Integrating cellular senescence with the concept of damage accumulation in aging: Relevance for clearance of senescent cells

Understanding the aging process and ways to manipulate it is of major importance for biology and medicine. Among the many aging theories advanced over the years, the concept most consistent with experimental evidence posits the buildup of numerous forms of molecular damage as a foundation of the aging process. Here, we discuss that this concept integrates well with recent findings on cellular senescence, offering a novel view on the role of senescence in aging and age‐related disease. Cellular senescence has a well‐established role in cellular aging, but its impact on the rate of organismal aging is less defined. One of the most prominent features of cellular senescence is its association with macromolecular damage. The relationship between cell senescence and damage concerns both damage as a molecular signal of senescence induction and accelerated accumulation of damage in senescent cells. We describe the origin, regulatory mechanisms, and relevance of various damage forms in senescent cells. This view on senescent cells as carriers and inducers of damage puts new light on senescence, considering it as a significant contributor to the rise in organismal damage. Applying these ideas, we critically examine current evidence for a role of cellular senescence in aging and age‐related diseases. We also discuss the differential impact of longevity interventions on senescence burden and other types of age‐related damage. Finally, we propose a model on the role of aging‐related damage accumulation and the rate of aging observed upon senescent cell clearance.     

Deep senescent human fibroblasts show diminished DNA damage foci but retain checkpoint capacity to oxidative stress

Critically shortened telomeres trigger a DNA damage response in replicatively senescent cells. Here we report that while DNA damage foci can be detected in newly senescent cells, these foci eventually diminished in deep senescent cells. However, DNA checkpoint signalling and repair machinery in response to oxidative stress remain uncompromised in these deep senescent cells. Activation of p53 by oxidative stress is unaffected despite a marked decrease in expression of platelet-derived growth factor alpha-receptor. These findings suggest that cellular senescence is not a static process hence care must be taken in the selection of biomarkers of senescence in studies of ageing.     

DNA damage, cellular senescence and organismal ageing: causal or correlative?

Cellular senescence has long been used as a cellular model for understanding mechanisms underlying the ageing process. Compelling evidence obtained in recent years demonstrate that DNA damage is a common mediator for both replicative senescence, which is triggered by telomere shortening, and premature cellular senescence induced by various stressors such as oncogenic stress and oxidative stress. Extensive observations suggest that DNA damage accumulates with age and that this may be due to an increase in production of reactive oxygen species (ROS) and a decline in DNA repair capacity with age. Mutation or disrupted expression of genes that increase DNA damage often result in premature ageing. In contrast, interventions that enhance resistance to oxidative stress and attenuate DNA damage contribute towards longevity. This evidence suggests that genomic instability plays a causative role in the ageing process. However, conflicting findings exist which indicate that ROS production and oxidative damage levels of macromolecules including DNA do not always correlate with lifespan in model animals. Here we review the recent advances in addressing the role of DNA damage in cellular senescence and organismal ageing.     

p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling

Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age‐related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage‐induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)‐κB kinase, leading to decreased cell survival. NF‐κB activation induced TNF‐α secretion and JNK activation to mediate death of senescent cells in a caspase‐ and JNK‐dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.     

The role of senescent T cells in immunopathology

The development of senescence in tissues of different organs and in the immune system are usually investigated independently of each other although during ageing, senescence in both cellular systems develop concurrently. Senescent T cells are highly inflammatory and secrete cytotoxic mediators and express natural killer cells receptors (NKR) that bypass their antigen specificity. Instead they recognize stress ligands that are induced by inflammation or infection of different cell types in tissues. In this article we discuss data on T cell senescence, how it is regulated and evidence for novel functional attributes of senescent T cells. We discuss an interactive loop between senescent T cells and senescent non‐lymphoid cells and conclude that in situations of intense inflammation, senescent cells may damage healthy tissue. While the example for immunopathology induced by senescent cells that we highlight is cutaneous leishmaniasis, this situation of organ damage may apply to other infections, including COVID‐19 and also rheumatoid arthritis, where ageing, inflammation and senescent cells are all part of the same equation.     

Elimination of senescent osteoclast progenitors has no effect on the age‐associated loss of bone mass in mice

Both an increase in osteoclast and a decrease in osteoblast numbers contribute to skeletal aging. Markers of cellular senescence, including expression of the cyclin inhibitor p16, increase with aging in several bone cell populations. The elimination of p16‐expressing cells in old mice, using the INK‐ATTAC transgene, increases bone mass indicating that senescent cells contribute to skeletal aging. However, the identity of the senescent cells and the extent to which ablation of p16‐expressing cells may prevent skeletal aging remain unknown. Using mice expressing the p16‐3MR transgene, we examined whether elimination of p16‐expressing cells between 12 and 24 months of age could preserve bone mass; and whether elimination of these cells from 20 to 26 months of age could restore bone mass. The activation of the p16‐3MR transgene by ganciclovir (GCV) greatly diminished p16 levels in the brain, liver, and osteoclast progenitors from the bone marrow. The age‐related increase in osteoclastogenic potential of myeloid cells was also abrogated by GCV. However, GCV did not alter p16 levels in osteocytes—the most abundant cell type in bone—and had no effect on the skeletal aging of p16‐3MR mice. These findings indicate that the p16‐3MR transgene does not eliminate senescent osteocytes but it does eliminate senescent osteoclast progenitors and senescent cells in other tissues, as described previously. Elimination of senescent osteoclast progenitors, in and of itself, has no effect on the age‐related loss of bone mass. Hence, other senescent cell types, such as osteocytes, must be the seminal culprits.     

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.     

A stochastic model of cell replicative senescence based on telomere shortening, oxidative stress, and somatic mutations in nuclear and mitochondrial DNA.

Human diploid fibroblast cells can divide for only a limited number of times in vitro, a phenomenon known as replicative senescence or the Hayflick limit. Variability in doubling potential is observed within a clone of cells, and between two sister cells arising from a single mitotic division. This strongly suggests that the process by which cells become senescent is intrinsically stochastic. Among the various biochemical mechanisms that have been proposed to explain replicative senescence, particular interest has been focussed on the role of telomere reduction. In the absence of telomerase--an enzyme switched off in normal diploid fibro-blasts-cells lose telomeric DNA at each cell division. According to the telomere hypothesis of cell senescence, cells eventually reach a critically short telomere length and cell cycle arrest follows. In support of this concept, forced expression of telomerase in normal fibroblasts appears to prevent cell senescence. Nevertheless, the telomere hypothesis in its basic form has some difficulty in explaining the marked stochastic variations seen in the replicative lifespans of individual cells within a culture, and there is strong empirical and theoretical support for the concept that other kinds of damage may contribute to cellular ageing. We describe a stochastic network model of cell senescence in which a primary role is played by telomere reduction but in which other mechanisms (oxidative stress linked particularly to mitochondrial damage, and nuclear somatic mutations) also contribute. The model gives simulation results that are in good agreement with published data on intra-clonal variability in cell doubling potential and permits an analysis of how the various elements of the stochastic network interact. Such integrative models may aid in developing new experimental approaches aimed at unravelling the intrinsic complexity of the mechanisms contributing to human cell ageing.     

Cellular senescence contributes to age‐dependent changes in circulating extracellular vesicle cargo and function

Extracellular vesicles (EVs) have emerged as important regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63⁺ particles mediate these effects. Treatment of macrophages with EV‐like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV‐like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging‐associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.     

Senescence chips for ultrahigh‐throughput isolation and removal of senescent cells

Cellular senescence plays an important role in organismal aging and age‐related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high‐throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh‐throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof‐of‐principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single‐cell resolution. After scale‐up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh‐throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.     

Organismal Aging and Oxidants beyond Macromolecules Damage

The relationship between oxidants and organismal aging was first articulated through the free radical theory of aging. One of the major predictions of the free radical theory of aging is that oxidative stress shortens organisms’ lifespan because of an increased level of oxidants, which are damaging to macromolecules. However, challenging the role of oxidants in age‐related diseases, there is now sufficient evidence that antioxidant supplements do not provide significant health benefits. Interestingly, in addition to an increase in oxidant‐mediated macromolecules damage, there is convincing experimental data to support the role of senescent cells in the process of aging. Here, the current knowledge regarding the role of oxidants and cellular senescence in organismal aging is reviewed and it is proposed that, in addition to the role of oxidants as inducers of macromolecular damage, oxidants may also function as regulators of signaling pathways involved in the establishment of cellular senescence. If this role for oxidants is established, it may be necessary to modify the free radical theory of aging from “Organisms age because cells accumulate reactive oxygen species‐dependent damage over time” to: “Organisms age because cells accumulate oxidants’‐dependent damage and oxidants’‐dependent senescent characteristics over time.”     

Attenuation of ataxia telangiectasia mutated signalling mitigates age‐associated intervertebral disc degeneration

Previously, we reported that persistent DNA damage accelerates ageing of the spine, but the mechanisms behind this process are not well understood. Ataxia telangiectasia mutated (ATM) is a protein kinase involved in the DNA damage response, which controls cell fate, including cell death. To test the role of ATM in the human intervertebral disc, we exposed human nucleus pulposus (hNP) cells directly to the DNA damaging agent cisplatin. Cisplatin‐treated hNP cells exhibited rapid phosphorylation of ATM and subsequent increased NF‐κB activation, aggrecanolysis, decreased total proteoglycan production and increased expression of markers of senescence, including p21, γH₂AX and SA‐ß‐gal. Treating cisplatin‐exposed hNP cells with an ATM‐specific inhibitor negated these effects. In addition, genetic reduction of ATM reduced disc cellular senescence and matrix proteoglycan loss in the progeroid Ercc1^?/? mouse model of accelerated ageing. These findings suggest that activation of ATM signalling under persistent genotoxic stress promotes disc cellular senescence and matrix homeostatic perturbation. Thus, the ATM signalling pathway represents a therapeutic target to delay the progression of age‐associated spine pathologies.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Senescence and apoptosis: dueling or complementary cell fates?

In response to a variety of stresses, mammalian cells undergo a persistent proliferative arrest known as cellular senescence. Many senescence‐inducing stressors are potentially oncogenic, strengthening the notion that senescence evolved alongside apoptosis to suppress tumorigenesis. In contrast to apoptosis, senescent cells are stably viable and have the potential to influence neighboring cells through secreted soluble factors, which are collectively known as the senescence‐associated secretory phenotype (SASP). However, the SASP has been associated with structural and functional tissue and organ deterioration and may even have tumor‐promoting effects, raising the interesting evolutionary question of why apoptosis failed to outcompete senescence as a superior cell fate option. Here, we discuss the advantages that the senescence program may have over apoptosis as a tumor protective mechanism, as well as non‐neoplastic functions that may have contributed to its evolution. We also review emerging evidence for the idea that senescent cells are present transiently early in life and are largely beneficial for development, regeneration and homeostasis, and only in advanced age do senescent cells accumulate to an organism's detriment.     

How might replicative senescence contribute to human ageing?

Cell senescence is the limited ability of primary human cells to divide when cultured in vitro. This eventual cessation of division is accompanied by a specific set of changes in cell physiology, morphology, and gene expression. Such changes in phenotype have the potential to contribute to human ageing and age-related diseases. Until now, senescence has largely been studied as an in vitro phenomenon, but recent data have for the first time directly demonstrated the presence of senescent cells in aged human tissues. Although a direct causal link between the ageing of whole organisms and the senescence of cells in culture remains elusive, a large body of data is consistent with cell senescence contributing to a variety of pathological changes seen in the aged. This review considers the in vitro phenotype of cellular senescence and speculates on the various possible routes whereby the presence of senescent cells in old bodies may affect different tissue systems.     

Senescence in cell oxidative status in two bird species with contrasting life expectancy

Oxidative stress occurs when the production of reactive oxygen species (ROS) by an organism exceeds its capacity to mitigate the damaging effects of the ROS. Consequently, oxidative stress hypotheses of ageing argue that a decline in fecundity and an increase in the likelihood of death with advancing age reported at the organism level are driven by gradual disruption of the oxidative balance at the cellular level. Here, we measured erythrocyte resistance to oxidative stress in the same individuals over several years in two free-living bird species with contrasting life expectancy, the great tit (known maximum life expectancy is 15.4 years) and the Alpine swift (26 years). In both species, we found evidence for senescence in cell resistance to oxidative stress, with patterns of senescence becoming apparent as subjects get older. In the Alpine swift, there was also evidence for positive selection on cell resistance to oxidative stress, the more resistant subjects being longer lived. The present findings of inter-individual selection and intra-individual deterioration in cell oxidative status at old age in free-living animals support a role for oxidative stress in the ageing of wild animals.     

The cellular hydration state: a critical determinant for cell death and survival

Alterations in cellular hydration not only contribute to metabolic regulation, but also critically determine the cellular response to different kinds of stress. Whereas cell swelling triggers anabolic pathways and protects cells from heat and oxidative challenge, cellular dehydration contributes to insulin resistance and catabolism and increases the cellular susceptibility to stress-induced damage. Intracellular accumulation of organic osmolytes, cell cycle delay and the expression of heat shock proteins provide cellular tolerance to hyperosmolarity and protect against stressors under dehydrating conditions. This article discusses some mechanisms by which alterations in cell hydration contribute to cytoprotection or cell damage. In addition, the close relationship between osmotic and oxidative stress and the contribution of isoosmotic shrinkage to apoptotic cell death are considered.     

Proteome Oxidative Modifications and Impairment of Specific Metabolic Pathways During Cellular Senescence and Aging

Accumulation of oxidatively modified proteins is a hallmark of organismal aging in vivo and of cellular replicative senescence in vitro. Failure of protein maintenance is a major contributor to the age‐associated accumulation of damaged proteins that is believed to participate to the age‐related decline in cellular function. In this context, quantitative proteomics approaches, including 2‐D gel electrophoresis (2‐DE)‐based methods, represent powerful tools for monitoring the extent of protein oxidative modifications at the proteome level and for identifying the targeted proteins, also referred as to the “oxi‐proteome.” Previous studies have identified proteins targeted by oxidative modifications during replicative senescence of human WI‐38 fibroblasts and myoblasts and have been shown to represent a restricted set within the total cellular proteome that fall in key functional categories, such as energy metabolism, protein quality control, and cellular morphology. To provide mechanistic support into the role of oxidized proteins in the development of the senescent phenotype, untargeted metabolomic profiling is also performed for young and senescent myoblasts and fibroblasts. Metabolomic profiling is indicative of energy metabolism impairment in both senescent myoblasts and fibroblasts, suggesting a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts and fibroblasts.