Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

Publisher Correction: Age and life expectancy clocks based on machine learning analysis of mouse frailty.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Age and life expectancy clocks based on machine learning analysis of mouse frailty.

The identification of genes and interventions that slow or reverse aging is hampered by the lack of non-invasive metrics that can predict the life expectancy of pre-clinical models. Frailty Indices (FIs) in mice are composite measures of health that are cost-effective and non-invasive, but whether they can accurately predict health and lifespan is not known. Here, mouse FIs are scored longitudinally until death and machine learning is employed to develop two clocks. A random forest regression is trained on FI components for chronological age to generate the FRIGHT (Frailty Inferred Geriatric Health Timeline) clock, a strong predictor of chronological age. A second model is trained on remaining lifespan to generate the AFRAID (Analysis of Frailty and Death) clock, which accurately predicts life expectancy and the efficacy of a lifespan-extending intervention up to a year in advance. Adoption of these clocks should accelerate the identification of longevity genes and aging interventions.

Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Resveratrol Improves Vascular Function and Mitochondrial Number but Not Glucose Metabolism in Older Adults.

BACKGROUND: Resveratrol, a plant-derived polyphenol, has been reported to improve glucose metabolism and vascular function and to extend life span in animal models, but studies in humans have been inconclusive. METHODS: In a randomized, double-blind crossover study, we treated older glucose-intolerant adults (n = 30) with resveratrol (2-3 g/daily) or placebo, each for 6 weeks. A standard mixed-meal test was used to assess insulin sensitivity (Matsuda index) and secretion (C-peptide deconvolution) and vascular function by reactive hyperemia peripheral arterial tonometry. Skeletal muscle samples were obtained for gene expression using RNA-Seq analysis and to assess mitochondrial morphology. RESULTS: There were no changes in glucose tolerance, insulin sensitivity, weight, blood pressure, or lipid profile following resveratrol treatment. Fasting reactive hyperemia index improved with resveratrol (2.02 ± 0.2 vs 1.76 ± 0.02, p = .002). RNA-Seq analysis yielded 140 differentially expressed transcripts (corrected p-value ≤ .05), predominantly associated with mitochondrial genes and noncoding RNA. Ingenuity Pathway Analysis confirmed that mitochondrial dysfunction (p = 2.77 × 10-12) and oxidative phosphorylation (p = 1.41 × 10-11) were the most significantly perturbed pathways. Mitochondrial number, but not size, was increased. CONCLUSIONS: Resveratrol treatment of older adults with impaired glucose regulation may have beneficial effects on vascular function, but not glucose metabolism or insulin sensitivity. Changes in gene expression suggest effects similar to those observed with caloric restriction, which has been shown to increase life and health span in animal models, although its significance for humans is uncertain. Future human studies should address the appropriate dose range and low bioavailability of resveratrol.

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+ Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.

Slowing ageing by design: the rise of NAD+ and sirtuin-activating compounds.

The sirtuins (SIRT1-7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacylases with remarkable abilities to prevent diseases and even reverse aspects of ageing. Mice engineered to express additional copies of SIRT1 or SIRT6, or treated with sirtuin-activating compounds (STACs) such as resveratrol and SRT2104 or with NAD+ precursors, have improved organ function, physical endurance, disease resistance and longevity. Trials in non-human primates and in humans have indicated that STACs may be safe and effective in treating inflammatory and metabolic disorders, among others. These advances have demonstrated that it is possible to rationally design molecules that can alleviate multiple diseases and possibly extend lifespan in humans.

Measuring aging rates of mice subjected to caloric restriction and genetic disruption of growth hormone signaling.

Caloric restriction and genetic disruption of growth hormone signaling have been shown to counteract aging in mice. The effects of these interventions on aging are examined through age-dependent survival or through the increase in age-dependent mortality rates on a logarithmic scale fitted to the Gompertz model. However, these methods have limitations that impede a fully comprehensive disclosure of these effects. Here we examine the effects of these interventions on murine aging through the increase in age-dependent mortality rates on a linear scale without fitting them to a model like the Gompertz model. Whereas these interventions negligibly and non-consistently affected the aging rates when examined through the age-dependent mortality rates on a logarithmic scale, they caused the aging rates to increase at higher ages and to higher levels when examined through the age-dependent mortality rates on a linear scale. These results add to the debate whether these interventions postpone or slow aging and to the understanding of the mechanisms by which they affect aging. Since different methods yield different results, it is worthwhile to compare their results in future research to obtain further insights into the effects of dietary, genetic, and other interventions on the aging of mice and other species.

Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition.

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein ß (C/EBPß) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Metabolic alterations due to caloric restriction and every other day feeding in normal and growth hormone receptor knockout mice.

Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice.

Deviation of innate circadian period from 24 h reduces longevity in mice.

The variation of individual life spans, even in highly inbred cohorts of animals and under strictly controlled environmental conditions, is substantial and not well understood. This variation in part could be due to epigenetic variation, which later affects the animal's physiology and ultimately longevity. Identification of the physiological properties that impact health and life span is crucial for longevity research and the development of anti-aging therapies. Here, we measured individual circadian and metabolic characteristics in a cohort of inbred F1 hybrid mice and correlated these parameters to their life spans. We found that mice with innate circadian periods close to 24 h (revealed during 30 days of housing in total darkness) enjoyed nearly 20% longer life spans than their littermates, which had shorter or longer innate circadian periods. These findings show that maintenance of a 24-h intrinsic circadian period is a positive predictor of longevity. Our data suggest that circadian period may be used to predict individual longevity and that processes that control innate circadian period affect aging.

Neuronal SIRT1 regulates endocrine and behavioral responses to calorie restriction.

Mammalian life span can be extended by both calorie restriction (CR) and mutations that diminish somatotropic signaling. Sirt1 is a mediator of many effects of CR in mammals, but any role in controlling somatotropic signaling has not been shown. Since the somatotropic axis is controlled by the brain, we created mice lacking Sirt1 specifically in the brain and examined the impacts of this manipulation on somatotropic signaling and the CR response. These mutant mice displayed defects in somatotropic signaling when fed ad libitum, and defects in the endocrine and behavioral responses to CR. We conclude that Sirt1 in the brain is a link between somatotropic signaling and CR in mammals.

The growth hormone receptor gene-disrupted mouse fails to respond to an intermittent fasting diet.

The interaction of longevity-conferring genes with longevity-conferring diets is poorly understood. The growth hormone receptor gene-disrupted (GHR-KO) mouse is long lived; and this longevity is not responsive to 30% caloric restriction, in contrast to wild-type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction, we subjected GHR-KO mice to a different dietary restriction regimen, an intermittent fasting diet. The intermittent fasting diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR-KO mice. From the results of two paradigms of dietary restriction, we postulate that GHR-KO mice would be resistant to any manner of dietary restriction; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan extending potential of an intervention.

Is altered expression of hepatic insulin-related genes in growth hormone receptor knockout mice due to GH resistance or a difference in biological life spans?

Growth hormone receptor knockout (GHRKO) mice live about 40%-55% longer than their normal (N) littermates. Previous studies of 21-month-old GHRKO and N mice showed major alterations of the hepatic expression of genes involved in insulin signaling. Differences detected at this age may have been caused by the knockout of the growth hormone receptor (GHR) or by differences in biological age between GHRKO and N mice. To address this question, we compared GHRKO and N mice at ages corresponding to the same percentage of median life span to see if the differences of gene expression persisted. Comparison of GHRKO and N mice at approximately 50% of biological life span showed significant differences in hepatic expression of all 14 analyzed genes. We conclude that these changes are due to disruption of GHR gene and the consequent suppression of growth hormone signaling rather than to differences in "biological age" between mutant and normal animals sampled at the same chronological age.

Alterations in oxygen consumption, respiratory quotient, and heat production in long-lived GHRKO and Ames dwarf mice, and short-lived bGH transgenic mice.

Growth hormone (GH) signaling influences longevity in mice, with decreased GH signaling associated with longer life span and increased GH signaling with shortened life span. A proposed mechanism through which GH signaling influences life span postulates that decreased GH signaling lowers metabolic rate, thus slowing aging by decreasing production of damaging free radicals. The influence of altered GH signaling on metabolism was tested by monitoring oxygen consumption (VO(2)), respiratory quotient (RQ), and heat production in long-lived GH receptor knockout (GHRKO) and Ames dwarf mice, and short-lived bovine GH-overexpressing transgenic (bGH TG) mice. Intriguingly, both GHRKO and Ames dwarf mice have increased VO(2) and heat per gram body weight, and decreased RQ, whereas bGH TG mice have decreased VO(2) and heat per gram body weight and increased RQ. In conclusion, decreased GH signaling associates with increased metabolism per body weight and may beneficially affect mitochondrial flexibility by increasing the capacity for fat oxidation; generally, GH excess produces opposite metabolic effects.

Insulin sensitivity as a key mediator of growth hormone actions on longevity.

Reduced insulin sensitivity and glucose intolerance have been long suspected of having important involvement in aging. Here we report that in studies of calorie restriction (CR) effects in mutant (Prop1(df) and growth hormone receptor knockout [GHRKO]) and normal mice, insulin sensitivity was strongly associated with longevity. Of particular interest was enhancement of the already increased insulin sensitivity in CR df/df mice in which longevity was also further extended and the lack of changes in insulin sensitivity in calorically restricted GHRKO mice in which there was no further increase in average life span. We suggest that enhanced insulin sensitivity, in conjunction with reduced insulin levels, may represent an important (although almost certainly not exclusive) mechanism of increased longevity in hypopituitary, growth hormone (GH)-resistant, and calorie-restricted animals. We also report that the effects of GH treatment on insulin sensitivity may be limited to the period of GH administration.