In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro-inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence-like state in mature postmitotic neurons in vivo. About 40-80% of Purkinje neurons and 20-40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL-6 production, heterochromatinization and senescence-associated β-galactosidase activity. Frequencies of these senescence-like neurons increased with age. Short-term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late-generation TERC-/- mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late-generation TERC-/-CDKN1A-/- mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence-like phenotype in neurons, as in senescing fibroblasts and other proliferation-competent cells. We conclude that a senescence-like phenotype is possibly not restricted to proliferation-competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence-like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.

Chronic dietary restriction (DR) has been shown to have beneficial effects on glucose homeostasis and insulin sensitivity. These factors show rapid and robust improvements when rodents were crossed over from an ad libitum (AL) diet to DR in mid life. We aimed to determine whether the beneficial effects induced by short-term exposure to DR can be retained as a 'metabolic memory' when AL feeding is resumed (AL-DR-AL) and vice versa: whether the effects of long-term DR can be reversed by a period of AL feeding (DR-AL-DR). C57BL/6 male and female mice were used to examine sex differences (N = 10/sex/group). Mice were fed AL or DR from 3 until 15 months (baseline) and each dietary crossover lasted approximately 5 months.

In the normal quiescent vasculature, only 0.01% of endothelial cells (ECs) are proliferating. However, this proportion increases dramatically following the angiogenic switch during tumor growth or wound healing. Recent evidence suggests that this angiogenic switch is accompanied by a metabolic switch. Here, we show that proliferating ECs increasingly depend on mitochondrial oxidative phosphorylation (OxPhos) for their increased energy demand. Under growth conditions, ECs consume three times more oxygen than quiescent ECs and work close to their respiratory limit. The increased utilization of the proton motif force leads to a reduced mitochondrial membrane potential in proliferating ECs and sensitizes to mitochondrial uncoupling. The benzoquinone embelin is a weak mitochondrial uncoupler that prevents neoangiogenesis during tumor growth and wound healing by exhausting the low respiratory reserve of proliferating ECs without adversely affecting quiescent ECs. We demonstrate that this can be exploited therapeutically by attenuating tumor growth in syngenic and xenograft mouse models. This novel metabolic targeting approach might be clinically valuable in controlling pathological neoangiogenesis while sparing normal vasculature and complementing cytostatic drugs in cancer treatment.

Senescent cells accumulate with age in multiple tissues and may cause age-associated disease and functional decline. In vitro, senescent cells induce senescence in bystander cells. To see how important this bystander effect may be for accumulation of senescent cells in vivo, we xenotransplanted senescent cells into skeletal muscle and skin of immunocompromised NSG mice. 3 weeks after the last transplantation, mouse dermal fibroblasts and myofibres displayed multiple senescence markers in the vicinity of transplanted senescent cells, but not where non-senescent or no cells were injected. Adjacent to injected senescent cells, the magnitude of the bystander effect was similar to the increase in senescence markers in myofibres between 8 and 32 months of age. The age-associated increase of senescence markers in muscle correlated with fibre thinning, a widely used marker of muscle aging and sarcopenia. Senescent cell transplantation resulted in borderline induction of centrally nucleated fibres and no significant thinning, suggesting that myofibre aging might be a delayed consequence of senescence-like signalling. To assess the relative importance of the bystander effect versus cell-autonomous senescence, we compared senescent hepatocyte frequencies in livers of wild-type and NSG mice under ad libitum and dietary restricted feeding. This enabled us to approximate cell-autonomous and bystander-driven senescent cell accumulation as well as the impact of immunosurveillance separately. The results suggest a significant impact of the bystander effect for accumulation of senescent hepatocytes in liver and indicate that senostatic interventions like dietary restriction may act as senolytics in immunocompetent animals.

This data article contains the results of molecular dynamics (MD) simulations performed to assess the stability of the previously computed complex between the hCES1 structure and the Amplex Red (AR) substrate (Miwa et al., 2015) [1] and to compare the dynamic behavior of this complex with that of the corresponding hCES1-deacetylAR product. The study involves both standard molecular dynamics (MD) and steered (SMD) simulations to offer a quantitative comparison of the stability for the two complexes. With regard the standard MD runs, the data article graphically reports the r.m.s.d. profile of the ligand׳s atoms as well as the dynamic behavior of key contacts involving the catalytic Ser221 residue. The SMD simulations provide a comparison of the pull forces required to undock the two ligands and reveal that Van der Waals and hydrophobic interactions play a key role in complex stabilization.

The incidence of non-alcoholic fatty liver disease (NAFLD) increases with age. Cellular senescence refers to a state of irreversible cell-cycle arrest combined with the secretion of proinflammatory cytokines and mitochondrial dysfunction. Senescent cells contribute to age-related tissue degeneration. Here we show that the accumulation of senescent cells promotes hepatic fat accumulation and steatosis. We report a close correlation between hepatic fat accumulation and markers of hepatocyte senescence. The elimination of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice or by treatment with a combination of the senolytic drugs dasatinib and quercetin (D+Q) reduces overall hepatic steatosis. Conversely, inducing hepatocyte senescence promotes fat accumulation in vitro and in vivo. Mechanistically, we show that mitochondria in senescent cells lose the ability to metabolize fatty acids efficiently. Our study demonstrates that cellular senescence drives hepatic steatosis and elimination of senescent cells may be a novel therapeutic strategy to reduce steatosis.

Dietary restriction (DR) extends the lifespan of a wide variety of species and reduces the incidence of major age-related diseases. Cell senescence has been proposed as one causal mechanism for tissue and organism ageing. We show for the first time that adult-onset, short-term DR reduced frequencies of senescent cells in the small intestinal epithelium and liver of mice, which are tissues known to accumulate increased numbers of senescent cells with advancing age. This reduction was associated with improved telomere maintenance without increased telomerase activity. We also found a decrease in cumulative oxidative stress markers in the same compartments despite absence of significant changes in steady-state oxidative stress markers at the whole tissue level. The data suggest the possibility that reduction of cell senescence may be a primary consequence of DR which in turn may explain known effects of DR such as improved mitochondrial function and reduced production of reactive oxygen species.

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level.

Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes.

Cellular senescence--the permanent arrest of cycling in normally proliferating cells such as fibroblasts--contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of 'deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFbeta. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro-inflammatory and pro-oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent-associated changes are dependent on mitochondria, particularly the pro-inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC-1β-dependent mitochondrial biogenesis, contributing to aROS-mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC-1β deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.

Mitochondrial function is an important determinant of the ageing process; however, the mitochondrial properties that enable longevity are not well understood. Here we show that optimal assembly of mitochondrial complex I predicts longevity in mice. Using an unbiased high-coverage high-confidence approach, we demonstrate that electron transport chain proteins, especially the matrix arm subunits of complex I, are decreased in young long-living mice, which is associated with improved complex I assembly, higher complex I-linked state 3 oxygen consumption rates and decreased superoxide production, whereas the opposite is seen in old mice. Disruption of complex I assembly reduces oxidative metabolism with concomitant increase in mitochondrial superoxide production. This is rescued by knockdown of the mitochondrial chaperone, prohibitin. Disrupted complex I assembly causes premature senescence in primary cells. We propose that lower abundance of free catalytic complex I components supports complex I assembly, efficacy of substrate utilization and minimal ROS production, enabling enhanced longevity.

Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs).

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Telomerase in its canonical function maintains telomeres in dividing cells. In addition, the telomerase protein TERT has non-telomeric functions such as shuttling to mitochondria resulting in a decreased oxidative stress, DNA damage and apoptosis. TERT protein persists in adult neurons and can co-localise to mitochondria under various stress conditions. We show here that TERT expression decreased in mouse brain during aging while release of reactive oxygen species (ROS) from the mitochondrial electron transport chain increased. Dietary restriction (DR) caused accumulation of TERT protein in mouse brain mitochondria correlating to decreased ROS release and improved learning and spatial short-term memory. Decreased mTOR signalling is a mediator of DR. Accordingly, feeding mice with rapamycin increased brain mitochondrial TERT and reduced ROS release. Importantly, the beneficial effects of rapamycin on mitochondrial function were absent in brains and fibroblasts from first generation TERT -/- mice, and when TERT shuttling was inhibited by the Src kinase inhibitor bosutinib. Taken together, our data suggests that the mTOR signalling pathway impinges on the mitochondrial localisation of TERT protein, which might in turn contribute to the protection of the brain by DR or rapamycin against age-associated mitochondrial ROS increase and cognitive decline.

Cellular senescence entails a stable cell-cycle arrest and a pro-inflammatory secretory phenotype, which contributes to aging and age-related diseases. Obesity is associated with increased senescent cell burden and neuropsychiatric disorders, including anxiety and depression. To investigate the role of senescence in obesity-related neuropsychiatric dysfunction, we used the INK-ATTAC mouse model, from which p16Ink4a-expressing senescent cells can be eliminated, and senolytic drugs dasatinib and quercetin. We found that obesity results in the accumulation of senescent glial cells in proximity to the lateral ventricle, a region in which adult neurogenesis occurs. Furthermore, senescent glial cells exhibit excessive fat deposits, a phenotype we termed "accumulation of lipids in senescence." Clearing senescent cells from high fat-fed or leptin receptor-deficient obese mice restored neurogenesis and alleviated anxiety-related behavior. Our study provides proof-of-concept evidence that senescent cells are major contributors to obesity-induced anxiety and that senolytics are a potential new therapeutic avenue for treating neuropsychiatric disorders.

There is an unmet need to develop and validate therapies that can treat or at least prevent premature therapy-induced frailty, multi-morbidity and mortality in long-term tumour survivors. In an approach to develop a first mouse model for therapy-induced long-term frailty, we irradiated male C57Bl/6 mice at 5-6 months of age sub-lethally with 3 × 3 Gy (whole body) and assessed subsequent frailty for up to 6 months using a Rockwood-type frailty index (FI). Frailty scorers were trained to obtain excellent inter- and intra-observer reproducibility. Irradiated mice developed progressive frailty approximately twice as fast as controls. This was premature frailty; it was phenotypically identical to that in non-irradiated mice at higher age. As expected, frailty was associated with decreased cognition and predicted mortality. In irradiated mice, frailty and neuromuscular performance, measured by Rotarod and Hanging Wire tests, were not associated with each other, probably because of long-term decreased body weights after irradiation. We conclude that progressive frailty following sub-lethal irradiation comprises a sensitive and easy to use test bed for interventions to stop premature ageing in long-term tumour survivors.

Neural activity is tightly coupled to energy consumption, particularly sugars such as glucose. However, we find that, unlike mature neurons and astrocytes, neural stem/progenitor cells (NSPCs) do not require glucose to sustain aerobic respiration. NSPCs within the adult subventricular zone (SVZ) express enzymes required for fatty acid oxidation and show sustained increases in oxygen consumption upon treatment with a polyunsaturated fatty acid. NSPCs also demonstrate sustained decreases in oxygen consumption upon treatment with etomoxir, an inhibitor of fatty acid oxidation. In addition, etomoxir decreases the proliferation of SVZ NSPCs without affecting cellular survival. Finally, higher levels of neurogenesis can be achieved in aged mice by ectopically expressing proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), a factor that increases cellular aerobic capacity by promoting mitochondrial biogenesis and metabolic gene transcription. Regulation of metabolic fuel availability could prove a powerful tool in promoting or limiting cellular proliferation in the central nervous system. Stem Cells 2015;33:2306-2319.

Inhibition of monocarboxylate transporter 1 has been proposed as a therapeutic approach to perturb lactate shuttling in tumor cells that lack monocarboxylate transporter 4. We examined the monocarboxylate transporter 1 inhibitor AZD3965, currently in phase I clinical studies, as a potential therapy for diffuse large B-cell lymphoma and Burkitt lymphoma. Whilst extensive monocarboxylate transporter 1 protein was found in 120 diffuse large B-cell lymphoma and 10 Burkitt lymphoma patients' tumors, monocarboxylate transporter 4 protein expression was undetectable in 73% of the diffuse large B-cell lymphoma samples and undetectable or negligible in each Burkitt lymphoma sample. AZD3965 treatment led to a rapid accumulation of intracellular lactate in a panel of lymphoma cell lines with low monocarboxylate transporter 4 protein expression and potently inhibited their proliferation. Metabolic changes induced by AZD3965 in lymphoma cells were consistent with a feedback inhibition of glycolysis. A profound cytostatic response was also observed in vivo: daily oral AZD3965 treatment for 24 days inhibited CA46 Burkitt lymphoma growth by 99%. Continuous exposure of CA46 cells to AZD3965 for 7 weeks in vitro resulted in a greater dependency upon oxidative phosphorylation. Combining AZD3965 with an inhibitor of mitochondrial complex I (central to oxidative phosphorylation) induced significant lymphoma cell death in vitro and reduced CA46 disease burden in vivo These data support clinical examination of AZD3965 in Burkitt lymphoma and diffuse large B-cell lymphoma patients with low tumor monocarboxylate transporter 4 expression and highlight the potential of combination strategies to optimally target the metabolic phenotype of tumors.