Aging Cell

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating cellular senescence and aging. Our recent studies identified a conserved C. elegans miRNA cluster (miR-229/64/65/66) that is required for normal adult lifespan, with overexpression significantly extending longevity. Notably, cel-miR-229 is evolutionarily conserved in humans, with hsa-miR-425 sharing an identical seed sequence. Here, we investigated the role of miR-425 in mammalian cellular senescence. We found that miR-425 expression is markedly reduced in pharmacologically induced senescence in human lung cancer cells. Restoration of miR-425 expression attenuates senescence and suppresses the expression of senescence-associated secretory phenotype (SASP) cytokines following senescence induction. We further observed that miR-425 levels decline during replicative senescence, whereas stable overexpression in WI-38 fibroblasts delays senescence accumulation and preserves proliferative capacity. Mechanistically, miR-425 suppresses TGF-{beta} signaling, leading to reduced expression of the cyclin-dependent kinase inhibitor p21/CDKN1A and increased phosphorylation of the retinoblastoma (RB) protein, thereby promoting cell-cycle progression. We further identify PPP2CB, the catalytic subunit of protein phosphatase 2A (PP2A), as a direct target of miR-425. PPP2CB expression is downregulated in miR-425-5p overexpressing cells, even under senescence induction. Knockdown of PPP2CB using siRNA phenocopies the effects of miR-425 overexpression, reducing senescence, enhancing proliferative potential, and increasing RB phosphorylation. Collectively, our findings identify miR-425 as a conserved regulator of cellular senescence that acts through upregulation of RB phosphorylation. These results establish a novel miR-425-PPP2CB-RB regulatory axis controlling proliferation and senescence and suggest miR-425 as a potential therapeutic target for mitigating senescence to promote extended health span.

Tendons are commonly injured, not only in athletes, but also during normal everyday activities, particularly in older age. Tendons are rich in extracellular matrix proteins, many of which are turned over extremely slowly during an individuals lifetime. As a result, tight regulation of the extracellular matrix is essential for tendons to remain resilient to the mechanical load placed on them. However, tendons are prone to age-associated functional decline, characterised by chronic inflammation, accumulation of damaged collagen, and matrix remodelling. These degenerative changes often precede injury, wherein additional inflammation and fibrotic tissue deposition make treatment difficult and reinjury highly likely. Understanding and preventing the causes of tendon functional decline is therefore vital to improving quality of life in the ageing population. Using an equine model, here we show that tendon fibroblasts, or tenocytes, isolated from aged tendons exhibit markers of senescence when cultured in vitro. Further, we describe how senescence in tenocytes drives inflammation, hypercontractility, and dysregulation of tendon matrix components. By replicatively senescing tenocytes isolated from young tendons, we observed that senescent tenocytes instigated proinflammatory signalling, had impaired capacity in wound healing assays, increased contractility, and secreted factors that induced senescence in healthy tenocytes. Further, tenocyte senescence dysregulated matrix turnover both at the gene and protein level. Finally, we demonstrate how senotherapeutic treatment of senescent tenocytes can reduce expression of senescence markers and restore proliferative capacity. Our results uncover systemic links between tendon ageing and cellular senescence, and identify mechanisms and therapeutic strategies for age-associated tendon degeneration.

Aging is accompanied by widespread metabolic change, but it remains unclear which features are shared across species with different physiology, lifespan, and sampling contexts. To address this, we employed a pathway-centered comparative metabolomics framework to evaluate age-associated metabolic remodeling across wild African savanna elephant, mouse, and Drosophila melanogaster. Drosophila provided a controlled adult time course to map age-associated metabolite trajectories, while mouse and elephant plasma datasets allowed us to test whether these pathway signatures extended to mammalian aging. Adult Drosophila showed extensive metabolomic remodeling, with significant metabolites organizing into distinct temporal trajectory classes. Although individual metabolite overlap across species was limited, robust correspondence at the pathway-level overlap was observed. Pathway scores derived from Drosophila increased progressively with fly age, successfully distinguished young and old mice, and captured age-associated stratification across the elephant lifespan. Notably, lipid metabolism, particularly carnitine and fatty acid metabolism, together with nucleotide-related pathways, consistently emerged as the core features of aging across analyses. These findings suggest pathway-level metabolic remodeling is a recurrent feature of cross-species aging.

BackgroundEstrogen-related receptor gamma (ERR{gamma}) drives an exercise mimicking aerobic gene program in the skeletal muscle that could be beneficial in aging. We have investigated the effect of chronic ERR{gamma} activation on minimizing sarcopenia. MethodsExperiments were performed in muscle specific ERR{gamma} transgenic (TG) mice and wild type (WT) littermates, at young (4-5 months) and old (24-26 months) age. In the skeletal muscle, global gene expression changes, as well as myofiber histological changes in fiber type, size, vascular supply and neuromuscular junction (NMJ), and mitochondrial content were measured. Functional analysis was performed using in vivo muscle contraction assay. Exercise fitness was measured using treadmill sprint and endurance test. Gene and protein expression was measured using QPCR and Westerns, respectively. ResultsERR{gamma} activates a pan-ERR aerobic program in the skeletal muscle to increase expression of 574 genes including ERR, mitochondrial homeostasis (e.g. Mfn1, Opa1, Drp1, Fis1, and Tfam), vascularization (e.g. Vegfa, Angpt1, Fgf1), and neuromuscular junction (NMJ) (e.g. Nrp1, Aspa, Ptprm, Cxcr4), simultaneously suppressing the expression of atrophy related genes (e.g. Atrogin1, Traf6, Nedd4, Myd88, p21). ERR{gamma} increases mitochondrial content [Mitochondrial area: old TG vs. WT, 2.00 fold; young TG vs. WT, 1.32 fold], oxidative capacity [NADH-TR activity: old TG vs. WT, 1.20 fold; young TG vs. WT, 1.22 fold] and myofiber type [2a: old TG (687{+/-}258) vs. WT (252{+/-}71); young TG (797{+/-}168) vs. WT (440{+/-}76); 2x: old TG 1348{+/-}87 vs. WT 976{+/-}219; young TG 1131{+/-}135 vs. WT 936{+/-}84; 2b: old TG (798{+/-}103) vs. WT (1628{+/-}148); young TG (967{+/-}133) vs. WT (1623{+/-}189)], and capillarity [capillary-to-myofiber ratio: old TG (3.25{+/-}0.19) vs. WT (2.41{+/-}0.16); young TG (3.41{+/-}0.21) vs WT (2.59{+/-}0.2)] and [NMJ number [old TG (67{+/-}8) vs. WT (40{+/-}9); young TG (77{+/-}11) vs WT (77{+/-}7)], mitigating age-related loss of NMJ and myofiber cross-sectional area [old TG (1570{+/-}147{micro}m2) vs. WT (1692.5{+/-}208{micro}m2) WT; young TG (1828.15{+/-}132.8{micro}m2) vs. WT (2109.7{+/-}296.8{micro}m2)]. ERR{gamma} overexpression preserves muscle contractility with aging [Fatigue resistance: 22.72% reduction in force in old vs. young WT; 3.11% reduction in force between old vs. young TG]. Furthermore, ERR{gamma} maintains exercise fitness in old mice [Running: old TG (2964.52{+/-}405m) vs. old WT (910.75{+/-}6034m); young TG (2232.43{+/-}193.64m) vs. young WT (1366.76{+/-}60.76m)]. ConclusionsERR{gamma} drives a pan-ERR and counter sarcopenic gene program enhancing oxidative myofiber type, mitochondrial content, vasculature, and NMJ in aging muscle. Consequently, ERR{gamma} minimizes myofiber atrophy, preserves contractility, and improves exercise fitness in old mice. Therefore, ERRs are potential translational targets for combating sarcopenia.

Ribosomal DNA (rDNA) is a highly repetitive and complex locus within the mammalian genome that exhibits substantial inter-individual variation in number of rDNA copies and epigenetic regulation. Nonetheless, our understanding of rDNA biology in skeletal muscle during periods of physiological stress is limited. Using publicly available whole genome and reduced representative bisulfite sequencing data sets, we identify a concurrent reduction in both the number of rDNA copies and the methylation profile of the rDNA in aged vs young mice, supported by large effect sizes and permutation testing, with significant reductions in methylation of the 18S coding unit in aged, compared to young controls (p = 0.024). We found a strong positive correlation between rDNA copy number and 18S methylation across both young and aged mice (p = 0.004; Spearman rho = 0.842). After analysing publicly available muscle (skeletal and cardiac) data sets following acute insult (endurance exercise, cancer cachexia, spinal cord injury), we do not observe a similarly coordinated epi-genetic modification in rDNA biology but uncover tissue and sex-specific differences in rDNA copy number or methylation status, in isolation. These findings suggest ageing as a unique physiological insult in which coordinated epi-genomic remodelling of the rDNA region appears, representing a previously underappreciated feature of the muscle ageing trajectory.

Aging is associated with a gradual and progressive decline in skeletal muscle mass and strength known as sarcopenia, which has been attributed to chronic low-grade inflammation. Dietary long-chain polyunsaturated fatty acids (LC-PUFAs), including omega-6 arachidonic acid (ARA) and omega-3 docosahexaenoic acid (DHA), are precursors to bioactive lipid mediators that regulate the initiation, propagation, and active resolution of inflammation. While traditionally considered a pro-inflammatory and catabolic factor, the ARA-derived eicosanoid prostaglandin E2 has recently emerged as a potential anti-sarcopenic molecule. DHA-derived specialized pro-resolving mediators may also act as immunomodulatory pro-regenerative molecules in muscle inflammaging. In the current study, we tested the effects of long-term dietary supplementation with either ARA or DHA on muscle health in aging mice. Twenty-two-month-old C57BL/6N mice were fed a control AIN-93M diet, or an AIN-93M diet supplemented with either ARA (0.48% w/w) or DHA (0.48% w/w) for 12 weeks. Both dietary interventions reduced total body weight, but only ARA reduced absolute fat mass and increased the percentage of lean mass. Despite these changes in body composition, ARA supplementation reduced absolute muscle strength and myofiber size. This functional decline was associated with increased neuromuscular junction fragmentation, elevated expression of pro-inflammatory cytokines/protein degradation markers, and suppressed ribosome biogenesis. In contrast, DHA uniquely reduced chronic inflammation of aged muscle and returned c-Myc expression to young levels but did not affect muscle mass or strength. These data demonstrate that long-term dietary intake of ARA and DHA have overall divergent effects on the structure and function of aging muscle.

IntroductionPeripheral nerve ageing leads to profound proteomic remodelling, with shifts in metabolic and inflammatory signalling pathways resembling changes that occur during nerve degeneration and regeneration following injury. Moreover, aged nerves exhibit impaired degeneration and regeneration, contributing to age-related peripheral neuropathies that show sex-specific differences in prevalence. However, it remains unclear whether these alterations arise from intrinsic nerve changes or an altered systemic environment. Therefore, we investigated the impact of sex on age-related proteome changes and nerve-intrinsic proteomic responses in young and aged male and female nerves using an ex vivo degeneration model. MethodsMass spectrometry-based proteomics were performed on young and old nerves from male and female animals, as well as on contralateral nerves after seven days of ex vivo nerve degeneration. A comparative bioinformatic analysis was then used to identify changes during ageing and ex vivo nerve degeneration that were independent of sex, as well as changes that were sex-specific. ResultsEx vivo nerve degeneration induced extensive proteome remodelling in mouse sciatic nerves that was largely independent of age and sex. Principal component and clustering analyses clearly separated intact from degenerated nerves, while revealing only subtle age- and sex-related effects, with more pronounced ageing-associated changes in males. Approximately 20% of age-regulated proteins and 7-10% of degeneration-regulated proteins exhibited sex-specific expression patterns. Degeneration was characterised by increased abundance of lysosomal and repair-associated proteins alongside reduced myelin and axonal proteins, consistent with active tissue remodelling. In aged nerves, impaired protein clearance and partial pre-activation of degeneration-associated pathways suggested altered injury responses. Comparative analyses demonstrated positive correlations of protein abundance changes between ex vivo and in vivo degeneration datasets, although the temporal dynamics were altered in aged nerves. Pathway enrichment analyses identified coordinated regulation of metabolic, RNA-processing and vesicular transport pathways, while ageing was associated with enhanced immune signalling and reduced lipid metabolism. Sex-specific analyses revealed stronger inflammatory signatures in males, whereas females exhibited enrichment of metabolic pathways, including folate biosynthesis. ConclusionThese findings reveal distinct sex-specific molecular features of peripheral nerve ageing, characterised by enhanced inflammatory signalling in males and metabolic adaptations that may confer resilience in females. Our datasets provide a comprehensive molecular resource of sex-dependent changes in peripheral nerve ageing and nerve-intrinsic injury responses, offering a foundation for identifying therapeutic strategies to promote healthy peripheral nerve ageing. Plain English summaryAge-related peripheral neuropathies are common disorders that can cause pain, numbness, weakness and reduced mobility, affecting millions of people worldwide. They become more common from around the age of 50 and affect men and women differently. These conditions are thought to result from age-related changes in the structure and function of peripheral nerves, which reduce their ability to repair themselves after injury. In this study, we used advanced protein analysis (proteomics) to investigate how ageing affects peripheral nerves in male and female mice. We also used an ex vivo model, in which nerves are studied outside the body, to examine how age and sex influence the molecular changes that occur during nerve degeneration. We found that degeneration caused widespread changes in the proteins present in the sciatic nerve in both young and old mice. Most of these changes were similar in males and females, but some important differences emerged. Male nerves showed stronger signs of inflammation, whereas female nerves showed increased activity of metabolic pathways, including those involved in folate metabolism. Ageing nerves also appeared less able to remove damaged material and showed signs of activating degeneration-related processes even before injury. Overall, the ex vivo model reproduced many of the molecular changes seen after nerve injury in living animals, although it did not fully capture the inflammatory response, suggesting that signals from the rest of the body, including factors carried in the blood, also contribute to nerve degeneration. HighlightsO_LIEx vivo nerve degeneration caused major protein changes in young and old mouse sciatic nerves. C_LIO_LIMost degeneration-related protein changes were shared between males and females. C_LIO_LIAgeing altered the nerve proteome, with stronger ageing-related shifts in males. C_LIO_LIMale nerves showed stronger inflammatory and immune-related signatures. C_LIO_LIFemale nerves showed enrichment of metabolic pathways, including folate biosynthesis, and ex vivo degeneration did not fully reproduce the inflammatory response seen after injury in vivo. C_LI

Cells release membrane-bound extracellular vesicles into the bloodstream laden with proteins that may reflect their physiological state. How this circulating EV proteome changes across life remains poorly understood. Identifying molecular signatures of aging in accessible biofluids could facilitate earlier intervention and monitoring of age-related disease. Many circulating aging proteome studies rely on affinity-based platforms which suffer from poor cross-species translation, ambiguous signal attribution, and inconsistent agreement between platforms. Here, we present a characterization of the aging plasma EV proteome from a cross-sectional cohort of 86 male and female C57BL/6J mice (5-31 months). We leveraged a species-agnostic EV enrichment (Mag-Net) and mass spectrometry to detect 2,575 protein groups from 15,969 peptides. Protein abundance heterogeneity increased with age and the abundance of 272 proteins were significantly correlated with chronological age including established senescence and frailty markers. Proteins increasing with age were enriched in genome maintenance pathways, while those decreasing were associated with the extracellular matrix organization and lipid metabolism. Notably, several of the strongest age-increased proteins converged on Alzheimer's and Parkinson's disease pathology. We observed sexual divergence in the aging EV proteome not previously characterized at this resolution. A proteomic clock built from this data accurately predicts chronological age, and peptide-level analysis reveals aging signals invisible at protein-level. These findings demonstrate that EV-enriched plasma proteomics can identify known aging markers, reveal novel sex-specific age-related changes, and generate predictive models of chronological age. This study provides a species-agnostic foundation for proteomic clocks that complement epigenetic approaches to monitor aging and evaluate healthspan.

Osteocytes are long-lived cells that play a central role in bone homeostasis, yet age-related changes in their functional states remain poorly understood, particularly because skeletal aging involves multiple processes beyond cellular senescence. We generated an osteocyte-specific MepeCre mouse line and combined osteocyte ablation in young and middle-aged mice with skeletal phenotyping, single-cell transcriptomics, and senolytic treatment. MepeCre-driven recombination was largely confined to osteocytes, with minimal off-target activity. Osteocyte ablation increased bone mass at both ages, indicating that osteocytes constrain bone accrual as part of their role in skeletal homeostasis. However, the accompanying remodeling changes differed with age: enhanced osteoblast activity predominated in young mice, whereas reduced osteoclast-mediated bone resorption predominated in middle-aged mice. Single-cell transcriptomics revealed distinct osteocyte subpopulations whose relative abundance shifted with age, from a predominantly matrix-enriched state in young mice to an expanded aging-transitional state in middle-aged mice. Although this state showed partial enrichment of senescence-associated transcriptional signatures, senolytic treatment failed to recapitulate the increase in bone mass induced by osteocyte ablation. Osteocyte therefore regulate bone mass through age-dependent mechanisms that coincide with shifts in osteocyte-state composition. These changes emerge by middle age and may contribute to early remodeling imbalance before overt cellular senescence during skeletal aging. Graphical AbstractGraphical summary of the findings of this study. AA, amino acids; NA, nucleic acid; UA, uric acid; TCA, tricarboxylic acid.

The female reproductive system is one of the first major organ systems to show signs of age-related decline, and menopause is associated with increased risk of several diseases, including osteoporosis and cardiovascular disease. Menstrual fluid contains a mixture of blood and endometrial tissue and is a noninvasive biological sample type that has immense potential for diagnostics related to female reproductive aging. However, existing epigenetic aging clocks show limited performance in hormone-dependent tissues such as the endometrium. At Xella Health, we collected menstrual fluid (MF) samples, from a diverse patient cohort (n=66) and quantified genome-wide 5mC methylation levels. We then developed a novel, deep learning-based epigenetic aging clock that is optimized for performance in menstrual fluid and endometrial tissue. Our model, the Xella Clock, outperforms other widely used epigenetic aging clocks at predicting chronological age from MF data and on endometrial tissue. The model is a useful tool for advancing the study of female reproductive aging and can be used to examine associations between endometrial age acceleration and clinical factors.

Muscle ageing is characterized by evolutionarily conserved subcellular alterations across diverse organisms. In Caenorhabditis elegans, the decline in sarcomeric gene expression is among the earliest detectable ageing-associated changes, emerging at the onset of adulthood. To identify causal regulators of muscle ageing in an unbiased manner, we developed a genetic screening strategy that enables visual monitoring of muscle ageing at both cellular and organismal scales. Using this approach, we identified a mutation that delays the age-associated loss of sarcomeric transcripts. Unexpectedly, the mutation maps to the troponin I gene unc-27, which encodes a conserved regulator of muscle contraction not previously implicated in gene regulation. The mutation alters a single amino acid within a predicted nuclear localization signal (NLS). We found that multiple NLS motifs mediate the active transport of UNC-27 into muscle nuclei from early adulthood onward. Disruption of UNC-27 nuclear localization preserves sarcomeric gene expression during ageing and delays early hallmarks of muscle decline, including proteostatic imbalance and mitochondrial fragmentation. Transcriptomic analyses further revealed that nuclear UNC-27 selectively regulates the expression of genes encoding structural components of the muscle apparatus in adult animals. These results support the existence of a homeostatic sarcomere surveillance pathway, in which a structural protein unexpectedly acquires a transcriptional regulatory role in response to age-associated physiological state. The conservation of NLS motifs in mammalian UNC-27 orthologues suggests that this mechanism may be evolutionarily conserved, with potential relevance to human muscle physiology and disease.

The pulmonary artery undergoes measurable structural and mechanical deterioration with age, but whether these changes can be integrated into a quantitative normative aging prediction model has not been demonstrated. Using two-photon imaging and paired vascular mechanical measurements from C57BL6 mice spanning 6 to 24 months, we developed a multimodal support vector regression (SVR) model integrating collagen fiber orientation, straightness, and hemodynamic mechanical parameters to predict normative age. Fiber orientation was encoded via the von Mises probability density function referenced to the circumferential and axial vessel wall axes providing a principled circular-variable encoding of both mean direction and concentration. The microstructure-only model achieved leave-one-out (LOO) R{superscript 2} = 0.596, Mean Absolute Error (MAE) = 3.43 months. Adding vascular mechanical parameters (PWV) raised a combined LOO R{superscript 2} to 0.834 (MAE = 2.26 months), a 40.1% improvement. Because pulmonary vascular and parenchymal aging are mechanistically coupled, lung mechanics were included as a complementary readout to assess whether airway mechanics contribute independent predictive signal beyond vascular microstructure alone. A sex dimorphism was observed, where females drove the majority of the collagen-based predictive signal (female-only R{superscript 2} = 0.960 vs. male-only R{superscript 2} = 0.658). These results establish a multimodal framework for vascular biological age quantification that integrates structural and mechanical aging signatures.

Aging of skeletal muscle is traditionally defined by progressive loss of mass and strength; however, the early events that precede these outcomes remain poorly characterized. Here, longitudinal analyses revealed that impaired glucose tolerance arises at 12 months of age in Wistar rats, before detectable changes in body composition, circulating damage markers, or muscle mass. Structural loss was preceded by functional decline and structural disorganization between 15 and 18 months. Animals exhibited marked reductions in strength, mobility, and motor coordination, accompanied by extensive remodeling of muscle architecture, including a shift toward glycolytic fiber composition, extracellular matrix expansion, reduced capillarization, and increased structural heterogeneity. Early supplementation with n-3 polyunsaturated fatty acids, initiated at midlife, significantly improved glucose tolerance, reduced adiposity, and enhanced neuromuscular performance without increasing muscle mass. These functional benefits were paralleled by reduced markers of muscle damage and attenuation of histopathological alterations, indicating preservation of tissue organization rather than hypertrophic effects. Notably, a substantial fraction of these benefits persisted after cessation of supplementation, with animals displaying sustained metabolic and structural advantages at 18 months compared to age-matched controls. Collectively, these findings support a model in which skeletal muscle aging is driven by early loss of functional and structural efficiency rather than mass decline, and demonstrate that transient nutritional intervention can durably reprogram the trajectory of muscle aging. These results highlight a critical window of intervention and position n-3 supplementation as a strategy to induce persistent resilience against age-related functional deterioration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/742308v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1f7ad39org.highwire.dtl.DTLVardef@18d8058org.highwire.dtl.DTLVardef@e552d4org.highwire.dtl.DTLVardef@1a0ee31_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neurodegenerative diseases, including Alzheimer's disease (AD), are strongly associated with aging. However, the molecular mechanisms underlying pathological brain aging remain incompletely understood. In this study, we used a mouse model of telomere attrition, a major driver of cellular senescence, to perform an unbiased analysis of how telomere-driven senescence affects cellular physiology and contributes to processes relevant to neurodegenerative conditions. After validating the presence of senescence hallmarks in telomerase-deficient brains, we characterized their transcriptomic and proteomic profiles. Mitochondrial function and associated energy metabolism emerged as the major dysregulated pathways, driven predominantly by proteomic rather than transcriptomic changes. Functional biochemical analyses on isolated brain mitochondria demonstrated impaired electron transport chain (ETC) complex activity and reduced energetic status, despite preserved ETC complex integrity and mitochondrial content. Further analyses in senescent primary neurons indicated an accumulation of dysfunctional mitochondria, characterized by increased reactive oxygen species (ROS) production and reduced ATP levels, although basal cellular respiration was maintained. At the tissue level, these alterations were associated with moderate reductions in neuronal density in the subiculum and cortical layer V, indicating region-specific vulnerability rather than widespread neurodegeneration. We propose that a major consequence of telomere dysfunction associated with pathological brain aging is the downregulation of mitochondrial activity, which contributes to the selective vulnerability of specific brain regions. These findings highlight mitochondrial pathways as attractive targets for interventions aimed at preserving brain health during aging.

The neuromuscular junction (NMJ) is a specialised synapse between motor neurons and skeletal muscle, and its progressive deterioration contributes to age-related and metabolic disease-associated declines in muscle function. Advanced glycation end-products (AGEs) accumulate in tissues during ageing, diabetes, and chronic metabolic dysfunction and have been implicated in neuromuscular degeneration, yet their effects on the intact NMJ have not previously been examined in a human model system. This study employed a fully human, serum-free, and neural growth factor-free NMJ co-culture system, combining neural progenitor cells with immortalised human myoblasts derived from an 83-year-old donor, to investigate the effects of AGE exposure on neuromuscular integrity across structural, metabolic, functional, and secretory outcomes. AGE exposure induced significant reductions in motor neuron axonal length, myotube remodelling with centralised nuclear positioning, mitochondrial membrane depolarisation, elevated mitochondrial superoxide production, mitochondrial uncoupling, and reductions in spontaneous contraction intensity and frequency. Neurotrophic and myogenic growth factor signalling was significantly downregulated in AGE-treated co-cultures. These findings identify the NMJ as a sensitive target of glycation stress and establish this fully human co-culture platform as a physiologically relevant model for investigating glycation-related neuromuscular pathology and evaluating candidate therapeutic interventions.

Some key inflammatory genes controlled by the RELA transcription factor are thought to be highly expressed in fibroblasts induced into senescence by ionizing radiation (IR) as part of the Senescent-Associated Secretory Phenotype (SASP). However, this view is based largely on studies of a limited number of fibroblast cell lines derived from fetal lung or neonatal foreskin. Here, we show that more than half of the primary adult fibroblast strains examined exhibit only weak induction of RELA-dependent inflammatory genes following IR-induced senescence. We define these fibroblasts as "low-responding" to distinguish them from fibroblasts that express high levels of inflammatory gene expression in response to IR. RNA-seq analysis indicated particularly weak IL1A and IL1B expression in low-responding fibroblasts. IL1-alpha and IL1-beta participate in a positive amplification loop for inflammatory gene expression in senescence. Addition of recombinant IL1-alpha or IL1-beta to these fibroblasts sufficed to induce high expression of inflammatory genes. Low-responding fibroblasts thus exhibit cell-autonomous defects in IL1A and IL1B gene activation in response to IR that explains their overall low expression of RELA-targeted inflammatory genes. This defect was correlated with reduced chromatin accessibility and H3-K27-acetylation at 2 putative enhancers in the intergenic region separating IL1A and IL1B, and deletion of either of these enhancers inhibited inflammatory gene expression in IR-induced senescence. Fibroblasts express distinct transcriptomes and we found that differential expression of the FOXF1 transcription factor gene in high-responding WI38 fetal lung fibroblasts contributes to inflammatory gene expression after IR. Our observations indicate that fibroblasts can be distinguished by their ability to manifest cell-autonomous induction of inflammatory genes under conditions of IR-induced senescence.

Aging is associated with proteome remodelling and progressive accumulation of insoluble proteins. Identifying age-enriched proteins that undergo aggregation and evaluating compounds capable of modulating their behaviour may provide insights into interventions that promote healthy aging. Here, we report a proteome-guided strategy to identify age-associated aggregation-prone proteins and evaluate phytochemicals targeting conserved proteins in Caenorhabditis elegans. We identified proteins whose abundance increased more than four-fold in aged worms compared with young worms, many of which also accumulated in the age-associated insoluble proteome, and subsequently identified their human orthologs for comparative analysis. Based on biological relevance, structural conservation, and availability of high-confidence structural models, glutamine-fructose-6-phosphate aminotransferase-2 (GFAT-2) was selected for molecular docking. Screening of fifteen phytochemicals against C. elegans GFAT-2 and its human ortholog GFPT1 identified quercetin as the strongest predicted binder, exhibiting conserved interactions with both proteins. However, treatment of worms with quercetin did not significantly alter global protein insolubility during aging. This may reflect its ability to modulate inappropriate protein-protein interactions without substantially affecting the overall aggregation burden. These findings underscore the need for experimental validation of favourable in silico docking predictions. More broadly, this study provides a proteome-guided framework for prioritizing age-associated aggregation-prone proteins as candidate therapeutic targets for preserving proteostasis during aging.

Transcriptome analyses are widely used for biomarker discovery and to gain insights into normal processes and diseases. Age-related changes in gene expression inferred from RNA-seq are typically reported relative to the transcriptome composition using library-size normalisation. As such, absolute changes in transcript abundance with age remain poorly characterised. Here, using external spike-in normalisation in the Tabula Muris Senis dataset, we quantify age-related variation in total mRNA content and gene expression across mouse cell types. We observe widespread changes in total mRNA abundance, with decreases predominantly in non-immune cell types and increases predominantly in immune cell types. In parallel, the number of genes expressed declines across most cell types, including immune populations. Differential expression analysis based on spike-in-normalised counts identifies genes consistently downregulated across cell types, enriched for functions in RNA metabolism and protein processing. Furthermore, genes downregulated during ageing and during proliferation arrest show partial overlap, suggesting that these transcriptional changes may share regulatory processes. Together, these results are consistent with a general repression of transcriptional and metabolic activity with age, modulated by immune-specific responses. More broadly, our results demonstrate that conclusions drawn from transcriptomic ageing studies can depend strongly on whether gene expression is interpreted in relative or absolute terms, highlighting the importance of absolute normalisation approaches for the analysis of age-related transcriptomic change

The liver is organized into tightly regulated zones with distinct metabolic functions but zonation erodes with age. Cellular senescence contributes to aging and liver diseases, however, its impact on aging biology is ill-defined. As part of The Cellular Senescence Network Consortium, we used multiple spatial transcriptomics approaches (GeoMx, Visium, CosMx) with snRNA-seq to profile senescence signatures, zonation markers, and metabolic pathways in livers from wild-type (WT) mice of multiple ages. We observed a loss of canonical zone signatures in aged mouse livers characterized by "expansion" of midlobular (zone 2) marker gene expression, accompanied by diminished expression of zone 3 marker genes by middle-age (18 months), indicative of loss of cell identity. Multiple analytic approaches identified distinct age-, zone- and sex-specific senescence signatures, which were significantly associated with zonation markers changes. This was recapitulated in Ercc1 mutant models of accelerated senescence, supporting a causal role of senescent cells in liver aging. A "no-zone" hepatocyte-like cluster expanded with age and with the strongest Senescence-Associated Secretory Phenotype (SASP) profile. Gene expression profiles from senescent hepatocytes implicate decreased WNT signaling and increased BMP as contributing to age-related loss of zonation. Together, these data elucidate the role of senescent cells in driving aging biology in non-diseased liver through disruption of cell:cell signaling and the loss of metabolic and cell identity gene expression necessary for hepatocyte function.

Skeletal muscle aging is characterized by the deterioration of muscle function, which can lead to negative quality-of-life outcomes including frailty and sarcopenia. While understanding the mechanisms of this process is increasingly important as the global population ages, previous molecular studies of skeletal muscle aging have been limited by statistical power and cell type resolution. In this study, we analyzed single-nucleus gene expression and chromatin accessibility data from 287 human skeletal muscle samples from individuals aged 20-79 years to explore sex- and cell type- specific aging effects. Across 467,126 nuclei from 13 cell types, we identify 384 age-associated genes and 4,061 age-associated chromatin regions. These age-associated molecular features are enriched for functional pathways, including metabolic processes, cell-to-cell communication, and senescence Kyoto Encyclopedia of Genes and Genomes KEGG terms. Age-associated closing chromatin was more common across fiber types and sexes than opening chromatin, and was enriched in active enhancer regions while depleted for active transcription start sites. We observe enrichment for specific transcription factor motifs in closing chromatin, including those of glucocorticoid and androgen receptors, both of which play a key role in the maintenance of healthy skeletal muscle. Together, these findings identify an age-associated regulatory shift, largely invisible in matched transcriptomic data, characterized by closing chromatin which reduces accessibility to hormone receptor binding sites and enhancer regions in the muscle fiber epigenome.