Identifying specific functional roles for senescence across cell types.

Cellular senescence plays critical roles in aging, regeneration, and disease; yet, the ability to discern its contributions across various cell types to these biological processes remains limited. In this study, we generated an in vivo genetic toolbox consisting of three p16Ink4a-related intersectional genetic systems, enabling pulse-chase tracing (Sn-pTracer), Cre-based tracing and ablation (Sn-cTracer), and gene manipulation combined with tracing (Sn-gTracer) of defined p16Ink4a+ cell types. Using liver injury and repair as an example, we found that macrophages and endothelial cells (ECs) represent distinct senescent cell populations with different fates and functions during liver fibrosis and repair. Notably, clearance of p16Ink4a+ macrophages significantly mitigates hepatocellular damage, whereas eliminating p16Ink4a+ ECs aggravates liver injury. Additionally, targeted reprogramming of p16Ink4a+ ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the functional diversity of p16Ink4a+ cells and offers insights for developing cell-type-specific senolytic therapies in the future.

Senescence in Health and Disease.

Many cellular stresses activate senescence, a persistent hyporeplicative state characterized in part by expression of the p16INK4a cell-cycle inhibitor. Senescent cell production occurs throughout life and plays beneficial roles in a variety of physiological and pathological processes including embryogenesis, wound healing, host immunity, and tumor suppression. Meanwhile, the steady accumulation of senescent cells with age also has adverse consequences. These non-proliferating cells occupy key cellular niches and elaborate pro-inflammatory cytokines, contributing to aging-related diseases and morbidity. This model suggests that the abundance of senescent cells in vivo predicts "molecular," as opposed to chronologic, age and that senescent cell clearance may mitigate aging-associated pathology.

Cellular senescence and cardiovascular diseases: moving to the "heart" of the problem.

Cardiovascular diseases (CVDs) constitute the prime cause of global mortality, with an immense impact on patient quality of life and disability. Clinical evidence has revealed a strong connection between cellular senescence and worse cardiac outcomes in the majority of CVDs concerning both ischemic and nonischemic cardiomyopathies. Cellular senescence is characterized by cell cycle arrest accompanied by alterations in several metabolic pathways, resulting in morphological and functional changes. Metabolic rewiring of senescent cells results in marked paracrine activity, through a unique secretome, often exerting deleterious effects on neighboring cells. Here, we recapitulate the hallmarks and key molecular pathways involved in cellular senescence in the cardiac context and summarize the different roles of senescence in the majority of CVDs. In the last few years, the possibility of eliminating senescent cells in various pathological conditions has been increasingly explored, giving rise to the field of senotherapeutics. Therefore, we additionally attempt to clarify the current state of this field with a focus on cardiac senescence and discuss the potential of implementing senolytics as a treatment option in heart disease.

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.

Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence.

Heterochromatin, a key component of the eukaryotic nucleus, is fundamental to the regulation of genome stability, gene expression and cellular functions. However, the factors and mechanisms involved in heterochromatin formation and maintenance still remain largely unknown. Here, we show that insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein, is indispensable for constitutive heterochromatin formation via liquid‒liquid phase separation (LLPS). In particular, IRTKS droplets can infiltrate heterochromatin condensates composed of HP1α and diverse DNA-bound nucleosomes. IRTKS can stabilize HP1α by recruiting the E2 ligase Ubc9 to SUMOylate HP1α, which enables it to form larger phase-separated droplets than unmodified HP1α. Furthermore, IRTKS deficiency leads to loss of heterochromatin, resulting in genome-wide changes in chromatin accessibility and aberrant transcription of repetitive DNA elements. This leads to activation of cGAS-STING pathway and type-I interferon (IFN-I) signaling, as well as to the induction of cellular senescence and senescence-associated secretory phenotype (SASP) responses. Collectively, our findings establish a mechanism by which IRTKS condensates consolidate constitutive heterochromatin, revealing an unexpected role of IRTKS as an epigenetic mediator of cellular senescence.

p21 regulates expression of ECM components and promotes pulmonary fibrosis via CDK4 and Rb.

Fibrosis and accumulation of senescent cells are common tissue changes associated with aging. Here, we show that the CDK inhibitor p21 (CDKN1A), known to regulate the cell cycle and the viability of senescent cells, also controls the expression of extracellular matrix (ECM) components in senescent and proliferating cells of the fibrotic lung, in a manner dependent on CDK4 and Rb phosphorylation. p21 knockout protects mice from the induction of lung fibrosis. Moreover, inducible p21 silencing during fibrosis development alleviates disease pathology, decreasing the inflammatory response and ECM accumulation in the lung, and reducing the amount of senescent cells. Furthermore, p21 silencing limits fibrosis progression even when introduced during disease development. These findings show that one common mechanism regulates both cell cycle progression and expression of ECM components, and suggest that targeting p21 might be a new approach for treating age-related fibrotic pathologies.

Genetic origins, regulators, and biomarkers of cellular senescence.

This review comprehensively examines the molecular biology and genetic origins of cellular senescence. We focus on various cellular stressors and pathways leading to senescence, including recent advances in the understanding of the genetic influences driving senescence, such as telomere attrition, chemotherapy-induced DNA damage, pathogens, oncogene activation, and cellular and metabolic stress. This review also highlights the complex interplay of various signaling and metabolic pathways involved in cellular senescence and provides insights into potential therapeutic targets for aging-related diseases. Furthermore, this review outlines future research directions to deepen our understanding of senescence biology and develop effective interventions targeting senescent cells (SnCs).

Cellular senescence induction leads to progressive cell death via the INK4a-RB pathway in naked mole-rats.

Naked mole-rats (NMRs) have exceptional longevity and are resistant to age-related physiological decline and diseases. Given the role of cellular senescence in aging, we postulated that NMRs possess unidentified species-specific mechanisms to prevent senescent cell accumulation. Here, we show that upon induction of cellular senescence, NMR fibroblasts underwent delayed and progressive cell death that required activation of the INK4a-retinoblastoma protein (RB) pathway (termed "INK4a-RB cell death"), a phenomenon not observed in mouse fibroblasts. Naked mole-rat fibroblasts uniquely accumulated serotonin and were inherently vulnerable to hydrogen peroxide (H2 O2 ). After activation of the INK4a-RB pathway, NMR fibroblasts increased monoamine oxidase levels, leading to serotonin oxidization and H2 O2 production, which resulted in increased intracellular oxidative damage and cell death activation. In the NMR lung, induction of cellular senescence caused delayed, progressive cell death mediated by monoamine oxidase activation, thereby preventing senescent cell accumulation, consistent with in vitro results. The present findings indicate that INK4a-RB cell death likely functions as a natural senolytic mechanism in NMRs, providing an evolutionary rationale for senescent cell removal as a strategy to resist aging.

The putative contribution of cellular senescence to driving tauopathies.

During mammalian aging, senescent cells accumulate in the body. Recent evidence suggests that senescent cells potentially contribute to age-related neurodegenerative diseases in the central nervous system (CNS), including tauopathies such as Alzheimer's disease (AD). Senescent cells undergo irreversible cell cycle arrest and release an inflammatory 'senescence-associated secretory profile' (SASP), which can exert devastating effects on surrounding cells. Senescent markers and SASP factors have been detected in multiple brain cells in tauopathies, including microglia, astrocytes, and perhaps even post-mitotic neurons, possibly contributing to the initiation as well as progression of these diseases. Here, we discuss the implications of presenting a senescent phenotype in tauopathies and highlight a potential role for the NOD-like receptor protein 3 (NLRP3) inflammasome as a newfound mechanism implicated in senescence and SASP formation.

Senolytic and senomorphic agent procyanidin C1 alleviates structural and functional decline in the aged retina.

Increased cellular senescence burden contributes in part to age-related organ dysfunction and pathologies. In our study, using mouse models of natural aging, we observed structural and functional decline in the aged retina, which was accompanied by the accumulation of senescent cells and senescence-associated secretory phenotype factors. We further validated the senolytic and senomorphic properties of procyanidin C1 (PCC1) both in vitro and in vivo, the long-term treatment of which ameliorated age-related retinal impairment. Through high-throughput single-cell RNA sequencing (scRNA-seq), we comprehensively characterized the retinal landscape after PCC1 administration and deciphered the molecular basis underlying the senescence burden increment and elimination. By exploring the scRNA-seq database of age-related retinal disorders, we revealed the role of cellular senescence and the therapeutic potential of PCC1 in these pathologies. Overall, these results indicate the therapeutic effects of PCC1 on the aged retina and its potential use for treating age-related retinal disorders.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Pharmacological CDK4/6 inhibition reveals a p53-dependent senescent state with restricted toxicity.

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti-cancer drugs can cause premature senescence of non-malignant cells. These therapy-induced senescent cells can have pro-tumorigenic and pro-disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin-dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence-like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non-malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i-induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro-inflammatory and NF-κB-driven components. We find that CDK4/6i-induced senescent cells do not acquire pro-tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress-dependent and a predictor for the biological functions of different senescence subsets.

2'-O-ribose methylation levels of ribosomal RNA distinguish different types of growth arrest in human dermal fibroblasts.

The 2'-O-methylation (2'-O-Me) of ribosomal RNA (rRNA) shows plasticity that is potentially associated with cell phenotypes. We used RiboMeth-seq profiling to reveal growth arrest-specific 2'-O-Me patterns in primary human dermal fibroblasts from three different donors. We exposed cells to hydrogen peroxide to induce cellular senescence and to high cell densities to promote quiescence by contact inhibition. We compared both modes of cell cycle arrest to proliferating cells and could indeed distinguish these conditions by their overall 2'-O-Me patterns. Methylation levels at a small fraction of sites showed plasticity and correlated with the expression of specific small nucleolar RNAs (snoRNAs) but not with expression of fibrillarin. Moreover, we observed subtle senescence-associated alterations in ribosome biogenesis. Knockdown of the snoRNA SNORD87, which acts as a guide for modification of a hypermethylated position in non-proliferating cells, was sufficient to boost cell proliferation. Conversely, depletion of SNORD88A, SNORD88B and SNORD88C, which act as guides for modification of a hypomethylated site, caused decreased proliferation without affecting global protein synthesis or apoptosis. Taken together, our findings provide evidence that rRNA modifications can be used to distinguish and potentially influence specific growth phenotypes of primary cells.

Dysfunction of programmed embryo senescence is linked to genetic developmental defects.

Developmental senescence is a form of programmed senescence that contributes to morphogenesis during embryonic development. We showed recently that the SIX1 homeoprotein, an essential regulator of organogenesis, is also a repressor of adult cellular senescence. Alterations in the SIX/EYA pathway are linked to the human branchio-oto-renal (BOR) syndrome, a rare congenital disorder associated with defects in the ears, kidneys and branchial arches. Here, we have used Six1-deficient mice, an animal model of the BOR syndrome, to investigate whether dysfunction of senescence underpins the developmental defects associated with SIX1 deficiency. We have focused on the developing inner ear, an organ with physiological developmental senescence that is severely affected in Six1-deficient mice and BOR patients. We show aberrant levels and distribution of senescence markers in Six1-deficient inner ears concomitant with defective morphogenesis of senescent structures. Transcriptomic analysis and ex vivo assays support a link between aberrant senescence and altered morphogenesis in this model, associated with deregulation of the TGFß/BMP pathway. Our results show that misregulation of embryo senescence may lead to genetic developmental disorders, significantly expanding the connection between senescence and disease.

Oxysterol Sensing Through GPR183 Triggers Endothelial Senescence in Hypertension.

BACKGROUND: Despite endothelial dysfunction being an initial step in the development of hypertension and associated cardiovascular/renal injuries, effective therapeutic strategies to prevent endothelial dysfunction are still lacking. GPR183 (G protein-coupled receptor 183), a recently identified G protein-coupled receptor for oxysterols and hydroxylated metabolites of cholesterol, has pleiotropic roles in lipid metabolism and immune responses. However, the role of GPR183 in the regulation of endothelial function remains unknown. METHODS: Endothelial-specific GPR183 knockout mice were generated and used to examine the role of GPR183 in endothelial senescence by establishing 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Echocardiography, transmission electron microscopy, blood pressure measurement, vasorelaxation response experiments, flow cytometry analysis, and chromatin immunoprecipitation analysis were performed in this study. RESULTS: Endothelial GPR183 was significantly induced in hypertensive mice, which was further confirmed in renal biopsies from subjects with hypertensive nephropathy. Endothelial-specific deficiency of GPR183 markedly alleviated cardiovascular and renal injuries in hypertensive mice. Moreover, we found that GPR183 regulated endothelial senescence in both hypertensive mice and aged mice. Mechanistically, GPR183 disrupted circadian signaling by inhibiting PER1 (period circadian regulator 1) expression, thereby facilitating endothelial senescence and dysfunction through the cAMP (cyclic adenosine monophosphate)/PKA (protein kinase A)/CREB (cAMP-response element binding protein) signaling pathway. Importantly, pharmacological inhibition of the oxysterol-GPR183 axis by NIBR189 or clotrimazole ameliorated endothelial senescence and cardiovascular/renal injuries in hypertensive mice. CONCLUSIONS: This study discovers a previously unrecognized role of GPR183 in promoting endothelial senescence. Pharmacological targeting of GPR183 may be an innovative therapeutic strategy for hypertension and its associated complications.

The transcription factor PAX5 activates human LINE1 retrotransposons to induce cellular senescence.

As a hallmark of senescent cells, the derepression of Long Interspersed Elements 1 (LINE1) transcription results in accumulated LINE1 cDNA, which triggers the secretion of the senescence-associated secretory phenotype (SASP) and paracrine senescence in a cGAS-STING pathway-dependent manner. However, transcription factors that govern senescence-associated LINE1 reactivation remain ill-defined. Here, we predict several transcription factors that bind to human LINE1 elements to regulate their transcription by analyzing the conserved binding motifs in the 5'-untranslated regions (UTR) of the commonly upregulated LINE1 elements in different types of senescent cells. Further analysis reveals that PAX5 directly binds to LINE1 5'-UTR and the binding is enhanced in senescent cells. The enrichment of PAX5 at the 5'-UTR promotes cellular senescence and SASP by activating LINE1. We also demonstrate that the longevity gene SIRT6 suppresses PAX5 transcription by directly binding to the PAX5 promoter, and overexpressing PAX5 abrogates the suppressive effect of SIRT6 on stress-dependent cellular senescence. Our work suggests that PAX5 could serve as a potential target for drug development aiming to suppress LINE1 activation and treat senescence-associated diseases.

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

Chromatin conformational changes at human satellite II contribute to the senescence phenotype in the tumor microenvironment.

Cellular senescence and senescence-associated secretory phenotype (SASP) in stromal cells within the tumor microenvironment promote cancer progression. Although cellular senescence has been shown to induce changes in the higher-order chromatin structure and abnormal transcription of repetitive elements in the genome, the functional significance of these changes is unclear. In this study, we examined the human satellite II (hSATII) loci in the pericentromere to understand these changes and their functional significance. Our results indicated that the hSATII loci decompact during senescence induction, resulting in new DNA-DNA interactions in distinct genomic regions, which we refer to as DRISR (Distinctive Regions Interacted with Satellite II in Replicative senescent Fibroblasts). Interestingly, decompaction occurs before the expression of hSATII RNA. The DRISR with altered chromatin accessibility was enriched for motifs associated with cellular senescence and inflammatory SASP genes. Moreover, DNA-fluorescence in situ hybridization analysis of the breast cancer tissues revealed hSATII decompaction in cancer and stromal cells. Furthermore, we reanalyzed the single-cell assay for transposase-accessible chromatin with sequencing data and found increased SASP-related gene expression in fibroblasts exhibiting hSATII decompaction in breast cancer tissues. These findings suggest that changes in the higher-order chromatin structure of the pericentromeric repetitive sequences during cellular senescence might directly contribute to the cellular senescence phenotype and cancer progression via inflammatory gene expression.

ATOH8 binds SMAD3 to induce cellular senescence and prevent Ras-driven malignant transformation.

The process of oncogene-induced senescence (OIS) and the conversion between OIS and malignant transformation during carcinogenesis is poorly understood. Here, we show that following overactivation of oncogene Ras in lung epithelial cells, high-level transforming growth factor ß1 (TGF-ß1)-activated SMAD3, but not SMAD2 or SMAD4, plays a determinant role in inducing cellular senescence independent of the p53/p16/p15 senescence pathways. Importantly, SMAD3 binds a potential tumor suppressor ATOH8 to form a transcriptional complex that directly represses a series of cell cycle-promoting genes and consequently causes senescence in lung epithelial cells. Interestingly, the prosenescent SMAD3 converts to being oncogenic and essentially facilitates oncogenic Ras-driven malignant transformation. Furthermore, depleting Atoh8 rapidly accelerates oncogenic Ras-driven lung tumorigenesis, and lung cancers driven by mutant Ras and Atoh8 loss, but not by mutant Ras only, are sensitive to treatment of a specific SMAD3 inhibitor. Moreover, hypermethylation of the ATOH8 gene can be found in approximately 12% of clinical lung cancer cases. Together, our findings demonstrate not only epithelial cellular senescence directed by a potential tumor suppressor-controlled transcriptional program but also an important interplay between the prosenescent and transforming effects of TGF-ß/SMAD3, potentially laying a foundation for developing early detection and anticancer strategies.