Senescent characteristics of human corneal endothelial cells upon ultraviolet-A exposure.

PURPOSE: The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. METHODS: We assessed cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. RESULTS: Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-ß-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. CONCLUSIONS: In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.

A Fully-Automated Senescence Test (FAST) for the high-throughput quantification of senescence-associated markers.

Cellular senescence is a major driver of aging and age-related diseases. Quantification of senescent cells remains challenging due to the lack of senescence-specific markers and generalist, unbiased methodology. Here, we describe the Fully-Automated Senescence Test (FAST), an image-based method for the high-throughput, single-cell assessment of senescence in cultured cells. FAST quantifies three of the most widely adopted senescence-associated markers for each cell imaged: senescence-associated ß-galactosidase activity (SA-ß-Gal) using X-Gal, proliferation arrest via lack of 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and enlarged morphology via increased nuclear area. The presented workflow entails microplate image acquisition, image processing, data analysis, and graphing. Standardization was achieved by i) quantifying colorimetric SA-ß-Gal via optical density; ii) implementing staining background controls; iii) automating image acquisition, image processing, and data analysis. In addition to the automated threshold-based scoring, a multivariate machine learning approach is provided. We show that FAST accurately quantifies senescence burden and is agnostic to cell type and microscope setup. Moreover, it effectively mitigates false-positive senescence marker staining, a common issue arising from culturing conditions. Using FAST, we compared X-Gal with fluorescent C12FDG live-cell SA-ß-Gal staining on the single-cell level. We observed only a modest correlation between the two, indicating that those stains are not trivially interchangeable. Finally, we provide proof of concept that our method is suitable for screening compounds that modify senescence burden. This method will be broadly useful to the aging field by enabling rapid, unbiased, and user-friendly quantification of senescence burden in culture, as well as facilitating large-scale experiments that were previously impractical.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Ex vivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Rutin is a potent senomorphic agent to target senescent cells and can improve chemotherapeutic efficacy.

Aging is a major risk factor for most chronic disorders, for which cellular senescence is one of the central hallmarks. Senescent cells develop the pro-inflammatory senescence-associated secretory phenotype (SASP), which significantly contributes to organismal aging and age-related disorders. Development of senotherapeutics, an emerging class of therapeutic agents to target senescent cells, allows to effectively delay aging and alleviate chronic pathologies. Here we report preliminary outputs from screening of a natural medicinal agent (NMA) library for senotherapeutic candidates and validated several agents with prominent potential as senomorphics. Rutin, a phytochemical constituent found in a number of plants, showed remarkable capacity in targeting senescent cells by dampening expression of the full spectrum SASP. Further analysis indicated that rutin restrains the acute stress-associated phenotype (ASAP) by specifically interfering with the interactions of ATM with HIF1α, a master regulator of cellular and systemic homeostasis activated during senescence, and of ATM with TRAF6, part of a key signaling axis supporting the ASAP development toward the SASP. Conditioned media produced by senescent stromal cells enhanced the malignant phenotypes of prostate cancer cells, including in vitro proliferation, migration, invasion, and more importantly, chemoresistance, while rutin remarkably downregulated these gain-of-functions. Although classic chemotherapy reduced tumor progression, the treatment outcome was substantially improved upon combination of a chemotherapeutic agent with rutin. Our study provides a proof of concept for rutin as an emerging natural senomorphic agent, and presents an effective therapeutic avenue for alleviating age-related pathologies including cancer.

Proteomic Analysis of the Senescence-Associated Secretory Phenotype: GDF-15, IGFBP-2, and Cystatin-C Are Associated With Multiple Aging Traits.

Cellular senescence, a hallmark of aging, results in a senescence-associated secretory phenotype (SASP) with an increased production of proinflammatory cytokines, growth factors, and proteases. Evidence from nonhuman models demonstrates that SASP contributes to tissue dysfunction and pathological effects of aging. However, there are relatively few human studies on the relationship between SASP and aging-related health outcomes. Proteins from the SASP Atlas were measured in plasma using aptamer-based proteomics (SomaLogic). Regression models were used to identify SASP protein associations with aging-related traits representing multiple aspects of physiology in 1 201 participants from 2 human cohort studies (BLSA/GESTALT and InCHIANTI). Traits examined were fasting glucose, C-reactive protein, interleukin-6, alkaline phosphatase, blood urea nitrogen, albumin, red blood cell distribution width, waist circumference, systolic and diastolic blood pressure, gait speed, and grip strength. Study results were combined with a fixed-effect inverse-variance weighted meta-analysis. In the meta-analysis, 28 of 77 SASP proteins were significantly associated with age. Of the 28 age-associated SASP proteins, 18 were significantly associated with 1 or more clinical traits, and 7 SASP proteins were significantly associated with 3 or more traits. Growth/differentiation factor 15, Insulin-like growth factor-binding protein 2, and Cystatin-C showed significant associations with inflammatory markers and measures of physical function (grip strength or gait speed). These results support the relevance of SASP proteins to human aging, identify specific traits that are potentially affected by SASP, and prioritize specific SASP proteins for their utility as biomarkers of human aging.

Chromosome instability and aneuploidy in the mammalian brain.

This review investigates the role of aneuploidy and chromosome instability (CIN) in the aging brain. Aneuploidy refers to an abnormal chromosomal count, deviating from the normal diploid set. It can manifest as either a deficiency or excess of chromosomes. CIN encompasses a broader range of chromosomal alterations, including aneuploidy as well as structural modifications in DNA. We provide an overview of the state-of-the-art methodologies utilized for studying aneuploidy and CIN in non-tumor somatic tissues devoid of clonally expanded populations of aneuploid cells.CIN and aneuploidy, well-established hallmarks of cancer cells, are also associated with the aging process. In non-transformed cells, aneuploidy can contribute to functional impairment and developmental disorders. Despite the importance of understanding the prevalence and specific consequences of aneuploidy and CIN in the aging brain, these aspects remain incompletely understood, emphasizing the need for further scientific investigations.This comprehensive review consolidates the present understanding, addresses discrepancies in the literature, and provides valuable insights for future research efforts.

PDK4-dependent hypercatabolism and lactate production of senescent cells promotes cancer malignancy.

Senescent cells remain metabolically active, but their metabolic landscape and resulting implications remain underexplored. Here, we report upregulation of pyruvate dehydrogenase kinase 4 (PDK4) upon senescence, particularly in some stromal cell lines. Senescent cells display a PDK4-dependent increase in aerobic glycolysis and enhanced lactate production but maintain mitochondrial respiration and redox activity, thus adopting a special form of metabolic reprogramming. Medium from PDK4+ stromal cells promotes the malignancy of recipient cancer cells in vitro, whereas inhibition of PDK4 causes tumor regression in vivo. We find that lactate promotes reactive oxygen species production via NOX1 to drive the senescence-associated secretory phenotype, whereas PDK4 suppression reduces DNA damage severity and restrains the senescence-associated secretory phenotype. In preclinical trials, PDK4 inhibition alleviates physical dysfunction and prevents age-associated frailty. Together, our study confirms the hypercatabolic nature of senescent cells and reveals a metabolic link between cellular senescence, lactate production, and possibly, age-related pathologies, including but not limited to cancer.

Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration following peripheral nerve injury.

Following peripheral nerve injury, successful axonal growth and functional recovery require Schwann cell (SC) reprogramming into a reparative phenotype, a process dependent upon c-Jun transcription factor activation. Unfortunately, axonal regeneration is greatly impaired in aged organisms and following chronic denervation, which can lead to poor clinical outcomes. While diminished c-Jun expression in SCs has been associated with regenerative failure, it is unclear whether the inability to maintain a repair state is associated with the transition into an axonal growth inhibition phenotype. We here find that reparative SCs transition into a senescent phenotype, characterized by diminished c-Jun expression and secretion of inhibitory factors for axonal regeneration in aging and chronic denervation. In both conditions, the elimination of senescent SCs by systemic senolytic drug treatment or genetic targeting improved nerve regeneration and functional recovery, increased c-Jun expression and decreased nerve inflammation. This work provides the first characterization of senescent SCs and their influence on axonal regeneration in aging and chronic denervation, opening new avenues for enhancing regeneration and functional recovery after peripheral nerve injuries.

Proteomic quantification of native and ECM-enriched mouse ovaries reveals an age-dependent fibro-inflammatory signature.

The ovarian microenvironment becomes fibrotic and stiff with age, in part due to increased collagen and decreased hyaluronan. However, the extracellular matrix (ECM) is a complex network of hundreds of proteins, glycoproteins, and glycans which are highly tissue specific and undergo pronounced changes with age. To obtain an unbiased and comprehensive profile of age-associated alterations to the murine ovarian proteome and ECM, we used a label-free quantitative proteomic methodology. We validated conditions to enrich for the ECM prior to proteomic analysis. Following analysis by data-independent acquisition (DIA) and quantitative data processing, we observed that both native and ECM-enriched ovaries clustered separately based on age, indicating distinct age-dependent proteomic signatures. We identified a total of 4,721 proteins from both native and ECM-enriched ovaries, of which 383 proteins were significantly altered with advanced age, including 58 ECM proteins. Several ECM proteins upregulated with age have been associated with fibrosis in other organs, but to date their roles in ovarian fibrosis are unknown. Pathways regulating DNA metabolism and translation were downregulated with age, whereas pathways involved in ECM remodeling and immune response were upregulated. Interestingly, immune-related pathways were upregulated with age even in ECM-enriched ovaries, suggesting a novel interplay between the ECM and the immune system. Moreover, we identified putative markers of unique immune cell populations present in the ovary with age. These findings provide evidence from a proteomic perspective that the aging ovary provides a fibroinflammatory milieu, and our study suggests target proteins which may drive these age-associated phenotypes for future investigation.

Gene expression signatures of human senescent corneal and conjunctival epithelial cells.

PURPOSE: This study aimed to investigate the senescent phenotypes of human corneal and conjunctival epithelial cells. METHODS: We examined cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation, and expression of senescence markers (p16 and p21). RNA sequencing analysis was conducted to compare gene expression profiles between senescent and non-senescent cells. Finally, the potential involvement of senescent cells in the pathogenesis of ocular surface diseases was investigated. RESULTS: X-irradiated corneal and conjunctival epithelial cells exhibited typical senescence phenotypes, i.e., flattened morphologies, increased SA-ß-gal activity, decreased cell proliferation, and increased expression of senescence markers, p16 and p21. RNA-seq analysis revealed substantial differences in gene expression profiles between senescent corneal (SCo) and conjunctival epithelial cells (SCj). Moreover, SCj were detected in pathological conjunctival tissues associated with limbal stem cell deficiency (LSCD) due to Stevens-Johnson syndrome or chemical burns, potentially being involved in abnormal differentiation. CONCLUSION: This study highlights the cellular and molecular characteristics of senescent ocular surface cells, particularly in SCj that show abnormal keratin expression, and their potential roles in severe ocular surface diseases and pathology.

Role of the Senescence-Associated Factor Dipeptidyl Peptidase 4 in the Pathogenesis of SARS-CoV-2 Infection.

During cellular senescence, persistent growth arrest and changes in protein expression programs are accompanied by a senescence-associated secretory phenotype (SASP). In this study, we detected the upregulation of the SASP-related protein dipeptidyl peptidase 4 (DDP4) in human primary lung cells rendered senescent by exposure to ionizing radiation. DPP4 is an exopeptidase that plays a crucial role in the cleavage of various proteins, resulting in the loss of N-terminal dipeptides and proinflammatory effects. Interestingly, our data revealed an association between severe coronavirus disease 2019 (COVID-19) and DDP4, namely that DPP4 levels increased in the plasma of patients with COVID-19 and were correlated with age and disease progression. Although we could not determine the direct effect of DDP4 on viral replication, mechanistic studies in cell culture revealed a negative impact on the expression of the tight junction protein zonula occludens-1 (ZO-1), which contributes to epithelial barrier function. Mass spectrometry analysis indicated that DPP4 overexpressing cells exhibited a decrease in ZO-1 and increased expression of pro-inflammatory cytokines and chemokines. By investigating the effect of DPP4 on the barrier function of human primary cells, we detected an increase in ZO-1 using DPP4 inhibitors. These results provide an important contribution to our understanding of DPP4 in the context of senescence, suggesting that DPP4 plays a major role as part of the SASP. Our results provide evidence that cellular senescence, a hallmark of aging, has an important impact on respiratory infections.

Cellular Senescence Contributes to Large Elastic Artery Stiffening and Endothelial Dysfunction With Aging: Amelioration With Senolytic Treatment.

BACKGROUND: Here, we assessed the role of cellular senescence and the senescence associated secretory phenotype (SASP) in age-related aortic stiffening and endothelial dysfunction. METHODS: We studied young (6-8 mo) and old (27-29 mo) p16-3MR mice, which allows for genetic-based clearance of senescent cells with ganciclovir (GCV). We also treated old C57BL/6N mice with the senolytic ABT-263. RESULTS: In old mice, GCV reduced aortic stiffness assessed by aortic pulse wave velocity (PWV; 477±10 vs. 382±7 cm/s, P<0.05) to young levels (old-GCV vs. young-vehicle, P=0.35); ABT-263 also reduced aortic PWV in old mice (446±9 to 356±11 cm/s, P<0.05). Aortic adventitial collagen was reduced by GCV (P<0.05) and ABT-263 (P=0.12) in old mice. To show an effect of the circulating SASP, we demonstrated that plasma exposure from Old-vehicle p16-3MR mice, but not from Old-GCV mice, induced aortic stiffening assessed ex vivo (elastic modulus; P<0.05). Plasma proteomics implicated glycolysis in circulating SASP-mediated aortic stiffening. In old p16-3MR mice, GCV increased endothelial function assessed via peak carotid artery endothelium-dependent dilation (EDD; Old-GCV, 94±1% vs. Old-vehicle, 84±2%, P<0.05) to young levels (Old-GCV vs. young-vehicle, P=0.98), and EDD was higher in old C57BL/6N mice treated with ABT-263 vs. vehicle (96±1% vs. 82±3%, P<0.05). Improvements in endothelial function were mediated by increased nitric oxide (NO) bioavailability (P<0.05) and reduced oxidative stress (P<0.05). Circulating SASP factors related to NO signaling were associated with greater NO-mediated EDD following senescent cell clearance. CONCLUSIONS: Cellular senescence and the SASP contribute to vascular aging and senolytics hold promise for improving age-related vascular function.

Elimination of senescent cells by treatment with Navitoclax/ABT263 reverses whole brain irradiation-induced blood-brain barrier disruption in the mouse brain.

Whole brain irradiation (WBI), a commonly employed therapy for multiple brain metastases and as a prophylactic measure after cerebral metastasis resection, is associated with a progressive decline in neurocognitive function, significantly impacting the quality of life for approximately half of the surviving patients. Recent preclinical investigations have shed light on the multifaceted cerebrovascular injury mechanisms underlying this side effect of WBI. In this study, we aimed to test the hypothesis that WBI induces endothelial senescence, contributing to chronic disruption of the blood-brain barrier (BBB) and microvascular rarefaction. To accomplish this, we utilized transgenic p16-3MR mice, which enable the identification and selective elimination of senescent cells. These mice were subjected to a clinically relevant fractionated WBI protocol (5 Gy twice weekly for 4 weeks), and cranial windows were applied to both WBI-treated and control mice. Quantitative assessment of BBB permeability and capillary density was performed using two-photon microscopy at the 6-month post-irradiation time point. The presence of senescent microvascular endothelial cells was assessed by imaging flow cytometry, immunolabeling, and single-cell RNA-sequencing (scRNA-seq). WBI induced endothelial senescence, which associated with chronic BBB disruption and a trend for decreased microvascular density in the mouse cortex. In order to investigate the cause-and-effect relationship between WBI-induced senescence and microvascular injury, senescent cells were selectively removed from animals subjected to WBI treatment using Navitoclax/ABT263, a well-known senolytic drug. This intervention was carried out at the 3-month post-WBI time point. In WBI-treated mice, Navitoclax/ABT263 effectively eliminated senescent endothelial cells, which was associated with decreased BBB permeability and a trend for increased cortical capillarization. Our findings provide additional preclinical evidence that senolytic treatment approaches may be developed for prevention of the side effects of WBI.

Systems biology and machine learning approaches identify metabolites that influence dietary lifespan and healthspan responses across flies and humans.

Dietary restriction (DR) is a potent method to enhance lifespan and healthspan, but individual responses are influenced by genetic variations. Understanding how metabolism-related genetic differences impact longevity and healthspan are unclear. To investigate this, we used metabolites as markers to reveal how different genotypes respond to diet to influence longevity and healthspan traits. We analyzed data from Drosophila Genetic Reference Panel strains raised under AL and DR conditions, combining metabolomic, phenotypic, and genome-wide information. Employing two computational methods across species-random forest modeling within the DGRP and Mendelian randomization in the UK Biobank-we pinpointed key traits with cross-species relevance that influence lifespan and healthspan. Notably, orotate was linked to parental age at death in humans and counteracted DR effects in flies, while threonine extended lifespan, in a strain- and sex-specific manner. Thus, utilizing natural genetic variation data from flies and humans, we employed a systems biology approach to elucidate potential therapeutic pathways and metabolomic targets for diet-dependent changes in lifespan and healthspan.

Accelerated replicative senescence of ataxia-telangiectasia skin fibroblasts is retained at physiologic oxygen levels, with unique and common transcriptional patterns.

The genetic disorder, ataxia-telangiectasia (A-T), is caused by loss of the homeostatic protein kinase, ATM, and combines genome instability, tissue degeneration, cancer predisposition, and premature aging. Primary fibroblasts from A-T patients exhibit premature senescence when grown at ambient oxygen concentration (21%). Here, we show that reducing oxygen concentration to a physiological level range (3%) dramatically extends the proliferative lifespan of human A-T skin fibroblasts. However, they still undergo senescence earlier than control cells grown under the same conditions and exhibit high genome instability. Comparative RNA-seq analysis of A-T and control fibroblasts cultured at 3% oxygen followed by cluster analysis of differentially expressed genes and functional enrichment analysis, revealed distinct transcriptional dynamics in A-T fibroblasts senescing in physiological oxygen concentration. While some transcriptional patterns were similar to those observed during replicative senescence of control cells, others were unique to the senescing A-T cells. We observed in them a robust activation of interferon-stimulated genes, with undetected expression the interferon genes themselves. This finding suggests an activation of a non-canonical cGAS-STING-mediated pathway, which presumably responds to cytosolic DNA emanating from extranuclear micronuclei detected in these cells. Senescing A-T fibroblasts also exhibited a marked, intriguely complex alteration in the expression of genes associated with extracellular matrix (ECM) remodeling. Notably, many of the induced ECM genes encode senescence-associated secretory phenotype (SASP) factors known for their paracrine pro-fibrotic effects. Our data provide a molecular dimension to the segmental premature aging observed in A-T patients and its associated symptoms, which develop as the patients advance in age.

Accelerated cerebromicrovascular senescence contributes to cognitive decline in a mouse model of paclitaxel (Taxol)-induced chemobrain.

Chemotherapy-induced cognitive impairment ("chemobrain") is a frequent side-effect in cancer survivors treated with paclitaxel (PTX). The mechanisms responsible for PTX-induced cognitive impairment remain obscure, and there are no effective treatments or prevention strategies. Here, we test the hypothesis that PTX induces endothelial senescence, which impairs microvascular function and contributes to the genesis of cognitive decline. We treated transgenic p16-3MR mice, which allows the detection and selective elimination of senescent cells, with PTX (5 mg/kg/day, 2 cycles; 5 days/cycle). PTX-treated and control mice were tested for spatial memory performance, neurovascular coupling (NVC) responses (whisker-stimulation-induced increases in cerebral blood flow), microvascular density, blood-brain barrier (BBB) permeability and the presence of senescent endothelial cells (by flow cytometry and single-cell transcriptomics) at 6 months post-treatment. PTX induced senescence in endothelial cells, which associated with microvascular rarefaction, NVC dysfunction, BBB disruption, neuroinflammation, and impaired performance on cognitive tasks. To establish a causal relationship between PTX-induced senescence and impaired microvascular functions, senescent cells were depleted from PTX-treated animals (at 3 months post-treatment) by genetic (ganciclovir) or pharmacological (treatment with the senolytic drug ABT263/Navitoclax) means. In PTX treated mice, both treatments effectively eliminated senescent endothelial cells, rescued endothelium-mediated NVC responses and BBB integrity, increased capillarization and improved cognitive performance. Our findings suggest that senolytic treatments can be a promising strategy for preventing chemotherapy-induced cognitive impairment.

A natural variation-based screen in mouse cells reveals USF2 as a regulator of the DNA damage response and cellular senescence.

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-κB, p53, and C/EBPß. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory Mus musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, upstream stimulatory factor 2 (USF2), for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program-shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.

Antiretroviral protease inhibitors induce features of cellular senescence that are reversible upon drug removal.

Antiretroviral drugs have dramatically improved the prognosis of HIV-infected patients, with strikingly reduced morbidity and mortality. However, long-term use can be associated with signs of premature aging. Highly active antiretroviral therapy generally comprises two nucleoside reverse transcriptase inhibitors (NRTIs), with one of three additional antiretroviral drug classes, including protease inhibitors (PIs). One commonality between mitochondrial dysfunction (induced by NRTIs) and defects in lamin A (induced by PIs) is they can cause or accelerate cellular senescence, a state of essentially irreversible growth arrest, and the secretion of many bioactive molecules collectively known as the senescence-associated secretory phenotype (SASP). We hypothesized that senescent cells increase following treatment with certain HIV therapies. We compared the effects of two distinct HIV PIs: ritonavir-boosted atazanavir (ATV/r) and ritonavir-boosted darunavir (DRN/r), used in combination treatments for HIV infection. Upon ATV/r, but not DRN/r, treatment, cells arrested growth, displayed multiple features of senescence, and expressed significantly upregulated levels of many SASP factors. Furthermore, mice receiving sustained ATV/r treatment showed an increase in senescent cells and age-related decline in physiological function. However, removing treatment reversed the features of senescence observed in vivo and cell culture. Given how these features disappeared with drug removal, certain features of senescence may not be prognostic as defined by an irreversible growth arrest. Importantly, for patients that are treated or have been treated with ATV/r, our data suggest that switching to another PI that does not promote premature aging conditions (DRN/r) may improve the associated age-related complications.