Intracellular acidification and glycolysis modulate inflammatory pathway in senescent cells.

Senescent cells accumulate in various organs with aging, and its accumulation induces chronic inflammation and age-related physiological dysfunctions. Several remodeling of intracellular environments has been identified in senescent cells, including enlargement of cell / nuclear size and intracellular acidification. Although these alterations of intracellular environments were reported to be involved in unique characteristics of senescent cells, the contribution of intracellular acidification to senescence-associated cellular phenotypes is poorly understood. Here, we identified that upregulation of TXNIP and its paralog ARRDC4 as a hallmark of intracellular acidification in addition to KGA-type GLS1. These genes were also upregulated in response to senescence-associated intracellular acidification. Neutralization of the intracellular acidic environment ameliorated not only senescence-related upregulation of TXNIP, ARRDC4, and KGA, but also inflammation-related genes, possibly through suppression of PDK-dependent anaerobic glycolysis. Furthermore, we found that expression of the intracellular acidification-induced genes, TXNIP and ARRDC4, correlated with inflammatory gene expression in heterogeneous senescent cell population in vitro and even in vivo, implying that the contribution of intracellular pH to senescence-associated cellular features, such as SASP.

Senescence and senolysis in cancer: The latest findings.

Aging is a life phenomenon that occurs in most living organisms and is a major risk factor for many diseases, including cancer. Cellular senescence is a cellular trait induced by various genomic and epigenetic stresses. Senescent cells are characterized by irreversible cell growth arrest and excessive secretion of inflammatory cytokines (senescence-associated secretory phenotypes, SASP). Chronic tissue microinflammation induced by SASP contributes to the pathogenesis of a variety of age-related diseases, including cancer. Senolysis is a promising new strategy to selectively eliminate senescent cells in order to suppress chronic inflammation, suggesting its potential use as an anticancer therapy. This review summarizes recent findings on the molecular basis of senescence in cancer cells and senolysis.

M2 macrophage-derived TGF-ß induces age-associated loss of adipogenesis through progenitor cell senescence.

OBJECTIVES: Adipose tissue is an endocrine and energy storage organ composed of several different cell types, including mature adipocytes, stromal cells, endothelial cells, and a variety of immune cells. Adipose tissue aging contributes to the pathogenesis of metabolic dysfunction and is likely induced by crosstalk between adipose progenitor cells (APCs) and immune cells, but the underlying molecular mechanisms remain largely unknown. In this study, we revealed the biological role of p16high senescent APCs, and investigated the crosstalk between each cell type in the aged white adipose tissue. METHODS: We performed the single-cell RNA sequencing (scRNA-seq) analysis on the p16high adipose cells sorted from aged p16-CreERT2/Rosa26-LSL-tdTomato mice. We also performed the time serial analysis on the age-dependent bulk RNA-seq datasets of human and mouse white adipose tissues to infer the transcriptome alteration of adipogenic potential within aging. RESULTS: We show that M2 macrophage-derived TGF-ß induces APCs senescence which impairs adipogenesis in vivo. p16high senescent APCs increase with age and show loss of adipogenic potential. The ligand-receptor interaction analysis reveals that M2 macrophages are the donors for TGF-ß and the senescent APCs are the recipients. Indeed, treatment of APCs with TGF-ß1 induces senescent phenotypes through mitochondrial ROS-mediated DNA damage in vitro. TGF-ß1 injection into gonadal white adipose tissue (gWAT) suppresses adipogenic potential and induces fibrotic genes as well as p16 in APCs. A gWAT atrophy is observed in cancer cachexia by APCs senescence, whose induction appeared to be independent of TGF-ß induction. CONCLUSIONS: Our results suggest that M2 macrophage-derived TGF-ß induces age-related lipodystrophy by APCs senescence. The TGF-ß treatment induced DNA damage, mitochondrial ROS, and finally cellular senescence in APCs.

Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts.

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence-stable cell cycle arrest contributing to organismal aging-is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III (SNF7) and AAA-ATPase (VPS4) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca2+-p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.

Potential role of Fbxo22 in resistance to endocrine therapy in breast cancer with invasive lobular carcinoma.

BACKGROUND: Invasive lobular carcinoma (ILC) is distinct from invasive ductal carcinoma (IDC) in terms of their hormonal microenvironments that may require different therapeutic strategies. We previously reported that selective estrogen receptor modulator (SERM) function requires F-box protein 22 (Fbxo22). Here, we investigated the role of Fbxo22 as a potential biomarker contributing to the resistance to endocrine therapy in ILC. METHODS: A total of 302 breast cancer (BC) patients including 150 ILC were recruited in the study. Fbxo22 expression and clinical information were analyzed to elucidate whether Fbxo22 negativity could be a prognostic factor or there were any correlations among clinical variables and SERM efficacy. RESULTS: Fbxo22 negativity was significantly higher in ILC compared with IDC (58.0% vs. 27.0%, P < 0.001) and higher in postmenopausal patients than premenopausal patients (64.1% vs. 48.2%, P = 0.041). In the ILC cohort, Fbxo22-negative patients had poorer overall survival (OS) than Fbxo22-positive patients, with 10-year OS rates of 77.4% vs. 93.6% (P = 0.055). All patients treated with SERMs, Fbxo22 negativity resulted in a poorer outcome, with 10-year OS rates of 81.3% vs. 92.3% (P = 0.032). In multivariate analysis regarding recurrence-free survival (RFS) in ILC patients, Fbxo22 status was independently predictive of survival as well as lymph node metastasis. CONCLUSION: Fbxo22 negativity significantly impacts on survival in BC patients with IDC and ILC, and the disadvantage was enhanced among ILC postmenopausal women or patients treated with SERMs. The findings suggest that different therapeutic strategies might be needed according to the different histopathological types when considering adjuvant endocrine therapy.

Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment.

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.

LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits.

Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to protein aggregates that are not diluted by mitosis. Therefore, post-mitotic cells may have a specific protein quality control system. Here, we show that LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds and ubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. Lonrf2-/- mice exhibit age-dependent TDP-43-mediated motor neuron (MN) degeneration and cerebellar ataxia. Mouse induced pluripotent stem cell-derived MNs lacking LONRF2 showed reduced survival, shortening of neurites and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in MNs from patients with amyotrophic lateral sclerosis is rescued by ectopic expression of LONRF2. Our findings reveal that LONRF2 is a protein quality control ligase whose loss may contribute to MN degeneration and motor deficits.

In vivo dynamics of senescence in rhabdomyolysis-induced acute kidney injury.

Cellular senescence is involved in the pathogenesis of various diseases, including acute kidney injury (AKI). AKI is defined as a sudden loss of kidney function. In severe AKI, irreversible loss of kidney cells can occur. Cellular senescence might contribute to this maladaptive tubular repair, though, its pathophysiological role in vivo is incompletely understood. In this study, we used p16-CreERT2-tdTomato mice in which cells with high p16 expression, a prototypical senescent marker, are labeled with tdTomato fluorescence. Then, we induced AKI by rhabdomyolysis and traced the cells with high p16 expression following AKI. We proved that the induction of senescence was observed predominantly in proximal tubular epithelial cells (PTECs) and occurred in a relatively acute phase within 1-3 days after AKI. These acute senescent PTECs were spontaneously eliminated by day 15. On the contrary, the generation of senescence in PTECs persisted during the chronic recovery phase. We also confirmed that the kidney function did not fully recover on day 15. These results suggest that the chronic generation of senescent PTECs might contribute to maladaptive recovery from AKI and lead to chronic kidney disease progression.

Recent Advances in Senolysis for Age-Related Diseases.

Cellular senescence plays a central role in aging and geriatric diseases. Senolysis is a promising new strategy that selectively kills and eliminates senescent cells to control aging. To date, various senolytic drugs have been discovered and shown efficacy. This review highlights how we can benefit from senolysis.

The chemotherapeutic drug doxorubicin does not exacerbate p16Ink4a-positive senescent cell accumulation and cardiometabolic disease development in young adult female LDLR-deficient mice.

Cancer survivors who received chemotherapy, such as the anthracycline doxorubicin (DOX), have an increased risk of developing complications later in life, including the development of chronic metabolic diseases. Although the etiology of this increased risk for late metabolic complications in cancer survivors is poorly understood, a causal role of therapy-induced senescent cells has been suggested. To study the role of cellular senescence in chemotherapy-induced metabolic complications, young adult female low-density lipoprotein receptor-deficient (Ldlr-/-)-p16-3MR mice, in which p16Ink4a-positive (p16Ink4a+) senescent cells can be genetically eliminated, were treated with four weekly injections of DOX (2.5 mg/kg) followed by a high-fat high-cholesterol diet for 12 weeks. While DOX treatment induced known short-term effects, such as reduction in body weight, gonadal fat mass, and adipose tissue inflammation, it was not associated with significant long-term effects on glucose homeostasis, hepatic steatosis, or atherosclerosis. We further found no evidence of DOX-induced accumulation of p16Ink4a+-senescent cells at 1 or 12 weeks after DOX treatment. Neither did we observe an effect of elimination of p16Ink4a+-senescent cells on the development of diet-induced cardiometabolic complications in DOX-treated mice. Other markers for senescence were generally also not affected except for an increase in p21 and Cxcl10 in gonadal white adipose tissue long-term after DOX treatment. Together, our study does not support a significant role for p16Ink4a+-senescent cells in the development of diet-induced cardiometabolic disease in young adult DOX-treated female Ldlr-/- mice. These findings illustrate the need of further studies to understand the link between cancer therapy and cardiometabolic disease development in cancer survivors.

Transcriptomic characterization of Lonrf1 at the single-cell level under pathophysiological conditions.

The LONRF family of proteins consists of three isozymes, LONRF1-3, which harbors RING (really interesting new gene) domain and Lon substrate binding domain. We have recently identified LONRF2 as a protein quality control ubiquitin ligase that acts predominantly in neurons. LONRF2 selectively ubiquitylates misfolded or damaged proteins for degradation. LONRF2-/- mice exhibit late-onset neurological deficits. However, the physiological implications of other LONRF isozymes remain unclear. Here, we analysed Lonrf1 expression and transcriptomics at the single-cell level under normal and pathological conditions. We found that Lonrf1 was ubiquitously expressed in different tissues. Its expression in LSEC and Kupffer cells increased with age in the liver. Lonrf1high Kupffer cells showed activation of regulatory pathways of peptidase activity. In normal and NASH (nonalcoholic steatohepatitis) liver, Lonrf1high LSECs showed activation of NF-kB and p53 pathways and suppression of IFNa, IFNg and proteasome signalling independent of p16 expression. During wound healing, Lonrf1high/p16low fibroblasts showed activation of cell growth and suppression of TGFb and BMP (bone morphogenetic protein) signalling, whereas Lonrf1high/p16high fibroblasts showed activation of WNT (wingless and Int-1) signalling. These results suggest that although Lonrf1 does not seem to be associated with senescence induction and phenotypes, LONRF1 may play a key role in linking oxidative damage responses and tissue remodelling during wound healing in different modes in senescent and nonsenescent cells.

Efficacy of commercial live vaccines against QX-like infectious bronchitis virus in Japan.

Infectious bronchitis, an acute and highly contagious disease that affects chickens, is caused by the infectious bronchitis virus (IBV). The antigenic variant QX-like IBV was first reported in China in 1996 and is now endemic in many countries. Our previous study reported the first detection and isolation of QX-like IBVs in Japan and that they were genetically related to the recently detected strains in China and South Korea. The pathogenicity of 2 Japanese QX-like IBV strains (JP/ZK-B7/2020 and JP/ZK-B22/2020) was evaluated by inoculating specific pathogen-free (SPF) chickens with 102 to 106 median embryo infectious dose. Both strains caused clinical signs of respiratory symptoms, gross tracheal lesions, and moderate-to-severe suppression of tracheal ciliostasis. To evaluate the efficacy of commercial IBV live vaccines against the JP/ZK-B7/2020 strain, vaccinated SPF chickens were challenged with the JP/ZK-B7/2020 strain at 104 EID50 (median embryo infectious dose). Only the JP-Ⅲ vaccine provided high levels of protection (reduced suppression of tracheal ciliostasis and reduced viral loads in organs), whereas the Mass vaccine showed little protective effect. Virus neutralization test results and comparisons between IBV genotypes based on the S1 gene suggested that QX-like and JP-III genotypes were closely related. These results suggest that the JP-III IBV vaccine, which has relatively high S1 gene homology with QX-like IBVs, is effective against Japanese QX-like IBV strain.

The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5.

UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, the mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for the termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.

CDCA7 is a hemimethylated DNA adaptor for the nucleosome remodeler HELLS.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, characterized by hypomethylation at heterochromatin. The unique zinc-finger domain, zf-4CXXC_R1, of CDCA7 is widely conserved across eukaryotes but is absent from species that lack HELLS and DNA methyltransferases, implying its specialized relation with methylated DNA. Here we demonstrate that zf-4CXXC_R1 acts as a hemimethylated DNA sensor. The zf-4CXXC_R1 domain of CDCA7 selectively binds to DNA with a hemimethylated CpG, but not unmethylated or fully methylated CpG, and ICF disease mutations eliminated this binding. CDCA7 and HELLS interact via their N-terminal alpha helices, through which HELLS is recruited to hemimethylated DNA. While placement of a hemimethylated CpG within the nucleosome core particle can hinder its recognition by CDCA7, cryo-EM structure analysis of the CDCA7-nucleosome complex suggests that zf-4CXXC_R1 recognizes a hemimethylated CpG in the major groove at linker DNA. Our study provides insights into how the CDCA7-HELLS nucleosome remodeling complex uniquely assists maintenance DNA methylation.

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

Structural basis for activation of DNMT1.

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved α-helix which engages a crucial "Toggle" pocket, displacing a previously described inhibitory linker, and allowing the DNA Recognition Helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.

Blocking PD-L1-PD-1 improves senescence surveillance and ageing phenotypes.

The accumulation of senescent cells is a major cause of age-related inflammation and predisposes to a variety of age-related diseases1. However, little is known about the molecular basis underlying this accumulation and its potential as a target to ameliorate the ageing process. Here we show that senescent cells heterogeneously express the immune checkpoint protein programmed death-ligand 1 (PD-L1) and that PD-L1+ senescent cells accumulate with age in vivo. PD-L1- cells are sensitive to T cell surveillance, whereas PD-L1+ cells are resistant, even in the presence of senescence-associated secretory phenotypes (SASP). Single-cell analysis of p16+ cells in vivo revealed that PD-L1 expression correlated with higher levels of SASP. Consistent with this, administration of programmed cell death protein 1 (PD-1) antibody to naturally ageing mice or a mouse model with normal livers or induced nonalcoholic steatohepatitis reduces the total number of p16+ cells in vivo as well as the PD-L1+ population in an activated CD8+ T cell-dependent manner, ameliorating various ageing-related phenotypes. These results suggest that the heterogeneous expression of PD-L1 has an important role in the accumulation of senescent cells and inflammation associated with ageing, and the elimination of PD-L1+ senescent cells by immune checkpoint blockade may be a promising strategy for anti-ageing therapy.

Sentinel p16INK4a+ cells in the basement membrane form a reparative niche in the lung.

We engineered an ultrasensitive reporter of p16INK4a, a biomarker of cellular senescence. Our reporter detected p16INK4a-expressing fibroblasts with certain senescent characteristics that appeared shortly after birth in the basement membrane adjacent to epithelial stem cells in the lung. Furthermore, these p16INK4a+ fibroblasts had enhanced capacity to sense tissue inflammation and respond through their increased secretory capacity to promote epithelial regeneration. In addition, p16INK4a expression was required in fibroblasts to enhance epithelial regeneration. This study highlights a role for p16INK4a+ fibroblasts as tissue-resident sentinels in the stem cell niche that monitor barrier integrity and rapidly respond to inflammation to promote tissue regeneration.

Detection and isolation of QX-like infectious bronchitis virus in Japan.

The antigenic variant QX-like infectious bronchitis virus (IBV) is endemic in several countries. In Japan, the QX-like genotype is classified as the JP-III genotype based on the partial S1 gene and as the GI-19 genotype based on the complete S1 gene. This study showed that QX-like IBVs and JP-III IBVs can be identified based on the amino acid polymorphism of the S1 glycoprotein. Furthermore, genetic analysis of several IBV field strains detected in commercial broiler farms across the Kyushu area in 2020 revealed Japanese QX-like IBVs, which are highly homologous to the QX-like IBVs recently detected in China and South Korea. Herein, QX-like IBV field strains were isolated for evaluating commercial vaccine efficacy in our future studies.