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.

Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline.

Cellular senescence, which is characterized by an irreversible cell-cycle arrest1 accompanied by a distinctive secretory phenotype2, can be induced through various intracellular and extracellular factors. Senescent cells that express the cell cycle inhibitory protein p16INK4A have been found to actively drive naturally occurring age-related tissue deterioration3,4 and contribute to several diseases associated with ageing, including atherosclerosis5 and osteoarthritis6. Various markers of senescence have been observed in patients with neurodegenerative diseases7-9; however, a role for senescent cells in the aetiology of these pathologies is unknown. Here we show a causal link between the accumulation of senescent cells and cognition-associated neuronal loss. We found that the MAPTP301SPS19 mouse model of tau-dependent neurodegenerative disease10 accumulates p16INK4A-positive senescent astrocytes and microglia. Clearance of these cells as they arise using INK-ATTAC transgenic mice prevents gliosis, hyperphosphorylation of both soluble and insoluble tau leading to neurofibrillary tangle deposition, and degeneration of cortical and hippocampal neurons, thus preserving cognitive function. Pharmacological intervention with a first-generation senolytic modulates tau aggregation. Collectively, these results show that senescent cells have a role in the initiation and progression of tau-mediated disease, and suggest that targeting senescent cells may provide a therapeutic avenue for the treatment of these pathologies.

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.

Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16(Ink4a) (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16(Ink4a)-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16(Ink4a)-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

Pak2 kinase promotes cellular senescence and organismal aging.

Cellular senescence defines an irreversible cell growth arrest state linked to loss of tissue function and aging in mammals. This transition from proliferation to senescence is typically characterized by increased expression of the cell-cycle inhibitor p16INK4a and formation of senescence-associated heterochromatin foci (SAHF). SAHF formation depends on HIRA-mediated nucleosome assembly of histone H3.3, which is regulated by the serine/threonine protein kinase Pak2. However, it is unknown if Pak2 contributes to cellular senescence. Here, we show that depletion of Pak2 delayed oncogene-induced senescence in IMR90 human fibroblasts and oxidative stress-induced senescence of mouse embryonic fibroblasts (MEFs), whereas overexpression of Pak2 accelerated senescence of IMR90 cells. Importantly, depletion of Pak2 in BubR1 progeroid mice attenuated the onset of aging-associated phenotypes and extended life span. Pak2 is required for expression of genes involved in cellular senescence and regulated the deposition of newly synthesized H3.3 onto chromatin in senescent cells. Together, our results demonstrate that Pak2 is an important regulator of cellular senescence and organismal aging, in part through the regulation of gene expression and H3.3 nucleosome assembly.

Biphasic Modeling of Mitochondrial Metabolism Dysregulation during Aging.

Organismal aging is classically viewed as a gradual decline of cellular functions and a systemic deterioration of tissues that leads to an increased mortality rate in older individuals. According to the prevailing theory, aging is accompanied by a continuous and progressive decline in mitochondrial metabolic activity in cells. However, the most robust approaches to extending healthy lifespan are frequently linked with reduced energy intake or with lowering of mitochondrial activity. While these observations appear contradictory, recent work and technological advances demonstrate that metabolic deregulation during aging is potentially biphasic. In this Opinion we propose a novel framework where middle-age is accompanied by increased mitochondrial activity that subsequently declines at advanced ages.

Cellular Senescence and the Immune System in Cancer.

In response to a variety of cancer-inducing stresses, cells may engage a stable cell cycle arrest mechanism, termed cellular senescence, to suppress the proliferation of preneoplastic cells. Despite this cell intrinsic tumor suppression, senescent cells have also been implicated as active contributors to tumorigenesis by extrinsically promoting many hallmarks of cancer, including evasion of the immune system. Here, we discuss these dual, and seemingly contradictory, roles of senescence during tumorigenesis. Furthermore, we highlight findings of how senescent cells can influence the immune system and discuss the possibility that immune cells themselves may be acquiring senescence-associated alterations. Lastly, we discuss how senescent cell avoidance or clearance may impact pathology.

Spartan deficiency causes accumulation of Topoisomerase 1 cleavage complexes and tumorigenesis.

Germline mutations in SPRTN cause Ruijs-Aalfs syndrome (RJALS), a disorder characterized by genome instability, progeria and early onset hepatocellular carcinoma. Spartan, the protein encoded by SPRTN, is a nuclear metalloprotease that is involved in the repair of DNA-protein crosslinks (DPCs). Although Sprtn hypomorphic mice recapitulate key progeroid phenotypes of RJALS, whether this model expressing low amounts of Spartan is prone to DPC repair defects and spontaneous tumors is unknown. Here, we showed that the livers of Sprtn hypomorphic mice accumulate DPCs containing Topoisomerase 1 covalently linked to DNA. Furthermore, these mice exhibited DNA damage, aneuploidy and spontaneous tumorigenesis in the liver. Collectively, these findings provide evidence that partial loss of Spartan impairs DPC repair and tumor suppression.

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.

Advanced age is the main risk factor for most chronic diseases and functional deficits in humans, but the fundamental mechanisms that drive ageing remain largely unknown, impeding the development of interventions that might delay or prevent age-related disorders and maximize healthy lifespan. Cellular senescence, which halts the proliferation of damaged or dysfunctional cells, is an important mechanism to constrain the malignant progression of tumour cells. Senescent cells accumulate in various tissues and organs with ageing and have been hypothesized to disrupt tissue structure and function because of the components they secrete. However, whether senescent cells are causally implicated in age-related dysfunction and whether their removal is beneficial has remained unknown. To address these fundamental questions, we made use of a biomarker for senescence, p16(Ink4a), to design a novel transgene, INK-ATTAC, for inducible elimination of p16(Ink4a)-positive senescent cells upon administration of a drug. Here we show that in the BubR1 progeroid mouse background, INK-ATTAC removes p16(Ink4a)-positive senescent cells upon drug treatment. In tissues--such as adipose tissue, skeletal muscle and eye--in which p16(Ink4a) contributes to the acquisition of age-related pathologies, life-long removal of p16(Ink4a)-expressing cells delayed onset of these phenotypes. Furthermore, late-life clearance attenuated progression of already established age-related disorders. These data indicate that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.

Vascular Cell Senescence Contributes to Blood-Brain Barrier Breakdown.

BACKGROUND AND PURPOSE: Age-related changes in the cerebrovasculature, including blood-brain barrier (BBB) disruption, are emerging as potential risks for diverse neurological conditions. Because the accumulation of senescent cells in tissues is increasingly recognized as a critical step leading to age-related organ dysfunction, we evaluated whether senescent vascular cells are associated with compromised BBB integrity. METHODS: Effects of vascular cell senescence on tight junction and barrier integrity were studied using an in vitro BBB model, composed of endothelial cells, pericytes, and astrocytes. In addition, tight junction coverage in microvessels and BBB integrity in BubR1 hypomorphic (BubR1(H/H)) mice, which display senescence cell-dependent phenotypes, were examined. RESULTS: When an in vitro BBB model was constructed with senescent endothelial cells and pericytes, tight junction structure and barrier integrity (evaluated by transendothelial electric resistance and tracer efflux assay using sodium fluorescein and Evans blue-albumin were significantly impaired. Endothelial cells and pericytes from BubR1(H/H) mice had increased senescent-associated ß-galactosidase activity and p16(INK4a) expression, demonstrating an exacerbation of senescence. The coverage by tight junction proteins in the cortical microvessels were reduced in BubR1(H/H) mice, consistent with a compromised BBB integrity from permeability assays. Importantly, the coverage of microvessels by end-feet of aquaporin 4-immunoreactive astrocytes was not altered in the cortex of the BubR1(H/H) mice. CONCLUSIONS: Our results indicate that accumulation of senescent vascular cells is associated with compromised BBB integrity, providing insights into the mechanism of BBB disruption related to biological aging.

Senescence and apoptosis: dueling or complementary cell fates?

In response to a variety of stresses, mammalian cells undergo a persistent proliferative arrest known as cellular senescence. Many senescence-inducing stressors are potentially oncogenic, strengthening the notion that senescence evolved alongside apoptosis to suppress tumorigenesis. In contrast to apoptosis, senescent cells are stably viable and have the potential to influence neighboring cells through secreted soluble factors, which are collectively known as the senescence-associated secretory phenotype (SASP). However, the SASP has been associated with structural and functional tissue and organ deterioration and may even have tumor-promoting effects, raising the interesting evolutionary question of why apoptosis failed to outcompete senescence as a superior cell fate option. Here, we discuss the advantages that the senescence program may have over apoptosis as a tumor protective mechanism, as well as non-neoplastic functions that may have contributed to its evolution. We also review emerging evidence for the idea that senescent cells are present transiently early in life and are largely beneficial for development, regeneration and homeostasis, and only in advanced age do senescent cells accumulate to an organism's detriment.

Endonucleases: new tools to edit the mouse genome.

Mouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modification of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology. However, this approach harbors a number of disadvantages, as it is time-consuming and quite laborious. Over the last decade a number of new genome editing technologies have been developed, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas). These systems are characterized by a designed DNA binding protein or RNA sequence fused or co-expressed with a non-specific endonuclease, respectively. The engineered DNA binding protein or RNA sequence guides the nuclease to a specific target sequence in the genome to induce a double strand break. The subsequent activation of the DNA repair machinery then enables the introduction of gene modifications at the target site, such as gene disruption, correction or insertion. Nuclease-mediated genome editing has numerous advantages over conventional gene targeting, including increased efficiency in gene editing, reduced generation time of mutant mice, and the ability to mutagenize multiple genes simultaneously. Although nuclease-driven modifications in the genome are a powerful tool to generate mutant mice, there are concerns about off-target cleavage, especially when using the CRISPR/Cas system. Here, we describe the basic principles of these new strategies in mouse genome manipulation, their inherent advantages, and their potential disadvantages compared to current technologies used to study gene function in mouse models. This article is part of a Special Issue entitled: From Genome to Function.

Reduced life- and healthspan in mice carrying a mono-allelic BubR1 MVA mutation.

Mosaic Variegated Aneuploidy (MVA) syndrome is a rare autosomal recessive disorder characterized by inaccurate chromosome segregation and high rates of near-diploid aneuploidy. Children with MVA syndrome die at an early age, are cancer prone, and have progeroid features like facial dysmorphisms, short stature, and cataracts. The majority of MVA cases are linked to mutations in BUBR1, a mitotic checkpoint gene required for proper chromosome segregation. Affected patients either have bi-allelic BUBR1 mutations, with one allele harboring a missense mutation and the other a nonsense mutation, or mono-allelic BUBR1 mutations combined with allelic variants that yield low amounts of wild-type BubR1 protein. Parents of MVA patients that carry single allele mutations have mild mitotic defects, but whether they are at risk for any of the pathologies associated with MVA syndrome is unknown. To address this, we engineered a mouse model for the nonsense mutation 2211insGTTA (referred to as GTTA) found in MVA patients with bi-allelic BUBR1 mutations. Here we report that both the median and maximum lifespans of the resulting BubR1(+/GTTA) mice are significantly reduced. Furthermore, BubR1(+/GTTA) mice develop several aging-related phenotypes at an accelerated rate, including cataract formation, lordokyphosis, skeletal muscle wasting, impaired exercise ability, and fat loss. BubR1(+/GTTA) mice develop mild aneuploidies and show enhanced growth of carcinogen-induced tumors. Collectively, these data demonstrate that the BUBR1 GTTA mutation compromises longevity and healthspan, raising the interesting possibility that mono-allelic changes in BUBR1 might contribute to differences in aging rates in the general population.

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.

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.

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.

The ATM-p53 pathway suppresses aneuploidy-induced tumorigenesis.

The spindle assembly checkpoint (SAC) is essential for proper sister chromatid segregation. Defects in this checkpoint can lead to chromosome missegregation and aneuploidy. An increasing body of evidence suggests that aneuploidy can play a causal role in tumorigenesis. However, mutant mice that are prone to aneuploidy have only mild tumor phenotypes, suggesting that there are limiting factors in the aneuploidy-induced tumorigenesis. Here we provide evidence that p53 is such a limiting factor. We show that aneuploidy activates p53 and that loss of p53 drastically accelerates tumor development in two independent aneuploidy models. The p53 activation depends on the ataxia-telangiectasia mutated (ATM) gene product and increased levels of reactive oxygen species. Thus, the ATM-p53 pathway safeguards not only DNA damage but also aneuploidy.

BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice.

Faithful segregation of replicated chromosomes is essential for maintenance of genetic stability and seems to be monitored by several mitotic checkpoints. Various components of these checkpoints have been identified in mammals, but their physiological relevance is largely unknown. Here we show that mutant mice with low levels of the spindle assembly checkpoint protein BubR1 develop progressive aneuploidy along with a variety of progeroid features, including short lifespan, cachectic dwarfism, lordokyphosis, cataracts, loss of subcutaneous fat and impaired wound healing. Graded reduction of BubR1 expression in mouse embryonic fibroblasts causes increased aneuploidy and senescence. Male and female mutant mice have defects in meiotic chromosome segregation and are infertile. Natural aging of wild-type mice is marked by decreased expression of BubR1 in multiple tissues, including testis and ovary. These results suggest a role for BubR1 in regulating aging and infertility.

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.