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Senolytics improve physical function and increase lifespan in old age
 
 
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Abstract
 
Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.
 
Main
 
New approaches to treat age-related diseases (e.g., cardiovascular disease) may yield lifespan extension but potentially at the cost of extending late-life morbidity1. A major question in biology is whether interventions can be devised that enhance healthspan in tandem with increasing remaining lifespan so that the period of morbidity near the end of life is not increased2. Physical dysfunction and incapacity to respond to stresses3,4 become increasingly prevalent toward the end of life5, with up to 45% of people over the age of 85 being frail6. This physical dysfunction is associated with considerable morbidity, including decreased mobility and increased burden of age-related chronic diseases, loss of independence, nursing home and hospital admissions, and mortality7. The cellular pathogenesis of age-related physical dysfunction has not been fully elucidated, and there are currently no root cause-directed, mechanism-based interventions for improving physical function in the elderly available for clinical application. Here we report a potential strategy for addressing this need: reducing senescent cell burden.
 
Cellular senescence is a cell fate involving extensive changes in gene expression and proliferative arrest. Senescence can be induced by such stresses as DNA damage, telomere shortening, oncogenic mutations, metabolic and mitochondrial dysfunction, and inflammation8,9,10. Senescent cell burden increases in multiple tissues with aging11, at sites of pathology in multiple chronic diseases, and after radiation or chemotherapy12. Senescent cells can secrete a range of proinflammatory cytokines, chemokines, proteases, and other factors; together, these are termed the senescence-associated secretory phenotype (SASP)13,14, which contributes to local and systemic dysfunction with aging and in a number of diseases15,16,17,18,19,20,21. A month or more of continuous treatment with Janus kinase 1 (JAK1) and JAK2 inhibitors or rapamycin can enhance physical function in older mice14,22. However, JAK1 and JAK2 inhibitors and rapamycin have an extensive range of effects on cellular function. Among these is reduced production of some SASP components. We hypothesized, on the basis of these points, that senescent cells might potentially contribute to age-related physical dysfunction and, if so, that targeting them therapeutically could improve life- and healthspan.
 
Discussion
 
Our study shows that health- and lifespan are curtailed by increased senescent cell abundance and, conversely, are enhanced by reducing proinflammatory, SCAP-dependent senescent cell burden in mice, even late in life. Collectively, evidence for a causal role of such senescent cells in physical dysfunction satisfies a modified set of Koch's postulates34: (i) senescent cell burden14 and physical dysfunction5 are associated with each other and with aging; (ii) transplanting small numbers of senescent cells into young mice that have few endogenous senescent cells is sufficient to cause physical dysfunction that lasts for months; (iii) senolytics prevent and alleviate this senescent cell transplantation-induced physical dysfunction in young mice; (iv) clearing senescent cells alleviates physical dysfunction and increases remaining lifespan in old mice; and (v) locally inducing senescence in the lung impaired physical function, and clearing senescent cells improved physical function in a bleomycin-induced pulmonary fibrosis mouse model25. Notably, we determined that the total cell number in recipient adipose tissue is approximately 350 million. Several studies have shown that the percentage of senescent cells in adipose tissue from aged animals can be up to 2-10% (refs 14,15,20), indicating that there might be at least 7 million endogenous senescent cells in adipose tissue of aged mice. Although other mechanisms might contribute, one reason why such a small number (0.5-1 million) of transplanted senescent cells could cause systemic dysfunction is spread of senescence and inflammation locally and to distant tissues.
 
We used preadipocytes for transplantation, as these cells appear to be less susceptible to immune rejection than other cell types26, a reason for their increasing clinical use in cell transplantation studies. To further test whether there is a role of immune rejection in the physical dysfunction caused by senescent cell transplantation, we performed autologous transplantation, and we also transplanted human senescent cells into immunodeficient (SCID-beige) mice. Senescent cells induced essentially identical physical dysfunction in both of these settings in which immune rejection is greatly reduced. These points, together with our finding that transplanted senescent and control cells disappear at the same rate following transplantation, plus the long-lasting phenotypes caused by transplanting senescent cells, indicate that immune-rejection or transient 'sickness' caused by transplantation might not be a primary cause for the physical dysfunction we found. The SCID-beige mouse senescent cell-transplantation model could pave the way for testing the effects of other senescent cell types that are more immunogenic than preadipocytes or autologous cells in future studies.
 
We originally discovered that D + Q are senolytic agents by using a hypothesis-driven bioinformatics approach. This approach was based on the hypotheses that senescent cells depend on prosurvival pathways to defend themselves against their proapoptotic microenvironment and that disabling these pathways would therefore selectively kill senescent cells35. That D + Q can specifically kill senescent cells in human adipose tissue is supported by the following evidence: (i) D + Q induce apoptosis only in senescent human cells, rather than in control cells in vitro35; (ii) D + Q significantly decreased senescent cells as determined in three different assessments, whereas D + Q did not reduce the overall number of cells in adipose tissue; and (iii) secretion of two major adipokines was not reduced by D + Q, effectively ruling out nonspecific effects on secretion or cell viability. We believe that these data indicate that occurrence of nonspecific cellular toxicity occurs as a result of D + Q is unlikely. Of note, we showed the effectiveness of senolytics in targeting naturally occurring senescent cells in human adipose tissue explants collected from obese subjects. Although similar to aging, obesity is highly associated with increased senescent cell burden27,32. In future studies, the efficacy of senolytics in tissues from older humans needs to be compared to that in explants from obese subjects.
 
We found that D + Q has little direct effect on macrophages. However, it is unlikely that the substantial reduction of proinflammatory cytokine secretion following D + Q was due to elimination of senescent cells alone. Interestingly, we found that senescent cells can induce adipose tissue cytokine production, potentially amplifying inflammation. By clearing senescent cells, this possible amplification of inflammation could be attenuated in adipose tissue, which includes macrophages. On the basis of these findings, we speculate that by decreasing senescent cell abundance, reduced inflammatory activity in adipose tissue could ensue and contribute to the reduction of inflammation after D + Q exposure, as well as to decreased spread of senescence.
 
Reducing p16INK4A-hi cells, some of which are senescent, in INK-ATTAC transgenic mice starting from midadulthood (age 12 months) reportedly extends median lifespan49. In our study, administration of D + Q or AP20187 under the same conditions to WT and INK-ATTAC mice began at age 24-27 months. Unlike D + Q, AP20187 did not enhance late-life survival. One reason could be that senolytics act by disabling the SCAP, a mechanism of action that is distinct from that in INK-ATTAC mice, in which p16INK4A-hi cells are targeted. We chose old age (24-27 months) rather than middle age (12 months) to start treatment for several reasons. First, 12-month-old mice are roughly equivalent to 40-year-old humans, and 24-month-old mice are equivalent to 75-year-old humans. We felt that beginning at 24 months of age is potentially a more translatable scenario. Second, AP20187 targets p16INK4A+ cells, some of which are senescent, but others of which are not51,52 (e.g., pancreatic β-cells, macrophages, etc.). Considering that 12-month-old mice have very few detectible senescent cells in adipose tissue, whereas these cells are abundant in 24-month-old mice20, beginning AP20187 administration at 12 months of age could have targeted a higher proportion of p16INK4A+ cells that are not senescent than initiating treatment at 24 months of age, when senescent cells are readily detectible in multiple tissues. Third, treating INK-ATTAC mice from 12-month-old mice led to an increase in median rather than maximum survival. This is consistent with our finding that AP20187 did not detectibly increase late-life survival. On the other hand, we show here that by initiating senolytics in later life, late-life survival is actually increased.
 
Age-related diseases were delayed as a group in old male and female mice treated with D + Q compared to vehicle-treated controls. There was no one disease that was delayed or prevented that alone accounted for this increase in survival. In further support of the hypothesis that senescent cells participate in predisposition to age-related dysfunction, we found that the converse is also the case. Age-related diseases occurred earlier as a group in senescent cell-transplanted mice, coupled with shorter survival, than in control mice not transplanted with senescent cells. No one disease accounted for this decreased survival. These findings are consistent with the geroscience hypothesis: by targeting a fundamental aging process, such as cellular senescence, age-related disorders will be delayed as a group, with no one condition predominating.
 
Quercetin has been found to alleviate a variety of disorders through diverse mechanisms of action53, almost all of which have been studied with uninterrupted dosing, resulting in quercetin being continuously present. This is consistent with the presumption that quercetin acts on specific enzymes or pathways to achieve these effects. However, in our study, we administered D + Q intermittently. Dasatinib and quercetin both have short elimination half-lives44,45, supporting the possibility that their beneficial effects on late-life function and survival were at least partially due to a mechanism that persists long after the drugs are no longer present, such as senescent cell elimination. Dasatinib can have side effects, occasionally including serious ones such as pulmonary edema, which can occur after 8-48 months of daily administration54. Although recognized side effects of drugs in mice often differ substantially from those in humans, in our study, mice given D + Q intermittently lived longer and had improved physical function compared to vehicle-treated mice. When they eventually died, the pathologies of D + Q-treated mice determined at autopsy and through histological analysis did not differ substantially from those of vehicle-treated mice. Possibly, intermittent treatment with senolytics may serve to reduce potential side effects55, an important consideration in the frail elderly, a population vulnerable to adverse drug reactions. We emphasize that preclinical studies to search for potential toxicities and optimized regimens are required before contemplating trials in relatively healthy humans. A search for possible side effects of senolytics as a class and of individual senolytics is especially important.
 
Our study provides proof-of-concept evidence indicating that targeting senescent cells can improve both health- and lifespan in mice. Because of the role of senescent cells in inducing physical dysfunction in mice demonstrated here (with some similarity to human frailty4,31,56), senolytics might prove to be effective in alleviating physical dysfunction and resulting loss of independence in older human subjects, depending on whether these agents turn out to be safe and effective in clinical trials. Depending on the outcomes of such ongoing (ClinicalTrials.gov identifier: NCT02848131) and future preclinical and clinical studies, intermittent administration of senolytics, including newer agents such as optimized derivatives of D + Q, could possibly be useful in enhancing healthspan and remaining survival not only in older subjects, but also in other individuals, such as cancer survivors treated with senescence-inducing radiation or chemotherapy57.
 
Results
 
Transplanting small numbers of senescent cells is sufficient to induce physical dysfunction in young mice
 
To test whether senescent cells can directly cause physical dysfunction, we first transplanted senescent or control (nonsenescent) preadipocytes (also termed adipocyte progenitors or adipose-derived stem cells23) isolated from luciferase-expressing transgenic (LUC+) mice intraperitoneally into syngeneic, young (6-month-old) wild-type (WT) mice (Fig. 1a). We induced cellular senescence using 10 Gy radiation, which resulted in more than 85% of cells becoming senescent (Supplementary Fig. 1a,b). We transplanted preadipocytes because: (i) the SASP of radiation-induced senescent mouse preadipocytes (Supplementary Fig. 1c,d) resembles that of endogenous senescent cells with aging (Supplementary Fig. 1e)20,24 and in idiopathic pulmonary fibrosis25; (ii) preadipocytes are less immunogenic or subject to rejection than other cell types26; and (iii) these cells are arguably the most abundant type of progenitors in humans27 that are subject to cellular senescence. We also used doxorubicin to induce senescence to ascertain whether the physiological impact of transplanted senescent cells is limited to the context of radiation-induced senescence: senescent cells induced by doxorubicin developed a SASP similar to that of radiation-induced senescent cells (Supplementary Fig. 1f). Five days after intraperitoneal transplantation of senescent or control preadipocytes, the cells were mainly located in visceral fat (Fig. 1b,c and Supplementary Fig. 2a,b). Both the senescent and control transplanted cells remained detectable through in vivo bioluminescence imaging (BLI) for up to 40 d following transplant (Supplementary Fig. 2c). Of note, we observed that senescent cells had higher luciferase activity than control cells, even though they were from the same LUC+ transgenic mice (Supplementary Fig. 2d).
 
To determine whether the transplanted senescent cells induced physical dysfunction in these mice, as determined by criteria used in clinical practice4, we measured maximal walking speed (via RotaRod), muscle strength (via grip strength), physical endurance (via hanging test and treadmill), daily activity, food intake, and body weight. Previously healthy young adult mice transplanted with 106 senescent cells had significantly lower maximal walking speed, hanging endurance, and grip strength by 1 month after transplantation compared to mice transplanted with control cells (Fig. 1d-f and Supplementary Fig. 3a).
 
Transplanting the same number of control cells had no effect as compared to injecting PBS. Daily activity, treadmill performance, food intake, and body weight were not statistically different among groups (Fig. 1g-j). Transplanting 0.5 x 106 senescent cells was sufficient to cause decreased grip strength (Fig. 1f) and maximal walking speed (Supplementary Fig. 3b), whereas transplanting 0.2 x 106 senescent cells had no detectible effects. Thus, senescent cells can impair physical function in a dose-dependent manner. We estimate that 6-month-old mice have 7 x 109-8 x 109 cells of all types and that the total cell number in intraperitoneal adipose tissue, where the transplanted cells were mainly located (Supplementary Fig. 2b), is ∼3.5 x 108-6 x 108 cells (Methods). Thus in young mice, if only 1 cell in the 7,000-15,000 (0.01-0.03%) throughout the body or 1 cell in 350 (0.28%) locally is a transplanted senescent cell, age-related impairment in physical function ensues.
 
Reduced walking speed began as early as 2 weeks following a single implantation of senescent cells (Supplementary Fig. 3c) and persisted for up to 6 months (Supplementary Fig. 3d), yet the transplanted cells survived in vivo for only approximately 40 d, consistent with the possibility that senescent cells might induce senescence in normal host cells28,29. We therefore tested whether senescent cells can indeed cause other cells to become senescent in vivo by transplanting constitutively LUC+ senescent cells and determining whether senescence occurs in the LUC- recipients' tissue. Most of the transplanted LUC+ senescent cells resided in visceral fat (Supplementary Fig. 2b). Two months after transplantation, we found more senescence-associated β-galactosidase (SA-βgal)+ cells and higher cyclin-dependent kinase inhibitor 2 A (Cdkn2a; also known as p16Ink4a) expression in visceral adipose tissue from senescent cell- than control cell-transplanted mice (Fig. 1k,l) beyond the time the transplanted senescent cells were present as reflected by luciferase signal (Supplementary Fig. 2c). Telomere-associated foci (TAFs) are sites of DNA damage within telomeres30. TAFs appear to be a more specific marker of senescence than some others, such as SA-βgal. Significantly more TAF+ cells were found in visceral adipose tissue of mice that had been injected with senescent than control cells (Fig. 1m). These TAF+ cells were LUC-, indicating that they were the recipients' own cells and not transplanted cells. F4/80+ macrophage accumulation was not induced in adipose tissue by the intraperitoneal senescent cell transplantation (Supplementary Fig. 3e,f). Consistent with spread of senescence not only locally but also to distant tissues, expression of the markers and mediators of senescence p16Ink4a, tumor necrosis factor alpha (Tnf╬▒), and interleukin 6 (Il6) was higher in the quadriceps muscles, a tissue where transplanted cells were not detected (Supplementary Fig. 2b), of senescent- than control-transplanted mice (Supplementary Fig. 4a). Similarly to adipose tissue, F4/80+ macrophage accumulation was not induced in muscle by senescent cell transplantation (Supplementary Fig. 4b). Thus, senescence spreading may explain how a small number of transplanted SEN cells caused such profound, long-lasting, and deleterious systemic effects.
 
To further test whether SASP-related factors can contribute to induction of cellular senescence in vivo, we examined IL-10 knockout mice, which have a genetically induced premature proinflammatory phenotype that resembles the SASP and which also prematurely develop physical dysfunction reminiscent of human frailty31. We found that these mice do indeed have more senescent cells than WT controls, supporting the possibility that the SASP can induce senescence spreading in vivo (Supplementary Fig. 5a-c).
 
Aging and high-fat diet exacerbate effects of senescent cell transplantation
 
Because aging is associated with senescent cell accumulation14, we tested whether increased recipient age potentiates the effects of transplanting senescent cells. We transplanted 0.5 x 106 senescent or control preadipocytes into older (17-month-old) mice, so that 0.007% of all cells in the recipients were transplanted senescent or control cells, and 1 month later, we measured various parameters of physical function (Fig. 2a). We found that mice transplanted with senescent cells had lower maximal walking speed, hanging endurance, and grip strength than control mice (Fig. 2b-d). These findings were consistent across several independent cohorts of male (Supplementary Fig. 6a-f) and female mice (Supplementary Fig. 6g-l). Body weight, treadmill performance, daily activity, and food intake were not statistically different after transplanting senescent cells into the older mice (Fig. 2e-h). Transplanting 0.5 x 106 senescent cells led to greater impairment in walking speed and hanging endurance in 17-month-old mice than 6-month-old mice (Fig. 2i), whereas other parameters showed no statistically significant difference. Notably, in the 17-month-old mice transplanted with senescent cells, survival for the following year was significantly lower than that of age-matched control mice, with a 5.2-fold higher risk of death (mortality hazard ratio, P = 0.006) (Fig. 2j). Tumor burden, disease burden at death, and causes of death were not significantly altered by transplantation with senescent cells compared to control cells (Fig. 2k,l), suggesting that a small number of senescent cells may shorten survival through a general process, such as hastening the progression of aging, rather than by inducing any one individual disease or a few individual diseases. Thus, augmenting senescent cell burden induces physical dysfunction, more so in middle-aged than younger individuals, and increases mortality.

 
 
 
 
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