Longevity Papers

Autophagy and cellular senescence are fundamental stress-response programs that critically shape aging and disease progression, yet their functional relationship has remained paradoxical. Autophagy is traditionally viewed as a cytoprotective process that preserves cellular homeostasis and delays senescence. In contrast, emerging evidence demonstrates that autophagy is also indispensable for the survival and pathological activity of established senescent cells. In this review, we propose a "threshold model" to reconcile these opposing roles and to provide a unified framework linking signal transduction, organelle quality control, and therapeutic intervention. According to this model, autophagy exerts stage-dependent functions governed by stress intensity and disease progression. Below a critical damage threshold, robust autophagic flux suppresses senescence initiation by maintaining mitochondrial integrity, limiting oxidative stress, and preserving proteostasis. Once this threshold is exceeded, autophagy is functionally reprogrammed to sustain the metabolic and biosynthetic demands of senescent cells, including production of the senescence-associated secretory phenotype (SASP). We highlight key signaling nodes that regulate this transition, including mTORC1, AMPK, p53, and p62, as well as spatial and organelle-specific mechanisms such as the TOR-autophagy spatial coupling compartment (TASCC), mitophagy failure, lipophagy blockade, and aberrant nucleophagy. These processes converge on innate immune pathways, notably cGAS-STING and NF-κB signaling, to drive chronic inflammation and tissue dysfunction. Importantly, we extend this mechanistic framework to clinical translation, synthesizing evidence from ongoing trials in cancer, neurodegeneration, metabolic liver disease, and fibrosis. We argue that effective targeting of the autophagy-senescence axis requires precision gerontology, integrating dynamic biomarkers to guide stage-specific interventions-autophagy activation for prevention and autophagy inhibition or senolysis for established disease. This threshold-based perspective provides a rational foundation for next-generation therapeutic strategies targeting aging and age-related disorders.

Somatic mutations in mitochondrial DNA (mtDNA) provide natural barcodes that enable engineering-free lineage tracing in human tissues, but the complex dynamics of mtDNA inheritance across cell divisions and incomplete sampling of mtDNA introduce uncertainty in reconstructed lineages. Here, we present MitoDrift, a probabilistic framework that integrates Wright-Fisher drift dynamics with sparse single-cell measurements to produce confidence-refined lineage trees enriched for accurate clonal relationships. Validation with gold-standard lentiviral barcoding and whole-genome sequencing demonstrates that MitoDrift outperforms existing tree reconstruction methods in precision while maintaining high clonal recovery, enabling robust analyses linking lineage to cell state. Applying MitoDrift to human hematopoiesis reveals an age-associated decline in clonal diversity with differential impact across cell types and identifies heritable regulatory programs in hematopoietic stem cells in vivo, linking AP-1/stress-associated programs to clonal expansions. In multiple myeloma, MitoDrift captures therapy-associated clonal remodeling undetectable by copy number analysis, revealing phenotypic transitions and linking gene regulatory programs to differential drug sensitivity. Collectively, MitoDrift enables high-precision lineage tracing at scale and establishes quantitative lineage-state analysis in primary human tissues, linking clonal history to transcriptional and epigenetic programs in tissue homeostasis, aging, and disease.

Aging is characterized by the progressive loss of physiological integrity, leading to impaired tissue function and increased vulnerability to chronic diseases. Although the Hedgehog (Hh) signaling pathway is well established as a key regulator of embryonic development and tumorigenesis, emerging evidence suggests it also plays vital roles in adult tissue maintenance, regeneration and immune modulation-processes that are intimately linked to aging. Here we synthesize recent findings demonstrating that the controlled activation of Hh signaling across diverse tissues, including the brain, liver, heart, lung, bone, skin and adipose tissue, can counteract hallmark features of aging such as stem cell exhaustion, mitochondrial dysfunction and chronic inflammation. In preclinical models, Hh pathway modulation enhances tissue regeneration, supports progenitor cell function and suppresses senescence-associated secretory phenotypes. Promising therapeutic strategies-ranging from gene delivery to pharmacological agonists-have shown efficacy in mitigating age-related decline, though challenges remain regarding tissue specificity, long-term safety and tumorigenic risk. By integrating insights from developmental biology, regenerative medicine and geroscience, this Review positions Hh signaling as a compelling target for anti-aging interventions aimed at preserving organ function and extending healthspan.

A mild impairment of mitochondrial function activates the hypoxia inducible factor (HIF-1)-mediated hypoxia stress response pathway leading to a HIF-1-dependent increase in lifespan. Lifespan extension resulting from HIF-1 stabilization is dependent on activation of flavin-containing monooxygenase-2 (FMO-2). In this work, we explored the role of fmo-2 in the long lifespan of genetic mitochondrial mutants in C. elegans. We found that fmo-2, but not other fmo genes, are specifically upregulated in the long-lived mitochondrial mutants clk-1, isp-1 and nuo-6. Disruption of fmo-2 through RNA interference or genetic mutation shortens the lifespan of these mitochondrial mutants indicating that fmo-2 is required for lifespan extension in these worms. Moreover, signaling molecules that have been shown to be involved in upregulation of fmo-2 are also required for the long life of clk-1, isp-1 and nuo-6 mutants including HLH-30, NHR-49 and MDT-15. Finally, we examined the effect of multiple lifespan-promoting pathways in clk-1 mutants on the expression of fmo-2. We found that in all cases, genes required for clk-1 longevity are also required for the upregulation of fmo-2 in clk-1 worms. These genes included DAF-16, PMK-1, SKN-1, CEH-23, AAK-2, HIF-1 and ELT-2. Combined, this work advances our understanding of the molecular mechanisms contributing to longevity in the long-lived mitochondrial mutants and identifies FMO-2 as a common downstream effector of multiple pathways that modulate longevity.

Age-related skeletal muscle decline (sarcopenia) is a major contributor to frailty and mortality during aging, yet the extent to which sex shapes muscle aging and its response to dietary interventions remains poorly understood. Here, we use the short-lived vertebrate Nothobranchius furzeri (African turquoise killifish; ATK) to investigate how sex and intermittent fasting (IF) interact to regulate lifespan and skeletal-muscle aging. We establish and optimize an IF regimen that significantly extends lifespan in both male and female killifish, albeit with classical trade-offs including reduced growth and reproductive output. Despite these costs, IF markedly improves swimming performance in aged animals of both sexes. Structural analyses of killifish on a normal diet reveal pronounced sexual dimorphism in muscle aging. Males exhibit age-associated myofiber hyperplasia, whereas females maintain fiber number but undergo hypertrophic remodeling. IF partially reverses both phenotypes, restoring a more youthful fiber size distribution in both males and females. Single-nucleus RNA sequencing uncovers sex-specific remodeling of muscle-fiber composition in killifish on a normal diet, with females displaying an age-associated shift toward oxidative slow-twitch fibers that is reversed by IF, while males show relatively stable fiber-type proportions under normal and IF feeding regimens. Cell-cell communication analyses further reveal a global decline in intercellular signaling with age, alongside sex-specific restoration of distinct pathways under IF, including axon guidance and IGF signaling in females and metabolic ANGPTL signaling in males. Finally, bulk transcriptomic profiling demonstrates that aging follows largely sexually dimorphic molecular trajectories, whereas IF induces both sex-specific and shared responses. Notably, under IF, both sexes exhibit upregulation of ribosome biogenesis and genes supporting myofibrillar organization and contraction, likely underlying preserved muscle function. Together, these findings demonstrate that IF promotes longevity and muscle health through conserved anabolic mechanisms alongside sex-specific cellular and molecular rejuvenation strategies. Our work highlights the importance of incorporating sex as a biological variable when designing dietary interventions to promote healthy aging.

Understanding the links between metabolism, ageing and age-related phenotypes may clarify the role of ageing in disease onset and improve risk prediction. We conducted a cross-cohort assessment of biological age using broad-spectrum LC-MS metabolomics in 2,295 participants, aged 20-89, from the UK Airwave study (N=960) and The Irish Longitudinal Study of Ageing (N=1,335). N2,N2-dimethylguanosine, C-glycosyltryptophan, bile acid glucuronides, and zeta-carotene were associated with chronological age, frailty, and mortality. We developed a metabolomic clock that was highly predictive of chronological age (r = 0.92) in test samples. Metabolomic age acceleration was strongly correlated between study visits (r > 0.6). Each standard deviation higher metabolomic age acceleration (~5 years) was associated with 43% higher mortality risk, 27% higher risk of mild cognitive impairment, and 10% increased risk of a higher frailty score in fully adjusted models. Our metabolomic clock provides a reproducible marker of generalised age-related disease risk.

Ageing is commonly viewed as a consequence of cumulative molecular damage and declining stress resilience. However, this perspective overlooks the possibility that ageing may primarily arise from an efficiency loss of integrated cellular operation. Here, we show that ageing can be systemically prevented by enhancing cellular efficiency using frequency-specific mid-infrared (MIR) light, as revealed by multiscale (organismal-, cellular- and molecular-level) analyses of Caenorhabditis elegans. Remarkably, super-weak 34-THz MIR light (~1 W mm-2) prolonged the median lifespan of worms by 60%, delaying ageing onset and preventing abrupt "cliff-edge" mortality, without detectable thermal effects. At the cellular level, MIR exposure enhanced global gene transcription in parallel with the establishment of a mitochondrial state of high-efficiency energy metabolism, together preserving youthful cellular homoeostasis during ageing. These cellular effects were associated with vibrational modes of phosphate groups (PO4) in nucleic acids and mitochondrial phospholipids within the 33-35 THz range, providing a frequency-matched molecular context for MIR modulation. Together, our results support an efficiency-first, systemic anti-ageing model in which frequency-specific MIR light resonantly enhances the integrated kinetic efficiency of cellular systems, spanning gene transcriptional dynamics and mitochondrial bioenergetic flux, rather than the activation of damage-repair or stress-response pathways. These findings advance our understanding of MIR light-matter interactions in living systems and establish a non-biochemical, physical strategy for modulating ageing.

Since the beginning of this century, the emergence of systems biology, driven by technological, informatic, and theoretical advances, has led to an unprecedented generation of data and information about biological systems at multiple levels of organization. We now have access not only to components of living systems but also to some of the underlying principles governing their organization within networks. This review focuses on the systems biology of aging, metabolism, and mitochondria, along with the integration of experimental and computational systems biology approaches as applied to multilayered biological networks, spanning from the molecular-subcellular to the whole organism. Sections 2 and 3 provide an overview of the insights gained from systems biology and multi-omics approaches as applied to aging and metabolism. Using the spatiotemporal dynamics of biological networks as a unifying thread, Sections 4 and 5 explore how systems biology and current methods can leverage the understanding of complex biological phenomena through integrated experimental-computational strategies, utilizing iterative, verification-validation loops between experiments and models. Section 6 concludes by highlighting the autonomously dynamic, self-organizing, and self-regulating integrative nature of living systems and the need to address these properties at the emerging convergence of biology, medicine, physics, and powerful computational technologies that include artificial intelligence.

Background: For decades, computational biology has failed to create unified models where metabolic state and regulatory control are bidirectionally coupled: metabolic models optimize flux but cannot represent dynamic regulation, while regulatory models treat ATP as a fixed parameter rather than a dynamic variable affected by pathway activity. This fundamental limitation prevents computational recapitulation of emergent threshold behaviors-spontaneous homeostasis, adaptive reorganization, pathway switching-observed in living organisms. The challenge requires formalisms where (1) metabolic state governs regulatory decisions AND (2) regulatory choices consume metabolic resources, producing emergent dynamics from feedback rather than programming. Methods: We introduce Signal Hierarchical Petri Nets, extending Hybrid Petri Nets with bidirectional metabolic-regulatory coupling through energy-dependent layer organization. Unlike classical approaches, ATP is simultaneously a regulatory signal (governing pathway availability through quantitative thresholds) and a material substrate (consumed by pathway activity). When ATP depletes below 1000 M, high-cost pathways automatically become unavailable; pathway activity consuming ATP creates feedback affecting subsequent pathway accessibility. This bidirectional coupling enables emergent threshold behaviors impossible in classical formalisms. We demonstrate the paradigm through macrocyclic peptide transport across 53 metabolic conditions, where drug accumulation depends on ATP-governed pathway reorganization. Results: The formalism produces three emergent behaviors never achieved in unified metabolic-regulatory models. (1) Spontaneous homeostasis without programming: Despite 113-fold permeability variation from N-methylation, ATP-replete cells maintain constant drug accumulation (CV=0.066%)-homeostatic compensation emerges from ATP-consumption feedback, not explicit control logic. (2) Threshold-triggered reorganization: ATP depletion to 300 M triggers 8533-fold active-to-passive transport shifts with paradoxical 141% accumulation increase from efflux collapse. (3) Tissue-specific dynamics from identical parameters: Tumor hypoxia (ATP=1200 M) versus normal tissue (ATP=5000 M) produces 6.62-fold selectivity differences from differential pathway accessibility-same model, different emergent outcomes. Computational predictions achieve r=0.911 correlation with experimental cyclosporin permeability (n=32). Conclusions: Signal Hierarchical Petri Nets represent the first computational formalism achieving emergent threshold dynamics through bidirectional metabolic-regulatory coupling. The paradigm enables in silico recapitulation of adaptive cellular behaviors previously impossible to model, with applications extending beyond drug transport to any biological system where metabolic state governs regulatory reorganization: cancer metabolism, ischemia, synthetic biology, and aging research.