Longevity Papers

Aging is the strongest risk factor for chronic diseases such as cardiovascular disease, Alzheimer's, and cancer. DNA methylation (DNAm) clocks offer a promising measure of biological age, but most rely on linear models that miss non-linear dynamics and CpG interactions. To address this, we developed a deep neural network (DNN)-based DNAm clock trained on 29,167 samples profiled on Illumina EPIC v1.0 and v2.0 arrays. Using 12,234 CpGs selected through sex- and age-stratified correlations, our model achieved high accuracy (1.89 years) and outperformed published deep learning and elastic net based epigenetic clocks in a separate validation cohort. Using Shapley Additive Explanations (SHAP), we further uncovered phase-structured, wave-like dynamics in age-influential CpGs: an early-life module, a midlife transition, and late-life remodeling, with distinct timings by sex. These epigenetic waves cohere with non-linear, multi-omic "aging waves" reported in proteomics and longitudinal omics. SHAP further enabled interpretable CpG attribution, revealing structured, sex-specific aging phases: early-life male clocks involved developmental pathways, while female clocks emphasized cytoskeletal regulation; late-life divergence included immune activation in males and transcriptional remodeling in females. Our framework thus unites accuracy with mechanistic interpretability, revealing sex-specific windows when molecular aging reconfigures most rapidly.

Mapping behavior of individual vertebrate animals across lifespan could provide an unprecedented view into the lifelong process of aging. We created a platform for high-resolution continuous behavioral tracking of the African killifish across natural lifespan from adolescence to death. We found that animals follow distinct individual aging trajectories. The behaviors of long-lived animals differed markedly from those of short-lived animals, even relatively early in life, and were linked to organ-specific transcriptomic shifts. Machine-learning models accurately inferred age and even forecasted an individual's future lifespan, given only behavior at a young age. Finally, we found that animals progressed through adulthood in a sequence of stable and stereotyped behavioral stages with abrupt transitions, revealing precise structure for an architecture of aging.

Cellular senescence, originally described as a finite proliferative arrest in cultured somatic cells, has since been recognized as a central mechanism underlying aging and the development of age-associated disorders. The progressive accumulation of senescent cells (SnCs) promotes chronic inflammation through the senescence-associated secretory phenotype (SASP) and circumvents immune-mediated clearance by upregulating pro-survival and immune checkpoint pathways. Early "first-generation" senolytics, including navitoclax (ABT-263) and the dasatinib-quercetin (D + Q) combination, provided proof-of-concept that selective removal of SnCs can alleviate certain fibrotic, metabolic, and cardiovascular pathologies in preclinical studies. However, these agents exhibited notable drawbacks, such as dose-dependent thrombocytopenia, variable therapeutic efficacy, and the emergence of resistance mechanisms. Consequently, current research has shifted toward precision senotherapy, though significant translational challenges remain. This review synthesizes three next-generation strategies developed to address limitations of early senolytic agents. (1) Immune-based senolysis: This approach applies immuno-oncology principles to counter immune evasion of SnCs. Strategies include blocking immunosuppressive ligands such as GD3 ganglioside, engineering chimeric antigen receptor (CAR) T cells to target senescence-specific surface markers like urokinase-type plasminogen activator receptor (uPAR), and exploiting metabolic vulnerabilities (e.g., glutaminolysis and ferroptosis) to sensitize SnCs to immune-mediated clearance. (2) Tissue-precision proteolysis-targeting chimeras (PROTACs): These agents recruit organ- or tissue-specific E3 ligases (e.g., von Hippel-Lindau (VHL)) to selectively degrade anti-apoptotic proteins such as BCL-xL. Localized activity may reduce systemic toxicity and mitigate dose-limiting effects observed with traditional inhibitors. (3) Microbiome-epigenetic interplay: This strategy modulates the gut-liver axis to enhance senolytic efficacy. Short-chain fatty acids (SCFAs), such as butyrate, epigenetically regulate drug transporter expression and suppress the SASP, while dietary interventions may create a microenvironment favorable to senolysis. These approaches offer potentially more targeted and personalized therapeutic options but face significant challenges, including immunopathology, manufacturing complexity, off-target effects, and long-term safety concerns. The ongoing shift from broad inhibition to precision reprogramming represents a promising but preliminary step in the treatment of age-related diseases.

Aging is accompanied by profound changes in both the gut microbiome and the immune system, which engage in continuous, bidirectional communication. Alterations in microbial diversity and metabolism, particularly reductions in short-chain fatty acid (SCFA) producers as well as shifts in bile acid and tryptophan-metabolizing species, can incite and worsen inflammation, damage barrier integrity, and accelerate immunosenescence. Concomitantly, immune aging and reduced mucosal IgA promote microbial dysbiosis, forming a self-reinforcing cycle that fuels chronic inflammation ("inflammaging"). Microbial metabolites such as SCFAs, secondary bile acids, and indole derivatives play central roles in this gut-immune dialog, influencing regulatory T-cell balance, epithelial repair, and neurological health through the gut-brain axis. Emerging evidence suggests that diet, probiotics, postbiotics, and microbiome transplantations can restore beneficial microbial and, consequently, immune functions, offering opportunities to promote healthy aging and potentially reverse adverse symptoms. Understanding and targeting the gut microbiome-immune feedback loops may reveal new strategies to modulate inflammaging and extend health span.

Ageing biomarkers can predict mortality risk beyond chronological age. Recently, plasma proteins were used to estimate the biological ages of eleven human organs, including the brain, heart, liver, kidneys, and pancreas. Accelerated organ ageing is linked to higher all-cause mortality; however, systematic benchmarking against established ageing biomarkers is lacking. Here, we pursued two complementary aims. First, we benchmarked proteomic organ ages against multimodal ageing biomarkers for all-cause mortality (444 deaths; [≤]17-year follow-up) using Cox regression in 861 Lothian Birth Cohort 1936 (LBC1936) participants. Ageing biomarkers included epigenetic age (GrimAge2), telomere length, neuroimaging, general cognitive function (g), and physical function (grip strength, walk time, and respiratory function). Among proteomic organ ageing biomarkers, accelerated liver (HRperSD [95% CI] = 1.43 [1.30-1.58]), immune (1.42 [1.29-1.57]), and heart (1.38 [1.25-1.53]) ageing were most strongly associated with higher mortality risk. However, GrimAge2 acceleration, total brain volume (TBV), grey matter volume, respiratory function, and g exhibited higher hazard estimates (HRperSD = 1.44-1.62) than organ ageing biomarkers. In a Cox model including all biomarkers, only TBV, white matter hyperintensity volume, g, and walk time associated with mortality. Second, survival analyses of SomaScan 11K plasma proteins identified 202 proteins associated with mortality and enriched for the liver and immune-related biological processes, with the strongest effects observed for GDF15 (HRperSD [95% CI] = 1.53 [1.37-1.72]), CST3 (1.48 [1.29-1.69]), and COL18A1 (1.47 [1.30-1.68]). These findings provide a systematic, cross-modal benchmarking of proteomic organ ages against established ageing biomarkers and highlight plasma proteomic signatures of mortality.

Aging impairs cartilage repair, with young animals exhibiting superior regenerative capacity due to enhanced tissue repairing and reduced inflammation compared to aged counterparts. This study employed single-cell omics to dissect age-dependent immune cell heterogeneity in cartilage injury, revealing a critical deficiency in anti-inflammation macrophage subsets in aged animals. We identified Arg-1 as a central regulator of macrophage polarization, demonstrating that its overexpression rescues impaired repair in aged animals. These findings establish Arg-1 as a novel therapeutic target to counteract age-related declines in cartilage regeneration, offering new insights into macrophage-driven tissue repair mechanisms. The integration of single-cell analysis with functional validation provides a framework for developing precision interventions for age-impaired tissue regeneration.

Critical segmental bone defects in elderly patients pose a formidable clinical challenge due to limited autograft availability, senescent bone dysfunction, and compromised healing from fibrous tissue invasion. Here, we present a development-based in vivo bioreactor strategy wherein BMP-2-loaded biomaterials trigger the body's intrinsic developmental programs, using the organism as a bioreactor to engineer bone. Distinct from classical developmental engineering, this in vivo bioreactor-derived bone (vBR-Bone) recapitulates native osseous architecture, including vasculature, cortical bone, trabeculae, and bone marrow niche. In aged murine models, the vBR-Bone exhibits a rejuvenated restoration of bone bioactivity lost in aging, with reduced senescence, elevated remodeling, and improved stem cell functionality. Capitalizing on its restored remodeling capacity of high bone turnover, the vBR-Bone fragments enclosed in an asymmetric biomimetic periosteum achieved 6-week repair of critical-sized 1/3 femoral shaft segmental defects. Through a "compartmentalized" approach that partitions the defect into manageable fragments, vBR-Bone progressively remodeled and integrated into functional trabecular bone, ultimately restoring bone mineral density, volume, and microstructure in defects of aged mice. The biomimetic periosteum inhibits fibrous invasion while permitting vascular ingrowth, thereby creating a space for regeneration. Mechanistically, the multifactors within vBR-Bone reconstitute a bone-remodeling microenvironment, wherein matrix-released TGF-β1 activates the PI3K/AKT/mTOR signaling axis via TRAF6-dependent ubiquitylation to promote robust osteogenesis. This strategy overcomes autograft shortage and senescence-associated dysfunction, offering a clinically translatable solution for critical age-related segmental bone defects.

The human testis, a crucial organ in male reproduction, is mainly responsible for spermatogenesis and androgen production. As men age, testicular function declines, yet the underlying molecular mechanisms are still not well understood. To elucidate the mechanisms of human testicular aging, we performed an integrated analysis of single-nucleus transcriptomes and chromatin accessibility (snRNA-seq and snATAC-seq) on pathologically confirmed non-tumor testicular tissues from young and aged individuals. Our integrated multi-omic analysis reveals significant age-induced alterations in the testis transcriptome, particularly affecting genes linked to spermatogenesis. Significant age-related changes were also observed in Sertoli and Leydig cells, with Sertoli cells exhibiting increased sensitivity to environmental influences as age. Furthermore, the expression of Wntless (WLS), a Wnt transporter, was substantially upregulated in Sertoli cells of aged testes, which was correlated with cellular senescence and the disruption of tight junctions. Overexpression of WLS in Sertoli cells significantly accelerated senescence in vitro, implying a potential role for WLS in testicular aging. Our study provides a detailed multi-omic map of the transcriptomic and chromatin accessibility changes in the human testis during aging, offering insights into the cellular and molecular mechanisms behind these changes, and identifying potential therapeutic targets for interventions against age-related declines in male reproductive health.

Brain vascular aging is increasingly recognized as a critical therapeutic target for age-related cognitive decline. Oxidative stress, bioenergetic dysfunction, and molecular damage play central roles in the progression of vascular aging, contributing to cerebrovascular dysfunction and impaired cognitive function. While naturally occurring polyphenols such as resveratrol (RSV) have demonstrated potential in mitigating aging-related pathologies, their poor bioavailability and limited brain targeting efficiency significantly constrain their therapeutic impact. As a result, high doses or advanced drug delivery strategies are necessary to achieve meaningful physiological effects. We introduce a novel nanocarrier system designed to enhance RSV delivery to the cerebral endothelium by leveraging the natural formation of an apolipoprotein E (ApoE)-enriched protein corona around fusogenic liposomes (FL) in vivo. These nanoparticles directly fuse with cytoplasmic cell membranes and thus evade endocytosis. We found that once in the circulation FL spontaneously acquire a protein corona, which is highly enriched in ApoE, a key ligand for brain endothelial low-density lipoprotein receptors (LDLR). Based on this observation, we engineered an ApoE-functionalized protein corona around FL (ApoE-FL) to systematically evaluate whether this mechanism could be exploited for targeted brain delivery. Following optimization and physicochemical characterization, the RSV-loaded liposomes were evaluated in vitro using human cerebral microvascular endothelial cells and in vivo C57BL/6 aged mice to assess their therapeutic potential. Both FL and engineered ApoE-FL liposomal delivery systems exhibited a strong affinity for endothelial cell membranes in vitro. The knockdown of the ApoE receptor, low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced liposomal docking. Microscopy analysis revealed that both ApoE-FL and non-functionalized FL directly fused with endothelial plasma membranes, thus bypassing intracellular organelles and minimizing lysosomal degradation. This suggests that the naturally formed ApoE corona in vivo may contribute to efficient cerebrovascular targeting, a property successfully replicated by the engineered ApoE corona strategy. In vivo biodistribution and kinetic studies demonstrated that especially ApoE-FL achieved enhanced brain-targeting efficiency, prolonged cerebrovascular retention, and extended targeting distance along the arteriovenous axis. This emphasizes that fusogenic liposomes effectively engage almost the entire microvascular network, including capillaries and post-capillary venules. Functionally, fusogenic liposome-delivered RSV improved blood-brain barrier (BBB) integrity, enhanced neurovascular coupling (NVC) responses, and promoted brain vascularization in aged mice. Single-cell RNA sequencing (scRNA-seq) revealed enhanced endothelial angiogenesis and barrier protective transcriptional profiles in cerebrovascular cells treated with ApoE-FL/RSV, suggesting a molecular basis for the observed vascular benefits. Liposomal RSV delivery achieved near-complete cerebrovascular and cognitive rejuvenation in aged mice applying a 2000-fold lower RSV dose than oral administration used as control sample. Thus, ApoE-FL liposomes exhibited exceptionally high delivery efficiency in deeper brain regions, further expanding their therapeutic potential. These findings underscore the importance of targeted drug delivery in optimizing therapeutic outcomes and establish ApoE-functionalized fusogenic liposomes as a promising strategy for mitigating brain vascular aging and cognitive decline.