Suppression of replicative senescence by rapamycin in rodent embryonic cells

The TOR (target of rapamycin) pathway is involved in aging in diverse organisms from yeast to mammals. We have previously demonstrated in human and rodent cells that mTOR converts stress-induced cell cycle arrest to irreversible senescence (geroconversion), whereas rapamycin decelerates or suppresses geroconversion during cell cycle arrest. Here, we investigated whether rapamycin can suppress replicative senescence of rodent cells. Mouse embryonic fibroblasts (MEFs) gradually acquired senescent morphology and ceased proliferation. Rapamycin decreased cellular hypertrophy, and SA-beta-Gal staining otherwise developed by 4-6 passages, but it blocked cell proliferation, masking its effects on replicative lifespan. We determined that rapamycin inhibited pS6 at 100-300 pM and inhibited proliferation with IC50 around 30 pM. At 30 pM, rapamycin partially suppressed senescence. However, the gerosuppressive effect was balanced by the cytostatic effect, making it difficult to suppress senescence without causing quiescence. We also investigated rat embryonic fibroblasts (REFs), which exhibited markers of senescence at passage 7, yet were able to slowly proliferate until 12–14 passages. REFs grew in size, acquired a large, flat cell morphology, SA-beta-Gal staining and components of DNA damage response (DDR), in particular, γH2AX/53BP1 foci. Incubation of REFs with rapamycin (from passage 7 to passage 10) allowed REFs to overcome the replicative senescence crisis. Following rapamycin treatment and removal, a fraction of proliferating REFs gradually increased and senescent phenotype disappeared completely by passage 24.     

Cellular lifespan and senescence: a complex balance between multiple cellular pathways

The study of cellular senescence and proliferative lifespan is becoming increasingly important because of the promises of autologous cell therapy, the need for model systems for tissue disease and the implication of senescent cell phenotypes in organismal disease states such as sarcopenia, diabetes and various cancers, among others. Here, we explain the concepts of proliferative cellular lifespan and cellular senescence, and we present factors that have been shown to mediate cellular lifespan positively or negatively. We review much recent literature and present potential molecular mechanisms by which lifespan mediation occurs, drawing from the fields of telomere biology, metabolism, NAD⁺ and sirtuin biology, growth factor signaling and oxygen and antioxidants. We conclude that cellular lifespan and senescence are complex concepts that are governed by multiple independent and interdependent pathways, and that greater understanding of these pathways, their interactions and their convergence upon specific cellular phenotypes may lead to viable therapies for tissue regeneration and treatment of age‐related pathologies, which are caused by or exacerbated by senescent cells in vivo.     

Sex‐ and tissue‐specific changes in mTOR signaling with age in C57BL/6J mice

Inhibition of the mTOR (mechanistic Target Of Rapamycin) signaling pathway robustly extends the lifespan of model organisms including mice. The precise molecular mechanisms and physiological effects that underlie the beneficial effects of rapamycin are an exciting area of research. Surprisingly, while some data suggest that mTOR signaling normally increases with age in mice, the effect of age on mTOR signaling has never been comprehensively assessed. Here, we determine the age‐associated changes in mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) signaling in the liver, muscle, adipose, and heart of C57BL/6J.Nia mice, the lifespan of which can be extended by rapamycin treatment. We find that the effect of age on several different readouts of mTORC1 and mTORC2 activity varies by tissue and sex in C57BL/6J.Nia mice. Intriguingly, we observed increased mTORC1 activity in the liver and heart tissue of young female mice compared to male mice of the same age. Tissue and substrate‐specific results were observed in the livers of HET3 and DBA/2 mouse strains, and in liver, muscle and adipose tissue of F344 rats. Our results demonstrate that aging does not result in increased mTOR signaling in most tissues and suggest that rapamycin does not promote lifespan by reversing or blunting such an effect.     

Gene Pathways That Delay Caenorhabditis elegans Reproductive Senescence

Reproductive senescence is a hallmark of aging. The molecular mechanisms regulating reproductive senescence and its association with the aging of somatic cells remain poorly understood. From a full genome RNA interference (RNAi) screen, we identified 32 Caenorhabditis elegans gene inactivations that delay reproductive senescence and extend reproductive lifespan. We found that many of these gene inactivations interact with insulin/IGF-1 and/or TGF-β endocrine signaling pathways to regulate reproductive senescence, except nhx-2 and sgk-1 that modulate sodium reabsorption. Of these 32 gene inactivations, we also found that 19 increase reproductive lifespan through their effects on oocyte activities, 8 of them coordinate oocyte and sperm functions to extend reproductive lifespan, and 5 of them can induce sperm humoral response to promote reproductive longevity. Furthermore, we examined the effects of these reproductive aging regulators on somatic aging. We found that 5 of these gene inactivations prolong organismal lifespan, and 20 of them increase healthy life expectancy of an organism without altering total life span. These studies provide a systemic view on the genetic regulation of reproductive senescence and its intersection with organism longevity. The majority of these newly identified genes are conserved, and may provide new insights into age-associated reproductive senescence during human aging.     

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism

Senescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild‐type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA‐β‐galactosidase (β‐gal) staining, senescence‐associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β‐gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β‐gal staining and pro‐inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.     

Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system

Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA‐approved drug rapamycin has been shown to promote lifespan and delay age‐related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long‐term prophylactic use of rapamycin as a therapy for age‐related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR‐containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA‐approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA‐approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs.     

Rapamycin improves healthspan but not inflammaging in nfκb1−/− mice

Increased activation of the major pro‐inflammatory NF‐κB pathway leads to numerous age‐related diseases, including chronic liver disease (CLD). Rapamycin, an inhibitor of mTOR, extends lifespan and healthspan, potentially via suppression of inflammaging, a process which is partially dependent on NF‐κB signalling. However, it is unknown if rapamycin has beneficial effects in the context of compromised NF‐κB signalling, such as that which occurs in several age‐related chronic diseases. In this study, we investigated whether rapamycin could ameliorate age‐associated phenotypes in a mouse model of genetically enhanced NF‐κB activity (nfκb1^?/?) characterized by low‐grade chronic inflammation, accelerated aging and CLD. We found that, despite showing no beneficial effects in lifespan and inflammaging, rapamycin reduced frailty and improved long‐term memory, neuromuscular coordination and tissue architecture. Importantly, markers of cellular senescence, a known driver of age‐related pathology, were alleviated in rapamycin‐fed animals. Our results indicate that, in conditions of genetically enhanced NF‐κB, rapamycin delays aging phenotypes and improves healthspan uncoupled from its role as a suppressor of inflammation.     

Hypothalamic mTORC2 is essential for metabolic health and longevity

The mechanistic target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that regulates growth and metabolism. mTOR is found in two protein complexes, mTORC1 and mTORC2, that have distinct components and substrates and are both inhibited by rapamycin, a macrolide drug that robustly extends lifespan in multiple species including worms and mice. Although the beneficial effect of rapamycin on longevity is generally attributed to reduced mTORC1 signaling, disruption of mTORC2 signaling can also influence the longevity of worms, either positively or negatively depending on the temperature and food source. Here, we show that loss of hypothalamic mTORC2 signaling in mice decreases activity level, increases the set point for adiposity, and renders the animals susceptible to diet‐induced obesity. Hypothalamic mTORC2 signaling normally increases with age, and mice lacking this pathway display higher fat mass and impaired glucose homeostasis throughout life, become more frail with age, and have decreased overall survival. We conclude that hypothalamic mTORC2 is essential for the normal metabolic health, fitness, and lifespan of mice. Our results have implications for the use of mTORC2‐inhibiting pharmaceuticals in the treatment of brain cancer and diseases of aging.     

The JAK1/2 inhibitor ruxolitinib delays premature aging phenotypes

Hutchinson–Gilford progeria syndrome (HGPS) is caused by an LMNA mutation that results in the production of the abnormal progerin protein. Children with HGPS display phenotypes of premature aging and have an average lifespan of 13 years. We found earlier that the targeting of the transmembrane protein PLA2R1 overcomes senescence and improves phenotypes in a mouse model of progeria. PLA2R1 is regulating the JAK/STAT signaling, but we do not yet know whether targeting this pathway directly would influence cellular and in vivo progeria phenotypes. Here, we show that JAK1/2 inhibition with ruxolitinib rescues progerin‐induced cell cycle arrest, cellular senescence, and misshapen nuclei in human normal fibroblasts expressing progerin. Moreover, ruxolitinib administration reduces several premature aging phenotypes: bone fractures, bone mineral content, grip strength, and a trend to increase survival in a mouse model of progeria. Thus, we propose that ruxolitinib, an FDA‐approved drug, should be further evaluated as a drug candidate in HGPS therapy.     

Insulin‐like signalling in neurons controls lifespan in C. elegans

Insulin‐like signalling controls C. elegans lifespan, development and metabolism. Mutations that weaken this insulin‐like signalling pathway extend lifespan. Severe mutations abolishing insulin‐like signalling cause animals to arrest development as dauer larvae, a larval form specialized for stress resistance and long‐term survival. A number of the genes acting in this pathway have been cloned, including daf‐2, which encodes a homolog of vertebrate insulin/IGF‐I receptors, and age‐1, encoding the C. elegans homolog of the PI(3)K p110 catalytic subunit. In order to identify cells from which insulin‐like signalling controls lifespan and development, transgenic animals were constructed which possessed insulin‐like signalling only in specific cell types. To achieve this, cell‐type specific promoters were used to drive expression of daf‐2 or age‐1 cDNAs in daf‐2(–/–) or age‐1(–/–) backgrounds, respectively. By utilizing this strategy, we could restore wild‐type daf‐2 or age‐1 activity only in cells that are capable of expressing each transgene. Restoring insulin‐like signalling to the nervous system of daf‐2 or age‐1 mutants could rescue long lifespan. This result was specific for transgenes restoring insulin‐like signalling to the nervous system. Expressing daf‐2 or age‐1 cDNAs from muscle‐ or intestinally‐restricted promoters was insufficient to rescue lifespan. In contrast, age‐1 and daf‐2 expression in either neuronal or non‐neuronal cell types rescued dauer larval arrest in the mutants. These findings demonstrate that insulin‐like signalling pathways in the nervous system control C. elegans lifespan.     

Autophagy and cardiovascular aging

The biological aging process is commonly associated with increased risk of cardiovascular diseases. Several theories have been put forward for aging-associated deterioration in ventricular function, including attenuation of growth hormone (insulin-like growth factors and insulin) signaling, loss of DNA replication and repair, histone acetylation and accumulation of reactive oxygen species. Recent evidence has depicted a rather unique role of autophagy as another important pathway in the regulation of longevity and senescence. Autophagy is a predominant cytoprotective (rather than self-destructive) process. It carries a prominent role in determination of lifespan. Reduced autophagy has been associated with aging, leading to accumulation of dysfunctional or damaged proteins and organelles. To the contrary, measures such as caloric restriction and exercise may promote autophagy to delay aging and associated comorbidities. Stimulation of autophagy using rapamycin may represent a novel strategy to prolong lifespan and combat aging-associated diseases. Rapamycin regulates autophagy through inhibition of the nutrient-sensing molecule mammalian target of rapamycin (mTOR). Inhibition of mTOR through rapamycin and caloric restriction promotes longevity. The purpose of this review is to recapitulate some of the recent advances in an effort to better understand the interplay between rapamycin-induced autophagy and decelerating cardiovascular aging.     

Studies of Caenorhabditis elegans DAF-2/insulin signaling reveal targets for pharmacological manipulation of lifespan

Much excitement has arisen from the observation that decrements in insulin‐like signaling can dramatically extend lifespan in the nematode, Caenorhabditis elegans, and fruitfly, Drosophila melanogaster. In addition, there are tantalizing hints that the IGF‐I pathway in mice may have similar effects. In addition to dramatic effects on lifespan, invertebrate insulin‐like signaling also promotes changes in stress resistance, metabolism and development. Which, if any, of the various phenotypes of insulin pathway mutants are relevant to longevity? What are the genes that function in collaboration with insulin to prolong lifespan? These questions are at the heart of current research in C. elegans longevity. Two main theories exist as to the mechanism behind insulin's effects on invertebrate longevity. One theory is that insulin programs metabolic parameters that prolong or reduce lifespan. The other theory is that insulin determines the cell's ability to endure oxidative stress from respiration, thereby determining the rate of aging. However, these mechanisms are not mutually exclusive and several studies seem to support a role for both. Here, we review recently published reports investigating the mechanisms behind insulin's dramatic effect on longevity. We also spotlight several C. elegans genes that are now known to interact with insulin signaling to determine lifespan. These insights into pathways affecting invertebrate lifespan may provide a basis for developing strategies for pharmacological manipulation of human lifespan.     

Autophagy and cardiovascular aging: Lesson learned from rapamycin

The biological aging process is commonly associated with increased risk of cardiovascular diseases. Several theories have been put forward for aging-associated deterioration in ventricular function, including attenuation of growth hormone (insulin-like growth factors and insulin) signaling, loss of DNA replication and repair, histone acetylation and accumulation of reactive oxygen species. Recent evidence has depicted a rather unique role of autophagy as another important pathway in the regulation of longevity and senescence. Autophagy is a predominant cytoprotective (rather than self-destructive) process. It carries a prominent role in determination of lifespan. Reduced autophagy has been associated with aging, leading to accumulation of dysfunctional or damaged proteins and organelles. To the contrary, measures such as caloric restriction and exercise may promote autophagy to delay aging and associated comorbidities. Stimulation of autophagy using rapamycin may represent a novel strategy to prolong lifespan and combat aging-associated diseases. Rapamycin regulates autophagy through inhibition of the nutrient-sensing molecule mammalian target of rapamycin (mTOR). Inhibition of mTOR through rapamycin and caloric restriction promotes longevity. The purpose of this review is to recapitulate some of the recent advances in an effort to better understand the interplay between rapamycin-induced autophagy and decelerating cardiovascular aging.     

Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects

Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10‐week RP or 40% CR. Protein abundance and turnover were measured in vivo using ²H₃–leucine heavy isotope labeling followed by LC‐MS/MS, and translation was assessed by polysome profiling. We observed 35–60% increased protein half‐lives after CR and 15% increased half‐lives after RP compared to age‐matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions.     

Testing the effect of early‐life reproductive effort on age‐related decline in a wild insect

The disposable soma theory of ageing predicts that when organisms invest in reproduction they do so by reducing their investment in body maintenance, inducing a trade‐off between reproduction and survival. Experiments on invertebrates in the lab provide support for the theory by demonstrating the predicted responses to manipulation of reproductive effort or lifespan. However, experimental studies in birds and evidence from observational (nonmanipulative) studies in nature do not consistently reveal trade‐offs. Most species studied previously in the wild are mammals and birds that reproduce over multiple discrete seasons. This contrasts with temperate invertebrates, which typically have annual generations and reproduce over a single season. We expand the taxonomic range of senescence study systems to include life histories typical of most temperate invertebrates. We monitored reproductive effort, ageing, and survival in a natural field cricket population over ten years to test the prediction that individuals investing more in early‐reproduction senesce faster and die younger. We found no evidence of a trade‐off between early‐life reproductive effort and survival, and only weak evidence for a trade‐off with phenotypic senescence. We discuss the possibility that organisms with multiple discrete breeding seasons may have greater opportunities to express trade‐offs between reproduction and senescence.     

Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence we examined the role of TOR pathway genes in regulating lifespan by using Drosophila. We show that inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2 (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. We find that overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction.