Age Factors Research Articles

The ESCRT-III complex is required for nuclear pore complex sequestration and regulates gamete replicative lifespan in budding yeast meiosis

ABSTRACT Cellular aging occurs as a cell loses its ability to maintain homeostasis. Aging cells eliminate damaged cellular compartments and other senescence factors via self-renewal. The mechanism that regulates cellular rejuvenation remains to be further elucidated. Using budding yeast gametogenesis as a model, we show here that the endosomal sorting complex required for transport (ESCRT) III regulates nuclear envelope organization. During gametogenesis, the nuclear pore complex (NPC) and other senescence factors are sequestered away from the prospore nuclei. We show that the LEM-domain protein Heh1 (Src1) facilitates the nuclear recruitment of ESCRT-III, which is required for meiotic NPC sequestration and nuclear envelope remodeling. Furthermore, ESCRT-III-mediated nuclear reorganization appears to be critical for gamete rejuvenation, as hindering this process curtails either directly or indirectly the replicative lifespan in gametes. Our findings demonstrate the importance of ESCRT-III in nuclear envelope remodeling and its potential role in eliminating senescence factors during gametogenesis.

The protective role of omentin-1 in IL-1β-induced chondrocyte senescence

Osteoarthritis is a common type of degenerative joint disease. Inflammation-related chondrocyte senescence plays a major role in the pathogenesis of osteoarthritis. Omentin-1 is a newly identified anti-inflammatory adipokine involved in lipid metabolism. In this study, we examined the biological function of omentin-1 in cultured chondrocytes. The presence of omentin-1 potently suppresses IL-1β-induced cellular senescence as revealed by staining with senescence-associated beta-galactosidase (SA-β-Gal). At the cellular level, omentin-1 attenuates IL-1β-induced G1 phase cell-cycle arrest. Mechanistically, we demonstrate that omentin-1 reduced IL-1β-induced expression of senescent factors including caveolin-1, p21, and PAI-1 as well as p53 acetylation through ameliorating SIRT1 reduction. Notably, silencing of SIRT1 abolishes IL-1β-induced senescence along with the induction of p21 and PAI-1, suggesting that the action of omentin-1 in chondrocytes is dependent on SIRT1. Collectively, our results revealed the molecular mechanism through which the adipokine omentin-1 exerts a beneficial effect, thereby protecting chondrocytes from senescence. Thus, omentin-1 could have clinical implication in the treatment of osteoarthritis.

FoxO3a is the Novel Anti-aging Factor for Age-related Skin Senescence and Pigmentation

FoxO3a is the Novel Anti-aging Factor for Age-related Skin Senescence and Pigmentation

The Sirt1/P53 Axis in Diabetic Intervertebral Disc Degeneration Pathogenesis and Therapeutics.

Intervertebral disc degeneration (IDD) is one of the major causes of low back pain. Diabetes is a risk factor for IDD and may aggravate IDD in rats; however, the mechanism is poorly understood. Previously, we demonstrated that apoptosis and senescence were increased in diabetic nucleus pulposus (NP) tissues; in the current study, we found that hyperglycaemia may promote the incidence of apoptosis and senescence in NP cells in vitro. Meanwhile, the acetylation of P53, a master transcription factor of apoptosis and senescence, was also found increased in diabetic NP tissues in vivo as well as in hyperglycaemic NP cells in vitro. Sirt1 is an NAD+-dependent deacetylase, and we showed that the expression of Sirt1 was decreased in NP tissues, while hyperglycaemia could suppress the expression and activity of Sirt1 in NP cells. Furthermore, we demonstrated that butein may inhibit acetylation of P53 and protect NP cells against hyperglycaemia-induced apoptosis and senescence through Sirt1 activation, as the Sirt1 inhibitor Ex527 may counteract the protective effect of butein in hyperglycaemic NP cells. An in vivo study showed that butein could ameliorate the IDD process in diabetic rats, while Sirt1 was increased and acetyl-p53 was decreased in NP tissues in butein-treated rats. These results indicate that the Sirt1/P53 axis is involved in the pathogenesis of diabetic IDD and may serve as a therapeutic target for diabetic IDD.

Meiotic cellular rejuvenation is coupled to nuclear remodeling in budding yeast.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation.

The Upregulation of Toll-Like Receptor 3 via Autocrine IFN-β Signaling Drives the Senescence of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Through JAK1.

Although mesenchymal stromal cells (MSCs) are among the most promising cell sources for cell-based therapies and regenerative medicine, the decline in their function with age due to cellular senescence limits their therapeutic applications. Unveiling the underlying mechanism of MSC senescence is therefore of substantial interest with regard to advancing MSC-based cell therapies. We here show that the induction of human umbilical cord blood-derived MSC (UCB-MSC) senescence causes the predominant upregulation of Toll-like receptor 3 (TLR3). Subsequent TLR3 activation by polyinosinic-polycytidylic acid triggers the prominent features of senescence. Using a clustered regularly interspaced short palindromic repeats/Cas9 library screening system, we identified Janus kinase 1 (JAK1) as the candidate regulatory factor for TLR3-mediated MSC senescence. A JAK1 deficiency blocked the MSC senescence phenotype upon TLR3 activation and TLR3 induction. Targeting the JAK1 pathway using chemical JAK1 inhibitors also significantly suppressed TLR3-mediated MSC senescence. Importantly, we further observed that UCB-MSC senescence is driven by a senescence-associated secretory phenotype (SASP) and that interferon-β (IFN-β) is a component of TLR3-dependent SASP, whereby its autocrine actions upregulate TLR3 and suppress cell proliferation. A JAK1 depletion significantly interrupted these effects of IFN-β, indicating that JAK1 is a signaling mediator linking IFN-β activity to TLR3 expression and the process of MSC senescence. Collectively, our findings provide new mechanistic insights into UCB-MSC senescence by revealing the role of an autocrine regulatory loop of SASP evoked by TLR3 activation.

Abstract 895: The identification of molecular mechanism underlying the progression of gastric cancer peritoneal dissemination by senescent fibroblasts

Abstract Background: Secreted factors from cancer associated fibroblast (CAFs) is likely to be implicated in cancer progression, but the precise mechanism is not fully understood. It is reported that senescent cells survive and secrete some cytokines, so called senescence-associated secretory phenotype (SASP). The purpose of current study is to identify the molecular mechanism underlying the progression of gastric cancer (GC) peritoneal dissemination by CAFs showing SASP. Methods: We examined the senescent state of CAFs activated NFkB signaling by inflammatory cytokines (iCAFs) and evaluated the expression of SASP factors. We investigated the growth ability of GC cell lines treated with iCAFs derived condition medium (CM). We transplanted GC cell lines with intraperitoneal administration to generate peritoneal metastasis mouse model and evaluated the influence of SASP factors on cancer progression. Results: We found that the growth ability of iCAFs decreased and their expression of p16 and p15, which was senescent markers increased. And the comprehensive genomic analysis revealed that iCAFs was highly enriched the senescent related genes and SASP factors related genes. Next, we also found that iCAFs derived CM significantly increased the growth ability of GC cells. Moreover, also in peritoneal metastasis mouse model, iCAFs derived CM remarkably enhanced intraperitoneal tumors. Conclusion: These findings suggest that SASP factors from senescent CAFs, which activated by inflammatory cytokines can promote peritoneal dissemination of GC cells. Citation Format: Tadahito Yasuda, Mayu Koiwa, Tomoyuki Uchihara, Atsuko Yonemura. The identification of molecular mechanism underlying the progression of gastric cancer peritoneal dissemination by senescent fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 895.

Identification of Transcription Factors Regulating Senescence in Wheat through Gene Regulatory Network Modelling.

Senescence is a tightly regulated developmental program coordinated by transcription factors. Identifying these transcription factors in crops will provide opportunities to tailor the senescence process to different environmental conditions and regulate the balance between yield and grain nutrient content. Here, we use ten time points of gene expression data along with gene network modeling to identify transcription factors regulating senescence in polyploid wheat (Triticum aestivum). We observe two main phases of transcriptional changes during senescence: early down-regulation of housekeeping functions and metabolic processes followed by up-regulation of transport and hormone-related genes. These two phases are largely conserved with Arabidopsis (Arabidopsis thaliana), although the individual genes underlying these changes are often not orthologous. We have identified transcription factor families associated with these early and later waves of differential expression. Using gene regulatory network modeling, we identified candidate transcription factors that may control senescence. Using independent, publicly available datasets, we found that the most highly ranked candidate genes in the network were enriched for senescence-related functions compared with all genes in the network. We validated the function of one of these candidate transcription factors in senescence using wheat chemically induced mutants. This study lays the groundwork to understand the transcription factors that regulate senescence in polyploid wheat and exemplifies the integration of time-series data with publicly available expression atlases and networks to identify candidate regulatory genes.

Influence of advanced glycation end products on rotator cuff

Most rotator cuff tears are the result of age-related degenerative changes, but the mechanisms underlying these changes have not been reported. Recently, advanced glycation end products (AGEs) have been regarded as an important factor in senescence. Therefore, we hypothesized that AGEs would have detrimental effects on rotator cuff-derived cells. In this study, we investigated the influence of AGEs on rotator cuff-derived cells invitro and exvivo. Rotator cuff-derived cells were obtained from human supraspinatus tendons. The cells were cultured in the following media: (1) regular medium with 500 μg/mL AGEs (High-AGEs), (2) regular medium with 100 μg/mL AGEs (Low-AGEs), and (3) regular medium alone (Control). Cell viability, secretion of vascular endothelial growth factor, and the expressions of hypoxia-inducible factor-1α, reactive oxygen species, and apoptosis were assessed after cultivation. An exvivo tissue culture with AGEs was also performed to measure the tensile strength. Cell viability in the High-AGEs group was significantly suppressed relative to that in the Controls. The amount of vascular endothelial growth factor secretion was significantly greater in the High- and Low-AGEs groups than in the Controls. Immunofluorescence stain demonstrated enhancement of hypoxia-inducible factor-1α and reactive oxygen species expressions and cell apoptosis in the High- and Low-AGEs groups relative to that in the Controls. In exvivo mechanical testing, tensile strength was significantly higher in the Control group than in the AGEs groups. These results indicated that AGEs caused age-related degenerative rotator cuff changes. The reduction of AGEs might prevent rotator cuff senescence-related degeneration.

Cell senescence, apoptosis and DNA damage cooperate in the remodeling processes accounting for heart morphogenesis.

During embryonic development, organ morphogenesis requires major tissue rearrangements that are tightly regulated at the genetic level. A large number of studies performed in recent decades assigned a central role to programmed cell death for such morphogenetic tissue rearrangements that often sculpt the shape of embryonic organs. However, accumulating evidence indicates that far from being the only factor responsible for sculpting organ morphology, programmed cell death is accompanied by other tissue remodeling events that ensure the outcome of morphogenesis. In this regard, cell senescence has been recently associated with morphogenetic degenerative embryonic processes as an early tissue remodeling event in development of the limbs, kidney and inner ear. Here, we have explored cell senescence by monitoring β‐galactosidase activity during embryonic heart development where programmed cell death is believed to exert an important morphogenetic function. We report the occurrence of extensive cell senescence foci during heart morphogenesis. These foci overlap spatially and temporally with the areas of programmed cell death that are associated with remodeling of the outflow tract to build the roots of the great arteries and with the septation of cardiac cavities. qPCR analysis allowed us to identify a gene expression profile characteristic of the so‐called senescence secretory associated phenotype in the remodeling outflow tract of the embryonic heart. In addition, we confirmed local upregulation of numerous tumor suppressor genes including p21, p53, p63, p73 and Btg2. Interestingly, the areas of cell senescence were also accompanied by intense lysosomal activation and non‐apoptotic DNA damage revealed by γH2AX immunolabeling. Considering the importance of sustained DNA damage as a triggering factor for cell senescence and apoptosis, we propose the coordinated contribution of DNA damage, senescence and apoptotic cell death to assure tissue remodeling in the developing vertebrate heart.

The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers

The plant hormone ethylene was identified as important triggering factor and primary regulator of flower senescence in many species. Consequently, application of chemical inhibitors of ethylene biosynthesis and action is used to extend the longevity of ethylene-sensitive flowers. Here, we show that the peptide NOP-1, a biological derived from the nuclear localization signal of ethylene regulator EIN2 tightly binds to the ethylene receptor of carnation plants - a model to study flower senescence. When applied on cut flowers the peptide biological delays petal senescence similar to previously identified and currently used chemical inhibitors, but offers significant advances to these chemicals in biodegradability, sustainability and ecotoxicity. Our bioinformatic analysis of a wide range of ethylene receptors indicates complete sequence conservation of the anticipated NOP-1 binding site in flower species supporting a widespread use of the peptide on flowering ornamentals to delay senescence and decay in cut flowers. We anticipate our innovative approach to extend flower longevity by a new class of biomolecules such as peptides, peptide analogues and peptide mimetics will significantly advance our technological capability to delay flower senescence and expand vase-life of cut flowers in a sustainable and environmentally friendly manner.

A new process-based model for predicting autumn phenology: How is leaf senescence controlled by photoperiod and temperature coupling?

Autumn phenological variation of temperate and northern ecosystems plays a major but poorly defined role in the global carbon cycle. Constructing robust process-based autumn phenology models is key for improving current ecosystem models to accurately simulate ecosystem productivity and carbon sequestration. In this study, we developed a new process-based autumn phenology model. The model was fitted and validated using 67 leaf coloration and brown-down time series collected for 27 woody and herbaceous species at 18 stations on the Qinghai–Tibetan Plateau from 1981 to 2012. Then, we used the model to analyze the spatial and interspecific differentiation of driving factors of leaf senescence. Moreover, we compared fitting and validation precisions of the new model and previously published models. Results show that leaf senescence processes were triggered by photoperiod shortening in 61.2% of time series but by daily minimum temperature decrease in 38.8% of time series. Photoperiod control of the leaf senescence start occurs predominantly at stations with shorter annual maximum daylength (88.9%), while daily minimum temperature control of the leaf senescence start appears mainly at stations with longer annual maximum daylength (71.0%), especially for the native species. The new model reveals coupling effects of shortened photoperiod and decreased daily minimum temperature on leaf senescence processes. Comparing with the representative existing process-based autumn phenology models, the new model is a more general model and more robust in fitting and predicting leaf coloration and brown-down dates. Our study has provided a new insight on the understanding of leaf senescence mechanisms in winter deciduous trees and herbaceous plants, and significantly improved the ability to predict climate change impacts on vegetation growth and carbon balance in the sensitive biogeographical region of global climate change.

Native low density lipoprotein increases the production of both nitric oxide and reactive oxygen species in the human umbilical vein endothelial cells.

Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells. The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2',7'-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader. NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs. These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.

Administration of hydrogen-rich water prevents vascular aging of the aorta in LDL receptor-deficient mice

The main cause of arteriosclerosis is atherosclerosis in the aorta. Atherosclerosis is recognized as a chronic inflammatory condition that begins with the dysfunction or activation of arterial endothelium. Low-density lipoprotein (LDL) and especially its oxidized form play a key role in endothelial dysfunction and atherogenesis. Recent studies showed that senescent cells are involved in the development and progression of atherosclerosis, and eliminating senescent cells suppresses the senescence-associated secretory phenotype. We previously reported that molecular hydrogen-rich water (HW) has antioxidant and anti-inflammatory effects in numerous diseases. Here, we used LDL receptor-deficient mice fed a high-fat diet (HFD) for 13 weeks as a model for atherosclerosis and evaluated the effects of continuous administration of HW. The numbers of endothelial cells in the atheroma expressing the senescence factors p16INK4a and p21 decreased in HFD-fed mice given HW compared with HFD-fed mice given control water. Furthermore, macrophage infiltration and Tnfα expression in the atheroma were also suppressed. These results suggest that vascular aging can be suppressed by HW.

Interaction of the hydrogen sulphide inhibitor, propargylglycine (PAG), with hydrogen sulphide on postharvest changes of the green leafy vegetable, pak choy

Propargylglycine (PAG) is an inhibitor of hydrogen sulphide (H2S) production and has been used to explore the mode of action of H2S in prolonging storage of horticultural produce but little attention has been given to how PAG and H2S interact when both are applied to produce. This study examined the effect of sequential application of PAG and H2S on a range of postharvest senescence factors of the leafy vegetable pak choy (Brassica rapa subsp. Chinensis) stored at 10 °C. The results showed differential responses between factors when compared to application of PAG or H2S alone. As expected, fumigation with H2S reduced the rate of loss of leaf green colour, respiration rate, ethylene production, ion leakage and enhanced antioxidant activity and leaves sprayed with PAG showing converse effects. If PAG acted solely by inhibiting endogenous H2S production then subsequent treatment with H2S should fully negate any effect induced by PAG. However, for the combined PAG + H2S treatment, respiration was similar leaves fumigated with H2S, loss of green leaf colour was similar to the PAG single treatment and less than the untreated control, antioxidant activity was less than for PAG but greater than for control leaves, and ethylene production and ion leakage were similar to control leaves. Thus, the concept that PAG is exclusively an inhibitor of endogenous H2S production was not validated, with PAG having effects on metabolism that are not linked to the action of endogenous H2S. The additional actions of PAG could be through its inhibition of pyridoxyal-5′-phosphate (PHP) which is a coenzyme for numerous enzyme systems.

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[BrafV600E] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.

Cardiac ageing: extrinsic and intrinsic factors in cellular renewal and senescence

Cardiac ageing manifests as a decline in function leading to heart failure. At the cellular level, ageing entails decreased replicative capacity and dysregulation of cellular processes in myocardial and nonmyocyte cells. Various extrinsic parameters, such as lifestyle and environment, integrate important signalling pathways, such as those involving inflammation and oxidative stress, with intrinsic molecular mechanisms underlying resistance versus progression to cellular senescence. Mitigation of cardiac functional decline in an ageing organism requires the activation of enhanced maintenance and reparative capacity, thereby overcoming inherent endogenous limitations to retaining a youthful phenotype. Deciphering the molecular mechanisms underlying dysregulation of cellular function and renewal reveals potential interventional targets to attenuate degenerative processes at the cellular and systemic levels to improve quality of life for our ageing population. In this Review, we discuss the roles of extrinsic and intrinsic factors in cardiac ageing. Animal models of cardiac ageing are summarized, followed by an overview of the current and possible future treatments to mitigate the deleterious effects of cardiac ageing.

P-118 - Protective effect of quercetin-quinone derivative in senescent human fibroblasts

Gradual accumulation of oxidatively damaged cell structures along with impairment of redox homeostasis has been suggested as a key factor in ageing and cellular senescence. Anti-ageing effects were reported for a number of phenol- and quinone-type compounds. By using a model of senescent human fibroblasts (strain VH-10), potential beneficial effects of semisynthetic compound, 4-O-(2-chloro-1,4-naphtoquinone-3-yloxy) quercetin (CHNQ) were investigated. CHNQ significantly suppressed senescence markers, SAβ-galactosidase activity and lipofuscin-related autofluorescence. Moreover, CHNQ, more efficiently than its precursors, restored mitochondrial membrane potential linked probably to observed decrease in ROS levels. In addition, CHNQ-treated cells showed increased LC3 turnover pointing to improvement of autophagy flux. By contrast, the compounds tested restored neither the gradual decline in proliferation, nor the cell cycle arrest at G2/M phase. Yet, modulation of p21 and p53 proteins dependently on the persistence time of senescent cells in culture was found. In conclusion, the result of our study point to protective effect of CHNQ on functionalities of senescent fibroblasts, without influencing their proliferative lifespan [VEGA 2/0041/17,2/0029/16,APVV-15–0308, ITMS 26240220040].

Mechanisms of neuroprotection against oxidative stress in the anoxia tolerant turtle Trachemys scripta

The detrimental effects of oxidative stress caused by the accumulation of Reactive Oxygen Species (ROS) have been acknowledged as major factors in aging, senescence, and several neurodegenerative conditions including Parkinson's Disease and stroke (ischemia/reperfusion). Mammalian models are extremely susceptible to these stresses and the restoration of oxygen after anoxia causes oxidative damage; however some organisms including the freshwater turtle Trachemys scripta can withstand such conditions without any apparent pathology. T. scripta thus provides us with a model in which we can investigate physiological mechanisms of neuroprotection in anoxia and reoxygenation without the damaging effects that come with oxidative stress.The ability of the turtle to survive these stressors is thought to be related to its high innate levels of antioxidants and its capacity to suppress ROS production. One potential antioxidant mechanism is the Methionine Sulfoxide Reductase System (Msr) that catalyzes the reduction of oxidized Methionine (Met) residues. Methionine residues are easily oxidized amino acids and oxidized Met is associated with decreased protein activity. Msr may also restore function to damaged proteins and it has been shown to scavenge ROS and thus ameliorate cellular damage.The present study investigated the role and regulation of MsrA in turtle primary neuronal cell cultures exposed to 4 hours of anoxia and 4 hours of anoxia followed by 4 hours of reoxygenation. Anoxia and anoxia/reoxygenation increase MsrA mRNA and protein levels, which suggests a protective role under these conditions. MsrA expression was then altered with target specific siRNA; we found that the knock down of MsrA expression resulted in decreased cell viability. As the factors that control MsrA expression in this model are still unknown, we also induced FOXO3a expression by transfecting a FOXO3a plasmid into the cells, as it has been shown to regulate MsrA levels in other animal models. The induction of FOXO3a protected neurons against cell death. Furthermore, treatment of cells with the green tea extract Epigallocatechin gallate (EGCG), which has antioxidant properties, results in increased expression of FOXO3a when the cells are exposed to oxidative stress. Together these results suggest that MsrA may play an important role in protection against anoxia and oxidative stress in the turtle brain, and may prove to be a target to treat diseases of ischemia and oxidative stress.Support or Funding InformationNIH‐NIA Project # 1R15AG033374‐01,FAU FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exposure of human melanocytes to UVB twice and subsequent incubation leads to cellular senescence and senescence-associated pigmentation through the prolonged p53 expression

BackgroundUltraviolet radiation (UVR) is a well-known factor in skin aging and pigmentation, and daily exposure to subcytotoxic doses of UVR might accelerate senescence and senescence-associated phenomena in human melanocytes. ObjectiveTo establish an in vitro melanocyte model to mimic the conditions of repeated exposure to subcytotoxic doses of UVB irradiation and to investigate key factor(s) for melanocyte senescence and senescence-associated phenomena. MethodsHuman epidermal melanocytes were exposed twice with 20 mJ/cm2 UVB over a 24-h interval and subsequently cultivated for 2 weeks. Senescent phenotypes were addressed morphologically, and by measuring the senescence-associated β-galactosidase (SA-β-Gal) activity, cell proliferation capacity with cell cycle analysis, and melanin content. ResultsThe established protocol successfully induced melanocyte senescence, and senescent melanocytes accompanied hyperpigmentation. Prolonged expression of p53 was responsible for melanocyte senescence and hyperpigmentation, and treatment with the p53-inhibitor pifithrin-α at 2-weeks post-UVB irradiation, but not at 48 h, significantly reduced melanin content along with decreases in tyrosinase levels. ConclusionMelanocyte senescence model will be useful for studying the long-term effects of UVB irradiation and pigmentation relevant to physiological photoaging, and screening compounds effective for senescence-associated p53-mediated pigmentation.