Pulmonary Alveolar Stem Cell Senescence, Apoptosis, and Differentiation by p53-Dependent and -Independent Mechanisms in Telomerase-Deficient Mice.

Pulmonary premature ageing and fibrogenesis as in idiopathic pulmonary fibrosis (IPF) occur with the DNA damage response in lungs deficient of telomerase. The molecular mechanism mediating pulmonary alveolar cell fates remains to be investigated. The present study shows that naturally occurring ageing is associated with the DNA damage response (DDR) and activation of the p53 signalling pathway. Telomerase deficiency induced by telomerase RNA component (TERC) knockout (KO) accelerates not only replicative senescence but also altered differentiation and apoptosis of the pulmonary alveolar stem cells (AEC2) in association with increased innate immune natural killer (NK) cells in TERC KO mice. TERC KO results in increased senescence-associated heterochromatin foci (SAHF) marker HP1γ, p21, p16, and apoptosis-associated cleaved caspase-3 in AEC2. However, additional deficiency of the tumour suppressor p53 in the Trp53-/- allele of the late generation of TERC KO mice attenuates the increased senescent and apoptotic markers significantly. Moreover, p53 deficiency has no significant effect on the increased gene expression of T1α (a marker of terminal differentiated AEC1) in AEC2 of the late generation of TERC KO mice. These findings demonstrate that, in natural ageing or premature ageing accelerated by telomere shortening, pulmonary senescence and IPF develop with alveolar stem cell p53-dependent premature replicative senescence, apoptosis, and p53-independent differentiation, resulting in pulmonary senescence-associated low-grade inflammation (SALI). Our studies indicate a natural ageing-associated molecular mechanism of telomerase deficiency-induced telomere DDR and SALI in pulmonary ageing and IPF.

Autophagy, TERT, and mitochondrial dysfunction in hyperoxia.

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1ß, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.

Functional characterization of miR-708 microRNA in telomerase positive and negative human cancer cells.

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.

Alternative splicing is a developmental switch for hTERT expression.

Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.

Saccharomyces cerevisiae Mus81-Mms4 prevents accelerated senescence in telomerase-deficient cells.

Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.

Nephrotoxic Effects in Zebrafish after Prolonged Exposure to Aristolochic Acid.

With the aim to explore the possibility to generate a zebrafish model of renal fibrosis, in this study the fibrogenic renal effect of aristolochic acid I (AAI) after immersion was assessed. This compound is highly nephrotoxic able to elicit renal fibrosis after exposure of rats and humans. Our results reveal that larval zebrafish at 15 days dpf (days post-fertilization) exposed for 8 days to 0.5 µM AAI showed clear signs of AKI (acute kidney injury). The damage resulted in the relative loss of the functional glomerular filtration barrier. Conversely, we did not observe any deposition of collagen, nor could we immunodetect α-SMA, a hallmark of myofibroblasts, in the tubules. In addition, no increase in gene expression of fibrogenesis biomarkers after whole animal RNA extraction was found. As zebrafish have a high capability for tissue regeneration possibly impeding fibrogenic processes, we also used a tert-/- zebrafish line exhibiting telomerase deficiency and impaired tissue homeostasis. AAI-treated tert-/- larvae displayed an increased sensitivity towards 0.5 µM AAI. Importantly, after AAI treatment a mild collagen deposition could be found in the tubules. The outcome implies that sustained AKI induced by nephrotoxic compounds combined with defective tert-/- stem cells can produce a fibrotic response.

Telomerase gene therapy ameliorates the effects of neurodegeneration associated to short telomeres in mice.

Neurodegenerative diseases associated with old age such as Alzheimer's disease present major problems for society, and they currently have no cure. The telomere protective caps at the ends of chromosomes shorten with age, and when they become critically short, they can induce a persistent DNA damage response at chromosome ends, triggering secondary cellular responses such as cell death and cellular senescence. Mice and humans with very short telomeres owing to telomerase deficiencies have an earlier onset of pathologies associated with loss of the regenerative capacity of tissues. However, the effects of short telomeres in very low proliferative tissues such as the brain have not been thoroughly investigated. Here, we describe a mouse model of neurodegeneration owing to presence of short telomeres in the brain as the consequence of telomerase deficiency. Interestingly, we find similar signs of neurodegeneration in very old mice as the consequence of physiological mouse aging. Next, we demonstrate that delivery of telomerase gene therapy to the brain of these mice results in amelioration of some of these neurodegeneration phenotypes. These findings suggest that short telomeres contribute to neurodegeneration diseases with aging and that telomerase activation may have a therapeutic value in these diseases.

Telomerase reverse transcriptase ameliorates lung fibrosis by protecting alveolar epithelial cells against senescence.

Mutations in the genes encoding telomerase reverse transcriptase (TERT) and telomerase's RNA components as well as shortened telomeres are risk factors for idiopathic pulmonary fibrosis, where repetitive injury to the alveolar epithelium is considered a key factor in pathogenesis. Given the importance of TERT in stem cells, we hypothesized that TERT plays an important role in epithelial repair and that its deficiency results in exacerbation of fibrosis by impairing this repair/regenerative process. To evaluate the role of TERT in epithelial cells, we generated type II alveolar epithelial cell (AECII)-specific TERT conditional knockout (SPC-Tert cKO) mice by crossing floxed Tert mice with inducible SPC-driven Cre mice. SPC-Tert cKO mice did not develop pulmonary fibrosis spontaneously up to 9 months of TERT deficiency. However, upon bleomycin treatment, they exhibited enhanced lung injury, inflammation, and fibrosis compared with control mice, accompanied by increased pro-fibrogenic cytokine expression but without a significant effect on AECII telomere length. Moreover, selective TERT deficiency in AECII diminished their proliferation and induced cellular senescence. These findings suggest that AECII-specific TERT deficiency enhances pulmonary fibrosis by heightening susceptibility to bleomycin-induced epithelial injury and diminishing epithelial regenerative capacity because of increased cellular senescence. We confirmed evidence for increased AECII senescence in idiopathic pulmonary fibrosis lungs, suggesting potential clinical relevance of the findings from our animal model. Our results suggest that TERT has a protective role in AECII, unlike its pro-fibrotic activity, observed previously in fibroblasts, indicating that TERT's role in pulmonary fibrosis is cell type-specific.

Pathogenic TERT promoter variants in telomere diseases.

PURPOSE: The acquisition of pathogenic variants in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. In nonmalignant diseases, TERTp variants have been reported only in patients with idiopathic pulmonary fibrosis (IPF) due to germline variants in telomere biology genes. METHODS: We screened patients with a broad spectrum of telomeropathies (n = 136), their relatives (n = 52), and controls (n = 195) for TERTp variants using a customized massively parallel amplicon-based sequencing assay. RESULTS: Pathogenic -124 and -146 TERTp variants were identified in nine (7%) unrelated patients diagnosed with IPF (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere biology genes. TERTp clone selection did not associate with peripheral blood counts, telomere length, and response to danazol treatment. However, it was specific for patients with telomeropathies, more frequently co-occurring with TERT germline variants and associated with aging. CONCLUSION: We extend the spectrum of nonmalignant diseases associated with pathogenic TERTp variants to marrow failure and liver disease due to inherited telomerase deficiency. Specificity of pathogenic TERTp variants for telomerase dysfunction may help to assess the pathogenicity of unclear constitutional variants in the telomere diseases.

Telomerase Mediates Lymphocyte Proliferation but Not the Atherosclerosis-Suppressive Potential of Regulatory T-Cells.

OBJECTIVE: Atherosclerosis is an age-related disease characterized by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence, a potential causative role of telomerase in atherogenesis is critically debated. APPROACH AND RESULTS: In this study, we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures, we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent because oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase knockout mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase deficiency had no effect on regulatory T-cell (Treg) numbers in vivo or suppressive function ex vivo. Adoptive transfer of telomerase reverse transcriptase-/- Tregs into Rag2-/- ApoE-/- (recombination activating gene 2/apolipoprotein E) double knockout mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. CONCLUSIONS: Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.

Epstein-Barr virus-encoded RNAs (EBERs) complement the loss of Herpesvirus telomerase RNA (vTR) in virus-induced tumor formation.

Marek's disease virus (MDV) is an alphaherpesvirus that causes fatal lymphomas in chickens and is used as a natural virus-host model for herpesvirus-induced tumorigenesis. MDV encodes a telomerase RNA subunit (vTR) that is crucial for efficient MDV-induced lymphoma formation; however, the mechanism is not completely understood. Similarly, Epstein Barr-virus (EBV) encodes two RNAs (EBER-1 and EBER-2) that are highly expressed in EBV-induced tumor cells, however their role in tumorigenesis remains unclear. Intriguingly, vTR and EBER-1 have interaction partners in common that are highly conserved in humans and chickens. Therefore, we investigated if EBER-1 and/or EBER-2 can complement the loss of vTR in MDV-induced tumor formation. We first deleted vTR (v∆vTR) and replaced it by either EBER-1 or EBER-2 in the very virulent RB-1B strain. Insertion of either EBER-1 or EBER-2 did not affect MDV replication and their expression levels were comparable to vTR in wild type virus. Intriguingly, EBER-2 restored tumor formation of MDV that lacks vTR. EBER-1 partially restored MDV oncogenicity, while tumor formation was severely impaired in chickens infected with v∆vTR. Our data provides the first evidence that EBERs possess tumor-promoting properties in vivo using this natural model for herpesvirus-tumorigenesis.

Telomerase reverse transcriptase protects against angiotensin II-induced microvascular endothelial dysfunction.

A rise in reactive oxygen species (ROS) may contribute to cardiovascular disease by reducing nitric oxide (NO) levels, leading to loss of NO's vasodilator and anti-inflammatory effects. Although primarily studied in larger conduit arteries, excess ROS release and a corresponding loss of NO also occur in smaller resistance arteries of the microcirculation, but the underlying mechanisms and therapeutic targets have not been fully characterized. We examined whether either of the two subunits of telomerase, telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC), affect microvascular ROS production and peak vasodilation at baseline and in response to in vivo administration to angiotensin II (ANG II). We report that genetic loss of TERT [maximal dilation: 52.0 ± 6.1% with vehicle, 60.4 ± 12.9% with Nω-nitro-l-arginine methyl ester (l-NAME), and 32.2 ± 12.2% with polyethylene glycol-catalase (PEG-Cat) ( P < 0.05), means ± SD, n = 9-19] but not TERC [maximal dilation: 79 ± 5% with vehicle, 10.7 ± 9.8% with l-NAME ( P < 0.05), and 86.4 ± 8.4% with PEG-Cat, n = 4-7] promotes flow-induced ROS formation. Moreover, TERT knockout exacerbates the microvascular dysfunction resulting from in vivo ANG II treatment, whereas TERT overexpression is protective [maximal dilation: 88.22 ± 4.6% with vehicle vs. 74.0 ± 7.3% with ANG II (1,000 ng·kg-1·min-1) ( P = not significant), n = 4]. Therefore, loss of TERT but not TERC may be a key contributor to the elevated microvascular ROS levels and reduced peak dilation observed in several cardiovascular disease pathologies. NEW & NOTEWORTHY This study identifies telomerase reverse transcriptase (TERT) but not telomerase RNA component as a key factor regulating endothelium-dependent dilation in the microcirculation. Loss of TERT activity leads to microvascular dysfunction but not conduit vessel dysfunction in first-generation mice. In contrast, TERT is protective in the microcirculation in the presence of prolonged vascular stress. Understanding the mechanism of how TERT protects against vascular stress represents a novel target for the treatment of vascular disorders.

Telomerase enzyme deficiency promotes metabolic dysfunction in murine hepatocytes upon dietary stress.

BACKGROUND & AIMS: Short telomeres and genetic telomerase defects are risk factors for some human liver diseases, ranging from non-alcoholic fatty liver disease and non-alcoholic steatohepatitis to cirrhosis. In murine models, telomere dysfunction has been shown to metabolically compromise hematopoietic cells, liver and heart via the activation of the p53-PGC axis. METHODS: Tert- and Terc-deficient mice were challenged with liquid high-fat diet. Liver metabolic contents were analysed by CE-TOFMS and liver fat content was confirmed by confocal and electronic microscopy. RESULTS: Tert-deficient but not Terc-deficient mice develop hepatocyte injury and frank steatosis when challenged with liquid high-fat diet. Upon high-fat diet, Tert-/- hepatocytes fail to engage the citric acid cycle (TCA), with an imbalance of NADPH/NADP+ and NADH/NAD+ ratios and depletion of intermediates of TCA cycle, such as cis-aconitic acid. Telomerase deficiency caused an intrinsic metabolic defect unresponsive to environmental challenge. Chemical inhibition of telomerase by zidovudine recapitulated the abnormal Tert-/- metabolic phenotype in Terc-/- hepatocytes. CONCLUSIONS: Our findings indicate that in telomeropathies short telomeres are not the only molecular trigger and telomerase enzyme deficiency provokes hepatocyte metabolic dysfunction, abrogates response to environmental challenge, and causes cellular injury and steatosis, providing a mechanism for liver damage in telomere diseases.

The genome-wide transcription response to telomerase deficiency in the thermotolerant yeast Hansenula polymorpha DL-1.

BACKGROUND: In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. RESULTS: Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. CONCLUSIONS: Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes.

OBJECTIVE: Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. APPROACH AND RESULTS: We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. CONCLUSIONS: These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC.

Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins.

Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress.

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.

Identification of novel noncoding transcripts in telomerase-negative yeast using RNA-seq.

Telomerase is a ribonucleoprotein that maintains the ends of linear chromosomes in most eukaryotes. Loss of telomerase activity results in shortening of telomeric DNA and eventually a specific G2/M cell-cycle arrest known as senescence. In humans, telomere shortening occurs during aging, while inappropriate activation of telomerase is associated with approximately 90% of cancers. Previous studies have identified several classes of noncoding RNAs (ncRNA) also associated with aging-related senescence and cancer, but whether ncRNAs are also involved in short-telomere-induced senescence in yeast is unknown. Here, we report 112 putative novel lncRNAs in the yeast Saccharomyces cerevisiae, 41 of which are only expressed in telomerase-negative yeast. Expression of approximately half of the lncRNAs is strongly correlated with that of adjacent genes, suggesting this subset may influence transcription of neighboring genes. Our results reveal a new potential mechanism governing adaptive changes in senescing and post-senescent survivor yeast cells.

Telomerase Deficiency Causes Alveolar Stem Cell Senescence-associated Low-grade Inflammation in Lungs.

Mutations of human telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) are associated with a subset of lung aging diseases, but the mechanisms by which TERC and TERT participate in lung diseases remain unclear. In this report, we show that knock-out (KO) of the mouse gene Terc or Tert causes pulmonary alveolar stem cell replicative senescence, epithelial impairment, formation of alveolar sacs, and characteristic inflammatory phenotype. Deficiency in TERC or TERT causes a remarkable elevation in various proinflammatory cytokines, including IL-1, IL-6, CXCL15 (human IL-8 homolog), IL-10, TNF-α, and monocyte chemotactic protein 1 (chemokine ligand 2 (CCL2)); decrease in TGF-ß1 and TGFßRI receptor in the lungs; and spillover of IL-6 and CXCL15 into the bronchoalveolar lavage fluids. In addition to increased gene expressions of α-smooth muscle actin and collagen 1α1, suggesting myofibroblast differentiation, TERC deficiency also leads to marked cellular infiltrations of a mononuclear cell population positive for the leukocyte common antigen CD45, low-affinity Fc receptor CD16/CD32, and pattern recognition receptor CD11b in the lungs. Our data demonstrate for the first time that telomerase deficiency triggers alveolar stem cell replicative senescence-associated low-grade inflammation, thereby driving pulmonary premature aging, alveolar sac formation, and fibrotic lesion.

Cell populations can use aneuploidy to survive telomerase insufficiency.

Telomerase maintains ends of eukaryotic chromosomes, telomeres. Telomerase loss results in replicative senescence and a switch to recombination-dependent telomere maintenance. Telomerase insufficiency in humans leads to telomere syndromes associated with premature ageing and cancer predisposition. Here we use yeast to show that the survival of telomerase insufficiency differs from the survival of telomerase loss and occurs through aneuploidy. In yeast grown at elevated temperatures, telomerase activity becomes limiting: haploid cell populations senesce and generate aneuploid survivors--near diploids monosomic for chromosome VIII. This aneuploidy results in increased levels of the telomerase components TLC1, Est1 and Est3, and is accompanied by decreased abundance of ribosomal proteins. We propose that aneuploidy suppresses telomerase insufficiency through redistribution of cellular resources away from ribosome synthesis towards production of telomerase components and other non-ribosomal proteins. The aneuploidy-induced re-balance of the proteome via modulation of ribosome biogenesis may be a general adaptive response to overcome functional insufficiencies.