Frontiers in Aging

Mitochondria are double membrane-bound organelles with pleiotropic roles in the cell, including energy production through aerobic respiration, calcium signaling, metabolism, proliferation, immune signaling, and apoptosis. Dysfunction of mitochondria is associated with numerous physiological consequences and drives various diseases, and is one of twelve biological hallmarks of aging, linked to aging pathology. There are many distinct changes that occur to the mitochondria during aging including changes in mitochondrial morphology, which can be used as a robust and simple readout of mitochondrial quality and function. Although mitochondrial morphology alone cannot be used to conclude the quality of mitochondria, it is highly correlated with mitochondrial function whereby mitochondria exhibit increased fragmentation with age in multiple cell types of the nematode C. elegans. Thus, C. elegans serve as a robust model for rapidly measuring mitochondrial morphology changes during aging. To standardize imaging methods for mitochondrial morphology in C. elegans, we provide a detailed comparative characterization of several transgenic constructs, highlighting benefits and caveats for aging biology studies. Summary BlurbThis study evaluates mitochondrial imaging in C. elegans during aging, comparing various transgenic constructs for tissue-specific mitochondrial visualization. The findings highlight technical considerations, imaging method standardization, and the utility of C. elegans as a robust model for studying mitochondrial dynamics.

BackgroundWhile immunologic aging impacts immune responses to vaccination, consistent biomarkers associated with aging of the immune system and suboptimal serologic response to influenza vaccination have not been well-studied. Identification of readily measurable biomarkers of immunosenescence may have predictive clinical utility and inform targeted influenza vaccination strategies and future research into aging of the immune system. MethodsWe quantified multiple serum/plasma and cell-based parameters related to immune aging (CMV serostatus, plasma cytokines/chemokines, TREC, TERT, NK cell functionality, and DNA methylation clock) at baseline in an adult (age range 18-85) cohort of 2019-2020 influenza vaccine recipients (n=337) and evaluated their associations with vaccine-induced HAI response to influenza A/H1N1, A/H3N2 and B/Victoria strains. ResultsCMV IgG titers were significantly positively correlated with vaccine-induced increases in HAI antibody titers to influenza A/H1N1 (p=0.02) and A/H3N2 (p=0.014). CMV IgG titers (p=0.00096) and CMV seropositivity (p=0.003) were also associated with Day 28 HAI seropositivity against influenza A/H3N2 in subjects seronegative at baseline. Conversely, plasma MCP-1 levels were negatively associated with HAI responses to the A/H3N2 (p=0.04) strain. These findings were significant independent of age, sex or vaccine type received (high vs standard-dose seasonal influenza vaccine) ConclusionsOur identification of significant relationships between easily quantifiable immune markers and HAI responses to influenza A vaccine strains across sex and age enhances our knowledge of specific links between immune aging and influenza vaccine-induced immunity. These markers could be leveraged for predicting response to influenza immunization.

BACKGROUNDAlong with advancing age comes declines in physical, cognitive, and cardiovascular function. This diminished capacity may lead to decreased ability to perform activities of daily living, disability onset, and loss of independence. Exercise is a regenerative medicine therapy that can mitigate this loss of function. High intensity interval training (HIIT) is an aerobic exercise paradigm consisting of intense activity periods interspersed with bouts of active recovery. Previously we demonstrated that HIIT preserved physical function in adult, middle-aged, and older male mice. However, whether HIIT preserves physical, cognitive, and cardiovascular function, mitigates frailty, and improves brain and heart health in older adult female mice remains unknown. HYPOTHESISCognitive, physical, and cardiovascular function in older adult female C57BL/6 will be preserved in exercised mice (HIIT) versus sedentary control (SED). METHODSMice (HIIT and SED, both n=9, 24m at end) were tested pre/post-intervention for physical (rotarod, treadmill, grip meter, inverted cling, voluntary wheel running, activity monitor), cognitive (open field, novel object recognition, puzzle box, y-maze), and cardiovascular (blood pressure, echocardiogram) function, body composition, and whole body calorimetry. The mice underwent 14-weeks of HIIT training with progressive volume and intensity. RESULTSHIIT significantly (p<0.05) increased or preserved function in many tests including: aerobic capacity (+71% HIIT versus, vs, no change, NC, in SED), four limb strength/endurance (-67% SED vs -28% HIIT), forelimb strength (-16% SED vs NC HIIT), overall motor function (NC SED vs +39% HIIT), executive function (NC SED vs +73% HIIT), and exploratory behavior, which improved across multiple tests with HIIT while remaining unchanged in SED. HIIT also reduced both systolic blood pressure by 12% (-17 mmHg) and mean arterial pressure by -16 mmHg. In addition, HIIT significantly reduced cardiac fibrosis, increased muscle fiber type 2a percentage, reduced IL-1{beta} expression in the hypothalamus, and mitigated frailty onset. CONCLUSIONHIIT significantly reduced age-related functional loss in all three domains assessed while preventing frailty onset in older adult females and improving markers of brain and heart health.

Finding new interventions that slow ageing and maintain human health is a huge challenge of our time. The nematode Caenorhabditis elegans, offers a rapid in vivo method to determine whether a compound extends its 2-3 week lifespan. However, the standard C. elegans lifespan assay is hard to scale for large screens. Lifespan analysis produces only one data point per animal with no information about health. Here we describe automated monitoring of movement from early to mid-adulthood as a healthspan-based alternative to measure ageing. Using our WormGazer technology, over 100 petri dishes containing C. elegans worms are imaged simultaneously and non-invasively by an array of cameras. This approach demonstrates that most functional decline in C. elegans occurs during the first week of adulthood. We find 7 days of imaging is sufficient to measure the dose-dependent efficacy of sulfamethoxazole to slow ageing, compared to 40 days required for a parallel lifespan experiment. Understanding any negative consequences of interventions that slow ageing is important. We show that the long-lived mutant age-1(hx546) stays active for longer than the wild type but it moves slower in early adulthood. Thus, continuous analysis of movement can rapidly identify interventions that slow ageing while simultaneously revealing any negative effects on health.

Aging-related muscle atrophy and weakness contribute to loss of mobility, falls and disability. Mitochondrial dysfunction is widely considered a key contributing mechanism to muscle aging. However, mounting evidence position physical activity as a confounding factor, making unclear whether muscle mitochondria accumulate bona fide defects with aging. To disentangle aging from physical activity-related mitochondrial adaptations, we functionally profiled skeletal muscle mitochondria in 51 inactive and 88 active men aged 20-93. Physical activity status conferred partial protection against age-related decline in physical performance. A trend for reduced muscle mitochondrial respiration with aging was observed in inactive but not in active participants, indicating that aging per se does not alter mitochondrial respiratory capacity. Mitochondrial reactive oxygen species (ROS) production was unaffected by aging and active participants displayed higher ROS production. In contrast, mitochondrial calcium retention capacity decreased with aging regardless of physical activity status and correlated with muscle mass, performance and the stress-responsive metabokine GDF15. Targeting mitochondrial calcium handling may hold promise for treating aging-related muscle impairments.

Declining functional capacity, both physical and cognitive, is a consequence of aging. However, exercise is a promising intervention to mitigate normal age-related decline. While numerous studies have elucidated the benefits of exercise per se, less well-studied is the effect of high intensity interval training (HIIT) on a middle-aged population. Our primary purpose was to assess the effect of three months of HIIT on physical and cognitive performance in middle-aged (17-month-old) male C57BL/6J mice, compared to sedentary controls. We hypothesized that exercised mice would be resistant to any decline in cognitive and physical ability, both measured pre- and post-intervention. To measure physical function, we used the well-validated CFAB (comprehensive functional assessment battery) scoring system comprised of determinants including voluntary wheel running, inverted cling, grip test, treadmill max speed, and rotarod. We measured cognition with open field, novel object recognition, y-maze, and puzzle box. Further measures of sarcopenia/frailty included body composition (MRI) and in vivo contractile physiology (plantar flexor torque). Training resulted in significant aerobic capacity improvements for the HIIT group, increasing treadmill time by 28%, while the SED group demonstrated a 41.4% decline in treadmill time. However, no significant differences in cognitive function were determined. Contrary to our previous research in other age groups, the current study found a negligible effect of HIIT on body composition. We note that at 17 months old, mice did not experience any evidence of cognitive deterioration in either group over the three-month period, thus explaining the lack of exercise effect. We found that HIIT had less influence on either physical or cognitive function than we expected, which may be because function in this age group is stable. Future work will investigate older adult cognitive response to HITT at ages where there is well-documented cognitive decline.

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.

In defiance of the paradigm that calories from all sources are equivalent, we and others have shown that dietary protein is a dominant regulator of healthy aging. The restriction of protein or the branched-chain amino acid isoleucine promotes healthspan and extends lifespan when initiated in young or adult mice. However, many interventions are less efficacious or even deleterious when initiated in aged animals. Here, we investigate the physiological, metabolic, and molecular consequences of consuming a diet with a 67% reduction of all amino acids (Low AA), or of isoleucine alone (Low Ile), in male and female C57BL/6J.Nia mice starting at 20 months of age. We find that both diet regimens effectively reduce adiposity and improve glucose tolerance, which were benefits that were not mediated by reduced calorie intake. Both diets improve specific aspects of frailty, slow multiple molecular indicators of aging rate, and rejuvenate the aging heart and liver at the molecular level. These results demonstrate that Low AA and Low Ile diets can drive youthful physiological and molecular signatures, and support the possibility that these dietary interventions could help to promote healthy aging in older adults.

Rodent gait analysis is crucial for modeling human aging, but the lack of comprehensive research on gait in elderly mice limits our ability to translate findings from animal models to human populations. Age-related changes in C57BL/10 strain remain unknown. The state of art protocol for gait analysis uses the CatWalk XT system that allows an understanding of the locomotion pattern by a variety of parameters. We aim to provide relevant information for experimental designs, presenting benchmark data on the performance of locomotion using healthy wild-type mice for future preclinical investigations of neurological and neuromuscular gait patterns. In this study, characterization of walking locomotion was demonstrated from complete gait analysis in aged C57BL/10ScCr/PasUnib mice using open-field, CatWalk, and treadmill tests. Mice were divided into the adult group (6 months; n = 9) and the aged group (20 months; n = 9). Aged mice demonstrated decreased mobility, distance traveled, and general speed in the open-field test. The spatiotemporal and kinetic parameters were altered in aged mice, with lower speed, higher stand time and stride length, and increased base of support and duty cycle in comparison with adult mice. Interlimb coordination has changed in elderly mice. To test whether speed alters the temporal parameters, we used a treadmill test and we demonstrated higher stand time in 20-month-old mice. We demonstrated that changes in gait parameters and mobility represent direct age-related singularities in the wild-type C57BL/10 mice. Overall, aged mice took more time in contact with the ground independently of the speed. These baseline gait results shed light on measures that allow the potential investigation of therapeutics and interventions in gerontology or neuromuscular diseases.

Aging encompasses the natural processes of birth, growth, and aging, during which the functional ability of muscles gradually decreases, leading to the loss of muscle size and reduced exercise performance known as sarcopenia. This condition is closely associated with weakness, osteoporosis, and degenerative diseases, increasing the risk of falls, fractures, metabolic diseases, and mortality due to limitations in physical performance among the elderly. This study investigated the effects of exercise intervention on biological markers related to skeletal muscle mass and functions in conjunction with aging. At age of four or twenty, the C57BL/6 mice were assigned to Young control (Y-Con, n = 10) or exercise training (Y--Exe, n = 10), and Aged control (A-Con, n=10) or exercise training (A-Exe, n = 10). Exercise intervention was performed on a rodent motor-driven treadmill with a frequency of 5 days per week for 8 weeks. As a consequence, exercise intervention in mice resulted in positive changes in IGF-1 signaling and muscle phenotype compared to mice that did not undergo exercise intervention, specifically showing prominent effects in the A-Exe group compared to the A-Con group. The mitigating effects of exercise intervention on age-related skeletal muscle dysfunction were accompanied by enhanced exercise performance and muscle function, as assessed by grip strength and the rotarod test. The current findings support previous studies that have reported the positive effect of exercise intervention in alleviating age-related declines in exercise performance and muscle function in older adults.

BackgroundSkeletal muscle aging exhibits substantial heterogeneity, with some individuals maintaining robust function into advanced age while others develop sarcopenia and frailty. Whether molecular signatures distinguishing these trajectories reflect biological aging or modifiable factors, such as physical activity, remains unclear. MethodsAn integrated discovery-validation study was conducted on skeletal muscle transcriptomes. Discovery analysis used the GSE144304 dataset comprising vastus lateralis biopsies from young adults (n=26, aged 18-30 years), fit elderly (n=30, aged 65-80 years with preserved function), and frail elderly (n=24, aged 65-80 years stratified by grip strength). Top 10 most significantly altered genes were validated across five independent transcriptomic studies (n=184 total) strategically selected to represent distinct activity contexts: activity-controlled aging, sedentary aging, mixed-activity aging, disease-impaired aging, and exercise intervention. Expression of two established atrogenes were examined (FBXO32/Atrogin-1 and TRIM63/MuRF-1) as benchmarks. ResultsDiscovery analysis identified 10 genes with profound age-related changes (adjusted p < 10-{superscript 2}{superscript 1}, |log2FC| > 1.3). Cross-dataset validation revealed striking activity-dependence: genes downregulated with aging in sedentary populations (MYORG, STRADB) showed maintained or increased expression in active elderly individuals (80% validation rate, r = 0.75-0.82 with activity level). In contrast, established atrogenes showed poor replication (25-50%) and context-dependent patterns. C4ORF54 expression strongly correlated with grip strength (r = 0.68, p < 0.001), with age effects disappearing after phenotype adjustment, indicating purely phenotype-mediated expression. Critically, sedentary versus active aging datasets showed opposing transcriptional patterns (r = -0.68), demonstrating that activity confounds conventional age-based signatures. ConclusionsMolecular signatures distinguishing fit from frail aging predominantly reflect physical activity levels rather than inevitable biological processes. MYORG and STRADB emerge as activity-responsive biomarkers of muscle health, while C4ORF54 serves as an indicator of functional capacity. These findings challenge conventional atrogene paradigms and suggest that exercise-responsive AMPK signaling pathways represent immediately translatable therapeutic targets for preserving muscle function in older adults.

Autophagy is a hallmark of aging, but autophagy-related proteins have not been exclusively targeted to attenuate the progressive decline in physical function associated with aging. Here, we combined Tat-Beclin1, an autophagy agonist, and endurance training to determine whether Tat-Beclin1 enhances exercise adaptation in old male mice. Tat-Beclin1 was administered intraperitoneally (TB group, 15 mg/kg, 2x/week) as a standalone therapy, or in combination with endurance training (TB+Exe group, 70% of maximal running speed 3x/week) for 1 month in 23-month-old male C57BL/6J mice. Control groups were age-matched cage controls and exercise-only groups. Animals were assessed for grip strength, endurance capacity on a treadmill, and balance and coordination on a rotarod. Gastrocnemius/plantaris (G/P) and tibialis anterior muscles were harvested for western blotting, myofiber typing, and proteomic profiling (G/P only). TB+Exe led to significant increases in grip strength, endurance capacity, and balance and coordination performance beyond those observed in the TB and Exe groups alone. Autophagy markers, including Beclin1, the LC3B-II/I ratio, and p62, did not differ among groups. A proteomic analysis of the G/P muscle revealed that TB upregulated biological processes involved in muscle contraction and adaptation, whereas TB+Exe increased mitochondrial bioenergetic processes and, surprisingly, upregulated acute inflammatory responses, including proteins such as haptoglobin and orosomucoid-1. We conclude that combining Tat-Beclin1 and endurance training may represent a new approach to attenuate aging-related decline in physical function. New & NoteworthyWe show evidence that combining Tat-Beclin1 and endurance training (TB+Exe) resulted in greater improvements in physical function in 24-month-old male mice than either standalone therapy. We also show that TB+Exe upregulates traditional exercise-like biological processes and unexpectedly upregulates acute-inflammatory proteins (e.g., orosomucoid-1), which are thought to improve physical function in preclinical studies. Our study suggests that TB may be a new drug enhancing physical function, especially when combined with endurance training in old male mice.

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.

Age-related reductions in whole-muscle function are attributed, in part, to pronounced atrophy of muscle fibers expressing the fast myosin heavy chain (MyHC) II isoforms. Senescence, a state of irreversible cell cycle arrest that can be characterized by DNA damage ({gamma}H2AX) and chromatin remodeling (loss of nuclear HMGB1), may contribute to skeletal muscle aging. Muscle nuclei (myonuclei) maintain fiber size and function and could exhibit senescence-associated features; however, the prevalence of senescent myonuclei and whether they contribute to fast fiber atrophy in older adults remains unknown. Vastus lateralis biopsies from 20 young (19-34yr; 10 females) and 20 older (65-84yr; 10 females) adults were analyzed via immunohistochemistry for myonuclei positive for {gamma}H2AX ({gamma}H2AX+) and negative for HMGB1 (HMGB1-). MyHC II cross-sectional area (CSA) was [~]70% larger in young compared with old, whereas MyHC I CSA did not differ with age. The relative abundance of {gamma}H2AX+/HMGB1- myonuclei did not differ with age and was not associated with CSA in either fiber type. Single-nucleus RNA-sequencing corroborated no age-related difference in the prevalence of myonuclei with senescence-associated features. Myonuclear content of MyHC II fibers was [~]30% higher in young compared with old and was closely associated with CSA in both fiber types. Size-cluster analysis revealed a pronounced age-related leftward shift in MyHC II CSA that paralleled the reductions in myonuclear number, consistent with myonuclear loss. These data suggest that age-related fast fiber atrophy is not attributed to an increased prevalence of senescent myonuclei but instead occurs concomitantly with fiber type-specific myonuclear loss across the lifespan.

Across normal aging, striated muscles undergo structural remodeling associated with loss of force production. However, it is unknown how the organization of contractile myofibrillar networks, linked to their efficiency, is modified during aging. Using serial block-face scanning electron microscopy (SBF-SEM), we assess myofibril size, shape, and connectivity across different muscle types in young and geriatric mice and humans. Regardless of skeletal muscle fiber type in mice, age was associated with increased myofibrillar connectivity, with 24 months of age, as compared to 3 months, displaying more sarcomere branches. Distinctive age-related trends in myofibril size and shape were observed among each muscle type. Notably, there was a decrease in myofibril circularity from 3 months of age to 24 months of age in the gastrocnemius muscles of mice, contrasting with an increase in circularity in the soleus muscles during the same time frame. Additionally, while the soleus myofibrils in an aged cohort had a higher cross-sectional area, a reduction was observed in the gastrocnemius muscles. Cardiac muscles displayed no changes in sarcomere connectivity from 3 months to 24 months, although myofibril circularity and cross-sectional area were increased during this time. In human vastus lateralis muscles, sarcomere branching was positively correlated with advanced age. However, there were no consistent changes in myofibril size or shape across a wide age range from 16 to 68 years old. Overall, these data suggest that aging is associated with increased connectivity of the contractile networks within mammalian skeletal muscle. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/615981v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1b06d92org.highwire.dtl.DTLVardef@1e99c7eorg.highwire.dtl.DTLVardef@2151b3org.highwire.dtl.DTLVardef@17ac3f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundGait is a clinically relevant indicator of functional decline in aging populations. However, most studies classify older adults by chronological rather than functional age, which may obscure early impairments detectable through kinematic profiling. This study examined whether stratifying older adults by functional status using the Short Physical Performance Battery (SPPB) enhances sensitivity in detecting gait abnormalities and instability-related compensatory patterns. MethodsA total of 190 adults completed gait trials on a pressure-sensitive walkway. Twenty-eight spatial, temporal, and variability-based gait parameters were derived. Participants were categorized as young adults or older adults, who were further stratified into high- and low-functioning groups based on SPPB scores. Analysis of covariance (ANCOVA) was performed, adjusting for habitual walking speed to isolate functional effects. FindingsAfter adjusting for speed, the low-functioning group demonstrated longer stance and double-support durations, wider step width, and greater step-to-step variability in both spatial and temporal domains compared with both the high-functioning and young reference groups. These findings indicate a compensatory, instability-driven control strategy that challenges the assumption of a "slower but steady" gait in aging. High-functioning older adults exhibited gait patterns more closely resembling those of younger adults. InterpretationFunctional classification using the SPPB provided greater sensitivity than chronological age in detecting early mobility decline. Gait variability emerged as a salient biomarker of impaired neuromuscular control. Integrating quantitative gait profiling with validated functional assessments may improve early screening, targeted intervention, and fall prevention strategies.

As epigenetic clocks have evolved from powerful estimators of chronological aging to predictors of mortality and disease risk, it begs the question of what role DNA methylation plays in the aging process. We hypothesize that while it has the potential to serve as an informative biomarker, DNA methylation could also be a key to understanding the biology entangled between aging, (de)differentiation, and epigenetic reprogramming. Here we use an unsupervised approach to analyze time associated DNA methylation from both in vivo and in vitro samples to measure an underlying signal that ties these phenomena together. We identify a methylation pattern shared across all three, as well as a signal that tracks aging in tissues but appears refractory to reprogramming, suggesting that aging and reprogramming may not be fully mirrored processes.

A post-hoc analysis of the phase 2 data was performed for the SARS-COV-2 subunit protein vaccine MVC-COV1901. Anti-spike IgG, neutralization assays with live virus and pseudovirus were used to demonstrate age-dependent vaccine-induced antibody response to the vaccine. Results showed that an association exists between age and immune responses to the vaccine, providing further support for the need of booster shots, especially for the older age groups.

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.

Autophagy is widely proposed to decline with age; however, direct evidence for this across cell and tissue types in humans remains limited. Furthermore, it remains unknown whether interventions that improve physiological health during aging can modify autophagic activity in humans. Here, we performed transcriptomic and functional autophagy analyses across subject-matched human cell types from a healthy aging cohort spanning the adult lifespan. RNA-seq of primary dermal fibroblasts and induced neurons (iNs) revealed increased transcription of many autophagy-related genes with age, most markedly in fibroblasts. The impact of age on autophagic activity, measured using autophagy flux assays, was cell type- and sex-dependent, and uncoupled from autophagy-gene transcription. Autophagy flux decreased with age in male fibroblasts, was unchanged in female fibroblasts, and increased in female iNs. In freshly isolated peripheral blood mononuclear cells (PBMCs), autophagy flux became more heterogeneous with age and trended higher in older individuals, independent of sex. Although autophagy flux levels did not match across different cell types, higher autophagy flux in all cell types was associated with reduced physical function in older adults ([&ge;]70 years). Importantly, autophagy flux decreased following 12 weeks of mild exercise in parallel with improved physical function. These findings indicate that autophagy is regulated in a cell type-, sex-and physiological function-dependent manner during human aging, and highlight PBMC autophagy flux as a potentially modifiable, blood-accessible readout of physiological state in older adults.