SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Senescent Macrophages Release Inflammatory Cytokines and RNA-Loaded Extracellular Vesicles to Circumvent Fibroblast Senescence.

Senescent cells, which accumulate with age, exhibit a pro-inflammatory senescence-associated secretory phenotype (SASP) that includes the secretion of cytokines, lipids, and extracellular vesicles (EVs). Here, we established an in vitro model of senescence induced by Raf-1 oncogene in RAW 264.7 murine macrophages (MΦ) and compared them to senescent MΦ found in mouse lung tumors or primary macrophages treated with hydrogen peroxide. The transcriptomic analysis of senescent MΦ revealed an important inflammatory signature regulated by NFkB. We observed an increased secretion of EVs in senescent MΦ, and these EVs presented an enrichment for ribosomal proteins, major vault protein, pro-inflammatory miRNAs, including miR-21a, miR-155, and miR-132, and several mRNAs. The secretion of senescent MΦ allowed senescent murine embryonic fibroblasts to restart cell proliferation. This antisenescence function of the macrophage secretome may explain their pro-tumorigenic activity and suggest that senolytic treatment to eliminate senescent MΦ could potentially prevent these deleterious effects.

Caloric Restriction Improves Spatial Learning Deficits in Tau Mice.

Background: Caloric restriction (CR) has been recognized for its benefits in delaying age-related diseases and extending lifespan. While its effects on amyloid pathology in Alzheimer's disease (AD) mouse models are well-documented, its effects on tauopathy, another hallmark of AD, are less explored. Objective: To assess the impact of a short-term 30% CR regimen on age-dependent spatial learning deficits and pathological features in a tauopathy mouse model. Methods: We subjected male PS19 tau P301S (hereafter PS19) and age-matched wildtype mice from two age cohorts (4.5 and 7.5 months old) to a 6-week 30% CR regimen. Spatial learning performance was assessed using the Barnes Maze test. Tau pathology, neuroinflammation, hippocampal cell proliferation, and neurogenesis were evaluated in the older cohort by immunohistochemical staining and RT-qPCR. Results: CR mitigated age-dependent spatial learning deficits in PS19 mice but exhibited limited effects on tau pathology and the associated neuroinflammation. Additionally, we found a decrease in hippocampal cell proliferation, predominantly of Iba1+ cells. Conclusions: Our findings reinforce the cognitive benefits conferred by CR despite its limited modulation of disease pathology. Given the pivotal role of microglia in tau-driven pathology, the observed reduction in Iba1+ cells under CR suggests potential therapeutic implications, particularly if CR would be introduced early in disease progression.

Senescence Targeting Methods Impact Alzheimer's Disease Features in 3xTg Mice.

Background: Cellular senescence has been associated with neurodegenerative disease and clearance of senescent cells using genetic or pharmaceutical strategies (senolytics) has demonstrated beneficial effects in mouse models investigating individual disease etiologies of Alzheimer's disease (AD). However, it has remained unclear if senescent cell clearance in a mouse model exhibiting both plaque and tau pathologies modifies the disease state (3xTg). Objective: To investigate the effects of senescent cell clearance in the 3xTg mouse model. Methods: 3xTg mice were treated with senolytics (ABT263 (navitoclax; NAVI), a combination of dasatinib and quercetin (D+Q)), or subjected to transgene-mediated removal of p16-expressing cells (via INK-ATTAC). Results: Senolytic treatments consistently reduced microgliosis and ameliorated both amyloid and tau pathology in 3xTg mice. Using RNA sequencing, we found evidence that synaptic dysfunction and neuroinflammation were attenuated with treatment. These beneficial effects were not observed with short-term senolytic treatment in mice with more advanced disease. Conclusions: Overall, our results further corroborate the beneficial effects senescent cell clearance could have on AD and highlight the importance of early intervention for the treatment of this debilitating disease.

Chronic social and psychological stress impact select neuropathologies in the PS19 mouse model of tauopathy.

OBJECTIVE: Despite advances toward understanding the etiology of Alzheimer's disease (AD), it remains unclear which aspects of this disease are affected by environmental factors. Chronic life stress increases risk for aging-related diseases including AD. The impact of stress on tauopathies remains understudied. We examined the effects of stress elicited by social (chronic subordination stress, CSS) or psychological/physical (chronic restraint stress, CRS) factors - on the PS19 mouse model of tauopathy. METHODS: Male PS19 mice (average age 6.3 months) were randomized to receive CSS, CRS, or to remain as singly-housed controls. Behavioral tests were used to assess anxiety-like behaviors and cognitive functions. Immunofluorescence staining and western blotting analysis were used to measure levels of astrogliosis, microgliosis and tau burden. Immunohistochemistry was used to assess glucocorticoid receptor expression. RESULTS: PS19 mice exhibit neuroinflammation (GFAP, t-tests; p = 0.0297; Iba1, t-tests; p = 0.006) and tau hyperphosphorylation (t-test, p = 0.0446) in the hippocampus, reduced anxiety (post hoc, p = 0.046), and cognitive deficits, when compared to wild type mice. Surprisingly, CRS reduced hippocampal levels of both total tau and phospho-tauS404 (t-test, p = 0.0116), and attenuated some aspects of both astrogliosis and microgliosis in PS19 mice (t-tests, p = 0.068 to p = 0.0003); however, this was not associated with significant changes in neurodegeneration or cognitive function. Anxiety-like behaviors were increased by CRS (post hoc, p = 0.046). Conversely, CSS impaired spatial learning in Barnes Maze without impacting tau phosphorylation or neurodegeneration and having a minimal impact on gliosis. CONCLUSIONS: Our results demonstrate that social or psychological stress can differentially impact anxiety-like behavior, select cognitive functions, and some aspects of tau-dependent pathology in PS19 male mice, providing entry points for the development of experimental approaches designed to slow AD progression.

An untapped window of opportunity for glioma: targeting therapy-induced senescence prior to recurrence.

High-grade gliomas are primary brain tumors that are incredibly refractory long-term to surgery and chemoradiation, with no proven durable salvage therapies for patients that have failed conventional treatments. Post-treatment, the latent glioma and its microenvironment are characterized by a senescent-like state of mitotic arrest and a senescence-associated secretory phenotype (SASP) induced by prior chemoradiation. Although senescence was once thought to be irreversible, recent evidence has demonstrated that cells may escape this state and re-enter the cell cycle, contributing to tumor recurrence. Moreover, senescent tumor cells could spur the growth of their non-senescent counterparts, thereby accelerating recurrence. In this review, we highlight emerging evidence supporting the use of senolytic agents to ablate latent, senescent-like cells that could contribute to tumor recurrence. We also discuss how senescent cell clearance can decrease the SASP within the tumor microenvironment thereby reducing tumor aggressiveness at recurrence. Finally, senolytics could improve the long-term sequelae of prior therapy on cognition and bone marrow function. We critically review the senolytic drugs currently under preclinical and clinical investigation and the potential challenges that may be associated with deploying senolytics against latent glioma. In conclusion, senescence in glioma and the microenvironment are critical and potential targets for delaying or preventing tumor recurrence and improving patient functional outcomes through senotherapeutics.

Advanced Age in Humans and Mouse Models of Glioblastoma Show Decreased Survival from Extratumoral Influence.

PURPOSE: Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a median age of onset of 68 to 70 years old. Although advanced age is often associated with poorer GBM patient survival, the predominant source(s) of maladaptive aging effects remains to be established. Here, we studied intratumoral and extratumoral relationships between adult patients with GBM and mice with brain tumors across the lifespan. EXPERIMENTAL DESIGN: Electronic health records at Northwestern Medicine and the NCI SEER databases were evaluated for GBM patient age and overall survival. The commercial Tempus and Caris databases, as well as The Cancer Genome Atlas were profiled for gene expression, DNA methylation, and mutational changes with varying GBM patient age. In addition, gene expression analysis was performed on the extratumoral brain of younger and older adult mice with or without a brain tumor. The survival of young and old wild-type or transgenic (INK-ATTAC) mice with a brain tumor was evaluated after treatment with or without senolytics and/or immunotherapy. RESULTS: Human patients with GBM ≥65 years of age had a significantly decreased survival compared with their younger counterparts. While the intra-GBM molecular profiles were similar between younger and older patients with GBM, non-tumor brain tissue had a significantly different gene expression profile between young and old mice with a brain tumor and the eradication of senescent cells improved immunotherapy-dependent survival of old but not young mice. CONCLUSIONS: This work suggests a potential benefit for combining senolytics with immunotherapy in older patients with GBM.

Senescent Microglia Represent a Subset of Disease-Associated Microglia in P301S Mice.

BACKGROUND: The existence and contribution of microglia with senescent-like alterations in the pathogenesis of age-related neurodegenerative diseases like Alzheimer's disease (AD) have been suggested in recent years. However, the identification of this distinct microglial population in vivo has proven challenging, largely due to overlaps in the inflammatory phenotype of activated and senescent microglia. Furthermore, attempts at recapitulating senescence in microglia in vitro are limited. OBJECTIVE: To identify and characterize senescent microglia that occur in vivo in an animal model of neurodegeneration driven by pathologic tau. METHODS: We analyzed the RNA expression patterns of individual microglia from normal mice and the pathogenic tau P301 S PS19 mouse model. We have previously demonstrated that p16-expressing senescent microglia occur in these mice when neurodegeneration has occurred. RESULTS: Here we identify a subset of disease-associated microglia with senescent features, notably characterized by the expression of Ccl4. This signature overlaps with established markers of senescence from other cell types. CONCLUSION: Our characterization of senescent microglia can be used to better understand the role of senescent microglia in various age-related contexts, including whether clearance of senescent microglia represents a viable therapeutic option.

Senescent cells as thermostats of antitumor immunity.

Harnessing the immunogenic potential of senescent cells may be a viable but context-dependent opportunity to boost antitumor immunity.

Senescent alveolar macrophages promote early-stage lung tumorigenesis.

Senescent cells play relevant but context-dependent roles during tumorigenesis. Here, in an oncogenic Kras-driven lung cancer mouse model, we found that senescent cells, specifically alveolar macrophages, accumulate early in neoplasia. These macrophages have upregulated expression of p16INK4a and Cxcr1, are distinct from previously defined subsets and are sensitive to senolytic interventions, and suppress cytotoxic T cell responses. Their removal attenuates adenoma development and progression in mice, indicating their tumorigenesis-promoting role. Importantly, we found that alveolar macrophages with these properties increase with normal aging in mouse lung and in human lung adenocarcinoma in situ. Collectively, our study indicates that a subset of tissue-resident macrophages can support neoplastic transformation through altering their local microenvironment, suggesting that therapeutic interventions targeting senescent macrophages may attenuate lung cancer progression during early stages of disease.

Hyperphosphorylated PTEN exerts oncogenic properties.

PTEN is a multifaceted tumor suppressor that is highly sensitive to alterations in expression or function. The PTEN C-tail domain, which is rich in phosphorylation sites, has been implicated in PTEN stability, localization, catalytic activity, and protein interactions, but its role in tumorigenesis remains unclear. To address this, we utilized several mouse strains with nonlethal C-tail mutations. Mice homozygous for a deletion that includes S370, S380, T382 and T383 contain low PTEN levels and hyperactive AKT but are not tumor prone. Analysis of mice containing nonphosphorylatable or phosphomimetic versions of S380, a residue hyperphosphorylated in human gastric cancers, reveal that PTEN stability and ability to inhibit PI3K-AKT depends on dynamic phosphorylation-dephosphorylation of this residue. While phosphomimetic S380 drives neoplastic growth in prostate by promoting nuclear accumulation of ß-catenin, nonphosphorylatable S380 is not tumorigenic. These data suggest that C-tail hyperphosphorylation creates oncogenic PTEN and is a potential target for anti-cancer therapy.

Transfer learning in a biomaterial fibrosis model identifies in vivo senescence heterogeneity and contributions to vascularization and matrix production across species and diverse pathologies.

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells' (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo-derived senescence signature (SenSig) using a foreign body response-driven fibrosis model in a p16-CreERT2;Ai14 reporter mouse. We identified pericytes and "cartilage-like" fibroblasts as senescent and defined cell type-specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34-CSF1R-TGFßR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.

Cellular senescence: beneficial, harmful, and highly complex.

The contribution of cellular senescence to a diverse range of biological processes, including normal physiology, ageing, and pathology were long overlooked but have now taken centre stage. In this Editorial, we will briefly outline the review and original work articles contained in The FEBS Journal's Special Issue on Senescence in Ageing and Disease. It is beginning to be appreciated that senescent cells can exert both beneficial and adverse effects following tissue injury. Additionally, while these cells play critical roles for maintaining a healthy physiology, they also promote ageing and certain pathological conditions (including developmental disorders). Progress has been made in re-defining and identifying senescent cells, especially in slow-proliferating or terminally differentiated tissues, such as the brain and cardiovascular system. Novel approaches and techniques for isolating senescent cells will greatly increase our appreciation for senescent properties in tissues. The inter-organ communication between senescent cells and other residents of the tissue microenvironment, via the senescence-associated secretory phenotype (SASP), is a focus of several reviews in this Special Issue. The importance of the SASP in promoting tumour development and the evolution of SARS CoV-2 variants is also highlighted. In one of the two original articles included in the issue, the impact of dietary macronutrients and the presence of senescent cells in mice is investigated. Lastly, we continue to deepen our understanding on the use of senolytics and senomorphics to specifically target senescent cells or their secreted components, respectively, which is discussed in several of the reviews included here.

Untangling senescent and damage-associated microglia in the aging and diseased brain.

Microglial homeostasis has emerged as a critical mediator of health and disease in the central nervous system. In their neuroprotective role as the predominant immune cells of the brain, microglia surveil the microenvironment for debris and pathogens, while also promoting neurogenesis and performing maintenance on synapses. Chronological ageing, disease onset, or traumatic injury promotes irreparable damage or deregulated signaling to reinforce neurotoxic phenotypes in microglia. These insults may include cellular senescence, a stable growth arrest often accompanied by the production of a distinctive pro-inflammatory secretory phenotype, which may contribute to age- or disease-driven decline in neuronal health and cognition and is a potential novel therapeutic target. Despite this increased scrutiny, unanswered questions remain about what distinguishes senescent microglia and non-senescent microglia reacting to insults occurring in ageing, disease, and injury, and how central the development of senescence is in their pivot from guardian to assailant. To intelligently design future studies to untangle senescent microglia from other primed and reactionary states, specific criteria must be developed that define this population and allow for comparisons between different model systems. Comparing microglial activity seen in homeostasis, ageing, disease, and injury allows for a more coherent understanding of when and how senescent and other harmful microglial subpopulations should be targeted.

p21 induces a senescence program and skeletal muscle dysfunction.

Recent work has established associations between elevated p21, the accumulation of senescent cells, and skeletal muscle dysfunction in mice and humans. Using a mouse model of p21 overexpression (p21OE), we examined if p21 mechanistically contributes to cellular senescence and pathological features in skeletal muscle. We show that p21 induces several core properties of cellular senescence in skeletal muscle, including an altered transcriptome, DNA damage, mitochondrial dysfunction, and the senescence-associated secretory phenotype (SASP). Furthermore, p21OE mice exhibit manifestations of skeletal muscle pathology, such as atrophy, fibrosis, and impaired physical function when compared to age-matched controls. These findings suggest p21 alone is sufficient to drive a cellular senescence program and reveal a novel source of skeletal muscle loss and dysfunction.

Current Challenges and Solutions for Clinical Management and Care of People with HIV: Findings from the 12th Annual International HIV and Aging Workshop.

People with HIV on combination antiretroviral therapy (ART) have longer life expectancy and are increasingly experiencing age-related comorbidities. Thus, aging with HIV has become a central issue in clinical care and research, which has been particularly challenging with the intersection of the ongoing coronavirus (COVID)-19 pandemic. Since 2009, the International Workshop on HIV and Aging has served as a multidisciplinary platform to share research findings from cross-disciplinary fields along with community advocates to address critical issues in HIV and aging. In this article, we summarize the key oral presentations from the 12th Annual International Workshop on HIV and Aging, held virtually on September 23rd and 24th, 2021. The topics ranged from basic science research on biological mechanisms of aging to quality of life and delivery of care under the COVID-19 pandemic. This workshop enriched our understanding of HIV and aging under the COVID-19 pandemic, identified challenges and opportunities to combat the impact of COVID-19 on HIV communities, and also provided updated research and future directions of the field to move HIV and aging research forward, with the ultimate goal of successful aging for older people with HIV.

Senescent cells limit p53 activity via multiple mechanisms to remain viable.

Super-enhancers regulate genes with important functions in processes that are cell type-specific or define cell identity. Mouse embryonic fibroblasts establish 40 senescence-associated super-enhancers regardless of how they become senescent, with 50 activated genes located in the vicinity of these enhancers. Here we show, through gene knockdown and analysis of three core biological properties of senescent cells that a relatively large number of senescence-associated super-enhancer-regulated genes promote survival of senescent mouse embryonic fibroblasts. Of these, Mdm2, Rnase4, and Ang act by suppressing p53-mediated apoptosis through various mechanisms that are also engaged in response to DNA damage. MDM2 and RNASE4 transcription is also elevated in human senescent fibroblasts to restrain p53 and promote survival. These insights identify key survival mechanisms of senescent cells and provide molecular entry points for the development of targeted therapeutics that eliminate senescent cells at sites of pathology.

p21 produces a bioactive secretome that places stressed cells under immunosurveillance.

Immune cells identify and destroy damaged cells to prevent them from causing cancer or other pathologies by mechanisms that remain poorly understood. Here, we report that the cell-cycle inhibitor p21 places cells under immunosurveillance to establish a biological timer mechanism that controls cell fate. p21 activates retinoblastoma protein (Rb)­dependent transcription at select gene promoters to generate a complex bioactive secretome, termed p21-activated secretory phenotype (PASP). The PASP includes the chemokine CXCL14, which promptly attracts macrophages. These macrophages disengage if cells normalize p21 within 4 days, but if p21 induction persists, they polarize toward an M1 phenotype and lymphocytes mount a cytotoxic T cell response to eliminate target cells, including preneoplastic cells. Thus, p21 concurrently induces proliferative arrest and immunosurveillance of cells under duress.

CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD+ and NMN levels.

Decreased NAD+ levels have been shown to contribute to metabolic dysfunction during aging. NAD+ decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD+ homeostasis. Here we show that an increase in CD38 in white adipose tissue (WAT) and the liver during aging is mediated by accumulation of CD38+ immune cells. Inflammation increases CD38 and decreases NAD+. In addition, senescent cells and their secreted signals promote accumulation of CD38+ cells in WAT, and ablation of senescent cells or their secretory phenotype decreases CD38, partially reversing NAD+ decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD+ through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that, through its ecto-enzymatic activity, decreases levels of NMN and NAD+.