Detecting the effect of genetic diversity on brain composition in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is broadly characterized by neurodegeneration, pathology accumulation, and cognitive decline. There is considerable variation in the progression of clinical symptoms and pathology in humans, highlighting the importance of genetic diversity in the study of AD. To address this, we analyze cell composition and amyloid-beta deposition of 6- and 14-month-old AD-BXD mouse brains. We utilize the analytical QUINT workflow- a suite of software designed to support atlas-based quantification, which we expand to deliver a highly effective method for registering and quantifying cell and pathology changes in diverse disease models. In applying the expanded QUINT workflow, we quantify near-global age-related increases in microglia, astrocytes, and amyloid-beta, and we identify strain-specific regional variation in neuron load. To understand how individual differences in cell composition affect the interpretation of bulk gene expression in AD, we combine hippocampal immunohistochemistry analyses with bulk RNA-sequencing data. This approach allows us to categorize genes whose expression changes in response to AD in a cell and/or pathology load-dependent manner. Ultimately, our study demonstrates the use of the QUINT workflow to standardize the quantification of immunohistochemistry data in diverse mice, - providing valuable insights into regional variation in cellular load and amyloid deposition in the AD-BXD model.

A prognostic molecular signature of hepatic steatosis is spatially heterogeneous and dynamic in human liver.

Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing in parallel with an obesity pandemic, calling for novel strategies for prevention and treatment. We defined a circulating proteome of human MASLD across ≈7000 proteins in ≈5000 individuals from diverse, at-risk populations across the metabolic health spectrum, demonstrating reproducible diagnostic performance and specifying both known and novel metabolic pathways relevant to MASLD (central carbon and amino acid metabolism, hepatocyte regeneration, inflammation, fibrosis, insulin sensitivity). A parsimonious proteomic signature of MASLD was associated with a protection from MASLD and its related multi-system metabolic consequences in >26000 free-living individuals, with an additive effect to polygenic risk. The MASLD proteome was encoded by genes that demonstrated transcriptional enrichment in liver, with spatial transcriptional activity in areas of steatosis in human liver biopsy and dynamicity for select targets in human liver across stages of steatosis. We replicated several top relations from proteomics and spatial tissue transcriptomics in a humanized "liver-on-a-chip" model of MASLD, highlighting the power of a full translational approach to discovery in MASLD. Collectively, these results underscore utility of blood-based proteomics as a dynamic "liquid biopsy" of human liver relevant to clinical biomarker and mechanistic applications.

Hippocampus Glutathione S Reductase Potentially Confers Genetic Resilience to Cognitive Decline in the AD-BXD Mouse Population.

Alzheimer's disease (AD) is a prevalent and costly age-related dementia. Heritable factors account for 58-79% of variation in late-onset AD, but substantial variation remains in age-of- onset, disease severity, and whether those with high-risk genotypes acquire AD. To emulate the diversity of human populations, we utilized the AD-BXD mouse panel. This genetically diverse resource combines AD genotypes with multiple BXD strains to discover new genetic drivers of AD resilience. Comparing AD-BXD carriers to noncarrier littermates, we computed a novel quantitative metric for resilience to cognitive decline in the AD-BXDs. Our quantitative AD resilience trait was heritable and genetic mapping identified a locus on chr8 associated with resilience to AD mutations that resulted in amyloid brain pathology. Using a hippocampus proteomics dataset, we nominated the mitochondrial glutathione S reductase protein (GR or GSHR) as a resilience factor, finding that the DBA/2J genotype was associated with substantially higher GR abundance. By mapping protein QTLs (pQTLs), we identified synaptic organization and mitochondrial proteins coregulated in trans with a cis-pQTL for GR. We found four coexpression modules correlated with the quantitative resilience score in aged 5XFAD mice using paracliques, which were related to cell structure, protein folding, and postsynaptic densities. Finally, we found significant positive associations between human GSR transcript abundance in the brain and better outcomes on AD-related cognitive and pathology traits in the Religious Orders Study/Memory and Aging project (ROSMAP). Taken together, these data support a framework for resilience in which neuronal antioxidant pathway activity provides for stability of synapses within the hippocampus.

Detecting the effect of genetic diversity on brain composition in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is characterized by neurodegeneration, pathology accumulation, and progressive cognitive decline. There is significant variation in age at onset and severity of symptoms highlighting the importance of genetic diversity in the study of AD. To address this, we analyzed cell and pathology composition of 6- and 14-month-old AD-BXD mouse brains using the semi-automated workflow (QUINT); which we expanded to allow for nonlinear refinement of brain atlas-registration, and quality control assessment of atlas-registration and brain section integrity. Near global age-related increases in microglia, astrocyte, and amyloid-beta accumulation were measured, while regional variation in neuron load existed among strains. Furthermore, hippocampal immunohistochemistry analyses were combined with bulk RNA-sequencing results to demonstrate the relationship between cell composition and gene expression. Overall, the additional functionality of the QUINT workflow delivers a highly effective method for registering and quantifying cell and pathology changes in diverse disease models.

Life-long dietary restrictions have negligible or damaging effects on late-life cognitive performance: A key role for genetics in outcomes.

Several studies report that caloric restriction (CR) or intermittent fasting (IF) can improve cognition, while others report limited or no cognitive benefits. Here, we compare the effects of 20% CR, 40% CR, 1-day IF, and 2-day IF feeding paradigms to ad libitum controls on Y-maze working memory (WM) and contextual fear memory (CFM) in a large population of Diversity Outbred mice that model the genetic diversity of humans. While CR and IF interventions improve lifespan, we observed no enhancement of working memory or CFM in mice on these feeding paradigms, and report 40% CR to be damaging to recall of CFM. Using Quantitative Trait Loci mapping, we identified the gene Slc16a7 to be associated with CFM outcomes in aged mice on lifespan promoting feeding paradigms. Limited utility of dieting and fasting on memory in mice that recapitulate genetic diversity in the human population highlights the need for anti-aging therapeutics that promote cognitive function, with the neuronal monocarboxylate transporter MCT2 encoded by Slc16a7 highlighted as novel target.

Sex differences in the genetic architecture of cognitive resilience to Alzheimer's disease.

Approximately 30% of elderly adults are cognitively unimpaired at time of death despite the presence of Alzheimer's disease neuropathology at autopsy. Studying individuals who are resilient to the cognitive consequences of Alzheimer's disease neuropathology may uncover novel therapeutic targets to treat Alzheimer's disease. It is well established that there are sex differences in response to Alzheimer's disease pathology, and growing evidence suggests that genetic factors may contribute to these differences. Taken together, we sought to elucidate sex-specific genetic drivers of resilience. We extended our recent large scale genomic analysis of resilience in which we harmonized cognitive data across four cohorts of cognitive ageing, in vivo amyloid PET across two cohorts, and autopsy measures of amyloid neuritic plaque burden across two cohorts. These data were leveraged to build robust, continuous resilience phenotypes. With these phenotypes, we performed sex-stratified [n (males) = 2093, n (females) = 2931] and sex-interaction [n (both sexes) = 5024] genome-wide association studies (GWAS), gene and pathway-based tests, and genetic correlation analyses to clarify the variants, genes and molecular pathways that relate to resilience in a sex-specific manner. Estimated among cognitively normal individuals of both sexes, resilience was 20-25% heritable, and when estimated in either sex among cognitively normal individuals, resilience was 15-44% heritable. In our GWAS, we identified a female-specific locus on chromosome 10 [rs827389, ß (females) = 0.08, P (females) = 5.76 × 10-09, ß (males) = -0.01, P(males) = 0.70, ß (interaction) = 0.09, P (interaction) = 1.01 × 10-04] in which the minor allele was associated with higher resilience scores among females. This locus is located within chromatin loops that interact with promoters of genes involved in RNA processing, including GATA3. Finally, our genetic correlation analyses revealed shared genetic architecture between resilience phenotypes and other complex traits, including a female-specific association with frontotemporal dementia and male-specific associations with heart rate variability traits. We also observed opposing associations between sexes for multiple sclerosis, such that more resilient females had a lower genetic susceptibility to multiple sclerosis, and more resilient males had a higher genetic susceptibility to multiple sclerosis. Overall, we identified sex differences in the genetic architecture of resilience, identified a female-specific resilience locus and highlighted numerous sex-specific molecular pathways that may underly resilience to Alzheimer's disease pathology. This study illustrates the need to conduct sex-aware genomic analyses to identify novel targets that are unidentified in sex-agnostic models. Our findings support the theory that the most successful treatment for an individual with Alzheimer's disease may be personalized based on their biological sex and genetic context.

Differential Regulation of Mouse Hippocampal Gene Expression Sex Differences by Chromosomal Content and Gonadal Sex.

Common neurological disorders, like Alzheimer's disease (AD), multiple sclerosis (MS), and autism, display profound sex differences in prevalence and clinical presentation. However, sex differences in the brain with health and disease are often overlooked in experimental models. Sex effects originate, directly or indirectly, from hormonal or sex chromosomal mechanisms. To delineate the contributions of genetic sex (XX v. XY) versus gonadal sex (ovaries v. testes) to the epigenomic regulation of hippocampal sex differences, we used the Four Core Genotypes (FCG) mouse model which uncouples chromosomal and gonadal sex. Transcriptomic and epigenomic analyses of ~ 12-month-old FCG mouse hippocampus, revealed genomic context-specific regulatory effects of genotypic and gonadal sex on X- and autosome-encoded gene expression and DNA modification patterns. X-chromosomal epigenomic patterns, classically associated with X-inactivation, were established almost entirely by genotypic sex, independent of gonadal sex. Differences in X-chromosome methylation were primarily localized to gene regulatory regions including promoters, CpG islands, CTCF binding sites, and active/poised chromatin, with an inverse relationship between methylation and gene expression. Autosomal gene expression demonstrated regulation by both genotypic and gonadal sex, particularly in immune processes. These data demonstrate an important regulatory role of sex chromosomes, independent of gonadal sex, on sex-biased hippocampal transcriptomic and epigenomic profiles. Future studies will need to further interrogate specific CNS cell types, identify the mechanisms by which sex chromosomes regulate autosomes, and differentiate organizational from activational hormonal effects.

Health benefits attributed to 17α-estradiol, a lifespan-extending compound, are mediated through estrogen receptor α.

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17α-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17α-estradiol elicits these benefits remain unresolved. Herein, we show that 17α-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor α (ERα) to that of 17ß-estradiol. In addition, we show that the ablation of ERα completely attenuates the beneficial metabolic effects of 17α-E2 in male mice. Our findings suggest that 17α-E2 may act through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17α-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17α-E2 are not limited to mice. Collectively, these studies suggest ERα may be a drug target for mitigating chronic diseases in male mammals.

Identifying Mechanisms of Normal Cognitive Aging Using a Novel Mouse Genetic Reference Panel.

Developing strategies to maintain cognitive health is critical to quality of life during aging. The basis of healthy cognitive aging is poorly understood; thus, it is difficult to predict who will have normal cognition later in life. Individuals may have higher baseline functioning (cognitive reserve) and others may maintain or even improve with age (cognitive resilience). Understanding the mechanisms underlying cognitive reserve and resilience may hold the key to new therapeutic strategies for maintaining cognitive health. However, reserve and resilience have been inconsistently defined in human studies. Additionally, our understanding of the molecular and cellular bases of these phenomena is poor, compounded by a lack of longitudinal molecular and cognitive data that fully capture the dynamic trajectories of cognitive aging. Here, we used a genetically diverse mouse population (B6-BXDs) to characterize individual differences in cognitive abilities in adulthood and investigate evidence of cognitive reserve and/or resilience in middle-aged mice. We tested cognitive function at two ages (6 months and 14 months) using y-maze and contextual fear conditioning. We observed heritable variation in performance on these traits (h 2 RIx̄ = 0.51-0.74), suggesting moderate to strong genetic control depending on the cognitive domain. Due to the polygenetic nature of cognitive function, we did not find QTLs significantly associated with y-maze, contextual fear acquisition (CFA) or memory, or decline in cognitive function at the genome-wide level. To more precisely interrogate the molecular regulation of variation in these traits, we employed RNA-seq and identified gene networks related to transcription/translation, cellular metabolism, and neuronal function that were associated with working memory, contextual fear memory, and cognitive decline. Using this method, we nominate the Trio gene as a modulator of working memory ability. Finally, we propose a conceptual framework for identifying strains exhibiting cognitive reserve and/or resilience to assess whether these traits can be observed in middle-aged B6-BXDs. Though we found that earlier cognitive reserve evident early in life protects against cognitive impairment later in life, cognitive performance and age-related decline fell along a continuum, with no clear genotypes emerging as exemplars of exceptional reserve or resilience - leading to recommendations for future use of aging mouse populations to understand the nature of cognitive reserve and resilience.

Cross-Species Analyses Identify Dlgap2 as a Regulator of Age-Related Cognitive Decline and Alzheimer's Dementia.

Genetic mechanisms underlying age-related cognitive decline and dementia remain poorly understood. Here, we take advantage of the Diversity Outbred mouse population to utilize quantitative trait loci mapping and identify Dlgap2 as a positional candidate responsible for modifying working memory decline. To evaluate the translational relevance of this finding, we utilize longitudinal cognitive measures from human patients, RNA expression from post-mortem brain tissue, data from a genome-wide association study (GWAS) of Alzheimer's dementia (AD), and GWAS results in African Americans. We find an association between Dlgap2 and AD phenotypes at the variant, gene and protein expression, and methylation levels. Lower cortical DLGAP2 expression is observed in AD and is associated with more plaques and tangles at autopsy and faster cognitive decline. Results will inform future studies aimed at investigating the cross-species role of Dlgap2 in regulating cognitive decline and highlight the benefit of using genetically diverse mice to prioritize novel candidates.

Genetic variants and functional pathways associated with resilience to Alzheimer's disease.

Approximately 30% of older adults exhibit the neuropathological features of Alzheimer's disease without signs of cognitive impairment. Yet, little is known about the genetic factors that allow these potentially resilient individuals to remain cognitively unimpaired in the face of substantial neuropathology. We performed a large, genome-wide association study (GWAS) of two previously validated metrics of cognitive resilience quantified using a latent variable modelling approach and representing better-than-predicted cognitive performance for a given level of neuropathology. Data were harmonized across 5108 participants from a clinical trial of Alzheimer's disease and three longitudinal cohort studies of cognitive ageing. All analyses were run across all participants and repeated restricting the sample to individuals with unimpaired cognition to identify variants at the earliest stages of disease. As expected, all resilience metrics were genetically correlated with cognitive performance and education attainment traits (P-values < 2.5 × 10-20), and we observed novel correlations with neuropsychiatric conditions (P-values < 7.9 × 10-4). Notably, neither resilience metric was genetically correlated with clinical Alzheimer's disease (P-values > 0.42) nor associated with APOE (P-values > 0.13). In single variant analyses, we observed a genome-wide significant locus among participants with unimpaired cognition on chromosome 18 upstream of ATP8B1 (index single nucleotide polymorphism rs2571244, minor allele frequency = 0.08, P = 2.3 × 10-8). The top variant at this locus (rs2571244) was significantly associated with methylation in prefrontal cortex tissue at multiple CpG sites, including one just upstream of ATPB81 (cg19596477; P = 2 × 10-13). Overall, this comprehensive genetic analysis of resilience implicates a putative role of vascular risk, metabolism, and mental health in protection from the cognitive consequences of neuropathology, while also providing evidence for a novel resilience gene along the bile acid metabolism pathway. Furthermore, the genetic architecture of resilience appears to be distinct from that of clinical Alzheimer's disease, suggesting that a shift in focus to molecular contributors to resilience may identify novel pathways for therapeutic targets.

Targeting cPLA2 derived lipid hydroperoxides as a potential intervention for sarcopenia.

Defects in neuromuscular innervation contribute significantly to the age-related decline in muscle mass and function (sarcopenia). Our previous studies demonstrated that denervation induces muscle mitochondrial hydroperoxide production (H2O2 and lipid hydroperoxides (LOOHs)). Here we define the relative contribution of mitochondrial electron transport chain (ETC) derived H2O2 versus cytosolic phospholipase A2 (cPLA2) derived LOOHs in neurogenic muscle atrophy. We show that denervation increases muscle cPLA2 protein content, activity, and metabolites downstream of cPLA2 including LOOHs. Increased scavenging of mitochondrial H2O2 does not protect against denervation atrophy, suggesting ETC generated H2O2 is not a critical player. In contrast, inhibition of cPLA2 in vivo mitigates LOOH production and muscle atrophy and maintains individual muscle fiber size while decreasing oxidative damage. Overall, we show that loss of innervation in several muscle atrophy models including aging induces generation of LOOHs produced by arachidonic acid metabolism in the cPLA2 pathway contributing to loss of muscle mass.

Identifying the molecular systems that influence cognitive resilience to Alzheimer's disease in genetically diverse mice.

Individual differences in cognitive decline during normal aging and Alzheimer's disease (AD) are common, but the molecular mechanisms underlying these distinct outcomes are not fully understood. We utilized a combination of genetic, molecular, and behavioral data from a mouse population designed to model human variation in cognitive outcomes to search for the molecular mechanisms behind this population-wide variation. Specifically, we used a systems genetics approach to relate gene expression to cognitive outcomes during AD and normal aging. Statistical causal-inference Bayesian modeling was used to model systematic genetic perturbations matched with cognitive data that identified astrocyte and microglia molecular networks as drivers of cognitive resilience to AD. Using genetic mapping, we identified Fgf2 as a potential regulator of the astrocyte network associated with individual differences in short-term memory. We also identified several immune genes as regulators of a microglia network associated with individual differences in long-term memory, which was partly mediated by amyloid burden. Finally, significant overlap between mouse and two different human coexpression networks provided strong evidence of translational relevance for the genetically diverse AD-BXD panel as a model of late-onset AD. Together, this work identified two candidate molecular pathways enriched for microglia and astrocyte genes that serve as causal AD cognitive biomarkers, and provided a greater understanding of processes that modulate individual and population-wide differences in cognitive outcomes during AD.

Early-life DNA methylation profiles are indicative of age-related transcriptome changes.

BACKGROUND: Alterations to cellular and molecular programs with brain aging result in cognitive impairment and susceptibility to neurodegenerative disease. Changes in DNA methylation patterns, an epigenetic modification required for various CNS functions are observed with brain aging and can be prevented by anti-aging interventions, but the relationship of altered methylation to gene expression is poorly understood. RESULTS: Paired analysis of the hippocampal methylome and transcriptome with aging of male and female mice demonstrates that age-related differences in methylation and gene expression are anti-correlated within gene bodies and enhancers. Altered promoter methylation with aging was found to be generally un-related to altered gene expression. A more striking relationship was found between methylation levels at young age and differential gene expression with aging. Highly methylated gene bodies and promoters in early life were associated with age-related increases in gene expression even in the absence of significant methylation changes with aging. As well, low levels of methylation in early life were correlated to decreased expression with aging. This relationship was also observed in genes altered in two mouse Alzheimer's models. CONCLUSION: DNA methylation patterns established in youth, in combination with other epigenetic marks, were able to accurately predict changes in transcript trajectories with aging. These findings are consistent with the developmental origins of disease hypothesis and indicate that epigenetic variability in early life may explain differences in aging trajectories and age-related disease.

Tamoxifen induction of Cre recombinase does not cause long-lasting or sexually divergent responses in the CNS epigenome or transcriptome: implications for the design of aging studies.

The systemic delivery of tamoxifen (Tam) to activate inducible CreERT2-loxP transgenic mouse systems is now widely used in neuroscience studies. This critical technological advancement allows temporal control of DNA-cre recombination, avoidance of embryonically lethal phenotypes, and minimization of residual cell labeling encountered in constitutively active drivers. Despite its advantages, the use of Tam has the potential to cause long-lasting, uncharacterized side effects on the transcriptome and epigenome in the CNS, given its mixed estrogen receptor (ER) agonist/antagonist actions. With the welcome focus on including both sexes in biomedical studies and efforts to understand sex differences, Tam administration could also cause sexually divergent responses that would confound studies. To examine these issues, epigenetic and transcriptomic profiles were compared in C57BL/6 J female and male hippocampus, cortex, and retina 1 month after a 5-day Tam treatment typical for cre induction, or vehicle control (sunflower seed oil). Cytosine methylation and hydroxymethylation levels, in both CG and non-CG contexts, were unchanged as determined by oxidative bisulfite sequencing. Long-lasting Tam transcriptomic effects were also not evident/minimal. Furthermore, there is no evidence of sexually divergent responses with Tam administration and Tam did not alter sex differences evident in controls. Combined with recently reported data that Tam alone does not cause long-lasting changes in behavior and neurogenesis, our findings provide confidence that Tam can be used as a cre-recombinase inducer without introducing significant confounds in transcriptomic and epigenomic neuroscience studies, particularly those focused on genomic and transcriptomic aspects of the aging brain.

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

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.

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-1ß (5.1-fold)], and 11 chemokines in old mice. DR attenuated the expression of IL-6, TNF-α, and IL-1ß 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.

Caloric restriction mitigates age-associated hippocampal differential CG and non-CG methylation.

Brain aging is marked by cognitive decline and susceptibility to neurodegeneration. Calorie restriction (CR) increases neurogenesis, improves memory function, and protects from age-associated neurological disorders. Epigenetic mechanisms, including DNA methylation, are vital to normal central nervous system 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 hippocampal DNA methylation changes. Hippocampal DNA from young (3 months) and old (24 months) male mice fed ad libitum and 24-month-old mice fed a 40% calorie-restricted 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 CG and ∼80,000 CH sites 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 DNA methyltransferase 1 and Tet methylcytosine dioxygenase 3 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 prolongevity effects of CR working through an epigenetic mechanism.

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.

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.