Inhibition of DAPK3 Suppresses Radiation-Induced Cellular Senescence by Activation of a PGC1α-Dependent Metabolism Pathway in Brain Endothelial Cells.

In the brain, environmental changes, such as neuroinflammation, can induce senescence, characterized by the decreased proliferation of neurons and dendrites and synaptic and vascular damage, resulting in cognitive decline. Senescence promotes neuroinflammatory disorders by senescence-associated secretory phenotypes and reactive oxygen species. In human brain microvascular endothelial cells (HBMVECs), we demonstrate that chronological aging and irradiation increase death-associated protein kinase 3 (DAPK3) expression. To confirm the role of DAPK3 in HBMVEC senescence, we disrupted DAPK3 activity using small interfering RNA (siRNA) or a dominant-negative mutant (DAPK3-P216S), which reduced cellular senescence phenotypes, as assessed by changes in tube formation, senescence-associated beta-galactosidase activity, and cell proliferation. In endothelial cells, DAPK3 promotes cellular senescence by regulating the phosphorylation and inactivation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) via the protein kinase B pathway, resulting in the decreased expression of mitochondrial metabolism-associated genes, such as ATP5G1, BDNF, and COX5A. Our studies show that DAPK3 is involved in cellular senescence and PGC1α regulation, suggesting that DAPK3 regulation may be important for treating aging-related brain diseases or the response to radiation therapy.

The effects of resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged adults.

Denervated myofibers and senescent cells are hallmarks of skeletal muscle aging. However, sparse research has examined how resistance training affects these outcomes. We investigated the effects of unilateral leg extensor resistance training (2 days/week for 8 weeks) on denervated myofibers, senescent cells, and associated protein markers in apparently healthy middle-aged participants (MA, 55 ± 8 years old, 17 females, 9 males). We obtained dual-leg vastus lateralis (VL) muscle cross-sectional area (mCSA), VL biopsies, and strength assessments before and after training. Fiber cross-sectional area (fCSA), satellite cells (Pax7+), denervated myofibers (NCAM+), senescent cells (p16+ or p21+), proteins associated with denervation and senescence, and senescence-associated secretory phenotype (SASP) proteins were analyzed from biopsy specimens. Leg extensor peak torque increased after training (p < .001), while VL mCSA trended upward (interaction p = .082). No significant changes were observed for Type I/II fCSAs, NCAM+ myofibers, or senescent (p16+ or p21+) cells, albeit satellite cells increased after training (p = .037). While >90% satellite cells were not p16+ or p21+, most p16+ and p21+ cells were Pax7+ (>90% on average). Training altered 13 out of 46 proteins related to muscle-nerve communication (all upregulated, p < .05) and 10 out of 19 proteins related to cellular senescence (9 upregulated, p < .05). Only 1 out of 17 SASP protein increased with training (IGFBP-3, p = .031). In conclusion, resistance training upregulates proteins associated with muscle-nerve communication in MA participants but does not alter NCAM+ myofibers. Moreover, while training increased senescence-related proteins, this coincided with an increase in satellite cells but not alterations in senescent cell content or SASP proteins. These latter findings suggest shorter term resistance training is an unlikely inducer of cellular senescence in apparently healthy middle-aged participants. However, similar study designs are needed in older and diseased populations before definitive conclusions can be drawn.

Evidence of a pan-tissue decline in stemness during human aging.

Despite their biological importance, the role of stem cells in human aging remains to be elucidated. In this work, we applied a machine learning methodology to GTEx transcriptome data and assigned stemness scores to 17,382 healthy samples from 30 human tissues aged between 20 and 79 years. We found that ~60% of the studied tissues exhibit a significant negative correlation between the subject's age and stemness score. The only significant exception was the uterus, where we observed an increased stemness with age. Moreover, we observed that stemness is positively correlated with cell proliferation and negatively correlated with cellular senescence. Finally, we also observed a trend that hematopoietic stem cells derived from older individuals might have higher stemness scores. In conclusion, we assigned stemness scores to human samples and show evidence of a pan-tissue loss of stemness during human aging, which adds weight to the idea that stem cell deterioration may contribute to human aging.

PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation.

Cellular senescence is a permanent cell cycle arrest that can be triggered by both internal and external genotoxic stressors, such as telomere dysfunction and DNA damage. The execution of senescence is mainly by two pathways, p16/RB and p53/p21, which lead to CDK4/6 inhibition and RB activation to block cell cycle progression. While the regulation of p53/p21 signaling in response to DNA damage and other insults is well-defined, the regulation of the p16/RB pathway in response to various stressors remains poorly understood. Here, we report a novel function of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase, as a potent inhibitor of p16 expression and senescence induction by ionizing radiation (IR), such as γ-rays. The results show that ectopic PR55α expression in normal pancreatic cells inhibits p16 transcription, increases RB phosphorylation, and blocks IR-induced senescence. Conversely, PR55α-knockdown by shRNA in pancreatic cancer cells elevates p16 transcription, reduces RB phosphorylation, and triggers senescence induction after IR. Furthermore, this PR55α function in the regulation of p16 and senescence is p53-independent because it was unaffected by the mutational status of p53. Moreover, PR55α only affects p16 expression but not p14 (ARF) expression, which is also transcribed from the same CDKN2A locus but from an alternative promoter. In normal human tissues, levels of p16 and PR55α proteins were inversely correlated and mutually exclusive. Collectively, these results describe a novel function of PR55α/PP2A in blocking p16/RB signaling and IR-induced cellular senescence.

Senescence Promotes the Recovery of Stemness among Cancer Cells via Reprograming.

Both the senescence of cancer cells and the maintenance of cancer stem cells seem to be mutually exclusive because senescence is considered a physiological mechanism that effectively suppresses tumor growth. Recent studies have revealed common signaling pathways between cellular senescence and the maintenance of stemness in cancer cells, thus challenging the conventional understanding of this process. Although the links between these processes have not yet been fully elucidated, emerging evidence indicates that senescent cancer cells can undergo reprograming to recover stemness. Herein, we provide a comprehensive overview of the close correlation between senescence and stemness reprograming in cancer cells, with a particular focus on the mechanisms by which senescent cancer cells recover their stemness in various tumor systems.

Cytokine profiling in senescent and reactive astrocytes: A systematic review.

Astrocytes play an important role in neuroinflammation by producing proinflammatory molecules. In response to various stressful stimuli, astrocytes can become senescent or reactive, both are present in age-associated cognitive impairment and other neurodegenerative diseases, and contribute to neuroinflammation. However, there are no studies that compare the cytokines secreted by these types of astrocytes in the brain during aging. Hence, we aimed to broaden the picture of the secretory profiles and to differentiate the variability between them. Therefore, a systematic review was conducted following the guidelines of the "Reporting Items for Systematic Review and Meta-Analyses". Only three studies that met the inclusion terms evaluated age-related cytokine secretion, however, no evaluation of senescence or gliosis was performed. Consequently, to increase the spectrum of the review, studies where those phenotypes were induced and cytokines determined were included. Although some cytokines were common for gliosis and senescence, some interesting differences were also found. The dissimilarities in cytokines secretion between these phenotypes could be studied in the future as potential markers.

Omilancor mitigates the senescence of nucleus pulposus cells induced by DDP through targeting MAP2K6.

PURPOSE: This study explores the potential of Omilancor in treating Intervertebral Disc Degeneration (IDD) through MAP2K6 targeting. METHODS: We analyzed mRNA microarray datasets to pinpoint MAP2K6 as a key regulator implicated in IDD progression. Follow-up studies demonstrated that cisplatin (DDP) could prompt cellular senescence in vitro by upregulating MAP2K6 expression. Through molecular docking and other analyses, we identified Omilancor as a compound capable of binding to MAP2K6. This interaction effectively impeded the cellular senescence induced by DDP. RESULTS: We further showed that administration of Omilancor could significantly alleviate the degeneration of IVDs in annulus fibrosus puncture-induced rat model. CONCLUSIONS: Omilancor shows promise as a treatment for IDD by targeting MAP2K6-mediated cellular senescence.

Cellular senescence in reproduction: a two-edged sword†.

Cellular senescence (CS) is the state when cells are no longer capable to divide even after stimulation with grown factors. Cells that begin to undergo CS stop in the cell cycle and enter a suspended state without committing to programmed cell death. These cells assume a specific phenotype and influence their microenvironment by secreting molecules and extracellular vesicles that are part of the so-called senescent cell-associated secretory phenotype (SASP). Cellular senescence is intertwined with physiological and pathological conditions in the human organism. In terms of reproduction, senescent cells are present from reproductive tissues and germ cells to gestational tissues, and participate from fertilization to delivery, going through adverse reproductive outcomes such as pregnancy losses. Furthermore, various SASP molecules are enriched in gestational tissues throughout pregnancy. Thus, the aim of this review is to provide a basis about the features and potential roles played by CS throughout the reproductive process, encompassing its implication in each step of it and proposing a way to manage it in adverse reproductive contexts.

Senolytic therapeutics: An emerging treatment modality for osteoarthritis.

Osteoarthritis (OA), a chronic joint disease affecting millions of people aged over 65 years, is the main musculoskeletal cause of diminished joint mobility in the elderly. It is characterized by lingering pain and increasing deterioration of articular cartilage. Aging and accumulation of senescent cells (SCs) in the joints are frequently associated with OA. Apoptosis resistance; irreversible cell cycle arrest; increased p16INK4a expression, secretion of senescence-associated secretory phenotype factors, senescence-associated ß-galactosidase levels, secretion of extracellular vesicles, and levels of reactive oxygen and reactive nitrogen species; and mitochondrial dysregulation are some common changes in cellular senescence in joint tissues. Development of OA correlates with an increase in the density of SCs in joint tissues. Senescence-associated secretory phenotype has been linked to OA and cartilage breakdown. Senolytics and therapeutic pharmaceuticals are being focused upon for OA management. SCs can be selectively eliminated or killed by senolytics to halt the pathogenesis and progression of OA. Comprehensive understanding of how aging affects joint dysfunction will benefit OA patients. Here, we discuss age-related mechanisms associated with OA pathogenesis and senolytics as an emerging modality in the management of age-related SCs and pathogenesis of OA in preclinical and clinical studies.

Mitochondrial injury induced by a Salmonella genotoxin triggers the proinflammatory senescence-associated secretory phenotype.

Bacterial genotoxins damage host cells by targeting their chromosomal DNA. In the present study, we demonstrate that a genotoxin of Salmonella Typhi, typhoid toxin, triggers the senescence-associated secretory phenotype (SASP) by damaging mitochondrial DNA. The actions of typhoid toxin disrupt mitochondrial DNA integrity, leading to mitochondrial dysfunction and disturbance of redox homeostasis. Consequently, it facilitates the release of damaged mitochondrial DNA into the cytosol, activating type I interferon via the cGAS-STING pathway. We also reveal that the GCN2-mediated integrated stress response plays a role in the upregulation of inflammatory components depending on the STING signaling axis. These SASP factors can propagate the senescence effect on T cells, leading to senescence in these cells. These findings provide insights into how a bacterial genotoxin targets mitochondria to trigger a proinflammatory SASP, highlighting a potential therapeutic target for an anti-toxin intervention.

Senescence and aging differentially alter key metabolic pathways in murine brain microglia.

Microglia, the resident immune cells of the central nervous system, are critically involved in maintaining brain homeostasis. With age, microglia display morphological and functional alterations that have been associated with cognitive decline and neurodegeneration. Although microglia seem to participate in an increasing number of biological processes which require a high energy demand, little is known about their metabolic regulation under physiological and pathophysiological conditions and during aging/senescence. Here, we determined mRNA expression levels of critical rate limiting enzymes in several key metabolic pathways including glycolysis, pentose phosphate pathway, fatty acid oxidation and synthesis in association with oxidative phosphorylation in microglia, both under aging and senescent conditions. We found strong evidence for different metabolic changes occuring in senescent vs. aged microglia cells. While senescent microglia display a hypermetabolic state as indicated by increased expression of key enzymes involved in glycolysis and pentose phosphate pathway, aging microglia are rather in a state of hypometabolism. Our findings indicate that studies involving aging and senescent microglia require a clear differentiation between these microglial states due to profound metabolic differences observed here. Understanding metabolic changes in senescent and aged microglia may lead to novel strategies to decrease over-activation of these cells due to aging, which is associated to the process of inflamm-aging and neurodegeneration.

Lessons from inducible pluripotent stem cell models on neuronal senescence in aging and neurodegeneration.

Age remains the central risk factor for many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Although the mechanisms of aging are complex, the age-related accumulation of senescent cells in neurodegeneration is well documented and their clearance can alleviate disease-related features in preclinical models. Senescence-like characteristics are observed in both neuronal and glial lineages, but their relative contribution to aging and neurodegeneration remains unclear. Human pluripotent stem cell-derived neurons provide an experimental model system to induce neuronal senescence. However, the extensive heterogeneity in the profile of senescent neurons and the methods to assess senescence remain major challenges. Here, we review the evidence of cellular senescence in neuronal aging and disease, discuss human pluripotent stem cell-based model systems used to investigate neuronal senescence and propose a panel of cellular and molecular hallmarks to characterize senescent neurons. Understanding the role of neuronal senescence may yield novel therapeutic opportunities in neurodegenerative disease.

Endothelial Senescence: From Macro- to Micro-Vasculature and Its Implications on Cardiovascular Health.

Endothelial cells line at the most inner layer of blood vessels. They act to control hemostasis, arterial tone/reactivity, wound healing, tissue oxygen, and nutrient supply. With age, endothelial cells become senescent, characterized by reduced regeneration capacity, inflammation, and abnormal secretory profile. Endothelial senescence represents one of the earliest features of arterial ageing and contributes to many age-related diseases. Compared to those in arteries and veins, endothelial cells of the microcirculation exhibit a greater extent of heterogeneity. Microcirculatory endothelial senescence leads to a declined capillary density, reduced angiogenic potentials, decreased blood flow, impaired barrier properties, and hypoperfusion in a tissue or organ-dependent manner. The heterogeneous phenotypes of microvascular endothelial cells in a particular vascular bed and across different tissues remain largely unknown. Accordingly, the mechanisms underlying macro- and micro-vascular endothelial senescence vary in different pathophysiological conditions, thus offering specific target(s) for therapeutic development of senolytic drugs.

Magnesium and the Hallmarks of Aging.

Magnesium is an essential ion in the human body that regulates numerous physiological and pathological processes. Magnesium deficiency is very common in old age. Age-related chronic diseases and the aging process itself are frequently associated with low-grade chronic inflammation, called 'inflammaging'. Because chronic magnesium insufficiency has been linked to excessive generation of inflammatory markers and free radicals, inducing a chronic inflammatory state, we formerly hypothesized that magnesium inadequacy may be considered among the intermediaries helping us explain the link between inflammaging and aging-associated diseases. We show in this review evidence of the relationship of magnesium with all the hallmarks of aging (genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, disabled autophagy, dysbiosis, and chronic inflammation), which may positively affect the human healthspan. It is feasible to hypothesize that maintaining an optimal balance of magnesium during one's life course may turn out to be a safe and economical strategy contributing to the promotion of healthy aging. Future well-designed studies are necessary to further explore this hypothesis.

Apigenin delays postovulatory oocyte aging by reducing oxidative stress through SIRT1 upregulation.

After ovulation, senescent oocytes inevitably experience reduced quality and defects in embryonic development. Apigenin (API) is a flavonoid with a wide range of pharmacological effects. Therefore, this study examined the protective effects of API on the quality of porcine oocytes during in-vitro ageing and the underlying mechanisms. The results showed that API treatment could reduce the activation rate after aging for 48 h. In addition, API significantly reduced reactive oxygen species, abnormal distribution of mitochondria, early apoptosis in ageing oocytes, increased glutathione, and mitochondrial adenosine triphosphate levels in ageing oocytes. Importantly, API increased the embryonic development rate in aged oocytes. We also examined molecular changes, finding decreased sirtuin 1 expression in in-vitro postovulatory oocytes, but API reversed this effect. Our results suggest that API attenuates the deterioration of oocyte quality during in-vitro ageing, possibly by reducing oxidative stress through the upregulation of sirtuin 1.

Astragalus polysaccharides attenuate rat aortic endothelial senescence via regulation of the SIRT-1/p53 signaling pathway.

BACKGROUND: Astragalus polysaccharides (APS) have been verified to have antioxidative and antiaging activities in the mouse liver and brain. However, the effect of APS on aortic endothelial senescence in old rats and its underlying mechanism are currently unclear. Here, we aimed to elucidate the effects of APS on rat aortic endothelial oxidative stress and senescence in vitro and in vivo and investigate the potential molecular targets. METHODS: Twenty-month-old natural aging male rats were treated with APS (200 mg/kg, 400 mg/kg, 800 mg/kg daily) for 3 months. Serum parameters were tested using corresponding assay kits. Aortic morphology was observed by staining with hematoxylin and eosin (H&E) and Verhoeff Van Gieson (VVG). Aging-related protein levels were evaluated using immunofluorescence and western blot analysis. Primary rat aortic endothelial cells (RAECs) were isolated by tissue explant method. RAEC mitochondrial function was evaluated by the mitochondrial membrane potential (MMP) measured with the fluorescent lipophilic cationic dye JC­1. Intracellular total antioxidant capacity (T-AOC) was detected by a commercial kit. Cellular senescence was assessed using senescence-associated-ß-galactosidase (SA-ß-Gal) staining. RESULTS: Treatment of APS for three months was found to lessen aortic wall thickness, renovate vascular elastic tissue, improve vascular endothelial function, and reduce oxidative stress levels in 20-month-old rats. Primary mechanism analysis showed that APS treatment enhanced Sirtuin 1 (SIRT-1) protein expression and decreased the levels of the aging marker proteins p53, p21 and p16 in rat aortic tissue. Furthermore, APS abated hydrogen peroxide (H2O2)-induced cell senescence and restored H2O2-induced impairment of the MMP and T-AOC in RAECs. Similarly, APS increased SIRT-1 and decreased p53, p21 and p16 protein levels in senescent RAECs isolated from old rats. Knockdown of SIRT-1 diminished the protective effect of APS against H2O2-induced RAEC senescence and T-AOC loss, increased the levels of the downstream proteins p53 and p21, and abolished the inhibitory effect of APS on the expression of these proteins in RAECs. CONCLUSION: APS may reduce rat aortic endothelial oxidative stress and senescence via the SIRT-1/p53 signaling pathway.

Detection of senescence using machine learning algorithms based on nuclear features.

Cellular senescence is a stress response with broad pathophysiological implications. Senotherapies can induce senescence to treat cancer or eliminate senescent cells to ameliorate ageing and age-related pathologies. However, the success of senotherapies is limited by the lack of reliable ways to identify senescence. Here, we use nuclear morphology features of senescent cells to devise machine-learning classifiers that accurately predict senescence induced by diverse stressors in different cell types and tissues. As a proof-of-principle, we use these senescence classifiers to characterise senolytics and to screen for drugs that selectively induce senescence in cancer cells but not normal cells. Moreover, a tissue senescence score served to assess the efficacy of senolytic drugs and identified senescence in mouse models of liver cancer initiation, ageing, and fibrosis, and in patients with fatty liver disease. Thus, senescence classifiers can help to detect pathophysiological senescence and to discover and validate potential senotherapies.

The Potential Role and Therapeutic Relevance of Cellular Senescence in Skeletal Pathophysiology.

Biological aging profoundly impairs the homeostasis of the skeletal system. Cellular senescence, a hallmark of biological aging, plays an instrumental role in bone disease. The underlying mechanisms of cellular senescence, triggered by both intracellular and extracellular stimuli, are multifaceted and yet to be uncovered. Recent research indicates that acute cellular senescence often serves beneficial roles, such as contributing to growth, development, and tissue regeneration. By contrast, chronic cellular senescence, primarily driven by the accumulation of senescent cells (SnCs) and the release of senescence-associated secretory phenotypes (SASP), has detrimental effects on the skeletal system by irreversibly disrupting bone homeostasis and promoting age-related disorders. Furthermore, the bone marrow is rich in immune cells and their exposure to SASP often leads to immune dysfunction, resulting in unresolved chronic inflammation and compromised adaptive immunity. Until now, the impact of SnCs and SASP on the skeleton has remained elusive. Meanwhile, extensive efforts are being made to combat age-related diseases through various strategies. Among them, SnCs and SASP are the primary targets for antiaging therapeutic clearance, resulting in the development of "senolytics" and "senomorphics," respectively. In this review, we summarize and highlight the role of SnCs and SASP in skeletal pathophysiology, the mechanism of cellular senescence in affecting bone metabolism, and potential therapeutic approaches, particularly senolytics and senomorphics, in treating cellular senescence-related bone diseases.

High-throughput assessment of cellular senescence.

Cellular senescence is a molecular process that is activated in response to a large variety of distinct stress signals. Mechanistically, cellular senescence is characterized by an arrest in cell cycle accompanied by phenotypic adaptations and physiological alterations including changes in the secretory profile of senescent cells termed the senescence-associated secretory phenotype (SASP). Here we describe a detailed, automation- compatible method for the detection of senescence-associated beta galactosidase (SA-ß-gal) activity as a hallmark of cellular senescence using a conventional fluorescent microscope equipped with a transmitted light module. Moreover, we outline a protocol for the automated analysis of cellular senescence using convolutional neural networks (CNNs) and mathematical morphology. In sum, we provide a toolset for the high throughput assessment of cellular senescence based on light microscopy and automated image analysis.

Insights into targeting cellular senescence with senolytic therapy: The journey from preclinical trials to clinical practice.

Interconnected, fundamental aging processes are central to many illnesses and diseases. Cellular senescence is a mechanism that halts the cell cycle in response to harmful stimuli. Senescent cells (SnCs) can emerge at any point in life, and their persistence, along with the numerous proteins they secrete, can negatively affect tissue function. Interventions aimed at combating persistent SnCs, which can destroy tissues, have been used in preclinical models to delay, halt, or even reverse various diseases. Consequently, the development of small-molecule senolytic medicines designed to specifically eliminate SnCs has opened potential avenues for the prevention or treatment of multiple diseases and age-related issues in humans. In this review, we explore the most promising approaches for translating small-molecule senolytics and other interventions targeting senescence in clinical practice. This discussion highlights the rationale for considering SnCs as therapeutic targets for diseases affecting individuals of all ages.