The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/Δ mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

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.

Aging is associated with increased cellular senescence, which is hypothesized to drive the eventual development of multiple co-morbidities1. Here, we investigate a role for senescent cells in age-related bone loss by multiple approaches. In particular, we used either genetic (i.e., the INK-ATTAC “suicide” transgene encoding an inducible caspase 8 expressed specifically in senescent cells2–4) or pharmacological (i.e., “senolytic” compounds5,6) means to eliminate senescent cells. We also inhibited the production of the pro-inflammatory secretome of senescent cells using a JAK inhibitor (JAKi)3,7. In old (20–22-months) mice with established bone loss, activation of the INK-ATTAC caspase 8 in senescent cells or treatment with senolytics or the JAKi for 2–4 months resulted in higher bone mass and strength and better bone microarchitecture compared to vehicle-treated mice. The beneficial effects of targeting senescent cells were due to lower bone resorption with either maintained (trabecular bone) or higher (cortical bone) bone formation as compared to vehicle-treated mice. In vitro studies demonstrated that senescent cell-conditioned medium impaired osteoblast mineralization and enhanced osteoclast progenitor survival, leading to increased osteoclastogenesis. Collectively, these data establish a causal role for senescent cells in bone loss with aging and demonstrate that targeting these cells has both anti-resorptive and anabolic effects on bone. As eliminating senescent cells and/or inhibiting their pro-inflammatory secretome also improves cardiovascular function4, enhances insulin sensitivity3, and reduces frailty7, targeting this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis but also for multiple age-related co-morbidities.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age-related bone loss.

While patients with type 2 diabetes (T2D) are at significant risk for well recognized diabetic complications, including macrovascular disease, retinopathy, nephropathy, and neuropathy, it is also clear that T2D patients are at increased risk for fragility fractures. Furthermore, fragility fractures in patients with T2D occur at higher bone mineral density (BMD) values compared to non-diabetic controls, suggesting abnormalities in bone material strength (BMS) and/or bone microarchitecture (bone “quality”). Thus, we performed in vivo microindentation testing of the tibia to directly measure BMS in 60 postmenopausal women (age range, 50–80 yrs) including 30 patients diagnosed with T2D for >10 yrs and 30 age-matched, non-diabetic controls. Regional BMD was measured by DXA; cortical and trabecular bone microarchitecture was assessed from HRpQCT images of the distal radius and tibia. Compared to controls, T2D patients had significantly lower BMS: unadjusted (−11.7%; p<0.001); following adjustment for BMI (−10.5%; p<0.001); and following additional adjustment for age, hypertension, nephropathy, neuropathy, retinopathy, and vascular disease (−9.2%; p=0.022). By contrast, after adjustment for confounding by BMI, T2D patients had bone microarchitecture and BMD that were not significantly different than controls; however, radial cortical porosity tended to be higher in the T2D patients. In addition, patients with T2D had significantly reduced serum markers of bone turnover (all p<0.001) compared to controls. Of note, in patients with T2D, the average glycated hemoglobin level over the previous 10 yrs was negatively correlated with BMS (r = −0.41; p=0.026). In conclusion, these findings represent the first demonstration of compromised bone material strength in patients with T2D. Furthermore, our results confirm previous studies demonstrating low bone turnover in patients with T2D and highlight the potential detrimental effects of prolonged hyperglycemia on bone quality. Thus, the skeleton needs to be recognized as another important target tissue subject to diabetic complications.

With the ageing of the global population, interest is growing in the 'geroscience hypothesis', which posits that manipulation of fundamental ageing mechanisms will delay (in parallel) the appearance or severity of multiple chronic, non-communicable diseases, as these diseases share the same underlying risk factor-namely, ageing. In this context, cellular senescence has received considerable attention as a potential target in preventing or treating multiple age-related diseases and increasing healthspan. Here we review mechanisms of cellular senescence and approaches to target this pathway therapeutically using 'senolytic' drugs that kill senescent cells or inhibitors of the senescence-associated secretory phenotype (SASP). Furthermore, we highlight the evidence that cellular senescence has a causative role in multiple diseases associated with ageing. Finally, we focus on the role of cellular senescence in a number of endocrine diseases, including osteoporosis, metabolic syndrome and type 2 diabetes mellitus, as well as other endocrine conditions. Although much remains to be done, considerable preclinical evidence is now leading to the initiation of proof-of-concept clinical trials using senolytics for several endocrine and nonendocrine diseases. Ageing is now generally accepted as the single largest risk factor for many of the major chronic diseases (for example, type 2 diabetes mellitus (T2DM), cardiovascular disease and cancer) that account for the bulk of morbidity, deaths and health costs in the USA and other developed countries 1. For these conditions, the predictive ability of advanced chronological age exceeds the predictive ability of all other risk factors combined. Enormous progress has been made over the years in the development of specific drugs to treat individual diseases associated with ageing, including metabolic dysfunction and T2DM (for example, GLP1R agonists and DPP4 inhibitors), skeletal fragility and osteoporosis (for example,

Although cellular senescence drives multiple age-related co-morbidities through the senescence-associated secretory phenotype, in vivo senescent cell identification remains challenging. Here, we generate a gene set (SenMayo) and validate its enrichment in bone biopsies from two aged human cohorts. We further demonstrate reductions in SenMayo in bone following genetic clearance of senescent cells in mice and in adipose tissue from humans following pharmacological senescent cell clearance. We next use SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from human and murine bone marrow/bone scRNA-seq data. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Using this senescence panel, we are able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways. SenMayo also represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

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