Mitochondrial dysfunction and cell senescence are hallmarks of aging and are closely interconnected. Mitochondrial dysfunction, operationally defined as a decreased respiratory capacity per ...mitochondrion together with a decreased mitochondrial membrane potential, typically accompanied by increased production of oxygen free radicals, is a cause and a consequence of cellular senescence and figures prominently in multiple feedback loops that induce and maintain the senescent phenotype. Here, we summarize pathways that cause mitochondrial dysfunction in senescence and aging and discuss the major consequences of mitochondrial dysfunction and how these consequences contribute to senescence and aging. We also highlight the potential of senescence-associated mitochondrial dysfunction as an antiaging and antisenescence intervention target, proposing the combination of multiple interventions converging onto mitochondrial dysfunction as novel, potent senolytics.
Cell senescence is a driver of ageing, frailty, age-associated disease and functional decline. In oncology, tumour cell senescence may contribute to the effect of adjuvant therapies, as it blocks ...tumour growth. However, this is frequently incomplete, and tumour cells that recover from senescence may gain a more stem-like state with increased proliferative potential. This might be exaggerated by the induction of senescence in the surrounding niche cells. Finally, senescence will spread through bystander effects, possibly overwhelming the capacity of the immune system to ablate senescent cells. This induces a persistent system-wide senescent cell accumulation, which we hypothesize is the cause for the premature frailty, multi-morbidity and increased mortality in cancer survivors.
Senolytics, drugs that selectively kill senescent cells, have been developed recently and have been proposed as second-line adjuvant tumour therapy. Similarly, by blocking accelerated senescence following therapy, senolytics might prevent and potentially even revert premature frailty in cancer survivors.
Adjuvant senostatic interventions, which suppress senescence-associated bystander signalling, might also have therapeutic potential. This becomes pertinent because treatments that are senostatic in vitro (e.g. dietary restriction mimetics) persistently reduce numbers of senescent cells in vivo, i.e. act as net senolytics in immunocompetent hosts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cell senescence is increasingly recognized as a major contributor to the loss of health and fitness associated with aging. Senescent cells accumulate dysfunctional mitochondria; oxidative ...phosphorylation efficiency is decreased and reactive oxygen species production is increased. In this review we will discuss how the turnover of mitochondria (a term referred to as mitophagy) is perturbed in senescence contributing to mitochondrial accumulation and Senescence-Associated Mitochondrial Dysfunction (SAMD). We will further explore the subsequent cellular consequences; in particular SAMD appears to be necessary for at least part of the specific Senescence-Associated Secretory Phenotype (SASP) and may be responsible for tissue-level metabolic dysfunction that is associated with aging and obesity. Understanding the complex interplay between these major senescence-associated phenotypes will help to select and improve interventions that prolong healthy life in humans.
Data for this review were identified by searches of MEDLINE, PubMed, and references from relevant articles using the search terms “mitochondria AND senescence”, “(autophagy OR mitophagy) AND senescence”, “mitophagy AND aging” and related terms. Additionally, searches were performed based on investigator names. Abstracts and reports from meetings were excluded. Articles published in English between 1995 and 2017 were included. Articles were selected according to their relevance to the topic as perceived by the authors.
•Perturbed mitophagy contributes to mitochondrial dysfunction in cell senescence.•Mitochondrial dysfunction drives multiple characteristic phenotypes of senescent cells.•Senescence-associated mitochondrial dysfunction may be an important cause for metabolic compromise in aging.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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 p16
-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.
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.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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.
Cancer survivors suffer from progressive frailty, multimorbidity, and premature morbidity. We hypothesise that therapy-induced senescence and senescence progression via bystander effects are ...significant causes of this premature ageing phenotype. Accordingly, the study addresses the question whether a short anti-senescence intervention is able to block progression of radiation-induced frailty and disability in a pre-clinical setting. Male mice were sublethally irradiated at 5 months of age and treated (or not) with either a senolytic drug (Navitoclax or dasatinib + quercetin) for 10 days or with the senostatic metformin for 10 weeks. Follow-up was for 1 year. Treatments commencing within a month after irradiation effectively reduced frailty progression (p<0.05) and improved muscle (p<0.01) and liver (p<0.05) function as well as short-term memory (p<0.05) until advanced age with no need for repeated interventions. Senolytic interventions that started late, after radiation-induced premature frailty was manifest, still had beneficial effects on frailty (p<0.05) and short-term memory (p<0.05). Metformin was similarly effective as senolytics. At therapeutically achievable concentrations, metformin acted as a senostatic neither via inhibition of mitochondrial complex I, nor via improvement of mitophagy or mitochondrial function, but by reducing non-mitochondrial reactive oxygen species production via NADPH oxidase 4 inhibition in senescent cells. Our study suggests that the progression of adverse long-term health and quality-of-life effects of radiation exposure, as experienced by cancer survivors, might be rescued by short-term adjuvant anti-senescence interventions.
Summary
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.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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‐TGFβ. 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.
Synopsis
The phenomenon of cellular ‘senescence’—the permanent arrest of division in normally proliferating mammalian cells such as fibroblasts—is thought to be a central component of the ageing process. Senescence contributes both to age‐related loss of tissue homeostasis, as the loss of division capacity leads to impaired cell renewal, and also to protect against cancer, because it acts to block the uncontrolled proliferation of cells that may give rise to a malignant tumour. Replicative senescence is triggered by uncapped telomeres or by ‘unrepairable’ non‐telomeric DNA damage. Both lesions initiate the same canonical DNA damage response (DDR) (d'Adda di Fagagna, 2008). This response is characterized by activation of sensor kinases (ATM/ATR, DNA‐PK), formation of DNA damage foci containing activated H2A.X (γH2A.X) and ultimately induction of cell cycle arrest through activation of checkpoint proteins, notably p53 (TP53) and the CDK inhibitor p21 (CDKN1A). This signalling pathway continues to contribute actively to the stability of the G0 arrest in fully senescent cells long after induction of senescence (d'Adda di Fagagna et al, 2003). However, senescence is more complex than mere CDKI‐mediated growth arrest. Senescent cells alter their expression of literally hundreds of genes (Shelton et al, 1999), prominent among these being pro‐inflammatory secretory genes (Coppe et al, 2008) and marker genes for a retrograde response induced by mitochondrial dysfunction (Passos et al, 2007a).
There is a growing evidence that multiple mechanisms interact to underpin ageing at the cellular level (Kirkwood, 2005; Passos et al, 2007b) necessitating a systems biology approach if the complex mechanisms of ageing are to be understood (Kirkwood, 2008). With respect to cell senescence, the two major unanswered questions are (i) How does a DNA lesion that can be repaired, at least in principle, induce and maintain irreversible growth arrest? and (ii) How does a growth arrest trigger a completely different cellular phenotype as soon as it becomes irreversible?
To understand those questions, we performed a kinetic analysis of the establishment phase of senescence initiated by DNA damage or telomere dysfunction, focussing on pathways downstream of the classical DDR. Using an approach that combined (i) in‐silico interactome analysis, (ii) functional target gene inhibition, (iii) stochastic modelling, and (iv) live cell microscopy, we identified a positive feedback loop between DDR and mitochondrial production of reactive oxygen species (ROS) as necessary and sufficient for long‐term maintenance of growth arrest. Using pathway log likelihood scores calculated by a quantitative in‐silico interactome analysis to guide siRNA and small molecule inhibition experiments, and using results of sequential and combined inhibition experiments to refine the predictions from the interactome analysis, we found that DDR triggered mitochondrial dysfunction leading to enhanced ROS activation through a linear signal transduction through TP53, CDKN1A, GADD45A, p38 (MAPK14), GRB2, TGFBR2 and TGFβ(Figure 2D). We hypothesized that these ROS stochastically generate novel DNA damage in the nucleus, thus forming a positive feedback loop contributing to the long‐term maintenance of DDR (Figure 3A). First confirmation came from static inhibitor experiments as before, showing that nuclear DNA damage foci frequencies in senescent cells were reduced if feedback signalling was suppressed. To formally establish the existence of a feedback loop and its relevance for senescence, we used live cell microscopy in combination with quantitative modelling.
We transformed the conceptual model shown in Figure 3A into a stochastic mechanistic model of the DDR feedback loop by extending the previously published model of the TP53/Mdm2 circuit (Proctor and Gray, 2008) to include reactions for synthesis/activation and degradation/deactivation/repair of CDKN1A, GADD45, MAPK14, ROS and DNA damage. The model replicated very precisely the kinetic behaviour of activated TP53, CDKN1A, ROS and DNA damage foci after initiation of senescence by irradiation. Having established its concordance with the experimental data, the model was then used to predict the effects of intervening in the feedback loop. The model predicted that any intervention reducing ROS levels by about half would decrease average DNA damage foci frequencies from six to four foci/nucleus within about 15 h. It further predicted that this would be sufficient to reduce CDKN1A to basal levels continuously for at least 6 h in about 20% of the treated cells, thus allowing a significant fraction of cells to escape from growth arrest and to resume proliferation. This should happen even if the intervention into the feedback loop was started at a late time point (e.g. 6 days) after induction of senescence.
To analyse DNA damage foci dynamics we used a reporter construct (AcGFP–53BP1c) that quantitatively reports single DNA damage foci kinetics in time‐resolved live cell microscopy (Nelson et al, 2009). Foci frequency measurements quantitatively confirmed the prediction from the stochastic model. More importantly, we found that many individual foci in both telomere‐ and stress‐dependent senescence had short lifespans with half‐lives below 15 h. Feedback loop inhibition reduced only the frequencies of short‐lived DNA damage foci in accordance with the hypothesis that ROS production contributed to DDR by constant replenishment of short‐lived DNA damage foci.
Finally, we inhibited signalling through the loop at different time points after induction of senescence by ionizing radiation and measured ROS levels, DNA damage foci frequencies and proliferation markers. Treatments with the MAPK14 inhibitor SB203580 or the free radical scavenger PBN were used to block the loop. The results quantitatively confirmed the model prediction and indicated that the feedback loop between DDR and ROS production was both necessary and sufficient to maintain cell cycle arrest for at least 6–10 days after induction of senescence. Interestingly, the loop was still active at later time points and in deep senescence, but proliferation arrest was then stabilized by additional factor(s). This indicated that certain features of the senescent phenotype‐like ROS production that might be responsible for the negative impact of senescent cells into their tissue environment can be successfully inhibited even in deep senescence. This may prove relevant for novel therapeutic studies aiming to modulate intracellular ROS levels in both aging and cancer.
The sustained activation of CDKN1A (p21/Waf1/Cip1) by a DNA damage response induces mitochondrial dysfunction and reactive oxygen species (ROS) production via signalling through CDKN1A‐GADD45A‐MAPK14‐ GRB2‐TGFBR2‐TGFbeta in senescing primary human and mouse cells in vitro and in vivo.
Enhanced ROS production in senescing cells generates additional DNA damage. Although this damage is repairable and transient, it elevates the average levels of DNA damage response permanently, thus forming a positive feedback loop.
This loop is necessary and sufficient to maintain the stability of growth arrest until a ‘point of no return’ is reached during establishment of senescence.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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