Association of Race and Poverty Status With DNA Methylation-Based Age.

Importance: The Dunedin Pace of Aging Calculated From the Epigenome (DunedinPACE) measure is a newly constructed DNA methylation (DNAm) biomarker associated with morbidity, mortality, and adverse childhood experiences in several cohorts with European ancestry. However, there are few studies of the DunedinPACE measure among socioeconomically and racially diverse cohorts with longitudinal assessments. Objective: To investigate the association of race and poverty status with DunedinPACE scores in a socioeconomically diverse middle-aged cohort of African American and White participants. Design, Setting, and Participants: This longitudinal cohort study used data from the Healthy Aging in Neighborhoods of Diversity Across the Life Span (HANDLS) study. HANDLS is a population-based study of socioeconomically diverse African American and White adults aged 30 to 64 years at baseline in Baltimore, Maryland, with follow-up approximately every 5 years. The current study was restricted to 470 participants with blood samples at 2 time points: August 14, 2004, to June 22, 2009 (visit 1), and June 23, 2009, to September 12, 2017 (visit 2). Genome-wide DNAm was assessed at visit 1 (chronological age, 30-64 years) and visit 2. Data were analyzed from March 18, 2022, to February 9, 2023. Main Outcomes and Measures: DunedinPACE scores were estimated for each participant at 2 visits. DunedinPACE scores are values scaled to a mean of 1, interpretable with reference to a rate of 1 year of biological aging per 1 year of chronological aging. Linear mixed-model regression analysis was used to examine the trajectories of DunedinPACE scores by chronological age, race, sex, and poverty status. Results: Among 470 participants, the mean (SD) chronological age at visit 1 was 48.7 (8.7) years. Participants were balanced by sex (238 [50.6%] were men and 232 [49.4%] were women), race (237 [50.4%] African American and 233 [49.6%] White), and poverty status (236 [50.2%] living below poverty level and 234 [49.8%] living above poverty level). The mean (SD) time between visits was 5.1 (1.5) years. Overall, the mean (SD) DunedinPACE score was 1.07 (0.14), representing a 7% faster pace of biological aging than chronological aging. Linear mixed-effects regression analysis revealed an association between the 2-way interaction between race and poverty status (White race and household income below poverty level: ß = 0.0665; 95% CI, 0.0298-0.1031; P < .001) and significantly higher DunedinPACE scores and an association between quadratic age (age squared: ß = -0.0113; 95% CI, -0.0212 to -0.0013; P = .03) and significantly higher DunedinPACE scores. Conclusions and Relevance: In this cohort study, household income below poverty level and African American race were associated with higher DunedinPACE scores. These findings suggest that the DunedinPACE biomarker varies with race and poverty status as adverse social determinants of health. Consequently, measures of accelerated aging should be based on representative samples.

Sex-specific transcriptome differences in a middle-aged frailty cohort.

BACKGROUND: Frailty is a clinical syndrome described as reduced physiological reserve and increased vulnerability. Typically examined in older adults, recent work shows frailty occurs in middle-aged individuals and is associated with increased mortality. Previous investigation of global transcriptome changes in a middle-aged cohort from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study demonstrated inflammatory genes and pathways were significantly altered by frailty status and race. Transcriptome differences in frailty by sex remain unclear. We sought to discover novel genes and pathways associated with sex and frailty in a diverse middle-aged cohort using RNA-Sequencing. METHODS: Differential gene expression and pathway analyses were performed in peripheral blood mononuclear cells for 1) frail females (FRAF, n = 4) vs non-frail females (NORF, n = 4), 2) frail males (FRAM, n = 4) vs non-frail males (NORM, n = 4), 3) FRAM vs FRAF, and 4) NORM vs NORF. We evaluated exclusive significant genes and pathways, as well as overlaps, between the comparison groups. RESULTS: Over 80% of the significant genes exclusive to FRAF vs NORF, FRAM vs NORM, and FRAM vs FRAF, respectively, were novel and associated with various biological functions. Pathways exclusive to FRAF vs NORF were associated with reduced inflammation, while FRAM vs NORM exclusive pathways were related to aberrant musculoskeletal physiology. Pathways exclusive to FRAM vs FRAF were associated with reduced cell cycle regulation and activated catabolism and Coronavirus pathogenesis. CONCLUSIONS: Our results indicate sex-specific transcriptional changes occur in middle-aged frailty, enhancing knowledge on frailty progression and potential therapeutic targets to prevent frailty.

APOE gene region methylation is associated with cognitive performance in middle-aged urban adults.

Apolipoprotein (APOE) ε4 allele is a strong risk factor for Alzheimer's disease (AD) and cognitive decline. Epigenetic modifications such as DNA methylation (DNAm) play a central role in cognition. This study sought to identify DNAm sites in the APOE genomic region associated with cognitive performance in a racially diverse middle-aged cohort (n = 411). Cognitive performance was measured by 11 standard neuropsychological tests. Two CpG sites were associated with the Card Rotation and Benton Visual Retention cognitive tests. The methylation level of the CpG site cg00397545 was associated with Card Rotation Test score (p = 0.000177) and a novel CpG site cg10178308 was associated with Benton Visual Retention Test score (p = 0.000084). Significant associations were observed among the dietary inflammatory index, which reflects the inflammatory potential of the diet, cognitive performance and the methylation level of several CpG sites. Our results indicate that DNAm in the APOE genomic area is correlated with cognitive performance and may presage cognitive decline.

The accelerated aging phenotype: The role of race and social determinants of health on aging.

The pursuit to discover the fundamental biology and mechanisms of aging within the context of the physical and social environment is critical to designing interventions to prevent and treat its complex phenotypes. Aging research is critically linked to understanding health disparities because these inequities shape minority aging, which may proceed on a different trajectory than the overall population. Health disparities are characteristically seen in commonly occurring age-associated diseases such as cardiovascular and cerebrovascular disease as well as diabetes mellitus and cancer. The early appearance and increased severity of age-associated disease among African American and low socioeconomic status (SES) individuals suggests that the factors contributing to the emergence of health disparities may also induce a phenotype of 'premature aging' or 'accelerated aging' or 'weathering'. In marginalized and low SES populations with high rates of early onset age-associated disease the interaction of biologic, psychosocial, socioeconomic and environmental factors may result in a phenotype of accelerated aging biologically similar to premature aging syndromes with increased susceptibility to oxidative stress, premature accumulation of oxidative DNA damage, defects in DNA repair and higher levels of biomarkers of oxidative stress and inflammation. Health disparities, therefore, may be the end product of this complex interaction in populations at high risk. This review will examine the factors that drive both health disparities and the accelerated aging phenotype that ultimately contributes to premature mortality.

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis.

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1.

Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF's receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

MeCP2 Deficiency Leads to Loss of Glial Kir4.1.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder usually caused by mutations in methyl-CpG-binding protein 2 (MeCP2). RTT is typified by apparently normal development until 6-18 mo of age, when motor and communicative skills regress and hand stereotypies, autonomic symptoms, and seizures present. Restoration of MeCP2 function selectively to astrocytes reversed several deficits in a murine model of RTT, but the mechanism of this rescue is unknown. Astrocytes carry out many essential functions required for normal brain functioning, including extracellular K+ buffering. Kir4.1, an inwardly rectifying K+ channel, is largely responsible for the channel-mediated K+ regulation by astrocytes. Loss-of-function mutations in Kir4.1 in human patients result in a severe neurodevelopmental disorder termed EAST or SESAME syndrome. Here, we evaluated astrocytic Kir4.1 expression in a murine model of Rett syndrome. We demonstrate by chromatin immunoprecipitation analysis that Kir4.1 is a direct molecular target of MeCP2. Astrocytes from Mecp2-deficient mice express significantly less Kir4.1 mRNA and protein, which translates into a >50% deficiency in Ba2+-sensitive Kir4.1-mediated currents, and impaired extracellular potassium dynamics. By examining astrocytes in isolation, we demonstrate that loss of Kir4.1 is cell autonomous. Assessment through postnatal development revealed that Kir4.1 expression in Mecp2-deficient animals never reaches adult, wild-type levels, consistent with a neurodevelopmental disorder. These are the first data implicating a direct MeCP2 molecular target in astrocytes and provide novel mechanistic insight explaining a potential mechanism by which astrocytic dysfunction may contribute to RTT.

RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome.

BACKGROUND: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation. METHODS: We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity® Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets. RESULTS: Our results indicate these two "omics" data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits. CONCLUSIONS: This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT.

NF-κB mediates Gadd45ß expression and DNA demethylation in the hippocampus during fear memory formation.

Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45ß gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45ß gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45ß expression. The learning-induced increases in Gadd45ß mRNA levels, Gadd45ß binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45ß expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45ß regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45ß expression and DNA demethylation in hippocampal neurons during fear memory.

The candidate splicing factor Sfswap regulates growth and patterning of inner ear sensory organs.

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.