Chronic mTOR inhibition in mice with rapamycin alters T,B, myeloid,and innate lymphoid cells and gut flora and prolongs life of immune‐deficient mice

The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age‐related debilities including increasing antigen‐specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long‐term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T‐ and B‐lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer‐ and infection‐prone mice supporting disease mitigation as a mechanism for mTOR suppression‐mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.     

Prevention of Neuromusculoskeletal Frailty in Slow-Aging Ames Dwarf Mice: Longitudinal Investigation of Interaction of Longevity Genes and Caloric Restriction

Ames dwarf (Prop1 ^df/df) mice are remarkably long-lived and exhibit many characteristics of delayed aging and extended healthspan. Caloric restriction (CR) has similar effects on healthspan and lifespan, and causes an extension of longevity in Ames dwarf mice. Our study objective was to determine whether Ames dwarfism or CR influence neuromusculoskeletal function in middle-aged (82 ± 12 weeks old) or old (128 ± 14 w.o.) mice. At the examined ages, strength was improved by dwarfism, CR, and dwarfism plus CR in male mice; balance/ motor coordination was improved by CR in old animals and in middle-aged females; and agility/ motor coordination was improved by a combination of dwarfism and CR in both genders of middle-aged mice and in old females. Therefore, extension of longevity by congenital hypopituitarism is associated with improved maintenance of the examined measures of strength, agility, and motor coordination, key elements of frailty during human aging, into advanced age. This study serves as a particularly important example of knowledge related to addressing aging-associated diseases and disorders that results from studies in long-lived mammals.     

Opposing effects on cardiac function by calorie restriction in different‐aged mice

Calorie restriction (CR) increases average and maximum lifespan and exhibits an apparent beneficial impact on age‐related diseases. Several studies have shown that CR initiated either in middle or old age could improve ischemic tolerance and rejuvenate the aging heart; however, the data are not uniform when initiated in young. The accurate time to initiate CR providing maximum benefits for cardiac remodeling and function during aging remains unclear. Thus, whether a similar degree of CR initiated in mice of different ages could exert a similar effect on myocardial protection was investigated in this study. C57BL/6 mice were subjected to a calorically restricted diet (40% less than the ad libitum diet) for 3 months initiated in 3, 12, and 19 months. It was found that CR significantly reversed the aging phenotypes of middle‐aged and old mice including cardiac remodeling (cardiomyocyte hypertrophy and cardiac fibrosis), inflammation, mitochondrial damage, telomere shortening, as well as senescence‐associated markers but accelerated in young mice. Furthermore, whole‐genome microarray demonstrated that the AMP‐activated protein kinase (AMPK)–Forkhead box subgroup ‘O’ (FOXO) pathway might be a major contributor to contrasting regulation by CR initiated in different ages; thus, increased autophagy was seen in middle‐aged and old mice but decreased in young mice. Together, the findings demonstrated promising myocardial protection by 40% CR should be initiated in middle or old age that may have vital implications for the practical nutritional regimen.     

Healthspan and longevity can be extended by suppression of growth hormone signaling

Average and maximal lifespan are important biological characteristics of every species, but can be modified by mutations and by a variety of genetic, dietary, environmental, and pharmacological interventions. Mutations or disruption of genes required for biosynthesis or action of growth hormone (GH) produce remarkable extension of longevity in laboratory mice. Importantly, the long-lived GH-related mutants exhibit many symptoms of delayed and/or slower aging, including preservation of physical and cognitive functions and resistance to stress and age-related disease. These characteristics could be collectively described as “healthy aging” or extension of the healthspan. Extension of both the healthspan and lifespan in GH-deficient and GH-resistant mice appears to be due to multiple interrelated mechanisms. Some of these mechanisms have been linked to healthy aging and genetic predisposition to extended longevity in humans. Enhanced insulin sensitivity combined with reduced insulin levels, reduced adipose tissue, central nervous system inflammation, and increased levels of adiponectin represent such mechanisms. Further progress in elucidation of mechanisms that link reduced GH action to delayed and healthy aging should identify targets for lifestyle and pharmacological interventions that could benefit individuals as well as society.     

Rapamycin persistently improves cardiac function in aged,male and female mice,even following cessation of treatment

Even in healthy aging, cardiac morbidity and mortality increase with age in both mice and humans. These effects include a decline in diastolic function, left ventricular hypertrophy, metabolic substrate shifts, and alterations in the cardiac proteome. Previous work from our laboratory indicated that short‐term (10‐week) treatment with rapamycin, an mTORC1 inhibitor, improved measures of these age‐related changes. In this report, we demonstrate that the rapamycin‐dependent improvement of diastolic function is highly persistent, while decreases in both cardiac hypertrophy and passive stiffness are substantially persistent 8 weeks after cessation of an 8‐week treatment of rapamycin in both male and female 22‐ to 24‐month‐old C57BL/6NIA mice. The proteomic and metabolomic abundance changes that occur after 8 weeks of rapamycin treatment have varying persistence after 8 further weeks without the drug. However, rapamycin did lead to a persistent increase in abundance of electron transport chain (ETC) complex components, most of which belonged to Complex I. Although ETC protein abundance and Complex I activity were each differentially affected in males and females, the ratio of Complex I activity to Complex I protein abundance was equally and persistently reduced after rapamycin treatment in both sexes. Thus, rapamycin treatment in the aged mice persistently improved diastolic function and myocardial stiffness, persistently altered the cardiac proteome in the absence of persistent metabolic changes, and led to persistent alterations in mitochondrial respiratory chain activity. These observations suggest that an optimal translational regimen for rapamycin therapy that promotes enhancement of healthspan may involve intermittent short‐term treatments.     

PAPP‐A: a new anti‐aging target?

This article focuses on the role of PAPP‐A in mammalian aging. It introduces PAPP‐A and a little of its history, briefly discusses the function of PAPP‐A in the insulin‐like growth factor (IGF) system and the regulators of PAPP‐A expression, and then reviews data concerning PAPP‐A in aging and age‐related diseases especially in regard to the PAPP‐A knockout (KO) mouse. The PAPP‐A KO mouse is a valuable new model to test hypotheses concerning the control of the tissue availability of IGF, independent from systemic levels, on healthspan as well as lifespan.     

A comprehensive approach to the molecular determinants of lifespan using a Boolean model of geroconversion

Altered molecular responses to insulin and growth factors (GF) are responsible for late‐life shortening diseases such as type‐2 diabetes mellitus (T2DM) and cancers. We have built a network of the signaling pathways that control S‐phase entry and a specific type of senescence called geroconversion. We have translated this network into a Boolean model to study possible cell phenotype outcomes under diverse molecular signaling conditions. In the context of insulin resistance, the model was able to reproduce the variations of the senescence level observed in tissues related to T2DM's main morbidity and mortality. Furthermore, by calibrating the pharmacodynamics of mTOR inhibitors, we have been able to reproduce the dose‐dependent effect of rapamycin on liver degeneration and lifespan expansion in wild‐type and HER2–neu mice. Using the model, we have finally performed an in silico prospective screen of the risk–benefit ratio of rapamycin dosage for healthy lifespan expansion strategies. We present here a comprehensive prognostic and predictive systems biology tool for human aging.     

Movement decline across lifespan of Caenorhabditis elegans mutants in the insulin/insulin‐like signaling pathway

Research in aging biology has identified several pathways that are molecularly conserved across species that extend lifespan when mutated. The insulin/insulin‐like signaling (IIS) pathway is one of the most widely studied of these. It has been assumed that extending lifespan also extends healthspan (the period of life with minimal functional loss). However, data supporting this assumption conflict and recent evidence suggest that life extension may, in and of itself, extend the frail period. In this study, we use Caenorhabditis elegans to further probe the link between lifespan and healthspan. Using movement decline as a measure of health, we assessed healthspan across the entire lifespan in nine IIS pathway mutants. In one series of experiments, we studied healthspan in mass cultures, and in another series, we studied individuals longitudinally. We found that long‐lived mutants display prolonged mid‐life movement and do not prolong the frailty period. Lastly, we observed that early‐adulthood movement was not predictive of late‐life movement or survival, within identical phenotypes. Overall, these observations show that extending lifespan does not prolong the period of frailty. Both genotype and a stochastic component modulate aging, and movement late in life is more variable than early‐life movement.     

Caloric restriction maintains OX40 agonist-mediated tumor immunity and CD4 T cell priming during aging

Immune responses wane during aging, posing challenges to the potential effectiveness of cancer immunotherapies. We previously demonstrated that in the context of a promising immunotherapeutic, OX40 agonist (αOX40), older animals exhibited impaired anti-tumor immune responses and diminished CD4 T cell effector differentiation. In this study, we hypothesized that tumor immune responses could be maintained during aging through caloric restriction (CR) or dietary supplementation with resveratrol (RES), a CR mimetic. Mice were placed on either a calorically restricted diet or a RES-formulated diet starting between 4 and 6 months of age and continued until mice reached 12 months of age. Tumor immune responses were assessed after challenging with either sarcoma or breast tumor cells followed by αOX40 treatment. Our results show that CR, but not RES, maintained OX40-mediated anti-tumor immunity. In addition, CR fully sustained antigen-specific CD4 T cell priming in aged hosts (12 months old), whereas tumor-specific CD8 T cell priming was not fully maintained compared to young reference animals (2 months old). Thus, CR appears to maintain immunological fitness of the CD4 T cell priming environment during aging, which is critical for optimal OX40-mediated responses.     

Neuronal‐specific proteasome augmentation via Prosβ5 overexpression extends lifespan and reduces age‐related cognitive decline

Cognitive function declines with age throughout the animal kingdom, and increasing evidence shows that disruption of the proteasome system contributes to this deterioration. The proteasome has important roles in multiple aspects of the nervous system, including synapse function and plasticity, as well as preventing cell death and senescence. Previous studies have shown neuronal proteasome depletion and inhibition can result in neurodegeneration and cognitive deficits, but it is unclear if this pathway is a driver of neurodegeneration and cognitive decline in aging. We report that overexpression of the proteasome β5 subunit enhances proteasome assembly and function. Significantly, we go on to show that neuronal‐specific proteasome augmentation slows age‐related declines in measures of learning, memory, and circadian rhythmicity. Surprisingly, neuronal‐specific augmentation of proteasome function also produces a robust increase of lifespan in Drosophila melanogaster. Our findings appear specific to the nervous system; ubiquitous proteasome overexpression increases oxidative stress resistance but does not impact lifespan and is detrimental to some healthspan measures. These findings demonstrate a key role of the proteasome system in brain aging.     

Transcriptome analysis reveals the difference between “healthy” and “common” aging and their connection with age‐related diseases

A key goal of aging research was to understand mechanisms underlying healthy aging and develop methods to promote the human healthspan. One approach is to identify gene regulations unique to healthy aging compared with aging in the general population (i.e., “common” aging). Here, we leveraged Genotype‐Tissue Expression (GTEx) project data to investigate “healthy” and “common” aging gene expression regulations at a tissue level in humans and their interconnection with diseases. Using GTEx donors' disease annotations, we defined a “healthy” aging cohort for each tissue. We then compared the age‐associated genes derived from this cohort with age‐associated genes from the “common” aging cohort which included all GTEx donors; we also compared the “healthy” and “common” aging gene expressions with various disease‐associated gene expressions to elucidate the relationships among “healthy,” “common” aging and disease. Our analyses showed that 1. GTEx “healthy” and “common” aging shared a large number of gene regulations; 2. Despite the substantial commonality, “healthy” and “common” aging genes also showed distinct function enrichment, and “common” aging genes had a higher enrichment for disease genes; 3. Disease‐associated gene regulations were overall different from aging gene regulations. However, for genes regulated by both, their regulation directions were largely consistent, implying some aging processes could increase the susceptibility to disease development; and 4. Possible protective mechanisms were associated with some “healthy” aging gene regulations. In summary, our work highlights several unique features of GTEx “healthy” aging program. This new knowledge could potentially be used to develop interventions to promote the human healthspan.     

Dietary fat composition influences glomerular and proximal convoluted tubule cell structure and autophagic processes in kidneys from calorie‐restricted mice

Calorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age‐related diseases in a wide range of animals, including non‐human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age‐related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents.     

Optimal window of caloric restriction onset limits its beneficial impact on T‐cell senescence in primates

We have recently shown in non‐human primates that caloric restriction (CR) initiated during adulthood can delay T‐cell aging and preserve naïve CD8 and CD4 T cells into advanced age. An important question is whether CR can be initiated at any time in life, and whether age at the time of onset would modulate the beneficial effects of CR. In the current study, we evaluated the impact of CR started before puberty or during advanced age on T‐cell senescence and compared it to the effects of CR started in early adulthood. Our data demonstrate that the beneficial effects of adult‐onset CR on T‐cell aging were lost by both early and late CR onset. In fact, some of our results suggest that inappropriate initiation of CR may be harmful to the maintenance of T‐cell function. This suggests that there may be an optimal window during adulthood where CR can delay immune senescence and improve correlates of immunity in primates.     

Lymph nodes as barriers to T‐cell rejuvenation in aging mice and nonhuman primates

In youth, thymic involution curtails production of new naïve T cells, placing the onus of T‐cell maintenance upon secondary lymphoid organs (SLO). This peripheral maintenance preserves the size of the T‐cell pool for much of the lifespan, but wanes in the last third of life, leading to a dearth of naïve T cells in blood and SLO, and contributing to suboptimal immune defense. Both keratinocyte growth factor (KGF) and sex steroid ablation (SSA) have been shown to transiently increase the size and cellularity of the old thymus. It is less clear whether this increase can improve protection of old animals from infectious challenge. Here, we directly measured the extent to which thymic rejuvenation benefits the peripheral T‐cell compartment of old mice and nonhuman primates. Following treatment of old animals with either KGF or SSA, we observed robust rejuvenation of thymic size and cellularity, and, in a reporter mouse model, an increase in recent thymic emigrants (RTE) in the blood. However, few RTE were found in the spleen and even fewer in the lymph nodes, and SSA‐treated mice showed no improvement in immune defense against West Nile virus. In parallel, we found increased disorganization and fibrosis in old LN of both mice and nonhuman primates. These results suggest that SLO defects with aging can negate the effects of successful thymic rejuvenation in immune defense.     

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.     

Lifelong calorie restriction affects indicators of colonic health in aging C57Bl/6J mice

Diminished colonic health is associated with various age-related pathologies. Calorie restriction (CR) is an effective strategy to increase healthy lifespan, although underlying mechanisms are not fully elucidated. Here, we report the effects of lifelong CR on indicators of colonic health in aging C57Bl/6J mice. Compared to an ad libitum control and moderate-fat diet, 30% energy reduction was associated with attenuated immune- and inflammation-related gene expression in the colon. Furthermore, expression of genes involved in lipid metabolism was higher upon CR, which may point towards efficient regulation of energy metabolism. The relative abundance of bacteria considered beneficial to colonic health, such as Bifidobacterium and Lactobacillus, increased in the mice exposed to CR for 28 months as compared to the other diet groups. We found lower plasma levels of interleukin-6 and lower levels of various metabolites, among which are bile acids, in the colonic luminal content of CR-exposed mice as compared to the other diet groups. Switching from CR to an ad libitum moderate-fat diet at old age (24 months) revealed remarkable phenotypic plasticity in terms of gene expression, microbiota composition and metabolite levels, although expression of a subset of genes remained CR-associated. This study demonstrated in a comprehensive way that CR affects indicators of colonic health in aging mice. Our findings provide unique leads for further studies that need to address optimal and feasible strategies for prolonged energy deprivation, which may contribute to healthy aging.