Niran Hadad

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.

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.

Caloric-restriction induced alterations in CG and non-CG methylation attenuate age-associated changes in the old brain.

Brain aging is marked by cognitive decline and susceptibility to neurodegeneration. Caloric-restriction (CR) increases neurogenesis, improves memory function, and protects from age-associated neurological disorders. Epigenetic mechanisms, including DNA methylation, are vital to normal CNS cellular and memory functions, and are dysregulated with aging. The beneficial effects of CR have been proposed to work through epigenetic processes, but this is largely unexplored. We therefore tested whether life-long CR prevents age-related DNA methylation changes in the brain. Hippocampal DNA from young (3 months) and old (24 months) male mice fed ad libitum and 24 month old mice fed a 40% calorierestricted diet from 3 months of age were examined by genome-wide bisulfite sequencing to measure methylation with base-specificity. Over 27 million CG and CH (non-CG) sites were examined. Of the ~40,000 differentially methylated CGs (dmCGs) and ~80,000 CHs (dmCHs) with aging, >1/3 were prevented by CR and were found across genomic regulatory regions and gene pathways. CR also caused alterations to CG and CH methylation at sites not differentially methylated with aging, and these CR-specific changes demonstrated a different pattern of regulatory element and gene pathway enrichment than those affected by aging. CR-specific DNMT1 and TET3 promoter hypermethylation corresponded to reduced gene expression. These findings demonstrate that CR attenuates age-related CG and CH hippocampal methylation changes, in combination with CR-specific methylation that may also contribute to the neuroprotective effects of CR. The prevention of age-related methylation alterations is also consistent with the pro-longevity effects of CR working through an epigenetic mechanism.Brain aging is marked by cognitive decline and increased susceptibility to neurodegeneration. Epigenetic mechanisms, including DNA methylation, are vital to CNS cellular function and memory formation, and are dysregulated with aging and age-related neurodegenerative disease. Caloric-restriction (CR), an established pro-longevity intervention, increases neurogenesis, improves memory function, and protects from age-associated pathologies. However, the molecular mechanisms promoting the neuroprotective effect of CR remain largely unknown. We tested the role of DNA methylation as a mechanism for CR-induced neuroprotection in the old hippocampus. Hippocampal DNA from young (3M) and old (24M) male mice fed ad libitum and 24M old mice fed 40% calorie-restricted diet from 3M of age were examined by genome-wide bisulfite sequencing to measure methylation levels at base-specific resolution. Over 22 million CG and CH (non-CG) sites were examined. Of the ~40,000 differentially methylated CGs (dmCGs) and ~80,000 CHs (dmCHs) observed with aging, 35% and 38%, respectively, were prevented by CR. Unique to dmCHs, CR preferentially prevented age-related hypermethylation. A diet-specific methylation response was observed in both CG and CH contexts. Diet-induced dmCHs were enriched in unexpected genomic locations including promoters and CG islands including hypermethylation of DNMT1 and Tet3 promoters corresponding to reduced gene expression. These findings demonstrate for the first time that caloric-restriction prevents age-induced cytosine methylation changes in the old brain in combination with diet-specific methylation changes that may function to maintain epigenetic homeostasis through epigenetic auto-regulation. The prevention of age-dmCGs/CHs by CR emphasizes the prominent role of DNA methylation as a driver of the aging process.Brain aging is marked by cognitive decline and increased susceptibility to neurodegeneration. Epigenetic mechanisms, including DNA methylation, are vital to CNS cellular function and memory formation, and are dysregulated with aging and age-related neurodegenerative disease. Caloric-restriction (CR), an established pro-longevity intervention, increases neurogenesis, improves memory function, and protects from age-associated pathologies. However, the molecular mechanisms promoting the neuroprotective effect of CR remain largely unknown. We tested the role of DNA methylation as a mechanism for CR-induced neuroprotection in the old hippocampus. Hippocampal DNA from young (3M) and old (24M) male mice fed ad libitum and 24M old mice fed 40% calorie-restricted diet from 3M of age were examined by genome-wide bisulfite sequencing to measure methylation levels at base-specific resolution. Over 22 million CG and CH (non-CG) sites were examined. Of the ~40,000 differentially methylated CGs (dmCGs) and ~80,000 CHs (dmCHs) observed with aging, 35% and 38%, respectively, were prevented by CR. Unique to dmCHs, CR preferentially prevented age-related hypermethylation. A diet-specific methylation response was observed in both CG and CH contexts. Diet-induced dmCHs were enriched in unexpected genomic locations including promoters and CG islands including hypermethylation of DNMT1 and Tet3 promoters corresponding to reduced gene expression. These findings demonstrate for the first time that caloric-restriction prevents age-induced cytosine methylation changes in the old brain in combination with diet-specific methylation changes that may function to maintain epigenetic homeostasis through epigenetic auto-regulation. The prevention of age-dmCGs/CHs by CR emphasizes the prominent role of DNA methylation as a driver of the aging process.

Exposure to environmental enrichment attenuates addiction-like behavior and alters molecular effects of heroin self-administration in rats

ABSTRACT Environmental factors profoundly affect the addictive potential of drugs of abuse and may also modulate the neuro‐anatomical/neuro‐chemical impacts of uncontrolled drug use and relapse propensity. This study examined the impact of environmental enrichment on heroin self‐administration, addiction‐related behaviors, and molecular processes proposed to underlie these behaviors. Male Sprague‐Dawley rats in standard and enriched housing conditions intravenously self‐administered similar amounts of heroin over 14 days. However, environmental enrichment attenuated progressive ratio, extinction, and reinstatement session responding after 14 days of enforced abstinence. Molecular mechanisms, namely DNA methylation and gene expression, are proposed to underlie abstinence‐persistent behaviors. A global reduction in methylation is reported to coincide with addiction, but no differences in total genomic methylation or repeat element methylation were observed in CpG or non‐CpG (CH) contexts across the mesolimbic circuitry as assessed by multiple methods including whole genome bisulfite sequencing. Immediate early gene expression associated with drug seeking, taking, and abstinence also were examined. EGR1 and EGR2 were suppressed in mesolimbic regions with heroin‐taking and environmental enrichment. Site‐specific methylation analysis of EGR1 and EGR2 promoter regions using bisulfite amplicon sequencing (BSAS) revealed hypo‐methylation in the EGR2 promoter region and EGR1 intragenic CpG sites with heroin‐taking and environmental enrichment that was associated with decreased mRNA expression. Taken together, these findings illuminate the impact of drug taking and environment on the epigenome in a locus and gene‐specific manner and highlight the need for positive, alternative rewards in the treatment and prevention of drug addiction. HIGHLIGHTSEnvironmental enrichment decreases the reinforcement efficacy of heroin.Genome‐wide methylation is not altered in mesolimbic structures with heroin self‐administration or environmental enrichment.Gene expression and base‐specific methylation responses to heroin are modified by environmental enrichment.

Necroptosis increases with age and is reduced by dietary restriction

Necroptosis is a newly identified programmed cell death pathway that is highly proinflammatory due to the release of cellular components that promote inflammation. To determine whether necroptosis might play a role in inflammaging, we studied the effect of age and dietary restriction (DR) on necroptosis in the epididymal white adipose tissue (eWAT), a major source of proinflammatory cytokines. Phosphorylated MLKL and RIPK3, markers of necroptosis, were increased 2.7‐ and 1.9‐fold, respectively, in eWAT of old mice compared to adult mice, and DR reduced P‐MLKL and P‐RIPK3 to levels similar to adult mice. An increase in the expression of RIPK1 (1.6‐fold) and MLKL (2.7‐fold), not RIPK3, was also observed in eWAT of old mice, which was reduced by DR in old mice. The increase in necroptosis was paralleled by an increase in 14 inflammatory cytokines, including the pro‐inflammatory cytokines IL‐6 (3.9‐fold), TNF‐α (4.7‐fold), and IL‐1β (5.1‐fold)], and 11 chemokines in old mice. DR attenuated the expression of IL‐6, TNF‐α, and IL‐1β as well as 85% of the other cytokines/chemokines induced with age. In contrast, inguinal WAT (iWAT), which is less inflammatory, did not show any significant increase with age in the levels of P‐MLKL and MLKL or inflammatory cytokines/chemokines. Because the changes in biomarkers of necroptosis in eWAT with age and DR paralleled the changes in the expression of pro‐inflammatory cytokines, our data support the possibility that necroptosis might play a role in increased chronic inflammation observed with age.