MDIBL UMPI-UMFK 2017 short course:

Genetics of Aging and Physiological Stress Resistance

Purpose:

To demonstrate that the aging process is regulated and modifiable by manipulating insulin-signaling pathways in C. elegans and observing its effect on age-related phenotypes, including stress resistance, mobility, and longevity.

Objectives:

1) To recognize that the process of aging is not a series of random or stochastic events, but is biologically regulated and modifiable through a variety of signaling pathways that are conserved across animal species.

2) To review the insulin signaling pathway in C. elegans nematodes and its homolog in humans.

3) To demonstrate in c. elegans that down-regulation of the insulin signaling pathway through mutation of the DAF-2 gene can increase DAF-16 (FOXO) translocation into the nucleus, activate a stress response, preserve mobility, and improve markers of longevity.

4) To show DAF-16 activation through attenuation of daf-2 gene expression can improve resistance to heat shock.

5) To extrapolate these observations into analogous human diseases and vulnerabilities associated with aging.

6) To learn about other biological and genetic manipulations that can increase longevity in lower organisms and their potential for translation into human interventions (e.g., caloric restriction, Sirtuins, mTOR inhibition with rapamycin, metformin, and the concept of hormesis).

7) To recognize that interventions to improve stress resistance in humans may have an impact on a person’s vulnerability to all age-related diseases – not just one. Therefore, understanding the biology of aging can ultimately make a profound impact on promoting a healthy human lifespan.

Background

Overview of Molecular and Genetic Mechanisms of Aging:

Insulin/IGF-1 Signaling pathway:

This lab will focus on the insulin/IGF-1 signaling pathway, which has been well demonstrated to affect the lifespan of c. elegans and other lower organisms. In this pathway the C. elegans DAF2 receptor is homologous to the insulin/IGF-1 receptor in humans, and exerts a downstream inhibitory influence on the DAF16 (FOXO) transcription factor. When insulin/IGF-1 signaling is reduced, DAF16 is translocated from the cytoplasm to the nucleus where it activates a cascade of genes that mediate oxidative stress, heat shock, innate immunity, metabolism, and autophagy. Accordingly, daf-2 mutation or daf-16 overexpression leads to an increase in lifespan, maintenance of mobility, and stress resistance.

Aging affects all organ systems and is one of the most complex phenotypes involving molecular, cellular and organ damage, leading to loss of function and increased vulnerability to disease and death. Major progress has recently been made in the identification of a large number of genes whose mutations affect life span in various organisms. Subsequent studies of their gene functions have revealed that the aging process, like so many other biological processes, is actively regulated by classical and highly conserved signaling pathways and transcription factors.

Figure 1. A simplified model of the influence of insulin-like signaling in C. elegans.

Insulin is the major hormone controlling critical energy functions such as glucose and lipid metabolism. Insulin elicits a diverse array of biological responses by binding to DAF-2/insulin/IGF-I receptor. This receptor mediates these effects by activation of signaling pathways, which utilize lipid kinases such as Phosphatidylinositol 3-Kinase (PI3K). It activates the serine/threonine kinase Protein Kinase-B (Akt/PKB). Then, Akt retains DAF-16/FOXO in the cytoplasm and inhibits its transcriptional activity. Akt also activates the Mammalian Target of Rapamycin (mTOR) pathway. Activation of mTOR results in the translation of ribosomal proteins, elongation factors and insulin-like growth factor to enhance protein synthesis. A Rapamycin interaction with mTOR inhibits its activity. In response to reduced insulin/IGF-I signaling or stress (heat, oxidative, starvation), DAF-16/FOXO enters the nucleus, where it turns on survival genes, including those that manage oxidative stress, heat shock, innate immunity, metabolism, autophagy, and xenobiotic response, among others. Indeed, over- or underexpression of such targets often impacts stress resistance and longevity.

Nutrient-sensing Pathways:

Nutrient-sensing pathways are fundamental to the aging process. Many of the mutations that extend life span decrease activity of nutrient-signaling pathways, such as the Insulin/IGF (insulin-like growth factor) and the TOR (target of rapamycin) pathways, suggesting that they may induce a physiological state similar to that resulting from periods of food shortage. Studies in several model organisms have shown that dietary restriction without malnutrition, or manipulation of nutrient-sensing pathways through mutations or drugs, can increase life span and reduce age-related disease. According to the principal of hormesis, the increase in longevity in response to food shortage (or its molecular equivalent), may enable an organism to delay reproduction and survive until conditions are more favorable to successfully breed offspring.

One of the best-characterized nutrient-sensing aging pathways across evolution is the insulin/IGF-1/PI3K signaling pathway (e.g. IGF1, IGF-1R, Klotho, IRS-1, p66SHC, PI3K, ATK1, FOXO, mTOR and S6K), which mediates the anti-aging effects of dietary restriction in mice. Reduced activity of the Insulin/IGF signaling pathway extends life span in C. elegans and other multicellular organisms. This increase in longevity requires the Forkhead FoxO transcription factor (daf-16 in C. elegans), which regulates genes involved in a wide range of defensive activities including cellular stress response, antimicrobial activity, and detoxification of xenobiotics and free radicals. In high nutrient conditions, FoxOs are phosphorylated and excluded from the nucleus; in low nutrient conditions, they relocalize to the nucleus.

Significantly, reduced activity of the insulin/IGF-1 signaling pathway not only increases life span, but also protects against cancer, Alzheimer’s Disease (AD), cardiovascular disease and Type 2 Diabetes in multicellular organisms such as mice and rhesus monkeys. In humans, an overrepresentation of heterozygotes for mutations in the IGF-1 receptor gene was found among Ashkenazi Jewish centenarians as compared to controls. Subjects with a genotype associated with reduced concentration of free IGF-1 in plasma were overrepresented among long-lived people indicating that specific polymorphisms that down-regulate the GH and IGF-1 signaling pathways may promote human longevity. Furthermore, genetic variants of FOXO transcription factors, orthologs of the key Insulin/IGF-1 effector daf-16 in C. elegans, have repeatedly been shown to be associated with human life span.

Nutrients increase the level of IGF-1 and activate the Insulin/IGF-1 signaling pathway, which, in turn, activates pro-aging pathways in various mammalian cells. In contrast, caloric restriction in humans causes changes that protect against age-related pathologies, including diabetes, cardiovascular disease, and cancer. Restricting calorie intake in mice or introducing mutations in nutrient-sensing pathways can extend lifespans by as much as 60%, in part by delaying the occurrence of many chronic diseases in these these ‘Methuselah mice’. Importantly, ~30% of animals on dietary restriction die without evidence of severe organ pathology. In other words, extending lifespan also seems to increase ‘healthspan’, the time lived without chronic age-related conditions. Longevity pathways identified in model organisms seem to be conserved in humans and can be manipulated in similar ways. Notably, genetic surveys of centenarians implicate hormonal and metabolic systems and long-term calorie restriction in humans induces drastic metabolic and molecular changes that resemble those of younger people, notably in inflammatory and nutrient-sensing pathways.

Other Longevity Pathways:

It is unlikely that a single, linear pathway mediates the effects of aging in any organism. Matching of metabolism, growth, and fecundity to food intake is crucial for survival and reproduction in nature, and parallel and redundant pathways appear to be involved. Indeed, not only the Insulin/IGF cascade but many other molecules (e.g. p53, sirtuins, AMP kinase, Notch, Wnt, β-catenin, telomerase) also have critical roles in affecting various aging processes. For example, SIRT1 (Sir2), the founding member of the sirtuin gene family, is an NAD+-dependent deacetylase regulating life span in many species as diverse as yeast, worms, flies and possibly mice. Similarly, age-associated telomere dysfunction and associated p53 activation have emerged as important instigators of a functional decline of tissue stem cells and of mitochondrial dysfunction that adversely affect renewal and bioenergetic support in diverse tissues. In fact, telomerase overexpression extends life span in cancer-resistant mice. Interestingly, many of these pathways or molecules interact with each other and often target some common downstream transcription factors such as the forkhead-related transcription factors (FoxO). Moreover, these pathways also respond to various cellular stresses (e.g. DNA damage, telomere loss, oxidation and dietary restriction). Finally and strikingly, manipulating many of these aging regulators in the neuron, gut, or adipose tissue is sufficient to modulate life span and other aging processes, as shown in IGF-1R, IRS2, SKN-1, SIRT1, p53 and Mgat1, indicating the central role of the brain and metabolic organs in aging.

Epigenetic Factors Influencing Lifespan

The discovery of long-lived mutants in animal model systems suggests that the ageing process can be genetically modulated. In addition to genetic inputs, emerging evidence implicates environmental factors in ageing, such as dietary manipulations, DNA damage, telomere loss or stress. Indeed, studies in humans have estimated the non-heritable portion of healthspan and longevity regulation to be approximately 70%. Recent advances in ultra-high-throughput technologies have revolutionized our knowledge of epigenetic factors and their relationships to gene regulation. Epigenetic mechanisms (modes of genomic regulation that are not directly encoded in DNA) and more specifically changes of chromatin, which are influenced by the environment, are now considered to act as an interface through which environmental signals interact with genetic components throughout lifespan. Also, stable changes in chromatin states could preserve memory of past environmental exposures, leading to long-lasting phenotypic effects that may be particularly relevant to ageing. Changes in the chromatin landscape influence transcription and seem to underlie the transcriptional changes that are observed with ageing. Reference epigenomes provided by the National Institutes of Health Roadmap Epigenomics Project and the Encyclopedia of DNA Elements (ENCODE) database aid our understanding of the remodelling of epigenomic patterns that occur during ageing.

Recently, DNA methylation has been linked to chronological as well as biological age and could thus represent a biomarker for aging. Furthermore, histone methylation which is associated with either active or repressed genome regions and is known to be dynamically regulated, changes at the global level in several organismal models of aging. For instance, the rare diseases Werner Syndrome and Hutchinson–Gilford progeria syndrome (HGPS), which are characterized by premature aging in humans, are associated with perturbed histone methylation and alterations in heterochromatin organization. Furthermore, histone acetylation, which influences the physical associations between histones and DNA, is a key, conserved player in longevity, and evidence suggests that its pattern changes during normal ageing. Both histone acetylases and histone deacetylases modulate lifespan and metabolic health.

One of the best studied classes of histone deacetylases associated with longevity are the NAD+-dependent deacetylase class of sirtuin proteins. Sirtuins like SIRT1, SIRT3 and SIRT6 are important mediators of dietary restriction-induced longevity across species and detect changes in metabolism and energy homeostasis. They coordinate cellular responses to maintain genome integrity, mainly through regulation of epigenetic mechanisms. Sirtuins target different histone marks, including H4K16Ac, H3K9Ac, H3K56Ac and H3K18Ac, and non-histone components of the chromatin machinery, and are activated by two major types of stress: metabolic stress (nutrient and calorie restriction) and genotoxic stress. Sirtuins are among the very few enzymes that participate in stress response at both the sensing and signaling levels. They are also direct effectors of the stress response by regulating numerous master regulators of stress, such as the transcription factors NF-κB, p53, HIF-1α, FOXOs, E2F1, PGC-1α and HSF1. For instance, SIRT1, and its orthologs, which deacetylate H4K16ac, are effectors of caloric restriction-mediated effects in promoting health and lifespan. They are also known to be activated by resveratrol found in red wine and to extend lifespan in metabolically abnormal obese mice.

Relevance of Longevity Pathways to Alzheimer’s Disease:

Alzheimer’s disease (AD) is characterized by a progressive loss of memory and other cognitive functions. It affects over 5 million people in the US alone and its incidence is expected to double over the next 30 years. Moreover, the total annual costs of AD in the United States are estimated at $236 billion. There is therefore an urgent need to understand the mechanisms underlying the degeneration of neuronal cells. Aging is the most important risk factor for late-onset AD. Although they remain controversial, head injury, low education levels, hyperlipidemia, hypertension, homocysteinemia, diabetes mellitus, and obesity are potential risk factors for late-onset AD. The two defining neuropathological features of AD are extracellular senile plaques and intracellular neurofibrillary tangles (NFTs). The senile plaques are made of amyloid-β (Aβ), cleaved products of the amyloid precursor protein (APP), whereas the neurofibrillary tangles mainly consist of the microtubule-associated protein tau. Many hypotheses have been proposed to explain the etiology and pathogenesis of AD and related disorders. Two dominant theories focus on increased production of Aβ and dysfunction of tau. In addition, apolipoprotein E4 is genetically linked to late-onset familial and sporadic AD.

Tau normally stabilizes the microtubule cytoskeletal network that functions to maintain a unique neuronal structure and to transport proteins and other molecules through neurons. Phosphorylation is a key regulatory mechanism, which disrupts the ability of tau to bind microtubules and to promote their assembly. In contrast to normal adult brains, tau in AD is hyperphosphorylated and aggregated into abnormal conformations, eventually leading to the NFT formation. Dephosphorylation of NFT-tau restores the ability of tau to bind microtubules and to promote their assembly, indicating the critical role of tau phosphorylation.

Mutations in proteins regulating Aβ production such as APP and presenilin, account for early-onset familial AD. There are two proteolytic pathways for APP processing. In the amyloidogenic pathway, β-secretase cleaves APP at the beginning of the sequence of Aβ, generating an extracellular soluble fragment called sAPPβ and an intracellular COOH-terminal fragment called CTFβ. Subsequently, -secretase cleaves CTFβ at residues 40/42/43 of the Aβ sequence, generating intact Aβ species. The non-amyloidogenic pathway involves the activity of -secretase at the plasma membrane. -secretase cleaves within the sequence of Aβ, resulting in sAPP. Unlike the amyloidogenic pathway, there is no release of intact Aβ or amyloidogenic products.

The insulin/IGF-1 and TOR signal transduction pathways have been found to regulate tau pathology and Aβ generation in a wide range of diverse organisms including C. elegans, fly, and mouse models. Reducing activity of the insulin/IGF-1 pathway can alter Aβ and tau protein homoeostasis towards less toxic protein conformations and can also improve cognitive function. For example, knockdown of DAF2 signaling reduces Aβ42 aggregation-induced toxicity through HSF1-mediated disaggregation and DAF16-induced assembly of small oligomers into larger less toxic structures. In addition, blocking TOR activity using rapamycin alleviates both Aβ and tau protein aggregation and their pathogenesis in AD animal models and restores cognitive function. Therefore, targeting this pathway to abrogate overactivation of the TOR pathway may be a viable therapeutic strategy, possibly in conjunction with promotion of normal insulin/IGF-1 signaling.

The current FDA approved drugs for the treatment of AD inhibit acetylcholine esterase to increase the levels of the neurotransmitter acetylcholine, which is depleted in AD brains, or antagonize NMDA-type glutamate receptors to prevent aberrant neuronal stimulation. Unfortunately, the impact of these drugs on AD manifestations is modest and transient. Attempts to develop AD drugs targeting Aβ, β- or -secretase, tau kinases, tau protein levels, and ApoE4 have failed due to the brain-blood barrier, side effects, and safety issues. Therefore, agents that can effectively and safely ameliorate NFTs and Aβ plaques represent promising therapeutic and potential prophylactic treatments in AD.

Relevance of Longevity Pathways to Obesity and Type 2 Diabetes:

Aging is the single largest risk factor for chronic diseases. More than 70% of people over 65 have two or more chronic conditions such as arthritis, diabetes, cancer, heart disease and stroke. Studies of diet, longevity genes and drugs indicate that delaying one age-related disease probably holds off others. In humans, dietary restriction provides sustained beneficial effects against many age-related pathologies including obesity, insulin resistance, inflammation, oxidative stress, and left ventricular diastolic dysfunction, in agreement with the metabolic and functional changes observed in dietary-restricted animal models (see above). In contrast, obesity increases the risk of chronic age-related diseases, such as Type 2 Diabetes (T2D), heart disease, osteoarthritis, and certain subtypes of cancer, and thus constitutes a major and rising global health problem. More specifically, the health consequences of increased visceral adiposity caused by a long-term, hypercaloric diet is a multi-systemic deterioration resulting in an increased risk for developing a metabolic syndrome. In fact, as we age, the chance of developing metabolic disorders rises dramatically and cross-sectional study data show that the prevalence of T2D among a nationally representative sample of US adults was highest in citizens aged 65 and over compared to younger cohorts.

Overall, T2D and obesity are recognized causes of accelerated aging. For example, leukocyte telomere length, which shortens with aging and is a widely used biomarker of aging in blood, has been found to be negatively correlated with body mass index (BMI). Furthermore, obesity is associated with an accelerated rate of epigenetic changes in the human liver associated with the age, and may play a role in insulin resistance as well as liver cancer. Obesity influences hormones, inflammation, and glucose homeostasis, which can lead to the development of T2D and subsequent characteristics of accelerated aging. More specifically, in obese states, macrophages infiltrate the adipose tissue, elevating cytokine levels (e.g. TNF-α and IL-6) and resulting in insulin resistance and T2D. These findings support the hypothesis that obesity is associated with accelerated aging effects and stresses the importance of maintaining a healthy weight.

In the early stages of T2D, insulin resistance is the dominant feature and as a result hyperinsulinemia occurs. Impaired glucose uptake and utilization follow this stage and hyperglycemia and hyperinsulinemia contribute to pancreatic β islet cell exhaustion and destruction as diabetes progresses. On the molecular level, the group of epigenetic histone deacetylases called sirtuins influence many steps of glucose metabolism in liver, pancreas, muscle and adipose tissue. The main regulator of these reactions is the deactylated form of PGC-1α in SIRT1 activated states, which induces gluconeogenesis and inhibits glycolysis in liver during fasting. Moreover, adipose tissue SIRT1 plays a key role in the regulation of whole body metabolic homeostasis, and downregulation of SIRT1 in visceral adipose tissue may contribute to the metabolic abnormalities that are associated with visceral obesity in diabetic and obese women. SIRT1 deficient mice also exhibit low levels of serum glucose and insulin.

Several molecular pathways that increase insulin sensitivity, glucose homeostasis and longevity in animals are affected by approved and experimental drugs. For example, the sirtuin proteins involved in metabolic cellular processes, are activated by high concentrations of naturally occurring compounds such as resveratrol found in red wine as well as metformin, a commonly used anti-diabetic drug. Metformin decreases insulin resistance and hyperglycemia by SIRT1 mediated AMPK activation and extends lifespan in metabolically abnormal obese mice. A clinical trial is currently being planned to test the effect of metformin on biomarkers of aging in humans.

Experimental Protocol:

Overview: In this heat stress tolerance (thermotolerance) experiment, students will compare what happens to survival when wild-type N2 animals have attenuated (daf-2 RNAi) or simulated high (daf-16 RNAi) insulin/insulin-like signaling. Students will also observe what happens to DAF-16 localization during heat stress using a transgenic animal in which green fluorescent protein (GFP) has been fused to the gene encoding DAF-16 protein (nomenclature: daf-16::gfp).

Animal physiology:

Procedures:

Module 1: Heat resistance

Provided Materials: Nematode strains, media plates, worm picks, dissecting scopes, fluorescence scopes, incubators. Students will need to record observations and take notes. Data will eventually be plotted, preferably using excel or other graphing software.

Thermotolerance, or resistance to heat stress, is a classical test of an organism’s ability to maintain and recover homeostasis under conditions that lead to unfolded intracellular proteins. The best chance of survival comes from activation of 2 transcription factors required for extended longevity in insulin-like signaling (ILS) attenuation mutants. These transcription factors are DAF-16 and HSF-1. The latter is the cytoplasmic heat shock factor 1 (classically considered cell autonomous, predating evolution of multicellular species). The former is analogous to foxo3a in humans and will be fluorescently tagged in the nematodes with which you will be working. It is part of the ILS pathway (classically considered cell nonautonomous, part of hormone signaling and thus orchestrating proper responses to environmental challenges between different tissues). Under standard laboratory (“good”) conditions, DAF-16 is mostly localized in the cytoplasm. Under various forms of physical stress that attenuate signaling through the ILS pathway, this transcription factor becomes dephosphorylated and translocates to the nucleus.

To carry out these stress response/survival assays, participants will use C. elegans wild-type N2 animals fed double-stranded RNA expressed in E. coli (C. elegans food source) by plasmids. In turn, double-stranded RNA is taken up through the intestine and passed between tissues through double-stranded (ds) RNA receptors/transporters. Within each cell, double-stranded RNA is processed by Dicer and Argonaute proteins into interfering RNA (RNAi) bearing sequences that are complementary to the gene being targeted for repression (See Zhuang and Hunter, 2011).

Adult nematodes will be fed bacteria containing dsRNA for several days before the experiment. In a blinded assay, your group will be given 12 nematode growth media plates labeled A-L for each of 4 time points (48 total). For each time point, the 12 plates will consist of 4 with bacteria bearing control dsRNA, 4 bearing daf-16 dsRNA, and 4 bearing daf-2 dsRNA. Each plate will have about 25-30 worms. I will place these animals in the incubator at 37 degrees Celsius early in the morning, a hot temperature for these nematodes. At each time point, each group member will randomly select 3 plates to score. Once you have your 3 plates, you will separate dead animals off to one side, recording the number of live versus dead. Once complete, each group member will check at least one other group member’s plates to get a second opinion. Death is scored when animals are no longer able to respond to touch provocation with a platinum wire. Scored plates will be discarded and should only be used for one time point.

A typical survival curve for this sort of experiment is shown below. For the purpose of the course, you can plot your results in Excel.

The example above shows a typical epistasis experiment, in which a wild-type C. elegans nematode (N2) is compared with a mutant with increased thermotolerance (ifg-1, a gene encoding a mRNA translation initiation factor). In this example, we determined its reliance on another gene encoding the heat shock transcription factor HSF-1. This result shows that, while hsf-1 is very important for survival under heat stress, the protection imparted by the ifg-1 mutation is not entirely dependent on the hsf-1 gene for its protective effect. In your experiment, you will only have 3 curves (one for each RNAi condition).

In between survival assay time points, students will observe what happens with a transgenic animal bearing a fluorescent reporter (GFP) fused to DAF-16 when it is exposed to 30 minutes of heat stress.

Questions:

1) Were you able to resolve differences in survival between plates to infer which conditions (RNAi) they were under?

2) What was the average survival for plates determined to carry the control strain? Was survival improved in the daf-2 The test (daf-2 mutant) strain?

3) Were differences in average strain survival statistically significant?

4) Using semi-quantitative analysis of nuclear GFP localization, were there differences between strains in the distribution of DAF-16 in unstressed animals? Stressed animals?

5) Once animals began to die from heat, how would you characterize the behavior of those animals that were still alive (e.g., movement, feeding according to pharyngeal pumping). Were there strain specific differences?

Extra suggested reading material:

Guarente L, Sirtuins, Aging, and Medicine. NEJM 2011;364: 2235-44.

Lapierre LR and Hansen M, Lessons from C. elegans: Signaling pathways for longevity. Trends in Endocrinol Metab 2012; 23: 637-644.

Kenyon C, Genes and Cells that determine the lifespan of C. elegans. Video on iBiology: http://www.ibiology.org/ibioseminars/development-stem-cells/cynthia-kenyon-part-1.html

Tatar, M, Bartke, A, Antebi A. The endocrine regulation of aging by insulin-like signals. Science 299. 1346. (2003)

Cynthia Kenyon, Jean Chang, Erin Gensch , Adam Rudner and Ramon Tabtiang. A C. elegans mutant that lives twice as long as wild type. Nature 366(6454), 461-464 (1993)

Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, Beasley TM, Allison DB, Cruzen C, Simmons HA, Kemnitz,JW, and Weindruch R, Caloric restriction delays disease onset and mortality in Rhesus monkeys. Science 2009; 325: 201-204.

Other References:

Ballatore, C., V.M. Lee, and J.Q. Trojanowski, Tau-mediated neurodegeneration in Alzheimer's disease and related disorders. Nat Rev Neurosci, 8, 663-672 (2007).

Douglas, P. M. & Dillin, A. Protein homeostasis and aging in neurodegeneration. J Cell Biol, 190, 719-729 (2010).

Hardy, J., and Selkoe, D.J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353–356 (2002).

Ittner, L.M. and J. Gotz, Amyloid-beta and tau--a toxic pas de deux in Alzheimer's disease. Nat Rev Neurosci, 12, 65-72 (2011)

Kenyon, C. J. The genetics of ageing. Nature 464, 504-512 (2010).

Fontana, L., Partridge, L. & Longo, V. D. Extending healthy life span — from yeast to humans. Science 328, 321–326 (2010).

Riedel, C. G. et al. DAF16 employs the chromatin remodeller SWI/SNF to promote stress resistance and longevity. Nat. Cell Biol. 15, 491–501 (2013).

Greer, E. L. et al. Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans. Nature 466, 383–387 (2010).

Weidner, C. I. et al. Aging of blood can be tracked by DNA methylation changes at just three CpG sites. Genome Biol. 15, R24 (2014).

Sun, D. et al. Epigenomic profiling of young and aged HSCs reveals concerted changes during aging that reinforce self-renewal. Cell Stem Cell 14, 673–688 (2014).

Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, R115 (2013).

Imai, S., Armstrong, C. M., Kaeberlein, M. & Guarente, L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800 (2000).

Horvath, S. et al. Obesity accelerates epigenetic aging of human liver. Proc. Natl Acad. Sci. USA 111, 15538–15543 (2014)

Turkmen, K., Karagoz, A., Kucuk, A., Sirtuins as novel players in the pathogenesis of diabetes mellitus. World J Diabetes, 5(6): 894–900 (2014).