Longevity Papers

Vascular aging contributes to multisystem diseases and limits health span. Although various animal models have contributed to aging research, their vasculatures poorly recapitulate human physiology. Even existing tissue-engineered blood vessels fail to mimic human vascular function and pathology, hindering translational advances in vascular aging studies. Here, we present a novel human physiological vascular model fabricated via the unique molding-induced circumferential alignment of human induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells with luminally seeded endothelial cells. This architecture enabled dynamic vasodiameter changes in response to vasoactive stimuli, including hormones and intraluminal pressure. Using iPSCs from a patient with Werner syndrome, the model recapitulated aging-associated phenotypes, such as hypercontractility and increased vascular compliance, possibly due to impaired nitric oxide bioavailability. Transcriptomic and metabolomic analyses revealed age-related dysregulation consistent with vascular senescence. As a key advantage of the vasculature, spatial transcriptomic analysis demonstrated upregulation of the aging marker CDKN1A near the lumen and downregulation of COL6A1 and TPM1 throughout the vessel. Treatment with mitochonic acid 5, a mitochondria-targeted compound, significantly reversed the aging phenotypes. These findings demonstrate that our engineered vascular model recapitulates key aspects of human vascular properties and provides a platform for mechanistic studies of vascular aging and drug discovery aimed at extending health span.

Epitalon, a naturally occurring tetrapeptide, is known for its anti-aging effects on mammalian cells. This happens through the induction of telomerase enzyme activity, resulting in the extension of telomere length. A strong link exists between telomere length and aging-related diseases. Therefore, telomeres are considered to be one of the biomarkers of aging, and increasing or maintaining telomere length may contribute to healthy aging and longevity. Epitalon has been the subject of several anti-aging studies however, quantitative data on the biomolecular pathway leading to telomere length increase, hTERT mRNA expression, telomerase enzyme activity, and ALT activation have not been extensively studied in different cell types. In this article, the breast cancer cell lines 21NT, BT474, and normal epithelial and fibroblast cells were treated with epitalon then DNA, RNA, and proteins were extracted. qPCR and Immunofluorescence analysis demonstrated dose-dependent telomere length extension in normal cells through hTERT and telomerase upregulation. In cancer cells, significant telomere length extension also occurred through ALT (Alternative Lengthening of Telomeres) activation. Only a minor increase in ALT activity was observed in Normal cells, thereby showing that it was specific to cancer cells. Our data suggests that epitalon can extend telomere length in normal healthy mammalian cells through the upregulation of hTERT mRNA expression and telomerase enzyme activity.

Mitochondrial sirtuins regulate metabolism and are emerging drug targets for metabolic and age-related diseases such as cancer, diabetes, and neurodegeneration. Yet, the extent of their functions remain unclear. Here, we uncover a physiological role for the C. elegans mitochondrial sirtuins, sir-2.2 and sir-2.3, in lifespan regulation. Using genetic alleles with deletions that destroy catalytic activity, we demonstrate that sir-2.2 and sir-2.3 mutants live an average of 25% longer than controls when fed the normal lab diet of live E. coli OP50. While decreased consumption of food is a known mechanism for lifespan extension, we did not find evidence of reduced pharyngeal pumping. Interestingly, lifespan extension effected by loss of sir-2.2 or sir-2.3 is sensitive to the diet. The lifespan extension of the sir-2.2 mutants is eliminated and that of sir-2.3 mutants is attenuated when the animals are fed the E. coli strain HT115, which is typically used for RNAi experiments. We used growth ability of the food source and a virulent pathogenic strain to ask if differences in pathogenicity are related to the mechanisms for lifespan extension. sir-2.3 deletion results in lifespan extension in all conditions. However, removing the ability of the food source to grow eliminated the sir-2-mediated effect. We also examine the response of the mutants to oxidative stress, and our results suggest that a hormetic response contributes to lifespan extension in both mutants. Our data suggest that sir-2.2 and sir-2.3 use overlapping yet distinct mechanisms for regulating lifespan.

Cellular senescence, a hallmark of aging, is characterized by irreversible, permanent cell cycle arrest accompanied by halted proliferation triggered by endogenous or exogenous stimuli. The accumulation of senescent cells in tissues or organs elicits detrimental effects on adjacent normal cells through their pathogenic senescence‑associated secretory phenotype (SASP), driving secondary senescence, disrupting tissue homeostasis and ultimately exacerbating age‑related pathologies such as types of cancer and neurodegenerative disorders. Hepatic disorders constitute a leading cause of global mortality, imposing considerable healthcare burdens. Robust clinical evidence has now demonstrated a strong correlation between cellular senescence and poor clinical outcomes in various hepatopathies. This intricate yet critical signaling network is dynamically regulated in both physiological homeostasis and chronic hepatic inflammatory conditions. Notably, recent years have witnessed extensive research into pharmacological strategies to deplete senescent cells, inhibit SASP, and target other senescence markers across diverse contexts, thereby establishing the field of senotherapeutics. The present review systematically summarized key molecular pathways and biomarkers of hepatic senescence, while outlining the emerging role of cellular senescence in inflammatory liver disorders. It also discussed the therapeutic potential of senescence‑regulating drugs for liver disease, which could alleviate hepatic inflammation and enhance clinical outcomes.

The accumulation of senescent cells (SNCs) contributes to tissue dysfunction and age-related diseases, creating an urgent need for effective senolytic strategies. We identified a metabolic vulnerability in SNCs characterized by marked downregulation of asparagine synthetase (ASNS), rendering them uniquely dependent on exogenous asparagine (Asn). This vulnerability was exploited through combined treatment with L-asparaginase (ASNase) and autophagy inhibitors, which synergistically deplete Asn via complementary mechanisms: ASNase degrades extracellular Asn pools, while autophagy inhibition blocks intracellular protein recycling as an alternative Asn source. This dual approach induced selective synthetic lethality across multiple SNC types in vitro. In aged mice, the combination therapy significantly reduced SNC burden in diverse tissues, improved physiological function, and attenuated progression of age-related conditions including osteoporosis, atherosclerosis, and non-alcoholic fatty liver disease. Our findings establish concurrent targeting of extracellular and intracellular Asn supplies as a potent, selective senolytic strategy with broad therapeutic potential for age-related disorders.

Smoking is one of the single most important preventable risk factors for cancer and other adverse health outcomes [1,2]. Smoking cessation represents a key public health intervention with the potential to reduce its negative health outcomes [2-4]. While epidemiological, cross-sectional, and individual longitudinal \'omic\' or biomarker studies have evaluated the impact of smoking cessation, no study to date has systematically profiled molecular and clinical changes in several organ systems or tissues longitudinally over the course of smoking cessation that could allow for more detailed assessment of response biomarkers and the identification of interindividual differences in the recovery of physiological functions. Here, we report the first human longitudinal multi-omic study of smoking cessation, evaluating 2,501 unique single or composite features from 1,094 longitudinal samples. Our comprehensive analysis, leveraging over half a million longitudinal data points, revealed a profound effect of smoking cessation on epigenetic biomarkers and microbiome features across multiple organ systems within 6 months of smoking cessation, alongside shifts in the immune and blood oxygenation system. Moreover, our multi-omic analysis provided unprecedented granularity that allows for identification of new cross-ome associations for mechanistic discovery. We anticipate that data and an interactive app from the Tyrol Lifestyle Atlas (eutops.github.io/lifestyle-atlas), comprising the current study and a parallel study arm evaluating the impact of diet on biomarkers of health and disease, will provide the basis for future discovery, biomarker benchmarking in their responsiveness to health-promoting interventions, and study of individualised response group, representing a major advance for personalised health monitoring using biomarkers.

Aging is a major risk factor for neurodegenerative diseases associated with protein aggregation, including Huntington's disease and amyotrophic lateral sclerosis (ALS). Although these diseases involve different aggregation-prone proteins, their common late onset suggests a link to converging changes resulting from aging. In this study, we found that age-associated hyperactivation of EPS8/RAC signaling in Caenorhabditis elegans promotes the pathological aggregation of Huntington's disease-related polyglutamine repeats and ALS-associated mutant FUS and TDP-43 variants. Conversely, knockdown of eps-8 or RAC orthologs prevents protein aggregation and subsequent deficits in neuronal function during aging. Similarly, inhibiting EPS8 signaling reduces protein aggregation and neurodegeneration in human cell models. We further identify the deubiquitinating enzyme USP4 as a regulator of EPS8 ubiquitination and degradation in both worms and human cells. Notably, reducing USP-4 upregulation during aging prevents EPS-8 accumulation, extends longevity and attenuates disease-related changes. Our findings suggest that targeting EPS8 and its regulatory mechanisms could provide therapeutic strategies for age-related diseases.

Age related diseases present disproportionately among African Americans and have been tied to broad social inequalities and accompanying stress. Yet, there is considerable variability among African Americans in susceptibility, highlighting potential connections to both intersectionality and stress-related biological processes. A growing body of research links exposure to racism and discrimination to telomere length (TL)-an indicator of biological aging that is increasingly implicated in explaining stress-related racial health disparities. However, few studies have examined links to accompanying stress processes that may precede TL shortening. This includes examining Uric Acid (UA), which growing evidence suggests may comprise a unique biological aspect of the acute stress response, with implications for both racial health disparities and within-race heterogeneity. In a secondary analysis of a sample of healthy African Americans (N = 103, 33 men; M age = 31.41 years), we assessed the relationship between salivary UA (sUA) and TL. With an eye towards within-group heterogeneity, we also considered the moderating role of age and gender. Our findings revealed a negative association between UA and TL that was most pronounced in African American men and among younger African Americans. We apply an intersectional lens to interpret these results, revealing that different intersections of identity operate through distinct mechanisms. Among men, UA consistently predicted shorter telomeres regardless of discrimination exposure, suggesting biological pathways may be primary. However, among women, the UA/TL relationship was moderated by discrimination-with UA positively predicting TL under low discrimination but showing negative associations under high discrimination conditions. These findings demonstrate that intersectionality operates through multiple pathways simultaneously, with some intersections characterized by biological vulnerabilities while others are defined by social moderation effects. Future research directions should consider the multifaceted influences of UA on TL, recognizing that different intersectional positions may require examination of distinct biological and social mechanisms including potential interventions targeting UA levels to mitigate age-related illnesses and address health disparities among African Americans. Additionally, future studies should examine how additional intersecting systems of oppression might moderate the relationship between UA and TL.

Vascular aging increases the susceptibility to cardio-cerebrovascular conditions, such as atherosclerotic diseases and hypertension, the leading causes of global disability and mortality. Dietary citrate extends the lifespan of Drosophila melanogaster and Caenorhabditis elegans as well as improves the memory of mice injured by a high-fat diet (HFD); whether it alleviates vascular aging and age-related vascular diseases; however, remains unknown. Here, we showed that dietary supplementation of citrate delayed vascular aging, as evidenced by maintaining the integrity of elastic fibers and decreasing the level of the aging-related marker, CDKN1A (p21). Functionally, citrate improved the sensitivity to endothelial-dependent vasodilators and lowered blood pressure, and in HFD-fed ApoE

Human and murine studies reveal that innate immune cells are able to mount enhanced responses to pathogens after primary inflammatory exposure. Innate immune memory has been shown to last for months to years, longer than the lifespan of most innate immune cells. Indeed, long-lived hematopoietic stem and progenitor cells (HSPCs) serve as a cellular reservoir for innate immune memory. In this review, we summarize the evidence that innate immune memory is epigenetically encoded in HSPCs, and we consider whether HSPC subpopulations with differentiation bias, cell autonomous epigenetic reprogramming, or both features underlie the phenomenon of central trained immunity. We further profile the significant implications of central trained immunity in stem cell transplant, aging, inflammatory diseases, and vaccination strategies for the future.

Dietary protein is a key regulator of metabolic health in humans and rodents. Many of the benefits of protein restriction are mediated by reduced consumption of dietary branched-chain amino acids (BCAAs; leucine, valine and isoleucine), and restriction of the BCAAs is sufficient to extend healthspan and lifespan in mice. While the BCAAs have often been considered as a group, it has become apparent that they have distinct metabolic roles, and we recently found that restriction of isoleucine is sufficient to extend the healthspan and lifespan of male and female mice. Here, we test the effect of lifelong restriction of the BCAA valine on healthy aging. We find that valine restriction (Val-R) improves metabolic health in C57BL/6J mice, promoting leanness and glycemic control in both sexes. To investigate the molecular mechanisms engaged by Val-R with aging, we conducted multi-tissue transcriptional profiling and gene network analysis. While Val-R had a significantly greater molecular impact in the liver, muscle, and brown adipose tissue of female mice than males, there was a stronger gene enrichment with phenotypic traits in male mice. Further, we found that phenotypic changes are associated with a multi-tissue downregulation of the longevity associated PI3K-Akt signaling pathway. Val-R reduces frailty in both sexes and extends the lifespan of male by 23%, but does not extend female lifespan, corresponding with a male-specific downregulation of PI3K-Akt signaling. Our results demonstrate that Val-R improves multiple aspects of healthspan in mice of both sexes and extends lifespan in males, suggests that interventions that mimic Val-R may have translational potential for aging and age-related diseases.

Over time, cells in the brain and in the body accumulate damage, which contributes to the ageing process

Research indicates a significant slowdown in life expectancy growth in the United States (US) post 2010, marking a departure from the consistent progress in longevity throughout the twentieth century. We extend this understanding, tracing the deceleration of US life expectancy back to the 1950s, after which average decadal change dropped from 3.80 to 1.61 years. Surprisingly, these mid-twentieth-century shifts were consistent across race and sex in the US and also in other high-income countries. Using a simple approach of quantifying potential life expectancy gains by eliminating mortality at specific ages, we find that the potential gains in life expectancy from reducing midlife mortality have been larger in the US than in other countries since 1900. The findings suggest that US life expectancy is unlikely to progress at the high speed observed between 1900 and the 1950s, with future advancements hinging on the reduction of old-age mortality, particularly from cardiovascular diseases and mental and nervous system diseases.

Despite the thousands of genes implicated in age-related phenotypes, effective interventions for aging remain elusive, a lack of advance rooted in the multifactorial nature of longevity and the functional interconnectedness of the molecular components implicated in aging. Here, we introduce a network medicine framework that integrates 2,358 longevity-associated genes onto the human interactome to identify existing drugs that can modulate aging processes. We find that genes associated with each hallmark of aging form a connected subgraph, or hallmark module, a discovery enabling us to measure the proximity of 6,442 clinically approved or experimental compounds to each hallmark. We then introduce a transcription-based metric, $pAGE$, which evaluates whether the drug-induced expression shifts reinforce or counteract known age-related expression changes. By integrating network proximity and $pAGE$, we identify multiple drug repurposing candidate that not only target specific hallmarks but act to reverse their aging-associated transcriptional changes. Our findings are interpretable, revealing for each drug the molecular mechanisms through which it modulates the hallmark, offering an experimentally falsifiable framework to leverage genomic discoveries to accelerate drug repurposing for longevity.

While aging impairs memory precision, its effects on engram dynamics and gene expression remain poorly understood. To address this, we used TRAP2 activity-reporter mice, nuclear tagging, and FOS-based activity mapping to track neurons activated during contextual fear memory encoding and reactivated during recall in young and aged mice. Across 378 brain regions, we quantified engram size, spatial distribution, and reactivation stability. We further applied fluorescence-activated nuclear sorting (FANS) combined with single-nucleus RNA sequencing (snRNA-seq) to characterize gene expression changes associated with memory encoding and recall across diverse cell types. In addition, we compared the transcriptional profiles of first-time versus second-time neuronal responder cells in the dentate gyrus. Aged brains exhibited altered engram allocation, reduced reactivation stability, and distinct gene expression patterns during memory retrieval. These findings reveal age-related changes in the organization and molecular identity of memory traces, providing mechanistic insight into cognitive decline and highlighting potential targets for intervention.

Evolution has given rise to lifespans in extant species ranging from days to centuries. Given that mechanisms of ageing are highly conserved, studying long-lived lineages across the animal kingdom could yield insights relevant for healthy ageing in humans. However, typical models of extended lifespan often live for decades, making them impractical for longitudinal studies. Ideal model systems would be organisms that are naturally long-lived compared to their close relatives, but have lifespans on experimentally tractable scales. Here, we present the Neotropical butterfly genus Heliconius as a novel model system for the evolution of extended longevity. We collate data from 27 species across the Heliconiini tribe to reveal a 25-fold variation in lifespan within the group, with our 348-day maximum for Heliconius hewitsoni longer than any butterfly species previously recorded in the scientific literature. While previous work has attributed this lifespan extension to a plastic response to enhanced nutrition, we conduct detailed survival and functional senescence analyses on two species representative of shorter- and longer-lived clades to show evidence of evolved, heritable mechanisms of slowed ageing in Heliconius. Our results add a new case study to the canon of noteworthy agers, and provide valuable insights into the evolution of increased longevity.

Osteoporosis (OP) is a metabolic bone disease, characterized by loss of bone mass and destruction of bone microstructure, which has a high incidence of disability. Identification of the key factors of pathogenesis is essential for diagnosis and therapy. In this study, we have identified the proton-sensing receptor GPR65, which is specifically expressed in osteoclasts and is significantly down-expressed in osteoclast differentiation, aging, ovariectomy (OVX)-, and tail suspension (TS)-induced osteoporotic bone tissue. In vivo experiments confirmed that knockout of GPR65 exacerbates bone loss and OP induced by TS, OVX, and aging. In vitro experiments demonstrated that silencing GPR65 or application of either endogenous or exogenous antagonist of GPR65 promotes osteoclast differentiation, whereas overexpression of GPR65 or application of either endogenous or exogenous agonist inhibits osteoclast differentiation, and knockout of Gpr65 mitigates this effect. Mechanistic studies revealed that GPR65 inhibits osteoclast differentiation by binding to Gαq, activating GSK3β, and suppressing its phosphorylation, thereby inhibiting the nuclear translocation of NFATc1 that mediates osteoclast differentiation. Furthermore, application of GPR65 agonist alleviated OVX-induced OP in vivo, indicating GPR65 as a novel therapeutic target for bone aging and OP.

Overexpression of the mRNA binding protein Ssd1 extends the yeast replicative lifespan. Using microfluidics to trap and image single cells throughout their lifespans, we find that lifespan extension by Ssd1 overexpression is accompanied by formation of cytoplasmic Ssd1 foci. The age-dependent Ssd1 foci are condensates that appear dynamically in a cell cycle-dependent manner and their failure to resolve during mitosis coincided with the end of lifespan. Ssd1 overexpression was epistatic with calorie restriction (CR) for lifespan extension and yeast overexpressing Ssd1 or undergoing CR were resistant to iron supplementation-induced lifespan shortening while their lifespans were reduced by iron chelation. The nuclear translocation of the Aft1 transcriptional regulator of the iron regulon occurred during aging in a manner that predicted remaining lifespan, but was prevented by CR. Accordingly, age-dependent induction of the Fit2 and Arn1 high-affinity iron transporters within the iron regulon was reduced by CR and Ssd1 overexpression. Consistent with age-dependent activation of the iron regulon, intracellular iron accumulated during aging but was prevented by CR and Ssd1 overexpression. Moreover, lifespan extension by Ssd1 overexpression or CR was epistatic to inactivation of the iron regulon. These studies reveal that CR and Ssd1 overexpression extend the yeast replicative lifespan by blocking deleterious age-dependent iron uptake, identifying novel therapeutic targets for lifespan extension.

Cellular senescence is a state of irreversible cell cycle arrest accompanied by a distinctive inflammatory secretory profile known as the senescence-associated secretory phenotype (SASP). While various biomarkers, such as senescence-associated beta-galactosidase (SA-βgal), EdU incorporation, p21 and p16, are used to identify senescent cells, no single biomarker universally defines cellular senescence and current methods often fail to address heterogeneity in biomarker expression levels. This study leverages single-cell fluorescence imaging to assess multiple senescence markers including SA-βgal enzymatic activity, p21 and IL-6 expression and nuclear and cell area in chemotherapy-induced (mitomycin C) and oxidative stress-induced (D-galactose) senescence models in human fibroblasts. Our findings reveal significant heterogeneity in SA-βgal activity and distinct sub-populations within senescent cells. Nuclear and cell area measurements emerged as robust indicators of cellular senescence, displaying similar variability across individual cells. Importantly, we identified specific nuclear area sub-populations that strongly correlate with IL-6 expression levels, demonstrating a relationship between the heterogeneous expression of senescence biomarkers and the SASP. To address this heterogeneity, we introduced an induction threshold method to more accurately quantify the percentage of cells expressing senescence biomarkers. Furthermore, in both senescence models, we observed that rapamycin, a well-known senomorphic agent, selectively targets specific biomarker-expressing sub-populations. This study underscores the value of assessing cellular heterogeneity in senescence research and provides an improved approach for analysing senescence markers in diverse cellular contexts.

Chaperone-mediated autophagy (CMA), a lysosome-dependent protein degradation pathway, plays a pivotal yet poorly understood role in cellular senescence-related degenerative diseases. Our study sheds light on a novel mechanism whereby UCHL1 plays a crucial role in mitigating nucleus pulposus cell (NPC) senescence and intervertebral disc degeneration (IVDD) by activating CMA to counteract autophagy-dependent ferroptosis. Through sequencing analysis of human samples, we identified UCHL1 as a potential factor influencing disc degeneration. Further research revealed that UCHL1 activates CMA by stabilizing HSPA8 through deubiquitination. HSPA8, in turn, recognizes and promotes the degradation of HPCAL1 via the CMA pathway by binding to its "KFERQ" motif, ultimately alleviating NPC senescence. Importantly, we demonstrated that engineered exosomes delivering

Muscle stem cells (MuSCs) are parenchymal cells in skeletal muscle regeneration and maintenance. With aging, MuSCs experience a decline in their regenerative function and reduction in their number. However, recent evidence points to substantial heterogeneity within the aged MuSC population, raising questions about the underlying mechanisms of age-associated dysfunction. Here, we used Pax7CreERT2;RosaYFP mice (MuSCYFP) to label Pax7-expressing MuSCs and chronologically traced MusCs until geriatric age. Genetic labeling and chronological tracing revealed that the number of YFP+ MuSC remained comparable between young, middle and geriatric ages. At geriatric age, YFP+ MuSCs exhibited reduced expression of traditional MuSC markers such as VCAM1 and PAX7. A previously unrecognized subpopulation emerged, characterized by loss of VCAM1 and low or absent PAX7. Despite their altered marker profile, these cells retained transcriptional signatures of quiescence and myogenic potential, but displayed significantly reduced proliferative and regenerative capacities. They displayed gene expression patterns indicative of senescence-like state and were selectively ablated by senolytic treatment. DHT restored regenerative function in aged mice and re-induced VCAM1 expression in YFP+/Pax7-/low/VCAM1- cells, indicating responsiveness to rejuvenation. Based on their emergence with aging, functional impairment and responsiveness to rejuvenation, we termed this population GERI-MuSCs (Geriatric Emerging Rejuvenation-responsive and Impaired MuSCs). CD63 and CD200 were identified as novel surface markers that together with VCAM1, reliably detect GERI-MuSCs as well as classical Pax7+/VCAM1High MuSCs, providing a tool for comprehensive isolation of MuSCs from aged wild-type mice. Together, our findings provide a refined framework for studying MuSC aging and offer new tools for isolating functionally distinct MuSC subsets from aged skeletal muscle.

Cerebrospinal fluid dynamics play an important role in maintaining brain health and clearing metabolic waste from the brain. In this issue of the JCI, Gursky et al. investigate how CSF distribution is affected when its primary efflux pathway - the deep cervical lymph nodes - is disrupted by cauterization. This timely study reveals compensatory fluid drainage routes from the skull, age-dependent adaptations in CSF homeostasis, and the emergence of neuroinflammation when an efflux pathway is occluded. The findings underscore the need to better understand the physiological mechanisms governing CSF clearance, how these pathways evolve with aging, and whether CSF influx and efflux exhibit region-specific dynamics shaped by neuroanatomy. Additionally, the study raises important questions about whether peripheral injury can influence central nervous system states. A more complete understanding of CSF flow regulation may offer new perspectives on the origins of neuropathology.

The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1β and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA-encoded library screen to identify novel NLRP3-binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses, we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950-binding pocket. Using humanized NLRP3 mice, we show that a derivative of BAL-0028, BAL-0598, inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that both BAL-0028 and BAL-0598 inhibit select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than MCC950. BAL-0028 and BAL-0598 thus represent a new modality for NLRP3 inhibition in inflammatory diseases.

Germline stem cells (GSCs), the source of gametes, are the only stem cells capable of passing genes to future generations and are therefore considered units of natural selection. Yet, the factors that influence GSC fitness, and thus govern GSC competition, which exist in both protochordates and mammals, remain poorly understood. We studied how aging affects GSC fitness in the protochordate Botryllus schlosseri, an evolutionary crosspoint between invertebrates and vertebrates. GSCs were isolated and distinguished from developing and mature gametes using flow cytometry and scRNA-Seq, facilitated by a new PacBio genome assembly. Moreover, their function was validated through a novel lineage tracing approach that combines membrane-labeled GSC transplantation with scRNA-Seq. Leveraging our method to isolate them, single-cell transcriptomics showed significant age-related changes between young and old GSCs. Spermatids and sperm, however, showed minimal changes, suggesting that reproductive aging is governed by GSCs rather than by gametes. Reduced expressions of markers like DDX4 and PIWIL1 in aged GSCs mirrored trends in mammalian datasets, pointing to a conserved GSC-driven aging mechanism across chordate evolution. This study provides new techniques that lay the foundation to investigate further drivers of GSC fitness and highlights fertility-related genes as promising targets for therapies to preserve reproductive health.

Age-related inflammation or 'inflammaging' increases disease burden and controls lifespan. Adipose tissue macrophages (ATMs) are critical regulators of inflammaging; however, the mechanisms involved are not well understood in part because the molecular identities of niche-specific ATMs are unknown. Using intravascular labeling to exclude circulating myeloid cells followed by single-cell sequencing with orthogonal validation via multiparametric flow cytometry, we define sex-specific changes and diverse populations of resident ATMs through lifespan in mice. Aging led to depletion of vessel-associated macrophages, expansion of lipid-associated macrophages and emergence of a unique subset of CD38

The growing prevalence of type 2 diabetes (T2D) among older adults represents a major public health concern, given its association with accelerated cognitive decline and increased risk of neurodegenerative diseases. Several diabetes-related mechanisms, including chronic hyperglycaemia, oxidative stress, vascular dysfunction, and insulin resistance in the brain, negatively impact key cognitive domains, including memory and executive functions. These neuropathophysiological alterations are also linked to structural brain changes, contributing to vulnerability to dementia. This narrative review examines both established and emerging strategies aimed at counteracting the cognitive impact of T2D in aging populations. Traditional interventions, especially structured physical activity programs, have consistently demonstrated benefits for global cognitive functioning. In parallel, new pharmacological treatments, such as GLP-1 receptor agonists (e.g., semaglutide), not only improve glycemic control but may also exert neuroprotective effects. Multidomain approaches integrating metabolic management, nutritional optimization, physical exercise, and social engagement, such as those tested in the J-MIND-Diabetes study, have yielded promising outcomes in preserving cognitive functions. We argue that combining pharmacological and behavioral strategies holds significant potential for supporting cognitive health in elderly individuals with T2D. Such multimodal interventions may enhance resilience to cognitive decline, improve quality of life, and promote healthy brain aging in this at-risk population.

The one-two-punch approach refers to the sequential administration of two different chemotherapies, the second of which targets cancer cells that resisted the initial treatment. To find such a second punch, we performed a chemical screen to find drugs that are preferentially toxic for cells with an activated DNA damage response (DDR). This screen identified the tyrosine kinase inhibitor GNF-7 as a top hit. Subsequent work revealed that GNF-7 is a potent senolytic, even when senescence is triggered by therapies that do not activate the DDR. Consistently, GNF-7 is highly efficacious to kill cancer cells previously treated with CDK4/6 inhibitors, including in patient-derived organoids and mouse xenografts. Surprisingly, the senolytic effect of GNF-7 is not mediated by the inhibition of a tyrosine kinase (TK), but rather by the activation of GCN2, an effect previously reported for other TK inhibitors. Together, our study reports the discovery of a novel senolytic agent that strongly synergizes with CDK4/6 inhibitors when applied sequentially and expands our understanding of the mechanisms behind the anticancer effects of TK inhibitors.

Extending human healthspan requires understanding how lifestyle interventions impact molecular systems across tissues and time. Here, we present the TirolGESUND Lifestyle Atlas (ClinicalTrials.gov: NCT05678426), a longitudinal, multi-modal resource profiling 156 healthy women (aged 30-60 years) undergoing 6-month intermittent fasting (n=114) or smoking cessation (n=42) interventions. Participants were sampled up to four times across seven tissues and fluids, generating >3,450 biospecimens with harmonised DNA methylation, metabolomics, microbiome, and immune profiling, alongside skin histology, barrier measurements, and rich clinical metadata. We demonstrate the utility of this dataset through: (i) multi-omics-wide association studies linking traits to molecular features; (ii) integrative factor modelling revealing coordinated cross-tissue signatures; (iii) epigenetic-biomarker cross-omic associations, and (iv) CpG-level variance decomposition mapping stable, individual-specific, tissue-restricted, and intervention-responsive methylation patterns. We further show that ageing-linked features are selectively malleable: highly compliant intermittent fasting participants exhibited attenuated or even age-opposing molecular trajectories within six months. The atlas enables unprecedented within-cohort comparisons across omic layers and tissues, supporting discovery of context-dependent biomarkers, cross-system coordination, and intervention responsiveness. Data are available via an interactive portal, with sensitive data under controlled access (https://eutops.github.io/lifestyle-atlas/). This resource provides a foundation for exploring biomarker association and multi-tissue epigenetics, enabling hypothesis generation and benchmarking for systems biology and human healthspan research.

Oct4, Sox2, and Klf4 (OSK) Yamanaka factors induce pluripotency and reverse age-related epigenetic changes, yet the mechanisms by which they promote rejuvenation remain poorly explored. Oxidative stress contributes to CNS aging and retinal pigmented epithelium (RPE) degeneration in age-related macular degeneration. We find that OSK expression in RPE restores retinal structure and visual function in aged mice and promotes oxidative resilience through a non-canonical, Tet2-independent pathway. Integrative functional genomics identifies GSTA4, a detoxifying enzyme that clears the lipid peroxidation byproduct 4-HNE, as a necessary and sufficient OSK effector. Dynamic GSTA4 regulation by OSK recapitulates a stem cell derived stress resilience program. GSTA4 overexpression alone enhances mitochondrial resilience, rejuvenates the aged RPE transcriptome, and reverses visual decline. GSTA4 is consistently upregulated across diverse lifespan-extending interventions suggesting a broader pro-longevity role. These findings uncover a previously unrecognized protective axis driven by Yamanaka factors that circumvents reprogramming, providing therapeutic insights for age-related diseases.

While most knowledge of animal DNA methylation comes from vertebrates, this epigenetic mark remains poorly understood in invertebrates, which comprise the majority of animal diversity. For instance, how promoter and gene body methylation contribute to gene regulation, and how methylation relates to aging, are still relatively unknown in most invertebrates. Focusing on the California mussel (Mytilus californianus), we paired whole-genome resequencing and whole-genome bisulfite sequencing from the same individuals and evaluated relationships among promoter methylation, gene body methylation, gene expression, and age. Using seven individuals spanning a range of body sizes from the Santa Barbara Channel, California, we found standing genetic variation levels similar to related species and a relatively small effective population size. CpG methylation was enriched in gene bodies, and gene body methylation was positively associated with expression. Promoter methylation was less frequent but showed a strong negative association with expression and remained the best predictor of repression after accounting for gene body methylation, aligning with patterns widely documented in vertebrates and adding to the limited evidence in invertebrates that promoter methylation can be regulatory. We identified thousands of age-associated differentially methylated loci with directional changes across age classes, providing candidate sites for epigenetic clocks that could enable assessment of biological age, health, and stress resilience in wild and cultured populations.

Repetitive transcranial magnetic stimulation (rTMS) is an attractive tool to promote healthy brain ageing in older adults and treat age-related neurological conditions. Despite its popularity, the neurological processes and plasticity mechanisms altered by rTMS in the aged brain, and where these changes occur in the brain are unknown. Furthermore, it is not known why different rTMS protocols induce different changes in the aged brain, or why rTMS is less effective in older adults compared to younger adults. Using spatial transcriptomics, we uncovered that rTMS primarily acts on genes related to synaptic plasticity in both cortical and subcortical circuits in aged mice, but the specific changes were dependent on the brain region and even down to individual cortical layers in the motor and somatosensory cortices. Comparing our results from aged mice to young adult mice revealed that rTMS acts on a larger variety of neural plasticity mechanisms in the young adult brain, and that rTMS was less effective at altering gene expression related to neural plasticity in the aged brain, but this varied between brain regions and the protocol of rTMS applied. These findings provide a comprehensive map of the mechanisms altered by rTMS across the aged brain and highlight the need to consider the effect of ageing when optimising rTMS protocols for older populations.