TNF‐α/IFN‐γ synergy amplifies senescence‐associated inflammation and SARS‐CoV‐2 receptor expression via hyper‐activated JAK/STAT1

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.     

Olive phenols preserve lamin B1 expression reducing cGAS/STING/NFκB‐mediated SASP in ionizing radiation‐induced senescence

Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress‐induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence‐associated secretory phenotype (SASP), which contributes to generate a pro‐inflammatory and pro‐tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti‐inflammatory and anti‐tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation‐induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ‐irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence‐associated (SA)‐β‐Gal‐staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP‐AMP Synthase (cGAS) activation. IL‐6, IL‐8, MCP‐1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation‐induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB‐mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy.     

NAD+ supplementation prevents STING‐induced senescence in ataxia telangiectasia by improving mitophagy

Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A‐T). Loss of mitochondrial function can drive age‐related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence‐associated secretory phenotype (SASP) occur in A‐T patient fibroblasts, and in ATM‐deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD⁺ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1‐dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm^−/− mice. Our findings suggest a central role for mitochondrial dysfunction‐induced senescence in A‐T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.     

Parkin drives pS65‐Ub turnover independently of canonical autophagy in Drosophila

Parkinson''s disease‐related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1‐Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1–Parkin pathway operates in vivo, we developed methods to detect Ser65‐phosphorylated ubiquitin (pS65‐Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1‐dependent pS65‐Ub production, while pS65‐Ub accumulates in unstimulated parkin‐null flies, consistent with blocked degradation. Additionally, we show that pS65‐Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65‐Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat‐induced pS65‐Ub in an Atg5‐null background. Thus, we have established that pS65‐Ub immunodetection can be used to analyse Pink1‐Parkin function in vivo as an alternative to reporter constructs. Moreover, our findings suggest that the Pink1‐Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.     

Prevalent intron retention fine‐tunes gene expression and contributes to cellular senescence

Intron retention (IR) is the least well‐understood alternative splicing type in animals, and its prevalence and function in physiological and pathological processes have long been underestimated. Cellular senescence contributes to individual aging and age‐related diseases and can also serve as an important cancer prevention mechanism. Dynamic IR events have been observed in senescence models and aged tissues; however, whether and how IR impacts senescence remain unclear. Through analyzing polyA⁺ RNA‐seq data from human replicative senescence models, we found IR was prevalent and dynamically regulated during senescence and IR changes negatively correlated with expression alteration of corresponding genes. We discovered that knocking down (KD) splicing factor U2AF1, which showed higher binding density to retained introns and decreased expression during senescence, led to senescence‐associated phenotypes and global IR changes. Intriguingly, U2AF1‐KD‐induced IR changes also negatively correlated with gene expression. Furthermore, we demonstrated that U2AF1‐mediated IR of specific gene (CPNE1 as an example) contributed to cellular senescence. Decreased expression of U2AF1, higher IR of CPNE1, and reduced expression of CPNE1 were also discovered in dermal fibroblasts with age. We discovered prevalent IR could fine‐tune gene expression and contribute to senescence‐associated phenotypes, largely extending the biological significance of IR.     

PINK1/Parkin‐mediated mitophagy enhances the survival of Staphylococcus aureus in bovine macrophages

Mitochondria are cellular organelles that are involved in various metabolic processes, and damage to mitochondria can affect cell health and even lead to disease. Mitophagy is a mechanism by which cells selectively wrap and degrade damaged mitochondria to maintain cell homeostasis. However, studies have not focused on whether mitophagy is involved in the occurrence of Staphylococcus aureus (S. aureus)‐induced mastitis in dairy cows. Here, we found that S. aureus infection of bovine macrophages leads to oxidative damage and mitochondria damage. The expression of LC3, PINK1 and Parkin was significantly increased after intracellular infection. We observed changes in the morphology of mitochondria and the emergence of mitochondrial autolysosomes in bovine macrophages by transmission electron microscopy and found that enhanced mitophagy promoted bacterial proliferation in the cell. In conclusion, this study demonstrates that S. aureus infection of bovine macrophages induces mitophagy through the PINK1/Parkin pathway, and this mechanism is used by the bacteria to avoid macrophage‐induced death. These findings provide new ideas and references for the prevention and treatment of S. aureus infection.     

AAV‐mediated expression of secreted and transmembrane αKlotho isoforms rescues relevant aging hallmarks in senescent SAMP8 mice

Senescence represents a stage in life associated with elevated incidence of morbidity and increased risk of mortality due to the accumulation of molecular alterations and tissue dysfunction, promoting a decrease in the organism''s protective systems. Thus, aging presents molecular and biological hallmarks, which include chronic inflammation, epigenetic alterations, neuronal dysfunction, and worsening of physical status. In this context, we explored the AAV9‐mediated expression of the two main isoforms of the aging‐protective factor Klotho (KL) as a strategy to prevent these general age‐related features using the senescence‐accelerated mouse prone 8 (SAMP8) model. Both secreted and transmembrane KL isoforms improved cognitive performance, physical state parameters, and different molecular variables associated with aging. Epigenetic landscape was recovered for the analyzed global markers DNA methylation (5‐mC), hydroxymethylation (5‐hmC), and restoration occurred in the acetylation levels of H3 and H4. Gene expression of pro‐ and anti‐inflammatory mediators in central nervous system such as TNF‐α and IL‐10, respectively, had improved levels, which were comparable to the senescence‐accelerated‐mouse resistant 1 (SAMR1) healthy control. Additionally, this improvement in neuroinflammation was supported by changes in the histological markers Iba1, GFAP, and SA β‐gal. Furthermore, bone tissue structural variables, especially altered during senescence, recovered in SAMP8 mice to SAMR1 control values after treatment with both KL isoforms. This work presents evidence of the beneficial pleiotropic role of Klotho as an anti‐aging therapy as well as new specific functions of the KL isoforms for the epigenetic regulation and aged bone structure alteration in an aging mouse model.     

NDP52 acts as a redox sensor in PINK1/Parkin‐mediated mitophagy

Mitophagy, the elimination of mitochondria via the autophagy‐lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin‐dependent mitophagy. We identified redox‐sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.     

Adipose tissue senescence is mediated by increased ATP content after a short‐term high‐fat diet exposure

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high‐fat diet (HFD, 1–10 weeks) in 5‐month‐old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence‐associated ß‐galactosidase activity and cyclin‐dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD‐derived preadipocytes, as compared with chow diet‐derived preadipocytes. One‐month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD‐induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

Senescence‐associated β‐galactosidase reveals the abundance of senescent CD8+ T cells in aging humans

Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second‐generation fluorogenic substrate for β‐galactosidase and multi‐parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence‐associated β‐galactosidase (SA‐βGal) activity with advancing donor age. The greatest age‐associated increases were observed in CD8+ T‐cell populations, in which the fraction of cells with high SA‐βGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA‐βGal activity, but not those with low SA‐βGal activity, were found to exhibit features of telomere dysfunction‐induced senescence and p16‐mediated senescence, were impaired in their ability to proliferate, developed in various T‐cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age.     

Senolytic treatment reduces cell senescence and necroptosis in Sod1 knockout mice that is associated with reduced inflammation and hepatocellular carcinoma

The goal of this study was to test the role cellular senescence plays in the increased inflammation, chronic liver disease, and hepatocellular carcinoma seen in mice null for Cu/Zn‐Superoxide dismutase (Sod1KO). To inhibit senescence, wildtype (WT) and Sod1KO mice were given the senolytics, dasatinib, and quercetin (D + Q) at 6 months of age when the Sod1KO mice begin exhibiting signs of accelerated aging. Seven months of D + Q treatment reduced the expression of p16 in the livers of Sod1KO mice to WT levels and the expression of several senescence‐associated secretory phenotype factors (IL‐6, IL‐1β, CXCL‐1, and GDF‐15). D + Q treatment also reduced markers of inflammation in livers of the Sod1KO mice, for example, cytokines, chemokines, macrophage levels, and Kupffer cell clusters. D + Q treatment had no effect on various markers of liver fibrosis in the Sod1KO mice but reduced the expression of genes involved in liver cancer and dramatically reduced the incidence of hepatocellular carcinoma. Surprisingly, D + Q also reduced markers of necroptosis (phosphorylated and oligomerized MLKL) in the Sod1KO mice to WT levels. We also found that inhibiting necroptosis in the Sod1KO mice with necrostatin‐1s reduced the markers of cellular senescence (p16, p21, and p53). Our study suggests that an interaction occurs between cellular senescence and necroptosis in the liver of Sod1KO mice. We propose that these two cell fates interact through a positive feedback loop resulting in a cycle amplifying both cellular senescence and necroptosis leading to inflammaging and age‐associated pathology in the Sod1KO mice.     

SIAH proteins regulate the degradation and intra‐mitochondrial aggregation of PINK1: Implications for mitochondrial pathology in Parkinson's disease

Parkinson''s disease (PD) is characterized by degeneration of neurons, particularly dopaminergic neurons in the substantia nigra. PD brains show accumulation of α‐synuclein in Lewy bodies and accumulation of dysfunctional mitochondria. However, the mechanisms leading to mitochondrial pathology in sporadic PD are poorly understood. PINK1 is a key for mitophagy activation and recycling of unfit mitochondria. The activation of mitophagy depends on the accumulation of uncleaved PINK1 at the outer mitochondrial membrane and activation of a cascade of protein ubiquitination at the surface of the organelle. We have now found that SIAH3, a member of the SIAH proteins but lacking ubiquitin‐ligase activity, is increased in PD brains and cerebrospinal fluid and in neurons treated with α‐synuclein preformed fibrils (α‐SynPFF). We also observed that SIAH3 is aggregated together with PINK1 in the mitochondria of PD brains. SIAH3 directly interacts with PINK1, leading to their intra‐mitochondrial aggregation in cells and neurons and triggering a cascade of toxicity with PINK1 inactivation along with mitochondrial depolarization and neuronal death. We also found that SIAH1 interacts with PINK1 and promotes ubiquitination and proteasomal degradation of PINK1. Similar to the dimerization of SIAH1/SIAH2, SIAH3 interacts with SIAH1, promoting its translocation to mitochondria and preventing its ubiquitin‐ligase activity toward PINK1. Our results support the notion that the increase in SIAH3 and intra‐mitochondrial aggregation of SIAH3‐PINK1 may mediate α‐synuclein pathology by promoting proteotoxicity and preventing the elimination of dysfunctional mitochondria. We consider it possible that PINK1 activity is decreased in sporadic PD, which impedes proper mitochondrial renewal in the disease.     

A circular intronic RNA ciPVT1 delays endothelial cell senescence by regulating the miR‐24‐3p/CDK4/pRb axis

Circular RNAs (circRNAs) have been established to be involved in numerous processes in the human genome, but their function in vascular aging remains largely unknown. In this study, we aimed to characterize and analyze the function of a circular intronic RNA, ciPVT1, in endothelial cell senescence. We observed significant downregulation of ciPVT1 in senescent endothelial cells. In proliferating endothelial cells, ciPVT1 knockdown induced a premature senescence‐like phenotype, inhibited proliferation, and led to an impairment in angiogenesis. An in vivo angiogenic plug assay revealed that ciPVT1 silencing significantly inhibited endothelial tube formation and decreased hemoglobin content. Conversely, overexpression of ciPVT1 in old endothelial cells delayed senescence, promoted proliferation, and increased angiogenic activity. Mechanistic studies revealed that ciPVT1 can sponge miR‐24‐3p to upregulate the expression of CDK4, resulting in enhanced Rb phosphorylation. Moreover, enforced expression of ciPVT1 reversed the senescence induction effect of miR‐24‐3p in endothelial cells. In summary, the present study reveals a pivotal role for ciPVT1 in regulating endothelial cell senescence and may have important implications in the search of strategies to counteract the development of age‐associated vascular pathologies.