Mitochondrial dysfunction leads to telomere attrition and genomic instability

Mitochondrial dysfunction and oxidative stress have been implicated in cellular senescence, apoptosis, aging and aging-associated pathologies. Telomere shortening and genomic instability have also been associated with replicative senescence, aging and cancer. Here we show that mitochondrial dysfunction leads to telomere attrition, telomere loss, and chromosome fusion and breakage, accompanied by apoptosis. An antioxidant prevented telomere loss and genomic instability in cells with dysfunctional mitochondria, suggesting that reactive oxygen species are mediators linking mitochondrial dysfunction and genomic instability. Further, nuclear transfer protected genomes from telomere dysfunction and promoted cell survival by reconstitution with functional mitochondria. This work links mitochondrial dysfunction and genomic instability and may provide new therapeutic strategies to combat certain mitochondrial and aging-associated pathologies.     

Monoamine oxidase‐A is a novel driver of stress‐induced premature senescence through inhibition of parkin‐mediated mitophagy

Cellular senescence, the irreversible cell cycle arrest observed in somatic cells, is an important driver of age‐associated diseases. Mitochondria have been implicated in the process of senescence, primarily because they are both sources and targets of reactive oxygen species (ROS). In the heart, oxidative stress contributes to pathological cardiac ageing, but the mechanisms underlying ROS production are still not completely understood. The mitochondrial enzyme monoamine oxidase‐A (MAO‐A) is a relevant source of ROS in the heart through the formation of H₂O₂ derived from the degradation of its main substrates, norepinephrine (NE) and serotonin. However, the potential link between MAO‐A and senescence has not been previously investigated. Using cardiomyoblasts and primary cardiomyocytes, we demonstrate that chronic MAO‐A activation mediated by synthetic (tyramine) and physiological (NE) substrates induces ROS‐dependent DNA damage response, activation of cyclin‐dependent kinase inhibitors p21^cip, p16^ink4a, and p15^ink4b and typical features of senescence such as cell flattening and SA‐β‐gal activity. Moreover, we observe that ROS produced by MAO‐A lead to the accumulation of p53 in the cytosol where it inhibits parkin, an important regulator of mitophagy, resulting in mitochondrial dysfunction. Additionally, we show that the mTOR kinase contributes to mitophagy dysfunction by enhancing p53 cytoplasmic accumulation. Importantly, restoration of mitophagy, either by overexpression of parkin or inhibition of mTOR, prevents mitochondrial dysfunction and induction of senescence. Altogether, our data demonstrate a novel link between MAO‐A and senescence in cardiomyocytes and provides mechanistic insights into the potential role of MAO‐dependent oxidative stress in age‐related pathologies.     

P66SHC deletion improves fertility and progeric phenotype of late‐generation TERC‐deficient mice but not their short lifespan

Oxidative stress and telomere attrition are considered the driving factors of aging. As oxidative damage to telomeric DNA favors the erosion of chromosome ends and, in turn, telomere shortening increases the sensitivity to pro‐oxidants, these two factors may trigger a detrimental vicious cycle. To check whether limiting oxidative stress slows down telomere shortening and related progeria, we have investigated the effect of p66SHC deletion, which has been shown to reduce oxidative stress and mitochondrial apoptosis, on late‐generation TERC (telomerase RNA component)‐deficient mice having short telomeres and reduced lifespan. Double mutant (TERC^?/? p66SHC^?/?) mice were generated, and their telomere length, fertility, and lifespan investigated in different generations. Results revealed that p66SHC deletion partially rescues sterility and weight loss, as well as organ atrophy, of TERC‐deficient mice, but not their short lifespan and telomere erosion. Therefore, our data suggest that p66SHC‐mediated oxidative stress and telomere shortening synergize in some tissues (including testes) to accelerate aging; however, early mortality of late‐generation mice seems to be independent of any link between p66SHC‐mediated oxidative stress and telomere attrition.     

Significant role for p16INK4a in p53-independent telomere-directed senescence

Telomere attrition in primary human fibroblasts induces replicative senescence accompanied by activation of the p53 and p16(INK4a)/RB tumor suppressor pathways. Although the contribution of p53 and its target, p21, to telomere-driven senescence have been well established, the role of p16(INK4a) is controversial. Attempts to dissect the significance of p16(INK4a) in response to telomere shortening have been hampered by the concomitant induction of p16(INK4a) by cell culture conditions. To circumvent this problem, we studied the role of p16(INK4a) in the cellular response to acute telomere damage induced by a dominant negative allele of TRF2, TRF2(Delta B Delta M). This approach avoids the confounding aspects of culture stress because parallel cultures with and without telomere damage can be compared. Telomere damage generated with TRF2(Delta B Delta M) resulted in induction of p16(INK4a) in the majority of cells as detected by immunohistochemistry. Inhibition of p16(INK4a) with shRNA or overexpression of BMI1 had a significant effect on the telomere damage response in p53-deficient cells. While p53 deficiency alone only partially abrogated the telomere damage-induced cell cycle arrest, combined inhibition of p16(INK4a) and p53 led to nearly complete bypass of telomere-directed senescence. We conclude that p16(INK4a) contributes to the p53-independent response to telomere damage.     

Significant Role for p16 in p53-Independent Telomere-Directed Senescence

Telomere attrition in primary human fibroblasts induces replicative senescence accompanied by activation of the p53 and p16(INK4a)/RB tumor suppressor pathways. Although the contribution of p53 and its target, p21, to telomere-driven senescence have been well established, the role of p16(INK4a) is controversial. Attempts to dissect the significance of p16(INK4a) in response to telomere shortening have been hampered by the concomitant induction of p16(INK4a) by cell culture conditions. To circumvent this problem, we studied the role of p16(INK4a) in the cellular response to acute telomere damage induced by a dominant negative allele of TRF2, TRF2(Delta B Delta M). This approach avoids the confounding aspects of culture stress because parallel cultures with and without telomere damage can be compared. Telomere damage generated with TRF2(Delta B Delta M) resulted in induction of p16(INK4a) in the majority of cells as detected by immunohistochemistry. Inhibition of p16(INK4a) with shRNA or overexpression of BMI1 had a significant effect on the telomere damage response in p53-deficient cells. While p53 deficiency alone only partially abrogated the telomere damage-induced cell cycle arrest, combined inhibition of p16(INK4a) and p53 led to nearly complete bypass of telomere-directed senescence. We conclude that p16(INK4a) contributes to the p53-independent response to telomere damage.     

MicroRNA-340-5p increases telomere length by targeting telomere protein POT1 to improve Alzheimer's disease in mice

Alzheimer′s disease (AD) is a chronic neurodegenerative disorder which is the primary cause of dementia in the elderly. Telomere attrition has been proposed as a hallmark of aging. Our study aimed to explore the mechanism of the protection of telomere 1 (POT1) in regulating telomere length and affecting cellular senescence in AD. The AD mouse model was established by d -galactose and aluminum chloride, and the water maze test and dark avoidance test were used to detect the behaviors of mice and confirm the success of AD mouse model. AD cell model was established with HT22 cells induced by Aβ₄₂ oligomers. POT1 expression in the AD model was detected by quantitative real-time polymerase chain reaction. Cellular telomere length in hippocampal tissue was analyzed by telomere restriction fragment. Localization of intracellular POT1, telomerase, and telomeres was analyzed by immunofluorescence and fluorescence in situ hybridization. Dual-luciferase assay was used to validate the targeted binding relationship between microRNA-340-5p (miR-340-5p) and POT1. After inhibiting POT1 expression, the symptoms of AD in mice were improved. Aβ_1–42 deposition was reduced, whereas telomere length and telomerase activity was increased. Dual-luciferase assay verified the binding relationship between miR-340-5p and POT1. An increase in miR-340-5p expression could alleviate cellular senescence and AD symptoms. miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms. This study made a conclusion that miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms in mice.     

CHK2‐independent induction of telomere dysfunction checkpoints in stem and progenitor cells

Telomere shortening limits the proliferation of primary human fibroblasts by the induction of senescence, which is mediated by ataxia telangiectasia mutated‐dependent activation of p53. Here, we show that CHK2 deletion impairs the induction of senescence in mouse and human fibroblasts. By contrast, CHK2 deletion did not improve the stem‐cell function, organ maintenance and lifespan of telomere dysfunctional mice and did not prevent the induction of p53/p21, apoptosis and cell‐cycle arrest in telomere dysfunctional progenitor cells. Together, these results indicate that CHK2 mediates the induction of senescence in fibroblasts, but is dispensable for the induction of telomere dysfunction checkpoints at the stem and progenitor cell level in vivo.     

Telomere attrition predicts reduced survival in a wild social bird,but short telomeres do not

Attempts to understand the causes of variation in senescence trajectories would benefit greatly from biomarkers that reflect the progressive declines in somatic integrity (SI) that lead to senescence. While telomere length has attracted considerable interest in this regard, sources of variation in telomere length potentially unrelated to declines in SI could, in some contexts, leave telomere attrition rates a more effective biomarker than telomere length alone. Here, we investigate whether telomere length and telomere attrition rates predict the survival of wild white‐browed sparrow‐weaver nestlings (Plocepasser mahali). Our analyses of telomere length reveal counterintuitive patterns: telomere length soon after hatching negatively predicted nestling survival to fledging, a pattern that appears to be driven by differentially high in‐nest predation of broods with longer telomeres. Telomere length did not predict survival outside this period: neither hatchling telomere length nor telomere length in the mid‐nestling period predicted survival from fledging to adulthood. Our analyses using within‐individual telomere attrition rates, by contrast, revealed the expected relationships: nestlings that experienced a higher rate of telomere attrition were less likely to survive to adulthood, regardless of their initial telomere length and independent of effects of body mass. Our findings support the growing use of telomeric traits as biomarkers of SI, but lend strength to the view that longitudinal assessments of within‐individual telomere attrition since early life may be a more effective biomarker in some contexts than telomere length alone.     

Mitochondrial function in skeletal myofibers is controlled by a TRF2‐SIRT3 axis over lifetime

Telomere shortening follows a developmentally regulated process that leads to replicative senescence of dividing cells. However, whether telomere changes are involved in postmitotic cell function and aging remains elusive. In this study, we discovered that the level of the TRF2 protein, a key telomere‐capping protein, declines in human skeletal muscle over lifetime. In cultured human myotubes, TRF2 downregulation did not trigger telomere dysfunction, but suppressed expression of the mitochondrial Sirtuin 3 gene (SIRT3) leading to mitochondrial respiration dysfunction and increased levels of reactive oxygen species. Importantly, restoring the Sirt3 level in TRF2‐compromised myotubes fully rescued mitochondrial functions. Finally, targeted ablation of the Terf2 gene in mouse skeletal muscle leads to mitochondrial dysfunction and sirt3 downregulation similarly to those of TRF2‐compromised human myotubes. Altogether, these results reveal a TRF2‐SIRT3 axis controlling muscle mitochondrial function. We propose that this axis connects developmentally regulated telomere changes to muscle redox metabolism.     

Mitochondrial dysfunction accounts for the stochastic heterogeneity in telomere-dependent senescence

Aging is an inherently stochastic process, and its hallmark is heterogeneity between organisms, cell types, and clonal populations, even in identical environments. The replicative lifespan of primary human cells is telomere dependent; however, its heterogeneity is not understood. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive UCP-2–dependent mitochondrial uncoupling. This mitochondrial dysfunction is accompanied by compromised [Ca²⁺]_i homeostasis and other indicators of a retrograde response in senescent cells. Replicative senescence of human fibroblasts is delayed by mild mitochondrial uncoupling. Uncoupling reduces mitochondrial superoxide generation, slows down telomere shortening, and delays formation of telomeric γ-H2A.X foci. This indicates mitochondrial production of reactive oxygen species (ROS) as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures, we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres, and telomeric γ-H2A.X foci. We propose that mitochondrial ROS is a major determinant of telomere-dependent senescence at the single-cell level that is responsible for cell-to-cell variation in replicative lifespan.     

The role of telomere trimming in normal telomere length dynamics

Telomeres consist of repetitive DNA and associated proteins that protect chromosome ends from illicit DNA repair. It is well known that telomeric DNA is progressively eroded during cell division, until telomeres become too short and the cell stops dividing. There is a second mode of telomere shortening, however, which is a regulated form of telomere rapid deletion (TRD) termed telomere trimming that is reviewed here. Telomere trimming appears to involve resolution of recombination intermediate structures, which shortens the telomere by release of extrachromosomal telomeric DNA. This has been detected in human and in mouse cells and occurs both in somatic and germline cells, where it sets an upper limit on telomere length and contributes to a length equilibrium set-point in cells that have a telomere elongation mechanism. Telomere trimming thus represents an additional mechanism of telomere length control that contributes to normal telomere dynamics and cell proliferative potential.     

The distribution pattern of critically short telomeres in human osteoarthritic knees

Introduction  

Telomere shortening is associated with a number of common age-related diseases. A role of telomere shortening in osteoarthritis (OA) has been suggested, mainly based on the assessment of mean telomere length in ex vivo expanded chondrocytes. We addressed this role directly in vivo by using a newly developed assay, which measures specifically the load of ultra-short single telomeres (below 1,500 base pairs), that is, the telomere subpopulation believed to promote cellular senescence.     

Telomeric injury by KML001 in human T cells induces mitochondrial dysfunction through the p53-PGC-1α pathway

Telomere erosion and mitochondrial dysfunction are prominent features of aging cells with progressive declines of cellular functions. Whether telomere injury induces mitochondrial dysfunction in human T lymphocytes, the major component of adaptive host immunity against infection and malignancy, remains unclear. We have recently shown that disruption of telomere integrity by KML001, a telomere-targeting drug, induces T cell senescence and apoptosis via the telomeric DNA damage response (DDR). In this study, we used KML001 to further investigate the role and mechanism of telomere injury in mitochondrial dysregulation in aging T cells. We demonstrate that targeting telomeres by KML001 induces mitochondrial dysfunction, as evidenced by increased mitochondrial swelling and decreased mitochondrial membrane potential, oxidative phosphorylation, mitochondrial DNA content, mitochondrial respiration, oxygen consumption, glycolysis, and ATP energy production. Mechanistically, we found that the KML001-induced telomeric DDR activated p53 signaling, which in turn repressed the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and nuclear respiratory factor 1 (NRF-1), leading to T cell mitochondrial dysfunction. These results, forging a direct link between telomeric and mitochondrial biology, shed new light on the human T cell aging network, and demonstrate that the p53-PGC-1α-NRF-1 axis contributes to mitochondrial dysfunction in the setting of telomeric DDR. This study suggests that targeting this axis may offer an alternative, novel approach to prevent telomere damage-mediated mitochondrial and T cell dysfunctions to combat a wide range of immune aging-associated human diseases.Subject terms: Immunology, Diseases     

Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells

Telomere shortening limits the regenerative capacity of primary cells in vitro by inducing cellular senescence characterized by a permanent growth arrest of cells with critically short telomeres. To test whether this in vitro model of cellular senescence applies to impaired organ regeneration induced by telomere shortening in vivo, we monitored liver regeneration after partial hepatectomy in telomerase-deficient mice. Our study shows that telomere shortening is heterogeneous at the cellular level and inhibits a subpopulation of cells with critically short telomeres from entering the cell cycle. This subpopulation of cells with impaired proliferative capacity shows senescence-associated beta-galactosidase activity, while organ regeneration is accomplished by cells with sufficient telomere reserves that are capable of additional rounds of cell division. This study provides experimental evidence for the existence of an in vivo process of cellular senescence induced by critical telomere shortening that has functional impact on organ regeneration.     

Accelerated fat cell aging links oxidative stress and insulin resistance in adipocytes

Telomere shortening is emerging as a biological indicator of accelerated aging and aging-related diseases including type 2 diabetes. While telomere length measurements were largely done in white blood cells, there is lack of studies on telomere length in relation to oxidative stress in target tissues affected in diabetes. Therefore, the aim of this study is to induct oxidative stress in adipocytes and to test whether these adipocytes exhibit shortened telomeres, senescence and functional impairment. 3T3-L1 adipocytes were subjected to oxidative stress and senescence induction by a variety of means for 2 weeks (exogenous application of H₂O₂, glucose oxidase, asymmetric dimethylarginine (ADMA) and glucose oscillations). Cells were probed for reactive oxygen species generation (ROS), DNA damage, mRNA and protein expression of senescent and pro-inflammatory markers, telomere length and glucose uptake. Compared to untreated cells, both ROS generation and DNA damage were significantly higher in cells subjected to oxidative stress and senescence. Adipocytes subjected to oxidative stress also showed shortened telomeres and increased mRNA and protein expression of p53, p21, TNFα and IL-6. Senescent cells were also characterized by decreased levels of adiponectin and impaired glucose uptake. Briefly, adipocytes under oxidative stress exhibited increased ROS generation, DNA damage, shortened telomeres and switched to senescent/pro-inflammatory phenotype with impaired glucose uptake.     

Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-3'-overhang

Telomeres shorten in human somatic cells with each round of DNA replication, and this shortening is thought to ultimately trigger replicative senescence. Telomere shortening is caused partly by the inability of semiconservative DNA replication to copy a linear strand of DNA to its very end. Post-replicative processing of telomeric ends, producing single-stranded G-rich 3' overhangs, has also been suggested to contribute to telomere shortening. This suggestion implies that a positive correlation should exist between the length of 3' overhangs and the rate of telomere shortening. We confirmed shortening of overhangs as human lung (MRC5) and foreskin (BJ) fibroblasts approach senescence by measuring overhang length using in-gel hybridization. However, a large study of fibroblast strains from 21 donors maintained under conditions which lead to two orders of magnitude of variation in telomere shortening rate failed to show any correlation between telomere overhang length and shortening rate, suggesting that overhang length is neither a cause nor a correlate of telomere shortening.     

p16INK4a as a Second Effector of the Telomere Damage Pathway

Telomere damage resulting from telomere shortening can potentially suppress tumorigenesis by permanently arresting or eliminating incipient cancer cells. Dysfunctional telomeres activate the canonical DNA damage signaling pathway, resulting in a p53-mediated G1/S arrest and senescence or apoptosis. Experimental induction of telomere damage through inhibition of the telomeric protein TRF2 recapitulates aspects of telomere attrition, including a p53-mediated cell cycle arrest. Using this system, we have shown that telomere damage can also elicit a G1/S arrest through the RB-regulator p16INK4a, especially in cells lacking p53 function. Here we discuss the significance of p16INK4a as a second effector of the telomere damage response.     

DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. It is triggered as a stereotypic response to unrepaired nuclear DNA damage or to uncapped telomeres. In addition to a direct role of nuclear DNA double-strand breaks as inducer of a DNA damage response, two more subtle types of DNA damage induced by physiological levels of reactive oxygen species (ROS) can have a significant impact on cellular senescence: Firstly, it has been established that telomere shortening, which is the major contributor to telomere uncapping, is stress dependent and largely caused by a telomere-specific DNA single-strand break repair inefficiency. Secondly, mitochondrial DNA (mtDNA) damage is closely interrelated with mitochondrial ROS production, and this might also play a causal role for cellular senescence. Improvement of mitochondrial function results in less telomeric damage and slower telomere shortening, while telomere-dependent growth arrest is associated with increased mitochondrial dysfunction. Moreover, telomerase, the enzyme complex that is known to re-elongate shortened telomeres, also appears to have functions independent of telomeres that protect against oxidative stress. Together, these data suggest a self-amplifying cycle between mitochondrial and telomeric DNA damage during cellular senescence.     

Telomere Length Dynamics in Telomerase-Positive Immortal Human Cell Populations

It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD)in vitro.Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.     

Extended interferon-alpha therapy accelerates telomere length loss in human peripheral blood T lymphocytes

Background

Type I interferons have pleiotropic effects on host cells, including inhibiting telomerase in lymphocytes and antiviral activity. We tested the hypothesis that long-term interferon treatment would result in significant reduction in average telomere length in peripheral blood T lymphocytes.

Methods/Principal Findings

Using a flow cytometry-based telomere length assay on peripheral blood mononuclear cell samples from the Hepatitis-C Antiviral Long-term Treatment against Cirrhosis (HALT-C) study, we measured T cell telomere lengths at screening and at months 21 and 45 in 29 Hepatitis-C virus infected subjects. These subjects had failed to achieve a sustained virologic response following 24 weeks of pegylated-interferon-alpha plus ribavirin treatment and were subsequently randomized to either a no additional therapy group or a maintenance dose pegylated-IFNα group for an additional 3.5 years. Significant telomere loss in naïve T cells occurred in the first 21 months in the interferon-alpha group. Telomere losses were similar in both groups during the final two years. Expansion of CD8⁺CD45RA⁺CD57⁺ memory T cells and an inverse correlation of alanine aminotransferase levels with naïve CD8⁺ T cell telomere loss were observed in the control group but not in the interferon-alpha group. Telomere length at screening inversely correlated with Hepatitis-C viral load and body mass index.

Conclusions/Significance

Sustained interferon-alpha treatment increased telomere loss in naïve T cells, and inhibited the accumulation of T cell memory expansions. The durability of this effect and consequences for immune senescence need to be defined.