Archana Unnikrishnan

Caloric restriction and genomic stability

Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents while increasing mean and maximum life spans. It has been suggested that CR extends longevity and reduces age-related pathologies by reducing the levels of DNA damage and mutations that accumulate with age. This hypothesis is attractive because the integrity of the genome is essential to a cell/organism and because it is supported by observations that both cancer and immunological defects, which increase significantly with age and are delayed by CR, are associated with changes in DNA damage and/or DNA repair. Over the last three decades, numerous laboratories have examined the effects of CR on the integrity of the genome and the ability of cells to repair DNA. The majority of studies performed indicate that the age-related increase in oxidative damage to DNA is significantly reduced by CR. Early studies suggest that CR reduces DNA damage by enhancing DNA repair. With the advent of genomic technology and our increased understanding of specific repair pathways, CR has been shown to have a significant effect on major DNA repair pathways, such as NER, BER and double-strand break repair.

A new role for oxidative stress in aging: The accelerated aging phenotype in Sod1−/− mice is correlated to increased cellular senescence

In contrast to other mouse models that are deficient in antioxidant enzymes, mice null for Cu/Zn-superoxide dismutase (Sod1−/− mice) show a major decrease in lifespan and several accelerated aging phenotypes. The goal of this study was to determine if cell senescence might be a contributing factor in the accelerated aging phenotype observed in the Sod1−/− mice. We focused on kidney because it is a tissue that has been shown to a significant increase in senescent cells with age. The Sod1−/− mice are characterized by high levels of DNA oxidation in the kidney, which is attenuated by DR. The kidney of the Sod1−/− mice also have higher levels of double strand DNA breaks than wild type (WT) mice. Expression (mRNA and protein) of p16 and p21, two of the markers of cellular senescence, which increased with age, are increased significantly in the kidney of Sod1−/− mice as is β-gal staining cells. In addition, the senescence associated secretory phenotype was also increased significantly in the kidney of Sod1−/− mice compared to WT mice as measured by the expression of transcripts for IL-6 and IL-1β. Dietary restriction of the Sod1−/− mice attenuated the increase in DNA damage, cellular senescence, and expression of IL-6 and IL-1β. Interestingly, the Sod1−/− mice showed higher levels of circulating cytokines than WT mice, suggesting that the accelerated aging phenotype shown by the Sod1−/− mice could result from increased inflammation arising from an accelerated accumulation of senescent cells. Based on our data with Sod1−/− mice, we propose that various bouts of increased oxidative stress over the lifespan of an animal leads to the accumulation of senescent cells. The accumulation of senescent cells in turn leads to increased inflammation, which plays a major role in the loss of function and increased pathology that are hallmark features of aging.

Curcumin is an early-acting stage-specific inducer of extended functional longevity in Drosophila

Larval feeding with curcumin induces an extended health span with significantly increased median and maximum longevities in the adult fly. This phenotype is diet insensitive and shows no additive effect on longevity when combined with an adult dietary restriction (DR) diet, suggesting that curcumin and DR operate via the same or overlapping pathways for this trait. This treatment significantly slows the aging rate so that it is comparable with that of genetically selected long lived animals. The larval treatment also enhances the adult animals geotactic activity in an additive manner with DR, suggesting that curcumin and DR may use different pathways for different traits. Feeding the drug to adults during only the health span also results in a significantly extended health span with increased median and maximum life span. This extended longevity phenotype is induced only during these stage-specific periods. Feeding adults with the drug over their whole life results in a weakly negative effect on median longevity with no increase in maximum life span. There are no negative effects on reproduction, although larval curcumin feeding increases development time, and also apparently accelerates the normal late-life neuromuscular degeneration seen in the legs. Gene expression data from curcumin-fed larvae shows that the TOR pathway is inhibited in the larvae and the young to midlife adults, although several other genes involved in longevity extension are also affected. These data support the hypothesis that curcumin acts as if it is a DR mimetic nutraceutical. These data also suggest that the search for DR mimetics may be enhanced by the use of stage-specific screening of candidate molecules.

FOLATE DEFICIENCY REGULATES EXPRESSION OF DNA POLYMERASE β IN RESPONSE TO OXIDATIVE STRESS

Folate deficiency has been shown to influence carcinogenesis by creating an imbalance in the base excision repair (BER) pathway, affecting BER homeostasis. The inability to mount a BER response to oxidative stress in a folate-deficient environment results in the accumulation of DNA repair intermediates, i.e., DNA strand breaks. Our data indicate that upregulation of β-pol expression in response to oxidative stress is inhibited by folate deficiency at the level of gene expression. Alteration in the expression of β-pol in a folate-deficient environment is not due to epigenetic changes in the core promoter of the β-pol gene, i.e., the CpG islands within the β-pol promoter remain unmethylated in the presence or absence of folate. However, the promoter analysis studies show a differential binding of regulatory factors to the -36 to -7 region (the folic acid-response region, FARR) within the core promoter of β-pol. Moreover, we observe a tight correlation between the level of binding of regulatory factors with the FARR and inhibition of β-pol expression. Based on these findings, we propose that folate deficiency results in an upregulation/stability of negative regulatory factors interacting with FARR, repressing the upregulation of the β-pol gene in response to oxidative stress.

Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female mice

BackgroundChanges to the epigenome with aging, and DNA modifications in particular, have been proposed as a central regulator of the aging process, a predictor of mortality, and a contributor to the pathogenesis of age-related diseases. In the central nervous system, control of learning and memory, neurogenesis, and plasticity require changes in cytosine methylation and hydroxymethylation. Although genome-wide decreases in methylation with aging are often reported as scientific dogma, primary research reports describe decreases, increases, or lack of change in methylation and hydroxymethylation and their principle regulators, DNA methyltransferases and ten-eleven translocation dioxygenases in the hippocampus. Furthermore, existing data are limited to only male animals.ResultsThrough examination of the hippocampus in young, adult, and old male and female mice by antibody-based, pyrosequencing, and whole-genome oxidative bisulfite sequencing methods, we provide compelling evidence that contradicts the genomic hypomethylation theory of aging. We also demonstrate that expression of DNA methyltransferases and ten-eleven translocation dioxygenases is not differentially regulated with aging or between the sexes, including the proposed cognitive aging regulator DNMT3a2. Using oxidative bisulfite sequencing that discriminates methylation from hydroxymethylation and by cytosine (CG and non-CG) context, we observe sex differences in average CG methylation and hydroxymethylation of the X chromosome, and small age-related differences in hydroxymethylation of CG island shores and shelves, and methylation of promoter regions.ConclusionThese findings clarify a long-standing misconception of the epigenomic response to aging and demonstrate the need for studies of base-specific methylation and hydroxymethylation with aging in both sexes.

A fish oil diet induces mitochondrial uncoupling and mitochondrial unfolded protein response in epididymal white adipose tissue of mice

Abstract White adipose tissue (WAT) mitochondrial dysfunction is linked to the pathogenesis of obesity driven insulin resistance. Dietary conditions that alter fat mass are known to affect white adipocyte mitochondrial function, however, the impact of high calorie diets on white adipocyte mitochondria is not fully understood. The aim of this study is to assess the effect of a diet rich in saturated or polyunsaturated fat on mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that maintains mitochondrial homeostasis, in epididymal WAT (eWAT). Mice were fed a low fat diet (LFD), saturated fat diet (SFD) or fish oil (unsaturated fat diet, UFD) and assessed changes in eWAT mitochondria. Compared to mice fed a LFD, SFD‐fed mice have reduced mitochondrial biogenesis markers, mitochondrial fatty acid oxidation enzymes and TCA cycle enzymes, suggesting an impaired mitochondrial function that could contribute to increased fat mass. In contrast, isocaloric UFD‐fed mice have increased expression of mitochondrial uncoupling protein 1 (UCP1) and peroxisomal fatty acid oxidation enzymes suggesting that elevated mitochondrial uncoupling and peroxisomal fatty acid oxidation could contribute to the reduction in fat mass. Interestingly, expression of UPRmt‐associated proteins caseinolytic peptidase (ClpP) and heat shock protein 60 (Hsp60) are induced by UFD, whereas SFD reduced the expression of ClpP. Based on our data, we propose that induction of UPRmt helps to preserve a functional mitochondria and efficient utilization of fat by UFD whereas a dampened UPRmt response might impair mitochondrial function and promote fat accumulation by SFD. Thus, our findings suggest a potential role of UPRmt in mediating the beneficial effects of fish oil. Graphical abstract Figure. No Caption available. HighlightsFish oil diet increases expression of UPRmt‐associated proteins in white adipose tissue.Fish oil diet increases expression of UCP1 and browning of white adipose tissue.A saturated fat diet reduces expression of UPRmt‐associated protein ClpP in white adipose tissue.A saturated fat diet reduces mitochondrial biogenesis markers and TCA cycle enzymes.

Significant life extension by ten percent dietary restriction

Although it is well documented that dietary restriction (DR) increases the life span of rodents and other animals, this increase is observed at relatively high levels of DR, in which rodents are typically fed 40% less than that consumed by rodents fed ad libitum. It is generally assumed that lower levels of DR will have a lesser impact on life span; however, there are very little published data on the effect of low levels of DR on life span. In this study, we show that 10% DR increased life span to almost the same extent as 40% DR. While both 10% and 40% DR resulted in similar changes in non‐neoplastic lesions, 10% DR had no significant effect on the incidence of neoplasia (except for pituitary adenoma), and 40% DR resulted in a significant reduction (40%) in neoplasia. These data clearly demonstrate that the life span of F344 rats does not increase linearly with the level of DR; rather, even a low level of DR can substantially affect life span. This rodent study has important translational implications because it suggests that a modest reduction in calories might have significant health benefits for humans.

Sexually divergent DNA methylation patterns with hippocampal aging

DNA methylation is a central regulator of genome function, and altered methylation patterns are indicative of biological aging and mortality. Age‐related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome‐wide analyses of age‐related methylation changes have considered the factor of sex in a controlled animal model. High‐depth, genome‐wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non‐CG (CH) contexts demonstrated age‐related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic and intronic regions and under‐represented in promoters, CG islands, and specific enhancer regions in both sexes, suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex‐specific. Lifelong sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome‐wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites was confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.

Analysis of DNA modifications in aging research

As geroscience research extends into the role of epigenetics in aging and age-related disease, researchers are being confronted with unfamiliar molecular techniques and data analysis methods that can be difficult to integrate into their work. In this review, we focus on the analysis of DNA modifications, namely cytosine methylation and hydroxymethylation, through next-generation sequencing methods. While older techniques for modification analysis performed relative quantitation across regions of the genome or examined average genome levels, these analyses lack the desired specificity, rigor, and genomic coverage to firmly establish the nature of genomic methylation patterns and their response to aging. With recent methodological advances, such as whole genome bisulfite sequencing (WGBS), bisulfite oligonucleotide capture sequencing (BOCS), and bisulfite amplicon sequencing (BSAS), cytosine modifications can now be readily analyzed with base-specific, absolute quantitation at both cytosine-guanine dinucleotide (CG) and non-CG sites throughout the genome or within specific regions of interest by next-generation sequencing. Additional advances, such as oxidative bisulfite conversion to differentiate methylation from hydroxymethylation and analysis of limited input/single-cells, have great promise for continuing to expand epigenomic capabilities. This review provides a background on DNA modifications, the current state-of-the-art for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.

Revisiting the genomic hypomethylation hypothesis of aging

The genomic hypomethylation hypothesis of aging proposes that an overall decrease in global DNA methylation occurs with age, and it has been argued that the decrease in global DNA methylation could be an important factor in aging, resulting in the relaxation of gene expression regulation and abnormal gene expression. Since it was initially observed that DNA methylation decreased with age in 1974, 16 articles have been published describing the effect of age on global DNA methylation in various tissues from rodents and humans. We critically reviewed the publications on the effect of age on DNA methylation and the expression of the enzymes involved in DNA methylation to evaluate the validity of the hypomethylation hypothesis of aging. On the basis of the current scientific literature, we conclude that a decrease in the global methylation of the genome occurs in most if not all tissues/cells as an animal ages. However, age‐related changes in DNA methylation in specific regions or at specific sites in the genome occur even though the global DNA methylation does not change.