Biology subjects

Gladyshev, V. N.

Publications and source records attributed to Gladyshev, V. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Transient transcriptomic rejuvenation during intestinal and liver regeneration in mice

Aging and regeneration are fundamental biological processes, but the relationship between them is poorly understood. During intestinal regeneration, adult epithelial cells reactivate developmental programs that support repair. To investigate if this plasticity alters tissue biological age, we developed and validated intestinal tissue and epithelium-specific transcriptomic aging clocks. Across various regeneration models, epithelial states were predicted by these clocks to be younger than controls. Their expression changes were also negatively associated with intestinal aging signatures. The resulting younger state was characterized by YAP-associated regeneration and reduced mature enterocyte identity. In a separate irradiation model, the predicted epithelial age first increased, then fell at the peak of regeneration and later increased again during recovery. The pace of this response differed with host age. In a chemotherapy-induced injury model, epithelium in young animals also entered this younger state earlier and then returned to its preinjury state during recovery. Old epithelium responded later, but also reversibly transitioned through a rejuvenated state. Further spatial transcriptomic analyses linked lower epithelial age with regeneration in selected injured and tumor-associated regions. In addition, liver regeneration after partial hepatectomy in old animals also led to a decreased transcriptomic age, accompanied by pathway changes negatively associated with aging signatures. Overall, these findings suggest that mammalian regeneration is associated with a transient reduction in biological age, whose timing and persistence depend on tissue, host age and the course of repair.

cell biology↗

bioRxiv2026

Systemic control and inter-organ communication of ovarian aging

Whether ovarian aging is intrinsic and unidirectional or dynamically regulated by systemic context remains an open question for fertility and women's healthspan. To address it, we developed a mouse ovarian transcriptomic aging clock (OvaAge) and applied it, together with published transcriptomic, epigenetic and pathway-resolved DNA methylation (PathAge) clocks, multi-omic analyses and reproductive outcomes, to new reciprocal heterochronic ovary transplantation and parabiosis experiments. Ovarian molecular age shifted in both directions: aged ovaries became younger in young hosts and young ovaries became older in aged hosts, and in each case the shift was partial across transcriptomic, epigenetic and pathway-resolved measures. Ovaries from 13-month-old C57BL/6J donors supported full-term pregnancies and live births at 17-18 months of age when transplanted into young hosts, but not when transplanted into old hosts, and young ovaries supported live births in 17-18-month-old recipients. Donor ovary age altered the transcriptomic age of the uterus in both directions, while young ovaries also lowered the transcriptomic age of aged liver and brain, identifying the ovary as an active node within an inter-organ aging network. Across unilateral and bilateral transplantation and parabiosis, an older environment increased inflammatory gene expression in the ovary, whereas a younger environment reduced it and increased mitochondrial and biosynthetic programs. Integration of methylation, expression and cell-type markers linked these changes to declining oocyte-associated programs and an expanding stromal compartment enriched for multinucleated giant cells (MNGCs). MNGC-rich regions formed a reversibly remodeled, focally aged niche with distinct spatial metabolic features, and a related macrophage expression signature was detected in human ovaries. Together, these findings identify ovarian aging as a modular, bidirectionally plastic process that is regulated by the systemic environment and point to tractable intervention points.

systems biology↗

bioRxiv2026

A genome-scale ORF screen for resilience factors that protect the RPE and restore vision in aged mice

Aging is a complex phenomenon, yet therapeutic discovery has largely relied on testing individual hypothesis-driven targets. Here, we performed a genome-scale open reading frame (ORF) screen for oxidative-stress resistance in retinal pigment epithelium (RPE) cells, which are vulnerable to oxidative damage during aging. The screen identified nine protective ORFs including antioxidant factors and an epithelial-to-mesenchymal transition regulator. Notably, we also identified three factors that reduce transcriptomic age including the cardiac transcription factor NKX2-5. NKX2-5 conferred robust oxidative resilience in vitro and in vivo. Domain dissection revealed that its homeodomain is dispensable for resilience, enabling development of NKX2-5{Delta}HD, which retained protective activity without detectable toxicity over 14 months. In middle-aged mice, subretinal NKX2-5{Delta}HD restored visual and electrophysiological function, and its systemic delivery improved grip strength and reduced frailty in late-aged mice. These findings highlight the value of unbiased, genome-scale gain-of-function screening, as resilience factors normally expressed in other contexts can be reengineered as potential therapeutics for age-related decline in other tissues.

genetics↗

bioRxiv2026

Transplanted hearts assimilate the recipient's biological age

Background. Despite the growing demand for transplantation, a shortage of donor organs persists, limiting access to this lifesaving procedure. To meet the increasing demand, organs are frequently transplanted across an age discrepancy between donor and recipients. Methods. We delineated biological age dynamics in heterochronic heart transplants performed in both mice and patients. We carried out multiomic profiling and applied advanced biomarkers of aging to understand the interactions between the transplanted organ and the host. Results. We show that the biological age of experimental heart transplants rapidly assimilates the age of recipients. Interestingly, this effect was limited to the grafted tissue without reciprocal effects on systemic biological age of the recipient. These effects were confirmed at the level of DNA methylation and gene expression. Extending our findings clinically using both omics and functional analyses, we show that the biological age of heterochronically transplanted hearts is strongly associated with the age of the recipient, rather than the donor, confirming the clinical relevance of our experimental findings. Conclusions. These data identify the systemic environment as a driving force on tissue biological age. The rejuvenation of older organs in young recipients suggests novel approaches to organ allocation that may expand the pool of transplantable organs.

systems biology↗

bioRxiv2026

Causally measuring aging and rejuvenation through transcriptomic damage

Aging is caused, fully in large part, by the progressive accumulation of damage, yet quantifying age-related damage across tissues and conditions remains a challenge. Here, we present a computational framework to quantify damage from standard RNA-sequencing data. It captures four classes of aberrant transcript structures, including premature termination upon intron retention, domain-disrupting splice variants, repeat elements, and gene fusion events, each reflecting distinct forms of RNA integrity loss. Using this method, we revealed a robust age-associated increase in transcriptomic damage across tissues. To integrate these measurements into a unified biomarker, we constructed a transcriptomic damage-based aging (tDamAge) clock using machine learning models trained across mouse tissues or human peripheral blood. It could predict age and detect transcriptomic shifts under both pro-aging and anti-aging conditions. Progeroid models exhibited accelerated tDamAge, whereas interventions such as caloric restriction, rapamycin, and methionine restriction lowered tDamAge. Cross-dataset analysis showed that diverse anti-aging interventions converge on shared transcriptomic signatures, particularly RNA processing and chromatin organization pathways, and these age-associated patterns could be reversed by interventions. We further identified elevated damage age acceleration in Alzheimers disease and observed rejuvenation-like reductions during embryonic development. Together, our findings establish transcriptomic damage as a causal, quantifiable and biologically interpretable feature of aging and demonstrate that tDamAge could detect age progression, acceleration, deceleration, and reversal.

bioinformatics↗

bioRxiv2026

Architectural fragility of gene regulatory networks underlies hematopoietic stem cell aging

Hematopoietic stem and progenitor cell (HSPC) aging contributes to immune dysfunction and age-associated disease, but its regulatory mechanisms remain unclear. Here, we present the largest single-cell multiome atlas of human circulating HSPCs to date, with >380,000 paired RNA and ATAC profiles across 77 donors. Beyond recapitulating established hallmarks of HSPC aging, we reconstructed a high-resolution gene regulatory network and identified a global rewiring in which stress-response and myeloid transcription factor (TF) programs expand, while self-renewal and lymphoid lineage-defining circuitry collapses. We associate these phenotypes with increased cis-regulatory entropy, including elevated transcriptomic noise, weakened peak-to-gene coupling, and chromatin peak broadening. This rewiring is selective, where TFs with GC-rich, promoter-proximal architectures are preserved or amplified, and complex, distal enhancer-dependent identity networks are eroded. Thus, these findings suggest that progressive entropic destabilization of gene regulatory architecture simultaneously drives stress hyperactivation, myeloid bias, and identity loss in aging HSPCs.

genomics↗

bioRxiv2026

Quantification of domain-specific intrinsic capacity using mortality data

Functional health is centered on five domains of Intrinsic Capacity (IC): locomotion, cognition, vitality, psychological and sensory capacity. Therefore, measuring IC at the domain-specific level is the cornerstone for developing preventive interventions to help individuals preserve their independence. In this study, we used 63 clinical features from the UK Biobank to develop IC age, an 18-year mortality risk estimator that approximates an individuals biological age associated with the decline of each IC domain. By establishing proteomic surrogates of IC age, we find immune system activation across domains and provide a proteomic framework that may facilitate scalable monitoring of functional health decline.

systems biology↗

bioRxiv2026

Thermodynamic scaling of canine aging and reversible clock deceleration by a reverse transcriptase inhibitor

Aging was tracked in a cohort of 99 "retired" sled dogs over four years to characterize the latent dynamics of physiological decline. Animals were randomized to receive either placebo or the reverse transcriptase inhibitor lamivudine for [~]30 months. We employed a variational autoregressive model to integrate longitudinal blood parameters and DNA methylation (DNAm) profiles. The model defines Biological Age (BA) as a signature of irreversible damage with Poissonian statistics, a feature conserved across mammalian scales. Lamivudine modulated age-independent latent variables and temporarily decelerated the DNAm clock in females, but these effects were reversible upon treatment discontinuation and did not alter the long-term BA trajectory. Critically, we show that physiological fluctuations are governed by a single systemic factor - an effective or phenotypic temperature representing an emergent (macroscopic) property. We show that while the rate of damage accumulation (the BA slope) is independent of this temperature, actuarial aging parameters (initial mortality and the Gompertz exponent) are strongly temperature dependent. This supports a model where mortality arises from effective activation across a protective free energy barrier that erodes with age. These findings identify phenotypic temperature as an important control variable governing the kinetics of organism-level failure, offering a compelling target for interventions aimed at extending healthspan by "squaring" the survival curve.

systems biology↗

bioRxiv2026

Balance between DNA repair, LINE1 suppression and lifespan in mice with SIRT6 Serine 10 mutations

Sirtuin 6 (SIRT6) is an important regulator of DNA repair, metabolism, chromatin maintenance and longevity. SIRT6 Serine 10 phosphorylation controls SIRT6 recruitment to the sites of DNA damage. To explore the effect of SIRT6 Serine 10 phosphorylation on lifespan, we generated two SIRT6 mutant mouse strains: phospho-null S10A and phosphomimetic S10E. The S10E mutant mice demonstrated enhanced DNA repair capacity, elevated LINE1 expression and reduced lifespan in male mice compared to the wild-type and S10A mice. This result suggests that SIRT6 S10E mutation enhances DNA repair capacity at the expense of reduced LINE1 silencing leading to shorter lifespan. While both SIRT6 functions in DNA repair and chromatin maintenance are important for longevity, our results suggest that when the balance between these functions is shifted, diminished of LINE1 control has a stronger impact on lifespan than enhanced DNA repair.

molecular biology↗

bioRxiv2025

Rejuvenation of white adipose tissue in a longitudinal heterochronic transplantation model

Exposure to a younger system can induce organismal rejuvenation, yet whether all tissues can be rejuvenated and by what mechanisms remains understudied. We performed heterochronic and isochronic transplantation of subcutaneous white adipose tissue (WAT) between young and old mice and longitudinally tracked changes in biological age. Transplantation accelerated tissue aging, and the molecular age of grafts shifted toward that of the host. Most importantly, old WAT was rejuvenated in a young body. Epigenetic and transcriptomic clocks revealed a reduction of predicted age, accompanied by coordinated activation of canonical and previously unrecognized thermogenic pathways. Molecular rejuvenation was further supported by architectural changes toward a youthful state, including reduced lipid droplet size and decreased cellular heterogeneity. Mitochondrial abundance and morphology remained unchanged, while collagen deposition increased. These results demonstrate that WAT biological age is partially reversible and identify molecular and cellular features underlying its rejuvenation

systems biology↗

bioRxiv2025

Conserved Master Regulators Orchestrate Cellular Reprogramming-Induced Rejuvenation

Partial somatic cell reprogramming has been proposed as a rejuvenation strategy, yet the regulatory architecture orchestrating age reversal remains unclear. Here, we performed gene regulatory network reconstruction across several independent systems to identify master regulators that coordinate reprogramming-induced rejuvenation (RIR). In mouse mesenchymal stem cells, mouse adipocytes, and human fibroblasts undergoing partial reprogramming, we identified genes showing opposite expression dynamics during aging and reprogramming. This approach revealed regulators governing rejuvenation rather than developmental programs. Despite divergent overall network architectures, nine transcription factors converged as master regulators across all three systems, including Ezh2, Parp1, and Brca1. These regulators undergo coordinated reorganization during reprogramming, characterized by broader target engagement and enhanced regulatory coherence. We further demonstrated that direct perturbation of Ezh2 bidirectionally modulates transcriptomic age. Notably, overexpression of a catalytically inactive Ezh2 mutant achieved rejuvenation, suggesting mechanisms distinct from canonical H3K27me3-mediated regulation are involved in RIR. Our findings reveal that cellular rejuvenation is orchestrated by conserved master regulators whose network coordination can be targeted independently of the reprogramming process.

systems biology↗

bioRxiv2025

IPSC-based modeling of resiliency in centenarians reveals longevity-specific signatures

Centenarians represent a human model of resilience to age-related decline, yet resiliency mechanisms remain elusive. Here, we establish an induced pluripotent stem cell (iPSC)-based platform to interrogate resilience signatures in centenarians. IPSC-derived neurons from centenarians exhibit transcriptional programs promoting synaptic integrity, calcium homeostasis, and cholesterol biosynthesis, while suppressing proteostatic stress pathways. Functionally, these neurons maintain stable calcium dynamics, reduced baseline mitochondrial activity, and energy-efficient homeostasis. Upon challenge, centenarian-derived neurons mount a robust stress response, in contrast to attenuated responses in non-centenarian controls. This resilience signature parallels adaptations in long-lived mammals and aligns with healthy brain aging, while showing erosion in Alzheimers disease and cancer. Our platform provides a scalable human model for dissecting resilience biology offering a framework to extend healthspan and mitigate age-related decline.

molecular biology↗

bioRxiv2025

Reprogramming Factors Activate a Non-Canonical Oxidative Resilience Pathway That Can Rejuvenate RPEs and Restore Vision

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. HIGHLIGHTSO_LIOSK-GSTA4 provides a dynamic, Tet2-independent stress-resilience axis. C_LIO_LIFunctional genomics pinpoints GSTA4 as a direct downstream effector activated by OSK. C_LIO_LIRPE aging involves progressive accumulation of 4-HNE that can be detoxified by GSTA4. C_LIO_LIEnhancing GSTA4 rejuvenates RPE cells, restores vision and is associated with lifespan-extending interventions. C_LI

genetics↗

bioRxiv2025

Comparative analysis of naked mole-rat thermogenesis and its potential to maintain euthermia in response to cold

The naked mole-rat (NMR) is a subterranean rodent known for its unique thermal biology, exceptional longevity and resistance to cancer and hypoxia. However, its thermal biology remains controversial, with various reports describing NMRs as poikilotherms, heterotherms, mesotherms or partial homeotherms. Here, we investigated whether the thermogenic potential of NMR brown adipose tissue and its UCP1 differ from those in mice and whether the lack of thermal insulation causes extreme changes in NMR body temperature upon cold exposure. Through longitudinal molecular, thermal, metabolic, and behavioral measurements, we found that NMRs initiated non-shivering thermogenesis and elevated body temperature but could not sustain it due to excessive heat loss and limits to substrate availability. Our results suggest that NMRs represent a unique thermoregulatory category that doesnt fit neatly into traditional classifications. In vitro and in vivo experiments showed that the NMR UCP1 is functional and can be activated and inhibited as expected for most other mammals. We further demonstrated that artificial insulation can partially restore thermoregulatory capabilities in NMRs. This study employs an advanced methodology to characterize the thermal biology of NMRs and helps resolve a long-standing controversy in the field. Significance StatementThis study provides critical insights into the thermal biology of naked mole-rats (NMRs), resolving long-standing controversies regarding their thermoregulatory strategies. We highlight the unique adaptations and limitations of NMR physiology by demonstrating that NMRs possess functional non-shivering thermogenesis and UCP1 but fail to maintain homeothermy due to excessive heat loss. Our findings suggest that artificial insulation can partially restore their thermoregulatory capabilities, offering a new perspective on the evolutionary and ecological significance of fur loss in NMRs. This research advances our understanding of mammalian thermal biology and presents an updated model for NMRs, bridging gaps between previous conflicting reports.

physiology↗

bioRxiv2025

Molecular signatures of longevity identify compounds that extendmouse lifespan and healthspan

Longevity interventions in mammals are typically discovered on a case-by-case basis, hindering systematic geroprotector development. We developed a platform for the identification of longevity interventions integrating longevity gene expression biomarkers within and across species, in silico chemical screening, analyses of selected compounds in cell culture, short-term dietary interventions coupled with omics profiling, and ultimately lifespan studies in mice. This approach identified compounds (selumetinib, vorinostat, celastrol, AZD-8055, LY-294002) that extended lifespan and/or healthspan in aged C57BL/6JN male mice, with limited effects in females. In addition, selumetinib and vorinostat increased lifespan when administered to young, genetically heterogeneous UM-HET3 mice. Our biomarker-driven platform accelerates geroprotector discovery, offering a scalable approach to target conserved longevity pathways.

systems biology↗

bioRxiv2025

In vivo chemical reprogramming is associated with a toxic accumulation of lipid droplets hindering rejuvenation

Partial reprogramming has emerged as a promising strategy to reset the epigenetic landscape of aged cells towards more youthful profiles. Recent advancements have included the development of chemical reprogramming cocktails that can lower the epigenetic and transcriptomic age of cells and upregulate mitochondrial biogenesis and oxidative phosphorylation. However, the ability for these cocktails to affect biological age in a mammalian aging model has yet to be tested. Here, we have analyzed the effects of partial chemical reprogramming on mitochondrial structure in aged mouse fibroblasts and tested its in vivo efficacy in genetically diverse male UM-HET3 mice. This approach increases the size of mitochondria, alters cristae morphology, causes an increased fusing of mitochondrial networks, and speeds up movement velocity. We also discover that partial chemical reprogramming upregulates the formation of intracellular lipid droplets. At lower doses, the chemical reprogramming cocktail can be safely administered to middle-aged mice using implantable osmotic pumps, albeit with no effect on the transcriptomic age of kidney or liver tissues, and only a modest effect on the expression of OXPHOS complexes. However, at higher doses, the cocktail causes a drastic reduction in body weight and body condition scores. In the livers and kidneys of these animals, we observe significant increases in oil red o staining indicative of excessive lipid droplet accumulation in these organs. Thus, the upregulation of lipid droplet formation during partial chemical reprogramming may cause toxicity hindering the rejuvenation of cells and tissues in aged mammals.

cell biology↗

bioRxiv2025

Non-DNA-damaging DNA-PK activation improving hearing and prolonging life due to NAD+ and SIRT upregulation

Emerging evidence strongly supports a close relationship between age-related hearing loss and frailty, highlighting the importance of early detection and intervention. Recently, we invented a mitochondria-homing drug named mitochonic acid 5 (MA-5), that increases the adenosine triphosphate (ATP) levels, rescue mitochondrial function, and protect tissue damages. Currently, the phase I clinical trial has been finished in Japan (jRCT2031210495) and the phase 2 clinical trial has already been approved by PMDA. Here we show that MA-5 improved various types of hearing loss in mouse models. Structural chemical bioanalysis revealed that MA-5 is a mixture of equal amount of S- and R- enantiomer and both S- and R- enantiomer increase ATP by binding mitochondrial protein, mitofilin. However, S-enantiomer significantly increased the NAD+ levels by binding to the NAD+-producing key enzyme nicotinamide phosphoribosyltransferase (NAMPT). Moreover, the S-enantiomer increased the sirtuin 1 protein by suppressing polyubiquitination induced by tripartite motif containing 28 (TRIM28) phosphorylation which was triggered by DNA-dependent protein kinase (DNA-PK) activation in the absence of DNA damage. Transcriptomic signatures showed that the signature of MA-5 shows an inverse correlation with aging and mortality and is oriented in the same direction as the OSKM-related iPSCs, suggesting the modification of aging pathways. Oral administration of MA-5 to mitochondrial disease model mouse showed increased survival. Our findings suggest that, in addition to enhancing ATP levels, the coordinated regulation of NAD+ metabolism, SIRT protein expression, and DNA-PK activity-constituting a novel therapeutic triad may contribute to the amelioration of hearing impairment and mitochondrial dysfunction, thereby improving life prognosis.

cell biology↗

bioRxiv2025

Boosting Cellular Longevity Through Intracellular ATP Modulation

Mitochondrial dysfunction and declining ATP production are common features of aging, yet whether ATP availability itself directly regulates cellular lifespan. However, the causal relationship between cellular ATP homeostasis and aging has not been established. Here, we developed a synthetic system to manipulate intracellular ATP independently of endogenous energy production by expressing a plasma membrane-targeted nucleotide transporter, NTT1, from the intracellular parasite Encephalitozoon cuniculi in Saccharomyces cerevisiae. NTT1 expression depleted intracellular ATP in the absence of extracellular ATP, whereas ATP supplementation produced robust NTT1-dependent ATP uptake and increased intracellular ATP abundance. ATP availability strongly influenced replicative lifespan: ATP depletion shortened lifespan, whereas ATP supplementation restored and extended lifespan in NTT1-expressing cells. Unexpectedly, extracellular ATP also extended lifespan in wild-type cells that lack ATP import, revealing an NTT1-independent response to extracellular ATP. Transcriptomic analyses showed that NTT1- mediated ATP import suppresses glucose uptake, carbohydrate catabolism, mitochondrial respiration, and autophagy, whereas extracellular ATP elicits a distinct transcriptional response in wild-type cells involving metabolic, mitochondrial, and signaling pathways. Single-cell aging analyses further showed that ATP supplementation extends lifespan across distinct aging trajectories and shifts cells away from the mitochondrial dysfunction-associated aging state. Finally, experiments in cells lacking mitochondrial DNA separated these effects mechanistically: NTT1-associated lifespan phenotypes and high-ATP toxicity required functional mitochondria, whereas extracellular ATP extended lifespan in wild-type cells independently of mitochondrial respiration. Together, these findings demonstrate that ATP availability is a direct regulator of cellular aging and reveal distinct metabolic and extracellular ATP-responsive routes through which cellular energy state influences longevity. SignificanceCellular energy homeostasis is a crucial factor in determining the health and longevity of organisms. While intracellular ATP levels are tightly regulated, the idea that cells can directly take in extracellular ATP to influence metabolism has not been thoroughly explored. In this study, we engineered yeast cells to import external ATP and demonstrated that this approach significantly alters mitochondrial function, metabolic flow, and aging processes. Our findings show that ATP uptake inhibits catabolic pathways and modulates mitochondrial bioenergetics function, thereby extending cellular lifespan through a novel and non-traditional mechanism. This research reveals an unexpected degree of metabolic flexibility and introduces a synthetic biology-based method to reprogram energy metabolism and longevity. The principles established in this study provide a new framework for understanding the role of cellular bioenergetics in aging, highlighting how the modulation of ATP availability can impact metabolic states and lifespan regulation.

molecular biology↗