicity is of relatively little importance to ‘‘man,’’ whereas aging is of major importance. Judging from the literature concerned with both aging and oxygen toxicity, this difference may not be appreciated by some.

Debacq-Chainiaux F., Pascal T., Boilan E., Bastin C., Bauwens E., Toussaint O.

Repeated exposures to sublethal concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of WI-38 human diploid fibroblasts. We found 16 replicative senescence-related genes with similar alterations in expression level in replicative senescence and two models of stress-induced premature senescence. Among these genes was IGFBP-3. Using a siRNA approach, we showed that IGFBP-3 regulates the appearance of several biomarkers of senescence after repeated exposures of WI-38 fibroblasts to tert-butylhydroperoxide and ethanol.

Lee B.Y., Han J.A., Im J.S., Morrone A., Johung K., Goodwin E.C., Kleijer W.J., DiMaio D., Hwang E.S.

Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence‐associated β‐galactosidase (SA‐β‐gal), which is defined as β‐galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA‐β‐gal and its cellular roles in senescence are not known. We demonstrate here that SA‐β‐gal activity is expressed from GLB1, the gene encoding lysosomal β‐D‐galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive GM1‐gangliosidosis, which have defective lysosomal β‐galactosidase, did not express SA‐β‐gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small‐hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA‐β‐gal. GLB1 mRNA depletion also prevented expression of SA‐β‐gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA‐β‐gal induction during senescence was due at least in part to increased expression of the lysosomal β‐galactosidase protein. These results also indicate that SA‐β‐gal is not required for senescence.

CRISTOFALO V.

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.

Toussaint O., Dumont P., Remacle J., Dierick J., Pascal T., Frippiat C., Magalhaes J.P., Zdanov S., Chainiaux F.

No consensus exists so far on the definition of cellular senescence. The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening counting cell generations (definition 1). Other authors gave an enlarged functional definition encompassing any kind of irreversible arrest of proliferative cell types induced by damaging agents or cell cycle deregulations after overexpression of proto-oncogenes (definition 2). As stress increases, the proportion of cells in “stress-induced premature senescence-like phenotype” according to definition 1 or “stress-induced premature senescence,” according to definition 2, should increase when a culture reaches growth arrest, and the proportion of cells that reached telomere-dependent replicative senescence due to the end-replication problem should decrease. Stress-induced premature senescence-like phenotype and telomere-dependent replicatively senescent cells share basic similarities such as irreversible growth arrest and resistance to apoptosis, which may appear through different pathways. Irreversible growth arrest after exposure to oxidative stress and generation of DNA damage could be as efficient in avoiding immortalisation as “telomere-dependent” replicative senescence. Probabilities are higher that the senescent cells (according to definition 2) appearing in vivo are in stress-induced premature senescence rather than in telomere-dependent replicative senescence. Examples are given suggesting these cells affect in vivo tissue (patho)physiology and aging.

Choi J., Shendrik I., Peacocke M., Peehl D., Buttyan R., Ikeguchi E.F., Katz A.E., Benson M.C.

Cellular senescence is a unique cellular response pathway thought to be closely associated with the aging process. The senescent phenotype is characterized by the loss of a cell's ability to respond to proliferative and apoptotic stimuli even while normal metabolic activity and vitality is maintained. Recently, a novel biomarker, senescent-associated beta-galactosidase (SA-beta-gal), was found to identify cells with the senescent phenotype. In the present study, we examined whether human prostatic epithelial cells adopt a senescence-associated phenotype after prolonged culture and analyzed a series of human benign prostatic hyperplasia (BPH) specimens to determine whether the cellular senescence process might be a factor in the development of BPH.A primary culture of epithelial cells was established from the normal tissue of the peripheral zone of a radical prostatectomy specimen and was serially passaged until senescence. Forty-three human prostate specimens were obtained subsequent to radical prostatectomy or transrectal ultrasound-guided biopsy. The cultured cells and tissue specimens were histochemically stained to reveal the expression of SA-beta-gal, the cellular senescence biomarker.As has been reported for other types of cultured cells, human prostatic epithelial cells demonstrated widespread expression of the cellular senescence marker, SA-beta-gal, on prolonged culture. In our survey of hypertrophied human prostate tissues, 17 specimens (40%) of the 43 analyzed demonstrated positive staining for SA-beta-gal. In these tissues, SA-beta-gal expression was noted only in the epithelial cells. No statistical correlation (P = 0.42) between the chronologic age of the patient donor and SA-beta-gal expression was found. However, a high prostate weight (greater than 55 g) was found to correlate strongly with the expression of the SA-beta-gal biomarker (P = 0. 0001).Cultured prostatic epithelial cells expressed SA-beta-gal on reaching replicative senescence in vitro. The survey of human BPH specimens for the senescent marker showed that prostatic epithelial cells in patients with BPH with more advanced enlargement of the prostate (greater than 55 g prostate weight) expressed SA-beta-gal, and the prostates from patients with BPH that weighed less than 55 g tended to lack senescent epithelial cells. On the basis of these results, we propose that the accumulation of senescent epithelial cells may play a role in the development of the prostatic enlargement associated with BPH.

Severino J., Allen R.G., Balin S., Balin A., Cristofalo V.J.

Cytochemically detectable beta-galactosidase (beta-gal) at pH 6.0 has been reported to increase during the replicative senescence of fibroblast cultures and has been used widely as a marker of cellular senescence in vivo and in vitro. In this study, we have characterized changes in senescence-associated (SA) beta-gal staining in early and late passage cultures, cultures established from donors of different ages, virally immortalized cells, and tissue slices obtained from donors of different ages. The effects of different culture conditions were also examined. While we confirm the previous report that SA beta-gal staining increased in low-density cultures of proliferatively senescent cells, we were unable to demonstrate that it is a specific marker for aging in vitro. Cultures established from donors of different ages stained for SA beta-gal activity as a function of in vitro replicative age, not donor age. We also failed to observe any differences in SA beta-gal staining in skin cells in situ as a marker of aging in vivo. The level of cytochemically detectable SA beta-gal was elevated in confluent nontransformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density, and in low-density, young, normal cultures oxidatively challenged by treatment with H2O2. Although we clearly demonstrate that SA beta-gal staining in cells is increased under a variety of different conditions, the interpretation of increased staining remains unclear, as does the question of whether the same mechanisms are responsible for the increased SA beta-gal staining observed in senescent cells and changes observed in cells under other conditions.

Yegorov Y.E., Akimov S.S., Hass R., Zelenin A.V., Prudovsky I.A.

Abstract Cytochemically detectable activity of endogenous β-galactosidase was found at pH 6.0 in Swiss 3T3 cells after long-term incubation in low serum or in the presence of heparin concentrations known to reversibly inhibit cell proliferation. A high percentage of β-galactosidase-positive cells were detected in U937 and HL60 cultures at the late stage of macrophage-like differentiation induced by TPA. Interestingly, a small number of β-galactosidase-positive cells were found even in the growing Swiss 3T3 cultures. These positive cells expressed morphological features similar to those of senescent cells. Thus, the activity of β-galactosidase at pH 6.0 cannot be considered an exclusive marker of senescent cells since it is expressed in other types of nonproliferating cells.

Tiwari I., Chauhan P., Singh A., Sharma N.K.

Senescence is the markup of events related to the exit of the cell cycle by some cells due to various factors, and accumulation of such senescent cells leads to the onset of aging, but when it occurs at early stages, it becomes the cause of many age-related pathologies. Many factors can be responsible for such events, such as oxidative stress, DNA damage, telomere shortening, and overexpression of lysosomal β-gal, forming it into senescence-associated β-gal (SA-β-gal). These cells activate the apoptotic pathway involving apoptotic factors like p53-p21-16 and begin to secrete pro-inflammatory factors like chemokines and cytokines, making other adjacent cells tumorigenic, thus leading to senescence-associated secretory phenotype (SASP). In another turn of events, due to some reasons, the folding process of proteins by cytosolic chaperones or in ER gets interrupted, causing the hydrophobic amino acids to come to the surface, but such misfolded proteins further undergo ubiquitinylation and proteasomal degradation. In case misfolded proteins do not get degraded, they then aggregate and form amyloid fibrils, especially in neurons, leading to various neurodegenerative diseases such as α-synuclein in the case of Alzheimer’s and Parkinson’s. The current chapter deals with cellular senescence and various neurodegenerative disorders other than Alzheimer’s and Parkinson’s and the mechanism and techniques of their diagnosis.

Wang H., Mu G., Cai X., Zhang X., Mao R., Jia H., Luo H., Liu J., Zhao C., Wang Z., Yang C.

AbstractNeoadjuvant radiotherapy (NAC), a preoperative intervention regimen for reducing the stage of primary tumors and surgical margins, has gained increasing attention in the past decade. However, radiation‐induced skin damage during neoadjuvant radiotherapy exacerbates surgical injury, remarkably increasing the risk of refractory wounds and compromising the therapeutic effects. Radiation impedes wound healing by increasing the production of reactive oxygen species (ROS) and inducing cell apoptosis and senescence. In this study, we prepared a self‐assembling peptide (R‐peptide) and hyaluronic acid (HA)‐based and cordycepin (Cor)‐loaded superstructure hydrogel (CHRgel) for surgical incision healing after neoadjuvant radiotherapy. Results showed that (i) R‐peptide co‐assembled with HA to form biomimetic fiber bundle microstructure, in which R‐peptide drives the assembly of single fiber through π‐π stacking and other forces and HA, as a single fiber adhesive, facilitates bunching through electrostatic interactions. (ii) The biomimetic superstructure contributes to the adhesion and proliferation of cells in the surgical wound. (iii) Aldehyde‐modified HA provides dynamic covalent binding sites for cordycepin to achieve responsive release, inhibiting radiation‐induced cellular senescence. (iv) Arginine in the peptides provides antioxidant capacity and a substrate for the endogenous production of nitric oxide (NO) to promote wound healing and angiogenesis of surgical wounds after neoadjuvant radiotherapy.This article is protected by copyright. All rights reserved

Fuentes‐Flores A., Geronimo‐Olvera C., Girardi K., Necuñir‐Ibarra D., Patel S.K., Bons J., Wright M.C., Geschwind D., Hoke A., Gomez‐Sanchez J.A., Schilling B., Rebolledo D.L., Campisi J., Court F.A.

AbstractFollowing peripheral nerve injury, successful axonal growth and functional recovery require Schwann cell (SC) reprogramming into a reparative phenotype, a process dependent upon c‐Jun transcription factor activation. Unfortunately, axonal regeneration is greatly impaired in aged organisms and following chronic denervation, which can lead to poor clinical outcomes. While diminished c‐Jun expression in SCs has been associated with regenerative failure, it is unclear whether the inability to maintain a repair state is associated with the transition into an axonal growth inhibition phenotype. We here find that reparative SCs transition into a senescent phenotype, characterized by diminished c‐Jun expression and secretion of inhibitory factors for axonal regeneration in aging and chronic denervation. In both conditions, the elimination of senescent SCs by systemic senolytic drug treatment or genetic targeting improved nerve regeneration and functional recovery, increased c‐Jun expression and decreased nerve inflammation. This work provides the first characterization of senescent SCs and their influence on axonal regeneration in aging and chronic denervation, opening new avenues for enhancing regeneration and functional recovery after peripheral nerve injuries.

Ma Y., Farny N.G.

Cellular senescence increases with aging. While senescence is associated with an exit of the cell cycle, there is ample evidence that post-mitotic cells including neurons can undergo senescence as the brain ages, and that senescence likely contributes significantly to the progression of neurodegenerative diseases (ND) such as Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Stress granules (SGs) are stress-induced cytoplasmic biomolecular condensates of RNA and proteins, which have been linked to the development of AD and ALS. The SG seeding hypothesis of NDs proposes that chronic stress in aging neurons results in static SGs that progress into pathological aggregates Alterations in SG dynamics have also been linked to senescence, though studies that link SGs and senescence in the context of NDs and the aging brain have not yet been performed. In this Review, we summarize the literature on senescence, and explore the contribution of senescence to the aging brain. We describe senescence phenotypes in aging neurons and glia, and their links to neuroinflammation and the development of AD and ALS. We further examine the relationships of SGs to senescence and to ND. We propose a new hypothesis that neuronal senescence may contribute to the mechanism of SG seeding in ND by altering SG dynamics in aged cells, thereby providing additional aggregation opportunities within aged neurons.

Fuentes-Flores A., Geronimo-Olvera C., Ñecuñir D., Patel S.K., Bons J., Wright M.C., Geschwind D., Hoke A., Gomez-Sanchez J.A., Schilling B., Campisi J., Court F.A.

AbstractAfter peripheral nerve injuries, successful axonal growth and functional recovery requires the reprogramming of Schwann cells into a reparative phenotype, a process dependent on the activation of the transcription factor c-Jun. Nevertheless, axonal regeneration is greatly impaired in aged organisms or after chronic denervation leading to important clinical problems. This regenerative failure has been associated to a diminished c-Jun expression by Schwann cells, but whether the inability of these cells to maintain a repair state is associated to the transition into a phenotype inhibitory for axonal growth, has not been evaluated so far. We find that repair Schwann cells transitions into a senescent phenotype, characterized by diminished c-Jun expression and secretion of factor inhibitory for axonal regeneration in both aging and chronic denervation. In both conditions, elimination of senescent Schwann cells by systemic senolytic drug treatment or genetic targeting improves nerve regeneration and functional recovery in aging and chronic denervation, associated with an upregulation of c-Jun expression and a decrease in nerve inflammation. This work provides the first characterization of senescent Schwann cells and their impact over axonal regeneration in aging and chronic denervation, opening new avenues for enhancing regeneration, and functional recovery after peripheral nerve injuries.

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.

Karimov D.D., Kudoyarov E.R., Mukhammadiyeva G.F., Ziatdinova M.M., Baigildin S.S., Yakupova T.G.

Aging is an individual, complex biological process, modulated by internal and external factors, characterized by a progressive loss of biological / physiological integrity, which leads to body dysfunction, increases vulnerability and death. Influence of activity type on aging rate has been convincingly shown in many studies, which makes it possible assess differences in aging rate of workers, exposed various occupational factors, conditions, work nature and intensity in certain professional and seniority groups, adequately reflects health state and can predict effectiveness of human labor activity. As integral indicator, it can help identify individuals at risk of age-related disorders, serving as a measure of relative fitness and predicting later life disability and mortality, regardless of chronological age. The article provides an overview of the main measuring ageing rate methods based on biomarkers, such as functional (“Kiev model”, WAI) and molecular genetic biomarkers (determination of telomere length, β-galactosidase enzyme activity) of human ageing, applicable in occupational medicine. The review discusses the main requirements for biomarker sets compilation, methods applicability and reliability, mathematical approaches to biological age calculating, and some workers biological age calculating problems. This allows assuming the great potential for using biological age to assess the impact of working conditions and work nature on workers’ ageing rate to prevent disability and improve quality of life.

Bagheri Y., Sadigh-Eteghad S., Fathi E., Mahmoudi J., Abdollahpour A., Namini N.J., Malekinejad Z., Mokhtari K., Barati A., Montazersaheb S.

Aging is a physiological process in which there is a progressive decline of function in multiple organs such as the liver. The development of natural therapies, such as sericin, for delaying age-associated diseases is of major interest in this regard. Twenty-seven mice were divided into three groups of nine, including young control group (8 weeks, received normal saline), aged control group (24 months, received normal saline), and sericin-treated aged mice (24 months, received sericin at dose 100 mg/kg/day) via oral administration for 14 days. The liver enzymes in serum and oxidative stress markers in liver tissue were evaluated using spectrophotometric/ELISA methods. Apoptotic proteins, pro-inflammatory cytokines, COX2, JNK, and P-38 levels were assessed by western blot analysis. β-galactosidase expression was determined by a qRT-PCR method. The findings showed that 100 mg/kg of sericin reduced liver enzymes in aged mice. Antioxidant capacity in treated aged mice showed an improvement in all indexes in the liver tissue. Also, sericin administration declined pro-inflammatory markers to varying degrees in aged-treated mice. Sericin also increased the expression level of Bcl-2 and decreased the expression level of Bax and cleaved caspase-3.In addition, treatment with sericin suppressed protein expression of p-JNK and p-JNK/JNK. Collectively, these findings would infer that sericin administration may have a hepatoprotective effect in aging-induced liver damage in mice.

Zayed M., Iohara K.

Aging, defined by a decrease in the physical and functional integrity of the tissues, leads to age-associated degenerative diseases. There is a relation between aged dental pulp and the senescence of dental pulp stem cells (DPSCs). Therefore, it is important to investigate the molecular processes underlying the senescence of DPSCs to elucidate the dental pulp aging mechanisms. p-Cresol (PC), a uremic toxin, is strongly related to cellular senescence. Here, age-related phenotypic changes including senescence, apoptosis, inflammation, and declining odontoblast differentiation in PC-treated canine DPSCs were investigated. Under the PC condition, cellular senescence was induced by decreased proliferation capacity and increased cell size, senescence-associated β-galactosidase (SA-β-gal) activity, and senescence markers p21, IL-1β, IL-8, and p53. Exposure to PC could stimulate inflammation by the increased expression of IL-6 and cause the distraction of the cell cycle by the increased level of Bax protein and decreased Bcl-2. The levels of odontoblast differentiation markers, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, and osterix, were decreased. Consistent with those findings, the alizarin red staining, alkaline phosphatase, and DSPP protein level were decreased during the odontoblast differentiation process. Taken together, these findings indicate that PC could induce cellular senescence in DPSCs, which may demonstrate the changes in aging dental pulp.

Van houcke J., Geeraerts E., Vanhunsel S., Beckers A., Noterdaeme L., Christiaens M., Bollaerts I., De Groef L., Moons L.

The development of effective treatments for age-related neurodegenerative diseases remains one of the biggest medical challenges today, underscoring the high need for suitable animal model systems to improve our understanding of aging and age-associated neuropathology. Zebrafish have become an indispensable complementary model organism in gerontology research, yet their growth-control properties significantly differ from those in mammals. Here, we took advantage of the clearly defined and highly conserved structure of the fish retina to study the relationship between the processes of growth and aging in the adult zebrafish central nervous system (CNS). Detailed morphological measurements reveal an early phase of extensive retinal growth, where both the addition of new cells and stretching of existent tissue drive the increase in retinal surface. Thereafter, and coinciding with a significant decline in retinal growth rate, a neurodegenerative phenotype becomes apparent,–characterized by a loss of synaptic integrity, an age-related decrease in cell density and the onset of cellular senescence. Altogether, these findings support the adult zebrafish retina as a valuable model for gerontology research and CNS disease modeling and will hopefully stimulate further research into the mechanisms of aging and age-related pathology.

Khokhlov A.N.

This is a brief overview of the ideas of the possibility of using the cell kinetic model developed by the author in the 1980s to test, in experiments on cell cultures, potential geroprotectors and geropromoters that slow down or accelerate, respectively, the aging process in animals and humans. The history of the evolution of this model—from estimation of only the cell reproduction rate and saturation density in a non-subcultured cell culture to constructing survival curves in the stationary phase of growth and to a further analysis of the possible interrelation between all parts of the curve of cells’ growth and subsequent dying out—is considered. Possible approaches to mathematical and statistical analysis of the data obtained within the framework of this model system are analyzed. It is emphasized that such studies can be carried out on cells of a very different nature (normal and transformed human and animal cells, plant cells, yeast, mycoplasmas, bacteria, etc.), which makes possible an evolutionary approach to the interpretation of the results obtained. At the same time, in the author’s opinion, the most promising experiments are those carried out on immortalized cells of humans and animals, since they are not cancerous on the one hand and have an unlimited mitotic potential on the other hand and, therefore, do not “age” in the process of numerous divisions, as, for example, normal human diploid fibroblasts do. It is assumed that the appropriate mathematical analysis of the entire growth and dying out curve of a non-subcultured cell culture (from seeding into a culture flask to the complete death of all cells) may allow the clarification of certain relationships between the development and aging of a multicellular organism and to increase the reliability of identifying promising geroprotectors.

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

In the review, the main types of autophagy (macroautophagy, microautophagy, and chaperonemediated autophagy) are shortly described. Data about the character of the influence of autophagy on the aging process and on the development of some neurodegenerative diseases in various organisms are analyzed. It is noted that this effect is usually (though not always) beneficial. Results of investigations of the phenomenon in experiments on mice, nematodes, fruit flies, bacteria, yeast, and cell cultures of higher organisms are considered. Obvious relationship between autophagy activation and cell proliferation restriction is emphasized. The latter, in our opinion, is the main cause of age-related accumulation of various defects (the most important of them is DNA damage) in cells and tissues, which leads to an increase in the death probability (i.e., to aging). It is concluded that studies of the role of autophagy in the aging process on the models of chronological aging in yeast or stationary phase aging of cell cultures could be considered as the most appropriate approach to the problem solution.