Aging Cell

Frailty, defined by progressive loss of physiological resilience, neuromuscular function, and cognitive capacity, is a central manifestation of biological aging yet remains difficult to quantify in scalable experimental systems. Here, we introduce a Composite Frailty Index (CFI) in the house cricket (Acheta domesticus) that integrates automated measures of locomotion, exploratory behavior, and freezing into a unified, quantitative framework of functional decline. Ten behavioral parameters derived from automated open-field tracking, including locomotor performance, exploratory behavior, and freezing were integrated into the CFI. Locomotor states were classified using k-means clustering (k = 2) of velocity distributions, and all features were normalized to age- or treatment-matched reference populations, discretized into quintiles, and summed to generate a 0-40 frailty score. Aging cohorts (young adult: 4-6 weeks; geriatric: 10-12 weeks, N = 103) and pharmacological cohorts treated at mid-life (8-10 weeks) with rapamycin (14 ppm), acarbose (1000 ppm), or phenylbutyrate (1000 ppm) were evaluated (N = 122). Across chronological aging cohorts, CFI increased from young adults to geriatrics in both females (d = 1.14 [95% CI: 0.53, 1.76], P = 0.0003) and males (d = -1.17 [95% CI: -1.75 to -0.59], P < 0.0001). Using pharmacological intervention cohorts, mid-life rapamycin treatment reduced late-life frailty relative to controls in both females (d = -1.31 [95% CI: -2.09, -0.53], P = 0.0017) and males (d = -1.33 [95% CI: -2.09, -0.58], P = 0.0004), whereas acarbose and phenylbutyrate produced inconclusive effects (ds = -0.54 to -0.03; Ps > 0.05). Together, these findings establish the cricket CFI as a scalable, high-throughput platform for quantifying multidimensional functional aging and prioritizing candidate geroprotective interventions based on clinically relevant endpoints beyond lifespan.

Ageing is characterised by the accumulation of senescent cells. Owing to their irreversible cell-cycle arrest, these cells lack the capacity to replenish the stem cell pool and regenerate tissue, while their pro-inflammatory secretome propagates senescence in a paracrine manner. Much of the senescent phenotype has been attributed to dysregulated mTORC1 signalling, a key regulator of protein synthesis implicated in organismal ageing. Nonetheless, the mechanism underlying this dysregulation is poorly understood and limited to a few selected cell types. Here, we show that mTORC1 dysregulation is also a characteristic of senescent muscle precursor cells, and in contrast to reports in other cell types, senescent myoblasts do not rely on lysosomal nutrient liberation to sustain mTORC1 activity. Instead, they appear to depend on the PI3K/Akt pathway, which is upregulated in these cells. Exogenous antioxidants were identified to alleviate PI3K/Akt/mTORC1 signalling, while exogenous ROS has the capacity to activate mTORC1, supporting a model in which ROS acts upstream of this pathway in senescent myoblasts. Moreover, antioxidants were able to suppress the expression of pro-inflammatory cytokines and enhance the differentiation of senescent myoblasts. Interestingly, prolonged antioxidant treatment led to increased cell death in senescent but not proliferating myoblasts, suggesting they are more prone to reductive stress-induced cell death. We propose that, in vitro, the antioxidant capacity of many plant-derived compounds may underlie their reported benefits as therapeutics targeting senescent cells (senotherapeutics). Together, our findings provide novel insights into mTORC1-dependent regulation of the senescent phenotype and highlight the role of redox modulation in senotherapeutic strategies.

In humans, aging is associated with an increased risk of developing neurodegenerative diseases such as Parkinsons disease and Alzheimers disease. In neurons, the effect of aging on intrinsic molecular processes, and how they tie to age-related neurodegeneration remains unclear. Animal studies have shown that mitochondrial function decline, autophagy impairment and defective elimination of damaged mitochondria by mitophagy are all central features of neuronal aging. However, very few studies have investigated such events in human neurons, due to a lack of models showing aging features, therefore leaving a crucial need for a better understanding of the effect of aging on neuronal health. Here, we use direct neuronal reprogramming, which maintains signatures of cellular aging, to study the effect of aging on mitochondrial health and mitophagy in human neurons. We show age-related mitochondrial impairment, as well as accumulation of mitochondria targeted for degradation in autophagosomes and unacidified autolysosomes following mitophagy induction in neurites of induced neurons (iNs) derived from older donors. These impairments culminate into incomplete elimination of damaged mitochondria. By showing age-dependant mitophagy impairment in human neurons, this study paves the way for more in-depth mechanistic studies that would allow for the identification of therapeutic targets for anti-aging treatment and in the context of age-associated neurodegenerative diseases.

Aging is associated with a progressive loss of skeletal muscle function, known as sarcopenia; however, the molecular mechanisms coordinating cellular stress responses and structural adaptations permissive of sarcopenia remain incompletely understood. In our previous studies, we found aging differentially impacted mitochondrial networks by muscle, suggesting unique stress thresholds and response activation. Here, we investigate the role of activating transcription factor 4 (ATF4), a master regulator of the integrated stress response (ISR), in aged quadriceps muscle using complementary patient and aging mouse models. Older adults exhibited a marked decrease in aerobic capacity, muscle strength, and endurance when compared with young participants. These results paralleled findings in aged mice, with significant loss of muscle mass across multiple hindlimb muscles. Ultrastructural analysis revealed substantial age-related changes in mitochondrial morphology, including increased volume, surface area, and branching index, as well as a shift toward larger, more complex mitochondria. Our data indicate that ATF4 binds directly to the promoter region of the gene encoding TFAM, suggesting a transcriptional regulatory relationship to support DNA stability. These structural and transcriptional changes likely impair oxidative capacity and drive a feed-forward cycle of mitochondrial dysfunction and ISR activation. Our findings indicate that ATF4 coordinates transcriptomic and structural adaptations in aging muscle, identifying the ISR pathway as a potential therapeutic target for preserving muscle function in older adults.

Atherosclerosis (AS) is a chronic inflammatory disease closely linked to vascular senescence, yet the specific molecular mechanisms connecting aging processes to AS pathogenesis remain incompletely understood. This study integrated transcriptomic data from GEO datasets (GSE100927 and GSE43292) to identify vascular aging-related differentially expressed genes (VARDEGs). Following batch effect correction, 28 VARDEGs were screened and subjected to functional enrichment, protein-protein interaction (PPI) network analysis, and immune infiltration assessment. Seven hub genes (MMP9, APOE, TNF, ICAM1, PPARG, CYBA, and NCF2) were identified and experimentally validated via qRT-PCR, confirming their significant upregulation in AS samples. Receiver operating characteristic (ROC) analysis demonstrated high diagnostic accuracy for six of these genes (AUC > 0.7), with TNF exhibiting superior performance. Immune infiltration analysis revealed profound alterations in 28 immune cell types, particularly monocytes and T cells, which correlated strongly with hub gene expression. Furthermore, single-cell RNA sequencing analysis (GSE184073) localized the expression of core genes predominantly to monocytes and T cells, highlighting TNF overexpression in T cells as a potential critical driver. Finally, molecular docking simulations suggested that curcumin exhibits strong binding affinity to these hub genes, particularly PPARG, providing a mechanistic basis for its therapeutic potential. Collectively, this study elucidates the landscape of vascular aging-related genes in AS, identifies novel diagnostic biomarkers, and proposes potential therapeutic targets involving immune modulation and natural compounds.

Age-related hearing loss (ARHL), or presbycusis, is one of the most prevalent sensory deficits in older adults and has been increasingly implicated in cognitive decline and dementia. This study characterizes ARHL in a rhesus macaque model by combining histological, physiological, and cognitive assessments. Aged macaques exhibited progressive cochlear degeneration, with marked outer hair cell loss at mid-to-high frequencies, elevated auditory thresholds, reduced distortion product otoacoustic emissions, and impaired auditory brainstem responses including amplitude reduction, latency prolongation, and diminished temporal precision. Despite modest reductions in inner hair cell ribbon synapse counts, hypertrophic changes were observed. These auditory deficits correlated with subtle impairments in visual working memory, as measured by a delayed match-to-sample task, underscoring a potential sensory-cognitive link. By capturing cross-domain aging markers in a translationally relevant primate model, this work lays a foundation for mechanistic studies and therapeutic interventions targeting both hearing and cognition in aging populations.

Understanding age-related cellular dysfunction in the brain is essential for developing strategies to promote healthy ageing. Towards this aim, we took advantage of a previously established mild dissociation method to profile cells in the cerebral cortex grey matter of adult and aged mice. This revealed glial cells with largely up-regulated and other glia and neurons with largely down-regulated gene expression upon ageing. Astrocytes were involved in increased interactions with microglia and decreased interaction with neurons, high-lighting potent age-induced changes in their regulatory roles. Single cell RNA-seq and single nuclei multiome analysis of astrocytes uncovered down-regulation of Wnt-signalling with increased expression of its inhibitors and reduced RNA and protein levels of its effectors JunB/D, acting downstream of Wnt signalling in ageing. This was confirmed by RNA-scope and immunostainings, as well as in human data. Notably, injection of JunD-expressing viral vectors in astrocytes increased their proliferation and HMGB1 levels in the aged brain, indicative of a more youthful astrocyte state. Main pointsO_LITranscriptomic analysis uncovers cell type-specific impact of ageing in the cortical grey matter, including altered intercellular communication networks. C_LIO_LIMultiomic profiling identifies dysregulated Wnt signalling in ageing cortical astrocytes. C_LIO_LIAgeing astrocytes exhibit upregulation of the Wnt signalling regulators Maml2 and Daam2, accompanied by downregulation of the AP-1 transcriptional complex component JunD. C_LIO_LIOverexpression of JunD increases proliferation after mild injury in aged astrocytes. C_LI

Age-related cognitive decline and depressive symptoms are prevalent in later life, yet the genetic determinants of vulnerability remain unclear. Here, we investigated how genetic and epigenetic regulation of the serotonin transporter gene SLC6A4 contributes to susceptibility to these age-related conditions in later life. In community-dwelling older adults in Japan (N = 1,317), functional stratification of the serotonin transporter-linked polymorphic region (5-HTTLPR) revealed that participants with low-activity genotypes showed a robust co-occurrence of cognitive decline and depressive symptoms, whereas this comorbid pattern was not observed in those with the high-activity genotype. The genotype-dependent co-occurrence was consistently replicated across seven independent population-based cohorts (total N = 7,889). DNA methylation at a functional promoter CpG site increased with age and partially mediated age-related cognitive decline specifically among low-activity genotypes. In contrast, the high-activity genotype was associated with relative resistance to these functional declines, partly mediated by a protective effect on hippocampal volume during aging. Notably, genotype-dependent effects on hippocampal volume were absent in adolescence, indicating that the influence of SLC6A4 emerges in an aging-specific manner. Together, these findings identify SLC6A4 promoter activity as a key genetic factor modulating vulnerability and resilience in later life.

Age-related hearing loss (ARHL) is a rapidly growing public health concern, affecting two-thirds of adults over 65 years old, with no effective therapeutics available. As the aging population grows at an unprecedented rate, the burden of ARHL will only increase. The causes of ARHL are multifactorial, but an understudied major contributor is glial dysfunction. The auditory nerve (AN) conducts sound from the cochlea to the brainstem and holds a diverse population of immune cells and myelinating glia. As the AN fibers bundle together within the cochlea to project to the brainstem, they are first myelinated by Schwann cells in the peripheral AN, then myelinated by oligodendrocytes in the central AN. The region where myelination shifts from Schwann cells to oligodendrocytes is the glial transition zone (GTZ), located in the cochlear modiolus, creating a unique biological niche. While central-peripheral interfaces are recognized in other cranial nerves, the AN GTZ is understudied. This region integrates the peripheral and central microenvironments within the confined bony cochlea, positioning it as a niche for glial dysfunction in pathological conditions, such as aging. We hypothesize that the GTZ is a site of enhanced glial dysfunction contributing to age-related AN demyelination, an important contributor to ARHL. We evaluated this in an ARHL mouse model combining RNA-sequencing, quantitative immunohistochemistry, and 3D high-resolution imaging. We examined the AN GTZ from human temporal bone donors. RNA-sequencing of the AN revealed age-associated increases in abnormal myelination/glial function and inflammation. There was a significant age-dependent increase in Iba1+ macrophages/microglia, with accumulation at the AN GTZ, and an increase in cellular volume and surface area, suggesting greater age-related activation. Macrophages/microglia contained significantly more internalized myelin debris in the AN (peripheral, central, and GTZ) with aging. More importantly, we found structurally intact myelin within macrophages/microglia only at the GTZ, suggesting a unique microenvironment at the GTZ altering phagocytic activity in aging. Together, our data suggest that the GTZ, a previously unrecognized central-peripheral interface, is a critical site of immune-glial interactions and especially vulnerable to age-related demyelination and neuroinflammation. This study highlights the GTZ as a potential target for preserving AN myelination and mitigating ARHL.

Immunosenescence, the age-associated decline in immune function, is a key feature of human aging. In human lymphoid organs, however, the specific immune cell populations that acquire senescence-associated phenotypes during aging and how they influence the surrounding tissue microenvironment remain poorly understood. A spatially resolved map of these senescence-associated immune states in human lymphoid tissues could help clarify their relationship with aging and their potential contributions to the progressive decline of immune function. Here, we integrated single-cell and spatial multi-omics to systematically characterize age-related senescence in human lymph nodes (LNs). Single-cell transcriptomics of lymphoid tissues from donors aged 18 to 100 years old identified 34 immune and stromal cell types and revealed age-associated upregulation of senescence signatures in specific populations. Spatial proteomic profiling of 99 LN sections from 51 donors (18-86 years) using high-plex immunofluorescence ([~]20 million cells) mapped senescence markers (p16, p21, HMGB1, -H2AX) at single-cell resolution, revealing diverse senescent-like cell types ("senotypes") and a stepwise shift from extrafollicular to germinal center (GC) localization with age. Notably, we observed focal clonal-like senescence in GC B cells in older donor LNs. Spatial transcriptomics, epigenomics, and metabolic imaging of selected samples further elucidate the multi-omics signatures and underlying mechanisms of functional impairment, metabolic remodeling, and distinct regulatory programs in senescent-like GC B cells. This study presents a comprehensive spatial atlas of senescence-associated immune states in human lymph nodes, revealing cell-type-specific and spatial heterogeneity that may contribute to immunosenescence and the decline of immune function during aging.

CD31+ T-cells reportedly possess angiogenic properties. These cells have recently been termed angiogenic T-cells (TANG). Advancing age is associated with altered circulating T-cell phenotypes, including TANG, and reduced angiogenesis. We examined various TANG subsets (CD3+, CD4+, CD8+), and their VEGF-A intracellular content in young (n=16, 18-30 years) and older (n=16, 50-65 years) male adults using flow cytometry. Cardiorespiratory fitness ([V]O2max) was quantified in all participants using a graded cycling ergometry test to volitional exhaustion. Resting blood samples were collected to measure circulating IL-6 and cytomegalovirus serostatus. CD31+ T-cells (TANG) contained more VEGF-A than CD31- T-cells (CD31+: 9374 {+/-} 8587 AU vs CD31-: 8722 {+/-} 8149 AU, p = 0.021) which was also exhibited in CD4+ and CD8+ subsets. Older adults possessed fewer CD4+ TANG cells as a proportion of total CD4+ T-cells than younger adults (young: 35 {+/-} 11%; older: 24 {+/-} 9%, p = 0.004), and CD3+ and CD4+ TANG subsets from older adults exhibited higher VEGF-A levels than younger adults (CD3+CD31+: young: 6081 {+/-} 4001 AU; older: 13426 {+/-} 10945 AU, p = 0.019; CD4+CD31+: young: 6373 {+/-} 3972 AU; older: 15660 {+/-} 12829 AU, p = 0.011). TANG cells were not associated with circulating IL-6, and TANG VEGF-A content was not associated with[V] O2max. Advancing age is associated with a pathological TANG phenotype, which may contribute to age-related inflammation and warrants further investigation as a potential therapeutic target.

{Delta}133p53 is a naturally occurring isoform of the human p53 protein that inhibits p53-mediated cellular senescence. We recently reported that transgenic expression of this senescence-inhibitory p53 isoform counteracts aging-associated pathological changes and extends lifespan in progeria model mice (heterozygous LmnaG609G/+). The anti-aging effect of {Delta}133p53 was attributed in part to reduced levels of the proinflammatory cytokine IL-6. To comprehensively profile {Delta}133p53-induced changes in cytokines and chemokines, we in this study performed a Luminex-based multiplex quantitative assay of mouse sera collected from transgenic {Delta}133p53-expressing LmnaG609G/+ mice and non-expressing controls. This assay not only confirmed the {Delta}133p53-mediated repression of IL-6 but also showed that {Delta}133p53 reduced the levels of CXCL1 (also known as KC), IL-1, and CXCL10 (also known as IP-10). Among these factors, we further characterized CXCL10, which has not previously been associated with progeria in mice or humans. Consistent with reduced serum CXCL10 levels, both young (15-week-old) and old (10-month-old) {Delta}133p53-expressing LmnaG609G/+ mice showed reduced Cxcl10 expression, compared with age-matched non-expressing controls, in the liver, spleen, and brain, major organs known to produce CXCL10. In naturally aged wild-type mice (2-year-old), Cxcl10 expression was also significantly repressed by transgenic {Delta}133p53 in the spleen and brain. Analysis of gene expression datasets from human tissues demonstrated an inverse association between CXCL10 and {Delta}133p53 levels, suggesting physiological relevance to human aging. This study defines CXCL10 as a proinflammatory chemokine elevated in both accelerated and natural aging and as a potential target of the anti-inflammatory activity of {Delta}133p53.

Frailty arising from loss of muscle function and mass is a significant health concern impacting quality of life and dramatically increasing health care costs as our population ages. Ameliorating frailty derived from reduced muscle function is thus a critical research priority to improve health span. Cell intrinsic defects in muscle stem cells (MuSC), or satellite cells, occur as skeletal muscle ages, reducing the capacity of MuSCs to maintain and repair skeletal muscle and are accompanied by cell nonautonomous changes. Although rejuvenating stem cells in aged tissues or organs has potential to improve muscle aging phenotypes, we found that the extracellular environment in aged mice abrogates rejuvenated muscle stem cell potential. MuSCs from young mice were unable to grow on extracellular matrix derived from aged mice that contains elevated collagen protein levels, establishing a critical role for the environment in contributing to muscle phenotypes in aging. Combining an inducible FGF receptor 1 (FGFR1) to rescue MuSC intrinsic aging defects with a drug to reduce fibrosis partially rescued muscle mass loss in aged mice. We conclude that aging affects tissues, and particularly skeletal muscle tissue, via complex multifactorial processes requiring multifaceted interventions to improve aging phenotypes.

Chronic oxidative stress is a major contributor to neuronal aging. Due to the lack of homologous recombination (HR) DNA damage repair, high oxygen consumption in neurons causes DNA damage accumulation with age, resulting in a decline in neuronal function, senescence-like phenotypes and onset of neurodegenerative diseases. Here, we identify increased PTBP1 as a stress-inducible negative regulator of neuronal gene expression and senescence-protectant genes. Oxidative stress robustly increases PTBP1 expression in ShSY-5Y differentiated neurons and primary mouse cortical neurons, coinciding with the loss of neuronal genes, including neuronal PTBP2, and activation of stress-responsive genes. Knockdown of PTBP1 in fibroblasts reduces the expression of key senescence genes. Transcriptomic analyses revealed that PTBP1 overexpression results in coordinated shift in gene expression characterized by repression of neuronal commitment genes and activation of stress and senescence genes. Mechanistically, PTBP1 induction is regulated by stress induced CTCF binding at the PTBP1 promoter. Together, our findings suggest that alteration in levels of PTBP1 acts as a molecular switch between neuronal function and survival, providing insight into transcriptional adaptations associated with aging. SUMMARYO_LILoss of PTBP1 in fibroblasts acts as a senescence protective gene C_LIO_LIxidative stress induces expression of PTBP1, reducing neuronal function gene expression and activating stress and cell cycle genes C_LIO_LIEctopic PTBP1 expression reprograms neuronal transcription, down-regulating cell fate commitment genes and activating a cell senescence program C_LIO_LIxidative stress induces PTBP1 and suppresses neuronal specific PTBP2 expression in primary cortical neurons C_LI

AbstractAs organisms age, mitochondrial metabolic activity declines, and disrupted gene expression regulation mediated by histone acetylation induces the emergence of senescent physiological phenotypes in tissues. In this study, we found that periodic exposure to red light significantly increased histone H3 Lys9 acetylation (H3K9ac) levels in the tissues and organs of aged mice. Following red light exposure, silent information regulation factor 4 (SIRT4) protein levels in keratinocytes were notably reduced, whereas glycolysis, fatty acid metabolism, and the tricarboxylic acid (TCA) cycle were significantly activated in keratinocytes. The reduction in mitochondrial SIRT4 levels enhances the acetylation of mitochondrial metabolic proteins, particularly malonyl-CoA decarboxylase (MCD), a potent inhibitor of the key rate-limiting enzyme carnitine palmitoyltransferase 1A (CPT1A) in fatty acid oxidation. This process promotes mitochondrial fatty acid oxidation and TCA cycle. Additionally, the decrease in SIRT4 activates SIRT1 through feedback mechanisms, thereby alleviating its inhibition on PPAR- in senescent keratinocytes and comprehensively activating the expression of genes related to lipid metabolism. This lipid metabolism activation ultimately facilitates the accumulation of acetyl-CoA within keratinocytes, increases H3K9ac levels, and reshapes the expression patterns of senescence-related genes. Eventually, cellular aging is effectively mitigated by the synergistic regulation of metabolism, inflammation, and gene expression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/717004v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@a3387dorg.highwire.dtl.DTLVardef@1d1b083org.highwire.dtl.DTLVardef@19ba6f0org.highwire.dtl.DTLVardef@1ecf20e_HPS_FORMAT_FIGEXP M_FIG Mechanism of anti-aging action of red light: Red light can reduce SIRT4 signalling in keratinocytes, thereby reactivating lipid metabolism and increasing levels of acetyl-CoA. This promotes histone acetylation, which in turn reverses the expression of age-related inflammatory factors and genes. C_FIG

The human ovary is among the first organs to show age-related functional decline, resulting in menopause. Beyond this transition, the postmenopausal ovary is often regarded as quiescent and remains poorly characterized. We analyzed the proteomes of healthy, non-pathological ovaries using mass spectrometry (data-independent acquisitions) from 28 postmenopausal women (50-75 years old), stratified into three age groups (50-59, 60-69, [&ge;]70). We quantified 5,812 protein groups and observed progressive age-associated shifts with 117 proteins significantly altered in the [&ge;]70 vs 50-59 age comparison. Multivariate analysis demonstrated clear separation between 50-59 and [&ge;]70-year-old age cohorts, with protein signatures shifting from RNA/gene-regulatory functions in younger ovaries to metabolic, trafficking, and innate immune/complement pathways in older ovaries. Across differential abundance, multivariate modelling, and covariate-adjusted linear modelling converged on a shared set of age-associated candidates, strengthening support for the gain of extracellular matrix remodeling, inflammatory signaling, and loss of structural/keratin components with age. Pathway enrichment further identified an increase in inflammatory, matrisome pathways, and increased abundance of damage-associated secretory factors decades following menopause. Secreted matrisome proteins WNT4 and Fibromodulin (FMOD) emerged as age-associated candidates and were validated by immunohistochemistry. These data fundamentally shift the notion of the postmenopausal ovary as an inert organ and instead demonstrate active and continuous molecular remodeling that has potential relevance to tissue signaling and implications for womens health.

BackgroundAgeing is a sex-specific process characterised by a progressive decline in physiological integrity. DNA methylation represents a primary epigenetic hallmark of ageing, yet sex-specific patterns of epigenetic ageing within and across tissues remain poorly understood. This study aims to address these gaps through an integrated analysis of sex-moderated epigenetic ageing across eight human tissues. MethodsA total of 137 DNA methylation datasets comprising over 36,000 individuals aged 10-114 years were analysed using a meta-analytic workflow to identify age-associated differentially methylated positions (aDMPs) and regions (aDMRs), meta-regression to assess sex moderation, and pathway enrichment analyses to interpret functional relevance. FindingsIndividual tissues displayed distinct age-related methylation trajectories, but some DMP sites showed consistent hyper- or hypomethylation across tissues. Across tissues, we identified 68,630 aDMPs (10%) robustly associated with ageing. Age-associated changes at the regional level were less common, with only 80 robust age-associated aDMRs detected across tissues, representing 0.09% of analysed regions. Sex moderation was observed for only 16 aDMPs (0.002%), indicating that sex effects on age-associated DNA methylation are largely tissue-specific rather than shared across tissues. InterpretationOur findings indicate that age-associated DNA methylation changes predominantly occur at isolated CpG sites rather than extended genomic regions and are strongly dependent on tissue and genomic context. The minimal overlap of sex-moderated methylation signals across tissues suggests that age-related sex differences at the epigenetic level are more likely attributable to tissue- and cell-type-specific variation rather than to broadly conserved epigenetic mechanisms shared across tissues. FundingThis study was funded by an Australian Research Council (ARC) Discovery project (DP200101830). Severine Lamon was funded by an ARC Future Fellowship (FT210100278). Nir Eynon was funded by NHMRC Investigator Grant (APP1194159), and a Hevolution/AFAR New Investigator Award in Aging Biology and Geroscience Research. Mandhri D. Abeysooryia was supported by an Australian Government Research Training Program (RTP) Scholarship. Research in context Evidence before this studyDNA methylation is widely recognised as a central epigenetic hallmark of ageing. Previous research has demonstrated that some age-related methylation changes are conserved across tissues, forming the basis of pan-tissue epigenetic clocks. Most studies to date have primarily examined age effects in isolation. Although biological sex influences ageing trajectories and susceptibility to nearly all age-related diseases, sex-moderated epigenetic ageing has received limited investigation. Specifically, pan-tissue clocks, including GrimAge and PhenoAge, are "sex-aware" but were trained and validated in mixed-sex cohorts, limiting their capacity to disentangle tissue-specific sex effects. Consequently, it remains unclear whether sex-moderated epigenetic ageing signals are shared across tissues or are tissue-specific. Added value of this studyThis study provides a large-scale, comprehensive multi-tissue analysis of sex-moderated epigenetic ageing, integrating 137 DNA methylation datasets across eight human tissues and more than 36,000 male and female individuals spanning the lifespan. Our findings show that while age-associated methylation changes are widespread at the CpG level, sex-moderated effects are rare and largely tissue-specific, with minimal overlap across tissues. Implications of all the available evidenceTogether, the available evidence indicates that epigenetic ageing is predominantly driven by shared, conserved age-related methylation changes, whereas sex differences in epigenetic ageing are modest and context dependent. These sex-related effects are more likely to reflect tissue- and cell-type-specific variation rather than widespread, shared mechanisms. This underscores the need to develop sex-specific epigenetic clocks and to conduct longitudinal cohort and intervention studies to more precisely characterise sex-specific dynamics of epigenetic ageing across tissues.

Aging at the subcellular level involves the simultaneous decline in the cells ability to maintain protein homeostasis and rise in misfolded proteins through a positive feedback loop. Here, we test if a widespread class of protein misfolding could contribute to proteome aging by examining if statistical associations exist between age-related changes in protein structure, measured by limited proteolysis mass spectrometry data of the aging Saccharomyces cerevisiae proteome, with structural annotations and molecular simulations. We find that globular proteins that are likely to exhibit entanglement misfolding are 121% more likely to exhibit age-related structural changes, and these changes are 59% more likely to be localized to natively entangled regions. Proteins containing native entanglements are seven-fold more likely to misfold, according to simulations, and populate long-lived, near-native misfolded states. Thus, the age-related structural changes in yeast proteins can be explained in part by the accumulation of misfolded proteins involving entanglements.

Declining spatial navigation abilities are a critical hallmark of aging, where the loss of spatial abilities precedes global cognitive impairment. While navigational decline is traditionally attributed to deficits in higher-order cognitive functions, emerging cognitive-motor frameworks suggest that age-related sensorimotor alterations play a significant, yet previously overlooked, role. Here, we investigate the coupling between locomotor integrity and navigation by combining an immersive virtual-reality path-integration paradigm with systematic manipulations of landmark availability and reliability, while recording gait kinematics alongside neural dynamics using high-density mobile-EEG from 30 young and 32 older adults. We demonstrate that older adults accumulate angular homing error more rapidly than younger adults, a deficit linked to altered gait dynamics. These age-dependent differences are reflected in increased mid-frontal theta activity, highlighting a robust coupling between gait-related sensorimotor alterations and decline in navigation. Older adults also exhibited increased reliance on visual landmarks, and particularly those with degraded gait, yet this compensatory reweighting of navigational cues remained less efficient and less precise than in younger adults. These findings highlight sensorimotor gait alterations as a central determinant of age-related navigation deficits, challenging the traditional separation of motor and cognitive domains and identifying locomotor integrity as a critical target for preserving spatial navigation abilities.

Age-related cognitive decline (ARCD) is driven by conserved biological mechanisms of aging, yet no gerotherapeutic directly targets these processes in the brain. Glycyl-L-histidyl-L-lysine complexed with copper (GHK-Cu) is an endogenous peptide with regenerative and anti-inflammatory properties that declines with age. Whether its effects on cognitive aging depend on delivery route or exposure duration remains unclear. Aged C57BL/6J mice (20-21 months) received GHK-Cu (15 mg/kg) via short-term intraperitoneal (IP; 5 days) or longer-term intranasal (IN; 8 weeks) administration. Hippocampal-dependent escape learning was assessed using a spatial navigation task. Molecular effects were evaluated using hippocampal immunohistochemistry and bulk RNA sequencing. Differential gene expression was analyzed using DESeq2 with false discovery rate (FDR) correction, and pathway-level changes were assessed via gene set enrichment analysis (GSEA). IN GHK-Cu improved escape latency across Trials 2-4 in both sexes (P < 0.05), whereas IP dosing produced a transient improvement in males during Trial 2 (P < 0.05) without sustained effects or improvement in females. IN treatment increased synaptophysin in females (P < 0.001) and decreased GFAP in both sexes (P < 0.01), while IP treatment reduced TGF-{beta}, GFAP, and MCP-1 in males (P < 0.05) and decreased p21 in females (P < 0.0001). Transcriptomic analysis revealed distinct molecular programs. IN GHK-Cu induced coordinated suppression of oxidative phosphorylation (male NES -5.44, female NES -4.20; FDR < 0.0001) and MYC target pathways (female NES -4.31, FDR < 0.0001), with additional attenuation of PI3K-AKT-mTOR signaling in females (NES -3.15, FDR = 0.062). In contrast, IP treatment activated oxidative phosphorylation (female NES 4.97, FDR < 0.001), DNA repair (NES 5.58, FDR < 0.001), and MYC targets (NES 4.34, FDR = 0.002), indicating engagement of acute stress-response and repair pathways. GHK-Cu improves hippocampal-dependent learning in aged mice through distinct biological modes: IP exposure activates repair and stress-response pathways, whereas IN delivery induces sustained suppression of growth and mitochondrial metabolic signaling associated with aging biology. These findings demonstrate that functional cognitive improvement can arise from divergent molecular states and identify administrative route and exposure duration as key determinants of gerotherapeutic response.