4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

Ribosomal DNA and the nucleolus at the heart of aging.

The rRNA genes [ribosomal DNA (rDNA)] are organized in a prominent nuclear compartment, the nucleolus. It is now well established that the nucleolus functions beyond ribosome biosynthesis, regulating several physiological cellular responses. The nucleoli constitute dynamic genomic/nuclear hubs and demonstrate unique inherent characteristics, rendering them ideal to sense, signal, and respond to various intrinsic and environmental insults. Here, we discuss emerging findings supporting direct links between rDNA/nucleolar instability and cellular senescence/organismal aging from yeast to mammals. Moreover, we highlight evidence that nucleolar functionality and rDNA architecture impact on meiotic/transgenerational rejuvenation, thus revealing causality underlying connections between rDNA/nucleolar instability and aging.

Extended replicative lifespan of primary resting T cells by CRISPR/dCas9-based epigenetic modifiers and transcriptional activators.

Extension of the replicative lifespan of primary cells can be achieved by activating human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths. In this work, we utilize CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to reactivate the endogenous TERT gene in unstimulated T cells in the peripheral blood mononuclear cells (PBMCs) by rewiring the epigenetic marks of the TERT promoter. Importantly, we have successfully expanded resting T cells and delayed their cellular senescence for at least three months through TERT reactivation, without affecting the expression of a T-cell marker (CD3) or inducing an accelerated cell division rate. We have also demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. TERT reactivation and replicative senescence delay were achieved without inducing malignancy transformation, as shown in various cellular senescence assays, cell cycle state, proliferation rate, cell viability, and karyotype analyses. Our chromatin immunoprecipitation (ChIP)-qPCR data together with TERT mRNA and protein expression analyses confirmed the specificity of CRISPR-based transcription activators in modulating epigenetic marks of the TERT promoter, and induced telomerase expression. Therefore, the strategy of cell immortalization described here can be potentially adopted and generalized to delay cell death or even immortalize any other cell types.

Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation.

Tissue regeneration therapy based on human dental pulp cells (hDPCs) faces the distinct challenge of cellular senescence during massive expansion in vitro. To further explore the regulatory mechanism of cellular senescence in hDPCs, we conduct experiments on young cells (Passage 5, P5) and replicative senescent (Passage 12, P12) hDPCs. The results confirm that hDPCs undergo replicative senescence with passaging, during which their ability to proliferate and osteogenic differentiation decreases. Notably, during replicative senescence, phosphoglycerate dehydrogenase (PHGDH), the key enzyme of the serine synthesis pathway (SSP), is significantly downregulated, as well as S-adenosylmethionine (SAM) levels, resulting in reduced H3K36me3 modification on Sirtuin 1 (SIRT1)and Runt-related transcription factor 2 (RUNX2) promoters. Inhibition of PHGDH leads to the same phenotype as replicative senescence. Serine supplementation fails to rescue the senescence phenotype caused by replicative senescence and inhibitors, in which folate metabolism-related genes, including serine hydroxymethyl transferase 2 (SHMT2), methylenetetrahydrofolate dehydrogenase 1(MTHFD1), methylenetetrahydrofolate dehydrogenase 2(MTHFD2), are notably decreased. Our research raised a possibility that PHGDH may be involved in cellular senescence by affecting folate metabolism and histone methylation in addition to serine biosynthesis, providing potential targets to prevent senescence.

Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction.

Short telomeres are a principal defining feature of telomere biology disorders, such as dyskeratosis congenita (DC), for which there are no effective treatments. Here, we report that primary fibroblasts from DC patients and late generation telomerase knockout mice display lower nicotinamide adenine dinucleotide (NAD) levels, and an imbalance in the NAD metabolome that includes elevated CD38 NADase and reduced poly(ADP-ribose) polymerase and SIRT1 activities, respectively, affecting many associated biological pathways. Supplementation with the NAD precursor, nicotinamide riboside, and CD38 inhibition improved NAD homeostasis, thereby alleviating telomere damage, defective mitochondrial biosynthesis and clearance, cell growth retardation, and cellular senescence of DC fibroblasts. These findings reveal a direct, underlying role of NAD dysregulation when telomeres are short and underscore its relevance to the pathophysiology and interventions of human telomere-driven diseases.

Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics.

BACKGROUND: Human mesenchymal stem cells have attracted interest in regenerative medicine and are being tested in many clinical trials. In vitro expansion is necessary to provide clinical-grade quantities of mesenchymal stem cells; however, it has been reported to cause replicative senescence and undefined dysfunction in mesenchymal stem cells. Quality control assessments of in vitro expansion have rarely been addressed in ongoing trials. Young small extracellular vesicles from the remnant pulp of human exfoliated deciduous teeth stem cells have demonstrated therapeutic potential for diverse diseases. However, it is still unclear whether young small extracellular vesicles can reverse senescence-related declines. RESULTS: We demonstrated that mitochondrial structural disruption precedes cellular dysfunction during bone marrow-derived mesenchymal stem cell replication, indicating mitochondrial parameters as quality assessment indicators of mesenchymal stem cells. Dynamin-related protein 1-mediated mitochondrial dynamism is an upstream regulator of replicative senescence-induced dysfunction in bone marrow-derived mesenchymal stem cells. We observed that the application of young small extracellular vesicles could rescue the pluripotency dissolution, immunoregulatory capacities, and therapeutic effects of replicative senescent bone marrow-derived mesenchymal stem cells. Mechanistically, young small extracellular vesicles could promote Dynamin-related protein 1 translocation from the cytoplasm to the mitochondria and remodel mitochondrial disruption during replication history. CONCLUSIONS: Our findings show that Dynamin-related protein 1-mediated mitochondrial disruption is associated with the replication history of bone marrow-derived mesenchymal stem cells. Young small extracellular vesicles from human exfoliated deciduous teeth stem cells alleviate replicative senescence by promoting Dynamin-related protein 1 translocation onto the mitochondria, providing evidence for a potential rejuvenation strategy.

The Combined Anti-Aging Effect of Hydrolyzed Collagen Oligopeptides and Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells on Human Skin Fibroblasts.

Stem cell-derived exosomes (SC-Exos) are used as a source of regenerative medicine, but certain limitations hinder their uses. The effect of hydrolyzed collagen oligopeptides (HCOPs), a functional ingredient of SC-Exos is not widely known to the general public. We herein evaluated the combined anti-aging effects of HCOPs and exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exos) using a senescence model established on human skin fibroblasts (HSFs). This study discovered that cells treated with HucMSC-Exos + HCOPs enhanced their proliferative and migratory capabilities; reduced both reactive oxygen species production and senescence-associated ß-galactosidase activity; augmented type I and type III collagen expression; attenuated the expression of matrix-degrading metalloproteinases (MMP-1, MMP-3, and MMP-9), interleukin 1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α); and decreased the expression of p16, p21, and p53 as compared with the cells treated with HucMSC-Exos or HCOPs alone. These results suggest a possible strategy for enhancing the skin anti-aging ability of HucMSC-Exos with HCOPs.

Cellular senescence in glioma.

INTRODUCTION: Glioma is the most common primary brain tumor and is often associated with treatment resistance and poor prognosis. Standard treatment typically involves radiotherapy and temozolomide-based chemotherapy, both of which induce cellular senescence-a tumor suppression mechanism. DISCUSSION: Gliomas employ various mechanisms to bypass or escape senescence and remain in a proliferative state. Importantly, senescent cells remain viable and secrete a large number of factors collectively known as the senescence-associated secretory phenotype (SASP) that, paradoxically, also have pro-tumorigenic effects. Furthermore, senescent cells may represent one form of tumor dormancy and play a role in glioma recurrence and progression. CONCLUSION: In this article, we delineate an overview of senescence in the context of gliomas, including the mechanisms that lead to senescence induction, bypass, and escape. Furthermore, we examine the role of senescent cells in the tumor microenvironment and their role in tumor progression and recurrence. Additionally, we highlight potential therapeutic opportunities for targeting senescence in glioma.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging.

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence.

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome-wide in two primary cell lines. We integrated ChIP-seq and Hi-C with graph theory to uncover clustering of HMGB1-marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross-Linking and Immuno-Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA-binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP-relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell-autonomous and paracrine senescence.

SHP2's gain-of-function in Werner syndrome causes childhood disease onset likely resulting from negative genetic interaction.

Prompt diagnosis of complex phenotypes is a challenging task in clinical genetics. Whole exome sequencing has proved to be effective in solving such conditions. Here, we report on an unpredictable presentation of Werner Syndrome (WRNS) in a 12-year-old girl carrying a homozygous truncating variant in RECQL2, the gene mutated in WRNS, and a de novo activating missense change in PTPN11, the major Noonan syndrome gene, encoding SHP2, a protein tyrosine phosphatase positively controlling RAS function and MAPK signaling, which have tightly been associated with senescence in primary cells. All the major WRNS clinical criteria were present with an extreme precocious onset and were associated with mild intellectual disability, severe growth retardation and facial dysmorphism. Compared to primary fibroblasts from adult subjects with WRNS, proband's fibroblasts showed a dramatically reduced proliferation rate and competence, and a more accelerated senescence, in line with the anticipated WRNS features occurring in the child. In vitro functional characterization of the SHP2 mutant documented its hyperactive behavior and a significantly enhanced activation of the MAPK pathway. Based on the functional interaction of WRN and MAPK signaling in processes relevant to replicative senescence, these findings disclose a unique phenotype likely resulting from negative genetic interaction.

Global spliceosome activity regulates entry into cellular senescence.

Cellular senescence is a state of permanent growth arrest that can ultimately contribute to aging. Senescence can be induced by various stressors and is associated with a myriad of cellular functions and phenotypic markers. Alternative splicing is emerging as a critical contributor to senescence and aging. However, it is unclear how the composition and function of the spliceosome are involved in senescence. Here, using replicative and oxidative stress-induced senescence models in primary human fibroblasts, we report a common shift in the expression of 58 spliceosomal genes at the pre-senescence stage, prior to the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity. Spliceosomal perturbation, induced by pharmacologic and genetic inhibition of splicesomal genes, triggered cells to enter senescence, suggesting a key role as a gatekeeper. Association analysis of transcription factors based on the 58 splicesomal genes revealed Sp1 as a key regulator of senescence entry. Indeed, Sp1 depletion suppressed the expression of downstream spliceosomal genes (HNRNPA3, SRSF7, and SRSF4) and effectively induced senescence. These results indicate that spliceosomal gene sets, rather than a single spliceosomal gene, regulate the early transition into senescence prior to SA-ß-gal expression. Furthermore, our study provides a spliceosome signature that may be used as an early senescence marker.

Human RAP1 specifically protects telomeres of senescent cells from DNA damage.

Repressor/activator protein 1 (RAP1) is a highly evolutionarily conserved protein found at telomeres. Although yeast Rap1 is a key telomere capping protein preventing non-homologous end joining (NHEJ) and consequently telomere fusions, its role at mammalian telomeres in vivo is still controversial. Here, we demonstrate that RAP1 is required to protect telomeres in replicative senescent human cells. Downregulation of RAP1 in these cells, but not in young or dividing pre-senescent cells, leads to telomere uncapping and fusions. The anti-fusion effect of RAP1 was further explored in a HeLa cell line where RAP1 expression was depleted through an inducible CRISPR/Cas9 strategy. Depletion of RAP1 in these cells gives rise to telomere fusions only when telomerase is inhibited. We further show that the fusions triggered by RAP1 loss are dependent upon DNA ligase IV. We conclude that human RAP1 is specifically involved in protecting critically short telomeres. This has important implications for the functions of telomeres in senescent cells.

Comparative evaluation of proliferative potential and replicative senescence associated changes in mesenchymal stem cells derived from dental pulp and umbilical cord.

Mesenchymal stem cells (MSC) have been widely studied for tissue regeneration and cell-based therapy. MSC can be isolated from different body tissues while several biological waste sources like dental pulp, umbilical cord, cord derived blood, amniotic fluid or urine have also emerged as potential sources of MSCs. Specifically, isolation of MSCs from such non-conventional sources show promising outcomes due to the non-invasiveness of the extraction process and high proliferation capacity of the isolated MSC. However, these stem cells also exhibit the limitation of replicative senescence in long-term culture condition. Inter-cellular reactive oxygen species is an important contributor for inducing cellular senescence under long-term culture conditions. For translational application, it becomes imperative to compare the stem cells isolated from these sources for their senescence and proliferative properties. In this study, MSC were extracted from two different sources of biological waste materials-dental pulp and umbilical cord, and compared for their proliferation capacity and replicative senescence at different passage numbers (i.e. P2 and P6). Intracellular ROS production was significantly (p < 0.001) less in dental pulp stem cells culture in comparison to umbilical cord-derived stem cells at P6. The ß-gal expression also showed significantly (p < 0.001) low expression in DPSC culture compared to that of UCSC at P6. The study indicates the source of stem cells influences the proliferation capacity as well as replicative senescence of MSCs. This study will thus pave the path of future research in selecting appropriate stem cell source for regenerative medicine application.

Identification of a Proteomic Signature of Senescence in Primary Human Mammary Epithelial Cells.

Senescence is a permanent cell cycle arrest that occurs in response to cellular stress and promotes age-related disease. Because senescence differs greatly depending on cell type and senescence inducer, continued progress in the characterization of senescent cells is needed. Here, we analyzed primary human mammary epithelial cells (HMECs), a model system for aging and cancer, using mass spectrometry-based proteomics. By integrating data from replicative senescence, immortalization by telomerase reactivation, and quiescence, we identified a robust proteomic signature of HMEC senescence consisting of 34 upregulated and 10 downregulated proteins. This approach identified known senescence biomarkers including ß-galactosidase (GLB1) as well as novel senescence biomarkers including catechol O-methyltransferase (COMT), synaptic vesicle membrane protein VAT-1 homolog (VAT1), and plastin-1/3 (PLS1/PLS3). Gene ontology enrichment analysis demonstrated that senescent HMECs upregulated lysosomal proteins and downregulated RNA metabolic processes. In addition, a classification model based on our proteomic signature successfully discriminated proliferating and senescent HMECs at the transcriptional level. Finally, we found that the HMEC senescence signature was positively and negatively correlated with proteomic alterations in HMEC aging and breast cancer, respectively. Taken together, our results demonstrate the power of proteomics to identify cell type-specific signatures of senescence and advance the understanding of senescence in HMECs.

From neural stem cells to glioblastoma: A natural history of GBM recapitulated in vitro.

Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal. Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that have complicated the development of effective therapeutic strategies. This complex pathogenesis makes necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies. In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate GBM when injected into the brain of C57BL/6 mice, or heterotopic tumours when injected IV. Overall, our results show that oncogenic transformation is the fate of NSC if cultured for long periods in vitro. In addition, as no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBM.

Gene regulatory network analysis defines transcriptome landscape with alternative splicing of human umbilical vein endothelial cells during replicative senescence.

BACKGROUND: Endothelial cell senescence is the state of permanent cell cycle arrest and plays a critical role in the pathogenesis of age-related diseases. However, a comprehensive understanding of the gene regulatory network, including genome-wide alternative splicing machinery, involved in endothelial cell senescence is lacking. RESULTS: We thoroughly described the transcriptome landscape of replicative senescent human umbilical vein endothelial cells. Genes with high connectivity showing a monotonic expression increase or decrease with the culture period were defined as hub genes in the co-expression network. Computational network analysis of these genes led to the identification of canonical and non-canonical senescence pathways, such as E2F and SIRT2 signaling, which were down-regulated in lipid metabolism, and chromosome organization processes pathways. Additionally, we showed that endothelial cell senescence involves alternative splicing. Importantly, the first and last exon types of splicing, as observed in FLT1 and ACACA, were preferentially altered among the alternatively spliced genes during endothelial senescence. We further identified novel microexons in PRUNE2 and PSAP, each containing 9 nt, which were altered within the specific domain during endothelial senescence. CONCLUSIONS: These findings unveil the comprehensive transcriptome pathway and novel signaling regulated by RNA processing, including gene expression and splicing, in replicative endothelial senescence.

The regulation of the DNA damage response at telomeres: focus on kinases.

The natural ends of linear chromosomes resemble those of accidental double-strand breaks (DSBs). DSBs induce a multifaceted cellular response that promotes the repair of lesions and slows down cell cycle progression. This response is not elicited at chromosome ends, which are organized in nucleoprotein structures called telomeres. Besides counteracting DSB response through specialized telomere-binding proteins, telomeres also prevent chromosome shortening. Despite of the different fate of telomeres and DSBs, many proteins involved in the DSB response also localize at telomeres and participate in telomere homeostasis. In particular, the DSB master regulators Tel1/ATM and Mec1/ATR contribute to telomere length maintenance and arrest cell cycle progression when chromosome ends shorten, thus promoting a tumor-suppressive process known as replicative senescence. During senescence, the actions of both these apical kinases and telomere-binding proteins allow checkpoint activation while bulk DNA repair activities at telomeres are still inhibited. Checkpoint-mediated cell cycle arrest also prevents further telomere erosion and deprotection that would favor chromosome rearrangements, which are known to increase cancer-associated genome instability. This review summarizes recent insights into functions and regulation of Tel1/ATM and Mec1/ATR at telomeres both in the presence and in the absence of telomerase, focusing mainly on discoveries in budding yeast.

nc886, a Non-Coding RNA, Is a New Biomarker and Epigenetic Mediator of Cellular Senescence in Fibroblasts.

Functional studies of organisms and human models have revealed that epigenetic changes can significantly impact the process of aging. Non-coding RNA (ncRNA), one of epigenetic regulators, plays an important role in modifying the expression of mRNAs and their proteins. It can mediate the phenotype of cells. It has been reported that nc886 (=vtRNA2-1 or pre-miR-886), a long ncRNA, can suppress tumor formation and photo-damages of keratinocytes caused by UVB. The aim of this study was to determine the role of nc886 in replicative senescence of fibroblasts and determine whether substances capable of controlling nc886 expression could regulate cellular senescence. In replicative senescence fibroblasts, nc886 expression was decreased while methylated nc886 was increased. There were changes of senescence biomarkers including SA-ß-gal activity and expression of p16INK4A and p21Waf1/Cip1 in senescent cells. These findings indicate that the decrease of nc886 associated with aging is related to cellular senescence of fibroblasts and that increasing nc886 expression has potential to suppress cellular senescence. AbsoluTea Concentrate 2.0 (ATC) increased nc886 expression and ameliorated cellular senescence of fibroblasts by inhibiting age-related biomarkers. These results indicate that nc886 has potential as a new target for anti-aging and that ATC can be a potent epigenetic anti-aging ingredient.

Hepatic Senescence Accompanies the Development of NAFLD in Non-Aged Mice Independently of Obesity.

Senescence is considered to be a cardinal player in several chronic inflammatory and metabolic pathologies. The two dominant mechanisms of senescence include replicative senescence, predominantly depending on age-induced telomere shortening, and stress-induced senescence, triggered by external or intracellular harmful stimuli. Recent data indicate that hepatocyte senescence is involved in the development of nonalcoholic fatty liver disease (NAFLD). However, previous studies have mainly focused on age-related senescence during NAFLD, in the presence or absence of obesity, while information about whether the phenomenon is characterized by replicative or stress-induced senescence, especially in non-aged organisms, is scarce. Herein, we subjected young mice to two different diet-induced NAFLD models which differed in the presence of obesity. In both models, liver fat accumulation and increased hepatic mRNA expression of steatosis-related genes were accompanied by hepatic senescence, indicated by the increased expression of senescence-associated genes and the presence of a robust hybrid histo-/immunochemical senescence-specific staining in the liver. Surprisingly, telomere length and global DNA methylation did not differ between the steatotic and the control livers, while malondialdehyde, a marker of oxidative stress, was upregulated in the mouse NAFLD livers. These findings suggest that senescence accompanies NAFLD emergence, even in non-aged organisms, and highlight the role of stress-induced senescence during steatosis development independently of obesity.