2017 Symposium on Healthy Aging
October 25-27, 2017, Mohonk Mountain House
- Peter Adams, University of Glasgow
The dynamic epigenome—a challenge to healthy agingHealthy aging depends on long-term maintenance of chromatin structure, for example in long-lived tumor suppressive senescent cells (e.g., nevus melanocytes), other post-mitotic cells, and quiescent stem cells. Stable chromatin is required to maintain cell phenotype, for example of a neuron, and to suppress tumorigenesis, for example in an oncogene-expressing senescent nevus melanocyte. Remarkably, in these post-mitotic cells, functional chromatin is typically maintained for several decades. This level of chromatin stability likely represents a challenge for these cells, since chromatin structure is known to be highly dynamic. Aside from chromatin disruption linked to fundamental processes such as gene transcription, the dynamic nature of chromatin is indicated by the phenomenon of position-effect variegation (PEV), fluorescence recovery after photobleaching (FRAP) experiments, and more recent whole-genome biochemical analyses performed as part of the ENCODE project. The Adams lab has been interested in cellular senescence, chromatin structure, cancer, and aging for several years. We are now working to understand the mechanisms by which chromatin structure is maintained, particularly in post-mitotic cells, to allow those cells to age healthily over the course of decades. However, we also hypothesize that eventual and progressive degeneration of chromatin structure with age contributes to the striking increased incidence of cancer, and other diseases, with age.
- Peter Arvan, University of Michigan
Mutant INS gene-induced diabetes of youth (MIDY)Healthy pancreatic ß-cells can synthesize 6000 proinsulin molecules every second. The dominant feature of ß-cells is the presence of stored insulin secretory granules, but mitochondria, Golgi stacks, and autophagosomes are also abundant. ß-cells do not reserve a specific cytoplasmic region for the endoplasmic reticulum (ER); nevertheless, this is where all proinsulin is initially made. Ordinarily, proinsulin in the ER rapidly folds to the native state, including three evolutionarily conserved disulfide bonds (B7-A7, B19-A20, and A6-A11) that are catalytically accelerated. In states of increased metabolic demand, as proinsulin synthesis is upregulated, there is an increase of misfolded proinsulin that is disposed of by mechanisms such as ER-associated degradation. However, beyond a certain threshold, accumulation of misfolded proinsulin molecules may interfere with normal intracellular transport of “bystander” proinsulin, leading to diminished insulin production and hyperglycemia, and provoking ER stress. We have bioengineered three proinsulin mutants, each with the potential to form only one native proinsulin disulfide bond. “Keep-B7/A7”, “keep-B19/A20”, and “keep-A6/A11” can never advance from the ER, but analysis from these mutants establishes that B19-A20 initiates covalent B-chain/A-chain interaction. Moreover, certain uncommitted Cys residues can function as “interlopers”, forming mispaired disulfides that disrupt the fidelity of native proinsulin disulfide pairings. These realizations provide the underpinnings of our studies on MIDY: an autosomal dominant form of diabetes that masquerades as several different diabetes syndromes. The INS gene coding sequence mutations causing this disease can involve various conserved residues of proinsulin, triggering misfolding, disulfide mispairing, aggregation, enhanced proinsulin interaction with ER molecular chaperones, and diminished insulin production. Folding defects may also occur in type 2 diabetes or other conditions when beta cells need to greatly increase proinsulin production. Our work provides several encouraging preclinical therapies that may ameliorate this problem.
- HM Brown-Borg, University of North Dakota School of Medicine; Health Sciences
The effects of dietary intervention on accelerated aging and age-related diseaseAging is the major risk factor for many diseases [Alzheimer’s (AD), diabetes, cancer]. Dietary interventions have been shown to delay aging and extend health and lifespans in model organisms. As such, these interventions exhibit the potential to reduce disease burden and health care costs by significantly postponing or preventing the pathology and symptoms associated with age-related disease. We are currently evaluating the effects of simple dietary interventions on the incidence of AD pathology and physiological decline in mouse models of AD and in mice that exhibit accelerated aging. Male and female APP/PS1, growth hormone transgenics, and corresponding wild type mice were subjected to one of four diets for 8 weeks beginning at 8 weeks of age: control, 80% methionine-restriction, rapamycin, or 80% methionine-restriction/rapamycin. Measures of behavior included tests for anxiety, working memory, and general locomotion. Liver, kidney, visceral fat, hind limb skeletal muscle, hippocampus, cortex, and plasma were collected to determine effects of diet on nutrient signaling, metabolism, detoxification, and stress resistance. Measures indicative of AD include APP, BACE, PSD95, pAKT, AKT, p-tau, and tau protein as well as immunohistochemistry to evaluate pathology. Differences in behavioral measures were apparent in mice on rapamycin and methionine restriction. Several components of methionine metabolism were affected by the diets in both the AD mice and the GH transgenic mice. A detailed description of the results will be presented. This study demonstrates that treating an age-related disease with interventions that slow and prevent aging processes also slows disease progression.
- Rochelle Buffenstein, Calico Life Sciences
Proteostatic mechanisms of the extremely long-lived naked mole-ratWhen compared to the four-year maximum life span (MLSP) of laboratory mice, the MLSP of more than thirty years for the naked mole-rat (Heterocephalus glaber) is astonishing. Strikingly, not only do naked mole-rats live an order of magnitude longer than similar-sized mice, they do so for most of their lives in cancer-free, good health. When compared to the deterioration of the human body during aging, this hairless rodent shows little age-associated physiological decline, maintaining heart health, bone quality, and reproductive capacity for more than 75% of its known lifetime. With a greater emphasis in aging research on mechanisms that may prolong healthspan, rather than lifespan, and an ever increasing demand to determine how to improve quality of life into old age, understanding how animals with exceptional longevity, like the naked mole-rat, are able to resist the vagaries of aging may provide pivotal insights into mechanisms that may prolong good health and attenuate age-related diseases. Here, we are review some of the idiosyncratic pedomorphic traits of naked mole-rats as well as the diverse suite of mechanisms that may contribute to the maintenance of protein homeostasis and genomic integrity. Newfound interest in their basic biology of aging serves to remind us that studying animals with unusual traits often sheds light on the more difficult biological and biomedical questions that have eluded scientists for years.
- Vishwa Deep Dixit, Yale School of Medicine
Caloric restriction in humans inhibits inflammation: Insights from CALERIE-IIChronic low-grade inflammation is a major driver of degenerative-chronic disease. To date, the cellular origin of age-related inflammation and the underlying molecular mechanisms are not well understood. Caloric restriction is the most robust intervention that reduces disease burden and extends lifespan; however, whether CR is relevant to human physiology remains unclear. CALERIE Phase 2 is a three-site (Pennington Biomedical Research Center, Tufts University, Washington University), single-protocol randomized clinical trial designed to test the effects of 2 years of sustained calorie restriction (CR) in healthy men (20-50 yr) and women (20-47 yr). We hypothesized that a reduction in energy intake to 75% of baseline requirement (25% CR) for 2 years will result in the same adaptive changes that occur in rodents subject to CR, with particular emphasis on the reduction in inflammation. This study investigated the impact of CR on inflammation through whole transcriptome and metabolome analyses in the s/c adipose tissue and their relationship with serum cytokines and metabolic outcomes. CR in humans did not impair the markers of adaptive immune response with no change in self-reported incidence of infections. In free living setting, instead of the intended 25% goal, only 15% CR was achieved in people. We found that the metabolic changes induced by 15% 2-year caloric restriction was sufficient to regulate the adipose-immune crosstalk within the adipose tissue to lower inflammation. These studies also reveal that CR in humans regulates several conserved longevity pathways previously identified from model organisms. Our findings suggest that drivers of CR-regulated immunometabolic response in can be harnessed for development of novel anti-inflammatory targets.
- F Brad Johnson, University of Pennsylvania
The potential for Wnt pathway agonists to ameliorate pathology driven by telomere dysfunctionThrough studies of the intestinal stem cell niche in mice and in cultured human intestinal organoids, we have identified a positive feedback loop through which the capping of telomeres and the expression of multiple components in the canonical Wnt signaling pathway support one another (Nat Commun 2017, 8:14766; Cell Stem Cell 2016, 19(3):397). Thus, on the one hand, if telomeres are in a capped state, Wnt signaling is active, and this, in turn, reinforces telomere capping. On the other hand, if telomeres begin to uncap, e.g., due to critical shortening, Wnt signaling declines, leading to further uncapping. The mechanisms underlying the loop include 1) upregulation of miR34a by uncapped telomeres, which inhibits expression of Wnt pathway factors, and 2) Wnt-dependent expression of several shelterins, including TRF2. We have exploited the Wnt-telomere loop to use Wnt pathway agonists to restore telomere capping and tissue homeostasis in the intestines of late generation mice lacking telomerase (mTR mutants) and in cultured human intestinal organoids derived from cells from people suffering from the telomerase deficiency disorder dyskeratosis congenita (DKC1 mutants). We will discuss the therapeutic potential of this approach for the treatment of dyskeratosis congenita, and describe our new efforts to use it to ameliorate pathology driven by telomere dysfunction in other tissues, including lung.
- JE Johnson, Orentreich Foundation for the Advancement of Science
Identification and characterization of methionine restriction mimetics that improve healthspan in yeast, cultured mammalian cells, and miceMethionine restriction (MR) is one of only a few dietary manipulations known to robustly extend lifespan in mammals. Despite this, the mechanistic basis for this extension has remained elusive. To address this, I previously developed genetically-tractable cell systems to model the benefits of MR in budding yeast and cultured mammalian cells. Using these, I have demonstrated that both dietary MR and impairment of the cell’s methionine biosynthetic machinery (“genetic MR”) significantly extend the chronological lifespan of yeast, while genetic MR extends the replicative lifespan of mammalian cells. In recent work, I have identified two autophagy genes that underlie the extended lifespan of methionine-restricted cells. Consistent with this finding, I have performed genetic studies that have revealed a positive epistatic relationship between MR and autophagy, suggesting that the primary benefit of MR to yeast lifespan extension is the activation of autophagy. Usefully, I have found that enzymatic elimination of methionine, as mediated by L-methionine gamma lyase, produces the methionine-restricted state, extends yeast lifespan, and represents a powerful tool for the study of MR. I have also observed that supplementation with either of two methionine-like amino acids can extend yeast lifespan. Recent and ongoing research in the laboratory has focused on A) dissecting the molecular mechanisms underlying the cellular benefits of MR, and B) identifying and characterizing interventions that phenocopy MR. Towards this end, we have performed genetic and biochemical studies to determine how supplementation with methionine analogues extends cellular lifespan, and whether administration of such compounds might recapitulate the benefits of MR to mice fed a methionine-replete diet. We anticipate that these studies will facilitate the eventual development of novel pharmacologic interventions that can be used to improve healthspan in humans.
- Matt Kaeberlein, University of Washington
Translational geroscience: Targeting mTOR signaling for mitochondrial disease and normative agingA primary goal of geroscience is to improve health, longevity, and quality of life for people through basic and translational research into the biology of aging. The FDA-approved drug rapamycin is currently the most effective pharmacological intervention to increase lifespan and improve measures of healthspan in mice. Nevertheless, important questions exist regarding the translational potential of rapamycin and other mTOR inhibitors for human aging, and the optimal dose, duration, and mechanisms of action remain to be determined. Here I will report on our studies examining the effects of chronic rapamycin treatment in mice and people with severe mitochondrial disease, as well as short-term treatment with rapamycin in middle-aged mice and dogs. Rapamycin effectively rescues mitochondrial disease progression in mice, enhances cellular measures of mitochondrial function, and improves clinical parameters in patients with MELAS syndrome. During normative aging, transient treatment with rapamycin is sufficient to increase life expectancy by more than 50% and improve measures of healthspan in middle-aged mice. In companion dogs, we have defined a dose of rapamycin that is well tolerated, and initial results are consistent with improvements in age-associated cardiac function similar to those observed in rapamycin-treated mice. These data suggest that rapamycin may be suitable for translational applications in both veterinary and human medicine to treat mitochondrial disorders and to improve healthy longevity during normative aging.
- Pankaj Kapahi, Buck Institute for Aging Research
Uncoupling of diet-related effects on longevity and healthspan in Drosophila melanogasterAging affects all individuals and is a risk factor for a myriad of diseases, as well as death. A number of interventions have been suggested to improve overall lifespan and health, with one of the most successful being in the form of dietary restriction. Despite the generally well-accepted health and longevity benefits of dietary restriction, cases remain in model organisms where some genotypes are not affected or are negatively impacted by dietary restriction. Further, it is often seen that individuals of different genotypes display a range of longevity and health phenotypes under different dietary conditions, indicating a role for natural genetic variation in the regulation of longevity and health as they pertain to diet. With the use of the Drosophila Genetic Reference Panel, we identify a new set of diet-dependent genes, the expressions of which vary naturally, which impact longevity and health. Further, our measures indicate that longevity and health, as measured by Drosophila climbing ability, are distinct traits that are not necessarily correlated and are regulated through genetically distinct mechanisms. Associated with diet-dependent longevity is the gene CG34351, which has a previously unreported function. Through case-control analysis for long-lived fly lines, we have also identified Fdxh, which is known to regulate iron-sulfur clustering in the mitochondria, as genes that regulate extreme longevity. We have also found that CG33690, a previously little-characterized gene, has a role in climbing fitness, and thus propose the new name “hiker”. Through alteration of the expression of these genes via gene disruption and RNAi, we have validated their roles in diet-dependent effects on lifespan or climbing ability. In addition to ascribing novel functions to these genes, our results underline that lifespan and healthspan may be regulated through distinct mechanisms in response to changes in diet.
- Robert A Koza, Maine Medical Center Research Institute
Cardioprotective effects of rapamycin treatment on adult female C57BLKS/J-db/db miceRapamycin (RAPA), an inhibitor of mTORC signaling, has been shown to extend lifespan in mice and other organisms. Recently, animal and human studies suggest that inhibition of mTORC signaling can alleviate or prevent the development of cardiomyopathy. In view of this, we used a murine model of type-2-diabetes (T2D), C57BLKS/J-db/db, to determine whether RAPA treatment can mitigate the development of T2D-induced cardiomyopathy in adult mice. Female C57BLKS/J-db/db fed diet supplemented with RAPA from 11 to 27 weeks of age showed reduced weight gain and significant reductions of fat and lean mass compared with untreated mice. No differences in plasma glucose or insulin levels were observed between groups; however, RAPA-treated mice were more insulin sensitive (P<0.01) than untreated mice. Kidney weights and urine ACR were lower in RAPA-treated mice, suggesting reduced diabetic nephropathy and improved kidney function. Echocardiography showed significantly reduced left ventricular wall thickness in mice treated with RAPA compared to untreated mice (P=0.02) that was consistent with reduced heart weight/tibia length ratios, reduced myocyte size measured by histomorphology, and reduced mRNA expression of Col1a1, a marker for cardiomyopathy. Our results suggest that inhibition of mTORC signaling is a plausible strategy for ameliorating complications of obesity and T2D, including cardiomyopathy.
- Sailendra N Nichenametla, Orentreich Foundation for Advancement of Science
Effect of methionine restriction on protein synthesisAlthough numerous studies confirm that a methionine-restricted (MR) diet confers multiple health benefits, such as lifespan extension and the amelioration of diabetes and obesity, the mechanisms by which it does so remain unclear. In addition to being a precursor for a number of metabolic intermediates (e.g., S-adenosylmethionine, homocysteine, cysteine, and glutathione), methionine is an essential and starting amino acid for the translation of mRNA into protein. Previous studies found MR induced changes in markers of protein synthesis and breakdown, such as total plasma protein, lean mass, and 3-methylhistidine. Despite a strong association between proteostasis and aging, there is no information on whether an MR diet affects the kinetics of proteostasis, and if such effects confer any benefits. We will discuss findings from our ongoing studies in rodents on the effect of an MR diet on rates of protein synthesis and if these changes confer any benefits.
- Arlan Richardson, University of Oklahoma Health Science Center
Effect of dietary restriction on DNA methylationDietary restriction (DR) to date is the most consistent nutritional intervention to increase lifespan and retard aging in a wide variety of organisms; however, the molecular basis of DR’s life-extending action is still unknown. Because early life DR has been shown to increase lifespan even when restriction is discontinued, we have explored whether DR retards aging through an epigenetic mechanism—DNA methylation. Alterations in DNA methylation at specific genes is critical during development and is a mechanism by which the transcriptional potential of cells can be altered for the life of an organism. In our first series of experiments, we measured the DNA methylation in the promoter regions of several genes whose expression is dramatically altered within months after the implementation of DR and whose expression remains altered after returning the mice to an ad libitum diet. We found that the methylation at three specific CG sites in the promotor of the Nts1 gene was correlated with the increased expression of Nts1 in the intestinal mucosa of mice fed a DR diet. Both the hypomethylation and increased expression of Nts1 gene persisted even when DR was discontinued and mice fed AL. The changes in DNA methylation in the Nts1 gene are likely to occur in intestinal stem cells and could play a role in preserving the intestinal stem cell pool in DR mice. In a second series of experiments, we used bisulfite oligonucleotide capture sequencing to measure DNA methylation genome-wide in base-specific sites in the hippocampus of young and old mice and old mice fed DR. Over 18,000 mCpGs and 30,000 mCpHs sites were significantly altered with age, and 34 to 40% of these sites of DNA methylation were prevented by DR. We also observed that DR induced specific changes in methylation at ~25,000 CpG sites and ~80,000 CpH sites, which were not significantly altered by age. Our data demonstrate that DR induces changes in DNA methylation, which has the potential of altering gene expression and having a memory effect.
- John P Richie, Jr, Penn State University College of Medicine
Dietary total sulfur amino acid restriction in healthy adults: A controlled feeding studyDietary methionine restriction (MR) delays the aging process and inhibits aging-related diseases and disorders in numerous laboratory animal models. These effects are accompanied by numerous metabolic alterations, including improvements in glucose and fat metabolism and a reduction in oxidative stress that may underlie MR’s beneficial health effects. Recent studies have indicated that a restriction in both methionine and cysteine (total sulfur amino acid restriction, SAAR) is required to obtain optimal beneficial effects. While SAAR holds promise as a possible intervention for prevention of aging-related impairments and diseases, little is known about the translation of these findings to humans. Thus, our objectives were to determine the short-term (1-mo) impact of feeding MR or SAAR diets to healthy adults on relevant anthropometric, metabolic, and oxidative stress biomarkers. In this controlled feeding study, twenty healthy adults (11 females/9 males) were randomized to either MR or SAAR diet arms. Each arm consisted of 3 controlled isocaloric 4-wk feeding periods: 1) control; 2) 70% MR or 50% SAAR; 3) 90% MR or 65% SAAR, separated by 3-4-wk washout periods. Biological samples were collected before and after each feeding period. Dietary intakes of methionine and cysteine prior to and during each feeding period were confirmed by unannounced 24-hr food recall assessments and chemical analyses of actual diet samples, respectively. SAAR diets were associated with dose-dependent reductions in body weight and, with 65% SAAR, an increase in body temperature. Likewise, during SAAR diet periods, decreases in plasma cholesterol, leptin, IGF-1, and 8-isoprostane levels were observed. No adverse effects were observed for either MR or SAAR diets. These results suggest that many of the short-term effects of SAAR observed in animal models are translatable to humans. Overall, these findings support the further clinical development of this dietary intervention for health promotion and disease prevention.
- Christian Sell, Drexel University College of Medicine
Metabolic interventions may delay or block key aspects of cellular senescenceThe process of cellular senescence contributes to aging and age-related dysfunction in multiple organ systems. The burden of senescent cells increases with age, and strategies to selectively target senescent cells (senolytics) improve late-life function in animal models. Although DNA damage has been classically associated with induction of senescence, multiple lines of evidence also support a connection between metabolic imbalance and activation of the senescence program. Mitochondrial stress can induce senescence, while altered metabolism and mitochondrial dysfunction are features of senescence induced by specific insults such as DNA damage. Metabolic interventions that extend lifespan, such as caloric restriction, methionine restriction, and rapamycin treatment, reduce the burden of senescent cells in model organisms. In human cell culture models, both rapamycin treatment and methionine restriction delay senescence; however, the molecular connections between metabolism and regulation of the senescence program have not been fully elucidated. Guided by a transcriptome-wide analysis, we examine the potential mechanisms that link metabolic pathways with the senescence program and discuss the possibility that senostatic therapies which block, rather than eliminate, senescent cells may be developed through a greater understanding of the intersection between metabolism and senescence.
- AK Shoveller, University of Guelph
Examining potential mechanisms of healthy aging with preventative nutrition approachesDietary restriction (DR) increases median lifespan and protects against age-related disease development. Extension of lifespan can be achieved by restricting intake of dietary energy, protein, or specific amino acids like methionine (Met) and tryptophan. We investigated a purported DR mimetic, mannoheptulose (MH), and its effects on metabolism. MH inhibits hexokinase and produces transient hyperglycemia with high doses of intravenous delivery. We predicted that limiting glucose availability via glycolytic inhibition would consequently increase fatty acid oxidation, which is beneficial for weight management. To support whole body outcomes, we hypothesized a role for AMP-activated protein kinase (AMPK) in the switch from oxidation of glucose to fatty acids. These studies focused on glucose metabolism in response to MH feeding in different breeds of dogs consuming diets of different macronutrient profiles. The impact of MH on protein metabolism and specifically on the control of the mechanistic target of rapamycin complex 1 (mTORC1) remains to be explored. The mTORC1 signaling network regulates protein synthesis and degradation, lipogenesis and cell growth, proliferation, differentiation, and metabolism in response to hormones, amino acids, and cellular energy status. Inhibition of mTORC1 signaling with rapamycin or genetic mutation increases median lifespan in model organisms, and mTORC1 inhibition may be responsible for some of the lifespan extending effects of energy, protein, and Met restriction. Met restriction also results in a reduction in mitochondrial production of reactive oxygen species and improved insulin sensitivity in adipose tissue. Low-Met diets can be formulated with high levels of cyst(e)ine in order to meet total sulfur amino acid requirements for growth. However, dietary cysteine supplementation reverses the lifespan extending effects of Met restriction. These results indicate that amino acid requirements need to consider more than just support of whole body protein synthesis and should include secondary measures related to tissue-specific and whole-body metabolism. Studies assessing energy and Met restriction need to explore energy, protein synthetic, and oxidative stress associated pathways concurrently.
- Yousin Suh, Albert Einstein College of Medicine
Enhancer mechanisms in human aging and diseaseGenome-wide association studies (GWAS) have achieved great success in identifying genetic variants robustly associated with increased disease risk. The vast majority (>90%) of risk variants detected by GWAS occur in non-coding genomic regions, suggesting that they impose risk by altering promoter and enhancer elements that regulate gene expression. Understanding how non-coding variants function in pathogenesis is critically important to translate the genetic association into molecular mechanisms and, ultimately, clinical applications. Our studies have centered on enhancer mechanisms underlying a non-coding region of the genome uncovered by GWAS, the so-called gene desert at the chromosome 9p21 locus. This locus is a GWAS hotspot associated with multiple age-related diseases, including cancer, heart disease, glaucoma, Alzheimer’s disease, and diabetes, suggesting a common underlying biology of aging. Indeed, the closest protein-coding genes are the two cyclin dependent kinase inhibitors, CDKN2A (encoding p16INK4A and p14ARF) and CDKN2B (encoding p15INK4B), known to be involved in tumor suppression and cellular senescence. We will discuss our approaches to elucidate the molecular mechanisms by which risk variants alter enhancer function and target gene expression, thereby conferring increased risk of aging and disease. In addition, we will present the roles of DNA binding transcription factor complexes recruited in trans in regulation of de novo enhancer networks using replicative senescence as a model of cellular aging.
- Jessica Tyler, Weill Cornell Medicine
An integrative analysis of replicative aging in budding yeastMitotic aging in budding yeast uses conserved aging pathways and serves as an excellent model for understanding the aging of human stem cells. We have been performing an integrative analysis of the replicative aging process, using the mother enrichment program to isolate unprecedented numbers of old yeast. Deep sequencing and mapping DNA damage sites by gammaH2A ChIPseq identified significant increases in chromosomal translocations, amplification of chromosomal arms, retrotransposition, and nuclear transfer of mitochondrial genomes in old cells. There are also more DNA double-strand breaks (DSBs) in old cells, which is partially due to the defect in DSB repair that we have uncovered in old cells. We have determined the mechanistic basis for this defect and reverse this defect to extend lifespan, demonstrating that the DNA repair defect is a cause of aging. We observe elevated rDNA instability during aging accompanied by insertion of the rDNA repeats into other chromosomes, and this rDNA instability leads to global genomic instability. We have uncovered a defect in sister chromatin cohesion in old cells, with the potential to cause chromosomal loss, aneuploidy, and further genomic instability. By mapping nucleosome occupancy over the aging genome, we find a loss of approximately half the nucleosomes with age, leading to transcriptional upregulation of the entire transcriptome. Conversely, our ribo-seq analyses show that most protein synthesis is reduced during aging, and we have discerned the molecular basis for this defect. Our metabolomics analyses find that amino acids are the most reduced metabolites during aging, indicating a defect in both amino acid uptake and amino acid synthesis in old cells, which activates the amino acid sensor Gcn2. Further mimicking the effect of amino acid depletion by tRNA overexpression was found to extend lifespan. Taken together, our studies are revealing novel molecular defects that occur during, and cause, replicative aging.