Longevity Papers

Cellular senescence is traditionally described through durable cell-cycle arrest, DNA-damage signaling, metabolic remodeling, mitochondrial and lysosomal dysfunction, and acquisition of a senescence-associated secretory phenotype (SASP). However, senescent cells also undergo prominent structural and biomechanical changes, including enlarged and flattened cell shape, altered stiffness and force transmission, cytoskeletal reorganization, defective nucleo-cytoskeletal coupling, impaired organelle positioning, and extracellular matrix (ECM) remodeling. In this review, we use the term Physical Senotype as a working framework to describe this recurrent but heterogeneous mechanical state. Rather than proposing a separate hallmark of senescence, this framework emphasizes loss of mechanical plasticity: the reduced capacity of senescent cells and tissues to sense, buffer, dissipate, and adapt to mechanical stress. We discuss how cytoskeletal maladaptation may amplify nuclear damage, impair mitochondrial and lysosomal quality control, reinforce inflammatory signaling, and interact with a mechanically altered extracellular niche. We also critically evaluate emerging physical and mechanical approaches to senotherapy, including pressure-based senolysis, remotely activated nanomaterials, ultrasound, exercise-associated immune surveillance, mechanical stimulation, cytoskeletal re-dynamization, and mechanically tuned biomaterials. Current evidence suggests that these interventions may produce distinct outcomes, including direct senescent-cell killing, immune-assisted clearance, or functional reprogramming of mechanically recoverable cells. However, most approaches remain early-stage, and major questions remain regarding specificity, tissue dependence, dosing thresholds, durability, and safety. We argue that integrating mechanical phenotyping with canonical senescence markers will be essential for distinguishing mechanically recoverable senescent states from irreversible states requiring clearance, and for predicting how the aged tissue niche shapes senotherapeutic response.

Although lifespan has long been the focus of ageing research, preventing functional decline late in life is a more pressing societal need. Here, we investigate the basis of senescence and declining fitness during replicative ageing in budding yeast, and describe a metabolic perturbation that preserves late-life fitness even on an unrestricted glucose diet. We show that senescence can be prevented by constitutive activation of AMPK, though only for approximately half the ageing population, and use genetic and functional assays to link this heterogeneous response with differences in cytosolic acetyl coenzyme A (Acetyl-CoA) metabolism. In one class of ageing cell, AMPK activity maintains fitness late in life through pathways that transport cytosolic Acetyl-CoA into mitochondria, but AMPK also inhibits fatty acid synthesis which leads to lipid starvation in the other class of ageing cell. Therefore, AMPK activity has both positive and negative effects, but we show that constitutive AMPK activity uncoupled from fatty acid synthesis inhibition (the A2A mutant) suppresses senescence and maintains fitness in both classes of ageing cell. Our findings support a model in which lipid starvation and excess Acetyl-CoA availability are major drivers of senescence in replicatively aged wild-type yeast. This work shows that ageing is not intrinsically associated with declining fitness, at least in yeast, and that re-engineering highly conserved metabolic pathways allows fitness to be preserved very late in life.

Cell death, DNA damage, and inflammation are closely interconnected processes implicated in ageing, cancer, and inflammatory disorders, yet the endogenous mechanisms linking them remain unclear. We previously identified cell-free chromatin particles (cfChPs), released from dying cells, as biologically active entities that enter neighboring cells and induce DNA damage and inflammation. Here, we show that serum-derived circulating cfChPs are rapidly internalized by human peripheral blood mononuclear cells and trigger a previously unrecognized biphasic STING signaling response. An early phase involves rapid STING trafficking to the perinuclear region and nucleus, with activation of IRF3 and NF-{kappa}B preceding detectable DNA damage. This is followed by a later phase characterized by STING phosphorylation, puncta formation, persistent DNA damage, and robust inflammatory cytokine production. Pharmacological inhibition or genetic deletion of STING markedly attenuated these responses. These findings identify extracellular cfChPs as endogenous DNA-damaging agents and reveal biphasic STING signaling as a mechanistic link between cell death, DNA damage, and sterile inflammation.

Ageing is the strongest risk factor for heart failure, yet the molecular mechanisms underlying cardiomyocyte (CM) ageing remain unclear. We aimed to map the transcriptomic and epigenomic landscape of CM ageing and to test whether DNA hypermethylation is a causal driver of diastolic dysfunction.

Lysosomal dysfunction is a well-recognized feature of aging. Here, we used a suite of tools for rapid lysosomal isolation to construct a multitissue atlas of the metabolite changes lysosomes undergo during aging. Aged lysosomes in brain, heart, muscle, and white adipose tissue accumulated glycerophosphodiesters and cystine, metabolites that are causally linked to juvenile lysosomal storage disorders, Batten disease, and cystinosis. Levels of these metabolites increased linearly with age, preceding organismal decline. Caloric restriction, a lifespan-extending intervention, mitigated these changes in the heart and muscle but not the brain. Our findings link lysosomal storage disorders to aging-related dysfunction and open avenues for the mechanistic investigation of how lysosomal functions deteriorate during aging and in age-associated diseases.

Cardiac fibrosis, a major pathological hallmark of aging that leads to heart failure, is characterized by excessive collagen deposition. Our knowledge of what sustains collagen synthesis in the aging heart is still very preliminary. Here, we uncover a central role for chaperone-mediated autophagy (CMA), a selective lysosomal degradation pathway, in this process. We demonstrate that CMA is suppressed in the aging heart, which promotes collagen overproduction in fibroblasts, whereas enhancing CMA activity ameliorates fibrosis and diastolic dysfunction. Mechanistically, we identify SHMT2 (serine hydroxymethyltransferase 2) as a CMA substrate whose accumulation with aging drives collagen synthesis by increasing glycine availability. Integrative omics revealed a systemic downregulation of the ketone body β-hydroxybutyrate (BHB) in aged mice. BHB supplementation - via a cyclic ketogenic diet - restored CMA, attenuated fibrosis, and improved cardiac function. This recovery was mediated through BHB-induced activation of the HCAR2 receptor and subsequent phosphorylation of HSPA8/HSC70, which systemically reactivates the CMA machinery. Furthermore, we show that

Aging is characterized by progressive physiological decline and accumulation of senescent cells that drive chronic "inflammaging" through the senescence-associated secretory phenotype (SASP). The complement system, traditionally viewed as a systemic extracellular defense mechanism, is now recognized as an essential intracellular network (the complosome). This review synthesizes current research on how intracellular C3 (intC3), and intracellular C5 (intC5) in certain contexts engage in extensive crosstalk with the mTOR, NF-κB, and AMPK pathways to modulate core cellular processes. We detail how intC3 intersects with multiple canonical hallmarks of aging-including cellular senescence, mitochondrial dysfunction, proteostasis loss, genomic instability, epigenetic alterations, altered intercellular communication, stem cell exhaustion, and deregulated nutrient sensing. Furthermore, we examine organ-specific consequences of intC3 dysregulation across the aging brain, liver, eye, kidney, vasculature, lung, and immune system, as well as cancer. Finally, we discuss therapeutic strategies-including complement inhibitors, senolytics, senomorphics, RNA interference, proteolysis targeting chimeras (PROTACs), and nanotechnology-enabled delivery-and outline critical gaps in compartment-specific tools, conditional knockouts, and longitudinal models. Precision strategies that preserve systemic complement function while selectively modulating intracellular pools will be essential to translate complosome biology into durable interventions for age-related diseases.

The aging brain exhibits a decline in the regenerative populations of neural stem cells (NSCs). While mechanisms that restore old NSC function have started to be identified, the role of lipids-especially complex lipids-in NSC aging remains largely unclear. Using lipidomic profiling by mass spectrometry, we identify age-related changes in complex lipids in quiescent NSCs in vitro and in vivo. Moreover, several polyunsaturated fatty acids increase across lipid classes in quiescent NSCs during aging. Using spatial lipidomics, we find that some of the changes in complex lipids are also observed in situ. Several age-related changes in complex lipids and side chain composition are occurring at the plasma membrane, as revealed by lipidomic profiling of isolated plasma membrane vesicles. Experimentally, we show that aging is accompanied by a decrease in plasma membrane order, a key membrane biophysical property, in old quiescent NSCs in vitro and in vivo. To determine the functional role of plasma membrane lipids in aging NSCs, we performed genetic and supplementation studies. Knocking out the phospholipid acyltransferase MBOAT2 exacerbates age-related lipidomic changes in old quiescent NSCs and impedes their ability to activate.

Importance: Preclinical and human observational studies suggest that metformin may decrease age-related pathology, including frailty. Objective: Determine whether metformin reduces frailty progression and biological age in older adults with glucose intolerance, a population at increased risk of becoming frail. Design, Setting and Participants: Randomized, double-blind, placebo-controlled trial of metformin in 145 non-frail or pre-frail older adults. Participants (72 +/-5 years, 48% female, 94% White, 35% Hispanic) were randomized to metformin vs. placebo for two years. Main Outcomes and Measures: Effect on frailty was primarily determined using generalized estimating equations by change in the Fried frailty phenotype score (based on weight loss, exhaustion, physical activity, gait speed, and grip strength). Because metformin can cause significant weight loss, effects on the Fried score were assessed with and without the weight loss criterion. Frailty also was assessed by change in the frailty index (composite of 95 deficits). Biological age was estimated by DNA methylation-based epigenetic clocks in blood. Results: Metformin led to a non-linear response in the Fried score rate of change, with an upward trajectory in year 1 (0.72 +/-0.22 per year vs. placebo, p=0.0011) and stabilization in year 2 (-0.33 +/-0.17 per year vs. placebo, p=0.056). Metformin led to more weight loss than placebo (-5.7 +/-5.2 vs. -2.3 +/-5.4 kg, p=0.0002); thus, when assessing effect on Fried score without the weight loss criterion, no difference was observed, indicating that weight loss in year 1 accounted for the change in Fried score. Notably, metformin caused a steady improvement in the frailty index (-0.006 +/-0.0026 per year vs. placebo, p=0.0222) that persisted with covariates adjustment including body mass index. Metformin reduced biological age estimated by PC-Horvath2 (-0.40 +/-0.16 per year, p=0.014) and PC-Hannum (-0.33 +/-0.16 per year, p=0.047) clocks. Metformin was well tolerated. Conclusions and Relevance: Metformin halts the progression of the deficit accumulation frailty index and reduces biological age, suggesting potential benefit for extending healthspan. Trial Registration: ClinicalTrials.gov: NCT02570672.

Cellular senescence contributes to neurodegeneration in Alzheimer's disease (AD), yet brain-penetrant senotherapeutic strategies remain limited. Here, we identify long interspersed nuclear element 1 (LINE-1) retrotransposons as key regulators of neuronal senescence and the senescence-associated-secretory-phenotype (SASP) in AD. Using transdifferentiated induced neurons (iNs) that preserve donor-specific aging-associated molecular signatures, we show that pharmacological inhibition of LINE-1 with nucleoside reverse transcriptase inhibitors (nRTIs) or antisense oligonucleotides reduces p16 expression, suppresses SASP and interferon-stimulated gene programs, and attenuates paracrine induction of reactive astrogliosis. Spatial transcriptomic analysis of human AD brain tissue further supports that senescent neurons with high LINE-1 expression are localized to inflammatory niches in the brain. Although bulk analysis finds no significant differences in LINE-1 expression between AD and control neurons, long-read single-cell RNA sequencing of iNs identifies a subset of neurons with elevated LINE-1 activity which display transcriptional signatures of neurodegeneration, immune activation, and senescence are enriched in AD relative to controls. RNA velocity analysis indicates that LINE-1 activation precedes the induction of canonical senescence markers, supporting a causal rather than consequential role. Mechanistically, LINE-1-derived cytoplasmic DNA activates the cGAS-STING innate immune pathway in post-mitotic neurons, and inhibition of cGAS phenocopies the effects of LINE-1 suppression. Together, these findings establish a LINE-1/cGAS-STING axis as a driver of neuronal senescence in AD and highlight LINE-1 inhibition as a tractable senomorphic strategy for neurodegenerative disease.