Aging Cell

Aging is accompanied by a loss of muscle mass and function, termed sarcopenia, which causes numerous morbidities and economic burdens in human populations. Mechanisms implicated in age-related sarcopenia include inflammation, muscle stem cell depletion, mitochondrial dysfunction and loss of motor neurons, but whether there are key drivers of sarcopenia is not yet known. To gain deeper insights into age-related sarcopenia, we performed transcriptome profiling on lower limb muscle biopsies from 72 young, old and sarcopenic subjects using bulk RNA-seq (N = 72) and single-nuclei RNA-seq (N = 17). This combined approach revealed novel changes in gene expression that occur with age and sarcopenia in multiple cell types comprising mature skeletal muscle. Notably, we found increased expression of the genes MYH8 and PDK4, and decreased expression of the gene IGFN1, in old muscle. We validated key genes in fixed human muscle tissue using digital spatial profiling. We also identified a small population of nuclei that express CDKN1A, present only in aged samples, consistent with p21-driven senescence in this subpopulation. Overall, our findings identify unique cellular subpopulations in aged and sarcopenic skeletal muscle, which will facilitate the development of new therapeutic strategies to combat age-related sarcopenia.

Aging is accompanied by a decline in physiological function and increased vulnerability to disease, with mitochondrial dysfunction and epigenetic alterations recognized as key hallmarks. Nicotinamide riboside (NR), a vitamin B3 precursor to NAD, and high-intensity interval training (HIIT) have both been proposed to ameliorate aging-related mitochondrial decline, but their effects on skeletal muscle epigenetic aging are not fully elucidated. Here, we assessed the impact of 5-month NR supplementation and 4-6 weeks HIIT on epigenetic age acceleration (EAA, via seven epigenetic clocks) in human skeletal muscle across three independent studies. NR supplementation was associated with reduced muscle EAA, particularly when measured with the PCHannum, MEAT, and DunedinPACE clocks, while HIIT produced opposite effects in some clocks, notably increasing pace of aging by DunedinPACE. Correlation analyses revealed that changes in skeletal muscle mitochondrial content correlated with changes in MEAT-derived EAA after NR and 6-weeks of HIIT. Together, these findings indicate that skeletal muscle epigenetic aging can be modulated by NR and HIIT interventions but in opposing directions, highlighting a potential link between mitochondria abundance and epigenetic clocks. Further studies are warranted to clarify how NR and exercise regulate epigenetic aging. These results offer new insights into development of strategies for promoting epigenetic outcomes and healthy aging.

Aging is the result of the accumulation of molecular damages that impair normal biochemical activities. We previously reported that aging-damaged amino acid sequence NGR (Asn-Gly-Arg) results in a gain-of-function conformational switching to isoDGR (isoAsp-Gly-Arg) motif. This integrin-binding motif activates leukocytes to induce chronic inflammation, which are characteristic features of age-linked cardiovascular disorders. We now report that anti-isoDGR immunotherapy doubles lifespan in mouse model of chronic inflammation. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1-KO and old WT animals, which could also be induced via injection of isoDGR-modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti-isoDGR mAb (1mg/kg) was sufficient to significantly reduce isoDGR-modified proteins and pro-inflammatory cytokine expression, improve behaviour and coordination, and double the average lifespan of Pcmt1-KO mice. Mechanistically, isoDGR-mAb mediated the immune clearance of damaged isoDGR-proteins by antibody-dependent cellular phagocytosis. These results indicate that immunotherapy targeting aging-damaged proteins may represent effective interventions for a range of age-linked degenerative disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/532237v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@81610borg.highwire.dtl.DTLVardef@a22aaorg.highwire.dtl.DTLVardef@169fe50org.highwire.dtl.DTLVardef@1b73d11_HPS_FORMAT_FIGEXP M_FIG Anti-isoDGR immunotherapy induces immune clearance of aging damaged isoDGR-proteins to reduce chronic inflammation, improve behaviour and coordination, and double lifespan in PCMT-/- mice. C_FIG

Peripheral nervous system (PNS) ageing is marked by structural and functional decline, yet it remains unclear whether ageing constitutes a distinct biological programme or reflects a chronic injury-like state. To address this, we performed an unbiased, comparative molecular analysis of PNS ageing, neuroprotective dietary restriction (DR), and nerve injury. We conducted transcriptomic and proteomic profiling of peripheral nerves from young, old and geriatric mice fed ad libitum or subjected to long-term DR, and proteomics of nerves collected at multiple time points following injury. Age-associated molecular changes followed both linear and non-linear trajectories, and DR partially attenuated these ageing-related alterations. Notably, ageing-and injury-induced proteomic signatures showed considerable similarities, supporting the concept that an aged nerve resembles an injured nerve. Together, our study provides the most comprehensive molecular resource of PNS changes during ageing, DR, and injury, enabling the definition of key molecular signatures underlying PNS physiology. All datasets are integrated into the "PNS-omics Viewer", a Shiny web application designed to facilitate data mining of the herein presented datasets (tba).

Primary sarcopenia is a progressive, age-related decline of skeletal muscle strength, size, and quality, the socio-economic and health impacts of which are set to increase due to global ageing. Despite differences in the physiology of female and male skeletal muscle being well characterised, their alteration with age is less clear. Here we report a striking sexual dimorphism in arm muscle ageing in 478,438 UK Biobank participants aged 40-82 yr. Although the sex difference in age-related arm muscle strength decline is modest, muscle mass loss is considerably more pronounced in males, both in absolute and percentage terms. We also present two alternative measures of muscle quality, each of which exhibits substantially greater age-related decline in females. These trends hold across independent analyses of separate cross-sectional and longitudinal participant groups, persist after accounting for systematic size differences between the sexes, and are apparent irrespective of female menopause status and hormone replacement therapy usage history, despite an sharp reduction in female strength during the perimenopause. Our findings confirm the importance of sex to effective diagnosis and mitigation of sarcopenia, and prompt consideration of the physiological basis of this pronounced sex difference in skeletal muscle ageing.

With the increasing proportion of elderly individuals, improving understanding of the biological mechanisms involved in healthy aging is of utmost importance. Retinal vascular complexity obtained from retinal fundus photographs, and evaluated as fractal dimension (ie, Df), a measure of the geometric pattern of the retinal vasculature, has recently emerged as a valuable indicator of diseases and aging processes. The purpose of our study was to elucidate possible mechanisms underlying those relationships. By integrating retinal Dfwith genomic and plasma proteome biomarkers, we uncovered novel pathways involved in determining Df and its links with cardiometabolic diseases and longevity. First, we performed a genome wide association study (GWAS) of retinal vascular Df by meta-analysing signals from 74,434 participants from three large epidemiological cohorts, the Canadian Longitudinal Study of Aging (CLSA), the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS), and the UK Biobank (UKBB). We replicated four known loci associated with Df, and identified one novel locus within or near DAAM1, as well as found seven suggestive associations within or near CSNK1A1L, MAST4, HIVEP1, LOC283038, FNDC3A, SP100, and SUGP1 genes. GWAS summary statistics confirmed a negative correlation between Df and cardiovascular disease, stroke, and inflammation, but a positive correlation with longevity. Next, employing Mendelian randomization which combined genetic determinants of Df, and cis-protein Quantitative Trait Loci from 1,159 circulating proteins from the Prospective Urban and Rural Epidemiological (PURE) cohort (n=12,066), we identified eight causal mediators for Df, namely IgG Fc receptorIIb, BST1, LILRB2, IL16, MMP12, PON2, ALPP, and PDL2. Notably, MMP12 and IgG Fc receptorIIb have been suggested to connect Df to cardiometabolic outcomes (i.e., coronary artery disease, stroke, peripheral artery disease, and type 2 diabetes) and longevity, respectively. Pathway enrichment analyses highlighted the role of IgG Fc receptorIIb in immune and inflammatory responses to aging processes, referred to as inflammaging. This multi-pronged approach unveils IgG Fc recIIb and MMP12 as key mediators in immune and inflammation pathways, linking lower Df to a higher risk of cardiometabolic diseases and a shorter lifespan. Therefore, retinal Df may be a convenient way to estimate inflammaging, such that lower retinal Df, may indicate a higher inflammaging status. Targeting MMP12 and IgG Fc ReceptorIIb may promote healthy aging and mitigating the occurrence of cardiometabolic diseases in the elderly population. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/24310153v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@15f8ec4org.highwire.dtl.DTLVardef@961efborg.highwire.dtl.DTLVardef@1cc2768org.highwire.dtl.DTLVardef@2f303a_HPS_FORMAT_FIGEXP M_FIG C_FIG

A wealth of literature in different model organisms have shown that proteostasis declines with age, which results in the accumulation of aggregated proteins that generally carry polyubiquitin chains. In Koyuncu et al. (Nature, 2021), the authors use a relatively mild homogenization approach to analyze total polyubiquitinated protein levels longitudinally in C. elegans and found that the overall level of polyubiquitinated proteins decreases in wild-type nematodes. Specifically, the substantial presence of polyubiquitinated proteins in early adulthood markedly decreases at day 10 and 15 of adulthood. This finding represents the central basis of their study where they subsequently suggest that age-dependent decrease in protein polyubiquitination in wild-type animals is due to enhanced deubiquitination. Here, we find that the mild homogenization of C. elegans used in Koyuncu et al. fails to properly extract all C. elegans proteins, because it largely omits relatively insoluble proteins in the pellet. When we perform complete homogenization of wild-type nematode proteins using SDS, sonication and heat, we unequivocally observe that polyubiquitinated protein levels do not decrease with age in C. elegans. Overall, the levels of polyubiquitinated proteins remains relatively constant post-reproduction. Therefore, our findings invalidate the main conclusion of Koyuncu et al. that the ubiquitinated proteome is rewired during ageing and demonstrate that C. elegans harbor polyubiquitinated proteins throughout their lifespan.

Sarcopenia, which diminishes lifespan and healthspan in the elderly, is commonly exacerbated by viral pneumonia, including influenza and COVID-19. In a study of influenza A pneumonia in mice, young mice fully recovered from sarcopenia, while older mice did not. We identified a population of tissue-resident skeletal muscle macrophages that form a spatial niche with satellite cells and myofibers in young mice but are lost with age. Mice with a gain-of-function mutation in the Mertk receptor maintained this macrophage-myofiber interaction during aging and fully recovered from influenza-induced sarcopenia. In contrast, deletion of Mertk in macrophages or loss of Cx3cr1 disrupted this niche, preventing muscle regeneration. Heterochronic parabiosis did not restore the niche in old mice. These findings suggest that age-related loss of Mertk in muscle tissue-resident macrophages disrupts the cellular signaling necessary for muscle regeneration after viral pneumonia, offering a potential target to mitigate sarcopenia in aging.

Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented1-3 however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the "cytokine storm" seen in severe COVID-194,5. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.

Semaglutide is a once-weekly GLP-1 receptor agonist that has been proposed as a gerotherapeutic, yet no data exist on its effects on epigenetic aging. We therefore conducted a post-hoc epigenetic analysis of a 32-week, double-blind, placebo-controlled phase 2b trial in adults with HIV-associated lipohypertrophy (semaglutide n = 45; placebo n = 39). Paired peripheral-blood methylomes were profiled to evaluate semaglutides impact across multiple generations of DNA-methylation clocks. After adjustment for sex, BMI, hsCRP, and sCD163, semaglutide significantly decreased epigenetic aging: PCGrimAge (-3.1 years, P = 0.007), GrimAge V1 (-1.4 years, P = 0.02), GrimAge V2 (-2.3 years, P = 0.009), PhenoAge (-4.9 years, P = 0.004), and DunedinPACE (-0.09 units, {approx}9 % slower pace, P = 0.01). Semaglutide also lowered the multi-omic OMICmAge clock (-2.2 years, P = 0.009) and the transposable element-focused RetroAge clock (-2.2 years, P = 0.030). Eleven organ-system clocks showed concordant decreased with semaglutide, most prominently inflammation, brain and heart, whereas an Intrinsic Capacity epigenetic clock was unchanged (P = 0.31). These findings provide, to our knowledge, the first clinical-trial evidence that semaglutide modulates validated epigenetic biomarkers of aging, justifying further evaluation of GLP-1 receptor agonists for health-span extension.

Genome-wide association studies of physical activity traits have mapped numerous loci, yet the molecular mechanisms through which exercise influences human biology remain poorly defined. Mechanistic progress has been limited by heritability-dominated signals, siloed single-omic analyses, and the lack of integrative models that connect genetic associations to causal, system-level pathways. We introduce the first deep learning, multi-omic framework for exercise genomics, unifying causal inference, molecular topology, protein structure, and functional context within a supervised Graph Neural Network (GNN) with experimental validation. Using linkage disequilibrium-aware Mendelian randomisation with accelerometer-derived vigorous physical activity as the exposure, we integrated four omic layers--plasma proteomics, blood CpG methylation, blood single-cell transcriptomics, and plasma glycomics. The GNN prioritised a coherent, multi-omic network of exercise-responsive genes spanning glycosylation and immunity, apoptosis/stress signalling, growth and transcription factors, proteostasis/autophagy, metabolism, oxidative stress/redox, mitochondrial/oxidative phosphorylation, chromatin/epigenetic, translation/ribosome, RNA splicing/processing, DNA damage and repair, cell cycle, and cytoskeleton/ECM functions. Pathway annotation and alignment with ageing biology using differentially methylated regions and principal component features from five biological ageing clocks (Horvath, Hannum, DunedinPACE, PhenoAge, Proteomic Clock), used as contextual markers of ageing-related regulation, mapped to: 1) autophagy (Fas signalling); 2) stress and DNA damage response pathways (Hypoxia-Inducible Factor, apoptosis, p53, p38, PI3K, Ras, oxidative stress, DNA replication, purine/pyrimidine metabolism and biosynthesis, pentose phosphate, Rho GTPase, G protein, ubiquitin proteasome, Epidermal Growth Factor Receptor); 3) immune and inflammatory pathways (cytokine and interleukin signalling, T and B cell activation, Toll-like receptor signalling); 4) physiological adaptation (VEGF, Wnt, GnRH, Fibroblast Growth Factor, Platelet-Derived Growth Factor, Thyrotropin-Releasing Hormone, glutamate receptor, plasminogen, endothelin, heme biosynthesis, cholecystokinin, Corticotropin-Releasing Hormone signalling); 5) neuroendocrine and neurotransmitter pathways (acetylcholine, dopamine, oxytocin, opioid, {beta}-adrenergic signalling); 6) neurodegeneration pathways (Alzheimers, Parkinsons, Huntingtons disease); 7) protein metabolism (leucine, isoleucine, valine biosynthesis); and exercise-responsive epigenetic regulatory pathways (S-adenosylmethionine, thiamine, vitamin D biosynthesis, bZIP transcription, and circadian rhythm). Finally, we partially validated GNN predictions in humans, demonstrating acute exercise-induced shifts in plasma glycomic markers consistent with GNN-predicted glycomic remodelling. This work establishes a deep learning, multi-omic map of exercise-responsive pathways in humans and identifies actionable regulators that couple habitual vigorous physical activity to stress-resilient immunometabolic regulation and healthy ageing trajectories.

Regular physical activity is a cornerstone of healthy aging, offering a wide range of benefits, including the modulation of immune regulation and reduction of chronic inflammation. With aging closely linked to persistent, low-grade inflammation, i.e. inflammaging, the effects of exercise intensity on acute immune responses in older adults remain not fully understood. In this study, we explored how moderate and intense acute continuous exercise impact immune cell activation, cytokine production and large extracellular vesicle (lEV) release in healthy elderly individuals. Fourteen participants completed a moderate continuous exercise intervention (60% VO2max for 30 minutes), while nineteen engaged in an intense continuous exercise session until exhaustion. Blood samples were collected at baseline, and at 1- and 24-hours post-exercise. Immune cell characterization by flow cytometry revealed distinct changes in monocyte subsets and NK cells activation across both exercise intensities. Intense exercise was associated with elevated proinflammatory TNF levels, accumulation of circulating plasma-derived lEV and changes in their surface marker expression after 24 hours. Additionally, we identified sex-specific differences, including distinct activation profiles in innate immunity, alterations in EV release from CD4+ and HLA+ cells, and an exercise-induced increase in IL-6 observed exclusively in females. These findings suggest that moderate continuous acute exercise enhances immune cell activation without altering cell counts, while intense continuous exercise triggers acute proinflammatory immune response. Further research should clarify the long-term implications and fundamental mechanisms of exercise-induced immune modulation in aging populations. Key points summaryO_LIDiminished immune function upon aging is increasing disease risk. This study examines how tailored acute exercise interventions stimulate immune regulation in older adults addressing age-related inflammatory challenges. C_LIO_LIAcute continuous moderate and intense exercise elicit distinct immune responses in elderly individuals with marked differences between sexes. Interestingly, IL-6 levels increased 30 min moderate exercise exclusively in females. C_LIO_LIExercise promotes the release of extracellular vesicles (EVs) and modulates peripheral immunity, suggesting a potent mechanism by which physical activity supports immune resilience in aging. C_LIO_LITailored acute exercise regimens for older adults may enhance immune health, mitigating age-related inflammatory risks and enhancing resilience. C_LIO_LIThis study emphasizes the need for further research on exercise-driven modulation focusing on sex differences and their implications for targeted interventions upon aging. C_LI

While genetic evidence robustly associates longevity in non-human primates with Klotho protein, such a direct correlation in humans remains elusive. To scrutinize the potential causal link between genetically predicted Klotho levels and human lifespan, we devised a meticulous two-sample Mendelian randomization (MR) analysis, just leveraging single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) and protein quantitative trait loci (pQTLs) as instrumental variables, meticulously analyzing the relationship between serum -klotho and human longevity. By integrating MR estimates across diverse data sources using the fixed-effects inverse-variance weighted (IVW) approach, we consolidated our findings with a fixed-effects meta-analysis, fortified by sensitivity analyses embracing the simple median, weighted median, MR-Egger regression, and MR-pleiotropy residual sum and outlier assessments. Surprisingly, our genome-wide association MR analyses failed to uncover a causal association between Klotho and human lifespan, holding for both experimental and validation cohorts. Furthermore, the analysis grounded in protein quantitative trait loci also yielded no evidence of a causal link, with the sensitivity analyses consistently reinforcing the robustness of our findings. Hence, while animal models suggest a correlation between circulating Klotho and lifespan, this study demonstrates that genetically predicted levels of circulating klotho do not exhibit a direct causal effect on human longevity.

De Barsy syndrome is a recessive progeroid disease classified under the group of cutis laxa syndromes. The disease is attributed to loss-of-function mutations in PYCR1 or ALDH18A1, leading to premature skin aging. Here we report five PYCR1 pathogenic alleles and a mouse knockout model of the disease. Through these investigations, we have confirmed the key role of PYCR1 in preventing dermal thinning and other connective tissue abnormalities. However, it remains unknown whether endogenous PYCR1 levels undergo changes during normal aging. To address this query, we examined its levels in cultured human cutaneous fibroblasts subjected to induced or replicative senescence. In both instances, PYCR1 levels dropped and correlated with the loss of proliferative capacity. Furthermore, we validated the relevance of these findings in vivo, by comparing young and chronologically aged human skin, and found that the levels of PYCR1 in the dermis, but not the epidermis, significantly decreased with age. Our results confirm that the loss of PYCR1 is a driver of human skin aging and that its levels in healthy individuals can serve as a biomarker for connective tissues undergoing normal chronological aging.

Aged skin is prone to viral infections, but the mechanisms responsible for this immunosenescent immune risk are unclear. We observed that aged murine and human skin expressed reduced antiviral proteins (AVPs) and circadian regulators including Bmal1 and Clock. Bmal1 and Clock were found to control rhythmic AVP expression in skin and such circadian-control of AVPs was diminished by disruption of immune cell interleukin 27 signaling and deletion of Bmal1/Clock genes in mouse skins, as well as siRNA-mediated knockdown of CLOCK in human primary keratinocytes. We found that treatment of circadian enhancing agents, nobiletin and SR8278, reduced infection of herpes simplex virus 1 (HSV1) in epidermal explants and human keratinocytes in a Bmal1/Clock-dependent manner. Circadian enhancing treatment also reversed susceptibility of aging murine skin and human primary keratinocytes to viral infection. These findings reveal an evolutionarily conserved and age-sensitive circadian regulation of cutaneous antiviral immunity, underscoring circadian restoration as an antiviral strategy in aging populations.

DYSF functions as a regulator of Ca{superscript 2} in the skeletal muscle and facilitates muscle repair following injury. It is also highly expressed in monocytes and macrophages and related to inflammation, immune regulation, and the mononuclear phagocyte system. Macrophages are pivotal in driving myogenesis, and specific cytokine-induced macrophage differentiation plays a role in maintaining skeletal muscle during disease and aging. Thus, a comprehensive understanding of the mechanisms by which DYSF operates in macrophages may inform the development of novel treatments for muscle atrophy. In this study, we demonstrated that DYSF expression is associated with monocyte differentiation and increases with age. Our findings indicate that DYSF overexpression induces the generation of M1-type macrophages, which subsequently secrete inflammatory cytokines and promote cell invasion. Furthermore, we observed that DYSF regulates Ca{superscript 2} influx into the cell and activates the STAT1 signaling pathway, whereas DYSF deficiency suppresses these processes. Macrophages overexpressing DYSF are associated with the inhibition of myoblast differentiation during myogenesis in a co-culture system involving macrophages and myoblasts. Therefore, the balance between the STAT1 signaling pathway and Ca{superscript 2}, which are regulated by DYSF abundance, may play a crucial role in myogenesis.

Metabolic ageing and associated changes in lipid mobilisation have been most heavily studied in humans and model taxa, yet remain poorly understood in farmed animals, with potentially important uncharacterised implications for health and welfare outcomes in food production systems. Here, we study both processes in domesticated Atlantic salmon (Salmo salar), the worlds most commercially valuable farmed fish, comparing three stages of aquaculture production. Our sampling captures a key life-cycle change where juvenile fish transition from freshwater into seawater (smoltification), followed by the ongoing ageing process during a final period of growth in seawater. Integrating lipidomics and proteomics of visceral adipose and skeletal muscle tissue, we firstly identified a metabolic-ageing profile akin to that observed in humans, which was distinct from lipid-associated remodelling associated with earlier smoltification. This was marked by impaired triglyceride storage, dysfunctional autophagy-lysosomal pathways, inflammation, fibrosis and reduced pathogen clearance pathways in visceral adipose tissue. In skeletal muscle, ageing was accompanied by reduced metabolic flexibility together with triglyceride and fatty acid accumulation, depletion of phospholipids, and a reduction in free fatty acids required for ATP production. We go on to provide experimental in vivo evidence that dietary spermidine supplementation suppresses adipose inflammation and reverses age-associated metabolic flexibility by re-establishing the buffering role of adipose tissue and enhancing fatty acid metabolism in skeletal muscle. Importantly, spermidine appears to reprogram lipid flux to counter metabolic ageing. As farmed Atlantic salmon exhibit key features of metabolic ageing observed in humans that appear linked to its recent domestication history, this species offers a novel model for ageing related studies of vertebrate metabolism.

How aging of human neurons affects dynamics of essential organelle such as mitochondria and autophagosomes remains largely unknown. MicroRNA-induced directly reprogrammed neurons (miNs) derived from adult fibroblasts retain age-associated signatures of the donor, enabling the study of age-dependent features in human neurons, including longitudinal isogenic samples. Transcriptomic analysis revealed that neurons derived from elderly individuals are characterized by gene expression changes associated with the regulation of autophagosomes, lysosomes, and mitochondria, compared to young counterparts. To clarify these changes at the cellular level, we performed live-cell imaging of cellular organelles in miNs from donors of different ages. Older donor miNs exhibit decreased mitochondrial membrane potential, which surprisingly co-occurs with a significant increase in mitochondrial fission and fusion events. We posit that the increased fission and fusion of mitochondria may reflect age-dependent compensation for impaired mitochondrial turnover, perhaps due to changes in autophagy. We subsequently identified a significant decrease in autophagosome acidification in neurons derived from individuals >65 years compared to younger donors, and a corresponding age-dependent reduction in neuritic lysosomes resulting in fewer lysosomes available to acidify autophagosomes. This age-dependent deficit in autolysosome flux was rescued by chemically promoting autophagosome generation, which also reversed the age-dependent increase in mitochondrial fission and fusion and improved mitochondrial health. Together, this work reveals a mechanism by which aging reduces autophagic flux secondary to a loss of neuritic lysosomes, resulting in in mitochondria-intrinsic mechanisms to avoid loss of energy production.

A loss of proteostasis is a primary hallmark of ageing that has emerged from mechanistic studies in model organisms, but little is currently known about changes to proteostasis in the muscle of older humans. We used stable isotope labelling (deuterium oxide; D2O) in vivo, and peptide mass spectrometry of muscle samples to investigate differences in proteome dynamics between the muscle of younger (28 {+/-} 5 y; n=4) and older (69 {+/-} 3 y; n=4) men during either habitual activity or resistance exercise training. We quantified the abundance of 1787 proteins and the turnover rate of 1046 proteins in bi-lateral samples of vastus lateralis (n=32 samples total) taken before and after a 15-day program including 5 sessions of unilateral leg-press exercise (3 sets of 10 repetitions at 90% of 10 RM). Our protein abundance profiling revealed a stoichiometric imbalance within the proteostasis network in aged skeletal muscle, including subunits of eIF3, subunits of 40S and 60S ribosomal proteins. The rate of bulk, mixed-protein synthesis was not different between younger and older men, but most ribosomal proteins were less abundant in the muscle of older participants, suggesting ribosomes in older muscle may exhibit increased translational efficiency to maintain similar levels of protein turnover compared to ribosomes in younger muscle. Resistance exercise partially restored age-related disruptions to the proteostasis network. In older skeletal muscle, resistance exercise specifically increased the absolute turnover rate (ATR) of mixed mitochondrial proteins, with increased fractional turnover rate (FTR) of prohibitin 1 (PHB1) and profilin-1 (PROF1), and increased abundance of prohibitin 2 (PHB2). These adaptations may suggest resistance exercise promotes mitochondrial proteostasis by facilitating the synthesis and maintenance of key mitochondrial proteins. Thus, our Dynamic Proteome Profiling data provide an impetus for further exploration of the role of proteostasis in maintaining skeletal muscle quality and supports resistance exercise as a potential therapeutic strategy to promote healthy skeletal muscle ageing in humans. In BriefNishimura et al. used Dynamic Proteome Profiling to uncover whether the distorted proteomic landscape of ageing skeletal muscle is associated with altered turnover of specific proteins. Basal muscle from older men exhibits a divergence in protein abundance between subunits of eIF3 and subunits of 40S and 60S ribosomal proteins, whereas resistance exercise partially restored age-related disruptions in the muscle proteome. In older muscle, protein-specific turnover generally increases after resistance exercise, independent of changes in protein abundance, suggesting improved protein quality and renewal. Created in BioRender. Nishimura, Y. (2025) https://BioRender.com/p2a1aio HighlightsO_LIDynamic Proteome Profiling in human skeletal muscle ageing C_LIO_LIAgeing alters muscle proteostasis C_LIO_LIMixed-muscle protein synthesis does not differ between younger and older men C_LIO_LIResistance exercise increased mitochondrial protein turnover specifically in older muscle C_LIO_LIProtein-specific responses to resistance exercise differed between age groups C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/684531v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@11d0c8corg.highwire.dtl.DTLVardef@22f451org.highwire.dtl.DTLVardef@d62118org.highwire.dtl.DTLVardef@16eda9_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.