Significant Telomere Shortening Research Articles

DNAR-15. GROUP 3 MEDULLOBLASTOMAS EXPLOIT A TRANSIENT MECHANISM OF TELOMERE REPAIR MEDIATED BY ZSCAN4 WHICH IS TARGETABLE FOR THERAPEUTIC BENEFIT WITH BRAIN-PENETRANT DRUGS

Abstract All cancers need some mechanism of telomere repair. However, it is unknown how Group 3 (G3) medulloblastomas (MB) repair their telomeres, even though MB is the most common malignant brain tumor in kids and G3 is its most aggressive subtype. With rare exceptions, G3 MBs lack telomerase gain-of-function alterations or evidence of constitutive alternative-lengthening-of-telomeres pathway activation. We profiled tumors from N=38 MB patients from UCSF via single-nucleus RNA sequencing; 13 cases were also subjected to single-cell assay for transposase-accessible chromatin by sequencing, and 4 were subjected to single-cell cleavage under targets and tagmentation for H3K27ac. These data identified a network of gene enhancers that were specifically active in rhombic-lip progenitor-like MB cells, the putative G3 MB cell of origin. This program was driven by zinc finger and SCAN domain containing 4 (ZSCAN4), a gene reported to drive transient telomere repair during zygotic genome activation in early embryos. CRISPR inhibition of ZSCAN4 led to significant telomere shortening, telomeric DNA damage, telomere fusions, decreased proliferation, and increased survival of orthotopic xenografts. Conversely, ZSCAN4 over-expression induced significant telomere elongation, increased proliferation, and decreased survival in vivo. Analysis of endogenous-reporter cells indicated that ZSCAN4 is transiently expressed in response to telomere shortening and increases global histone acetylation by inducing chromatin-remodeling factors. Rebastinib targeted ZSCAN4, histone acetylation, and telomere repair, was brain penetrant, and significantly enhanced orthotopic-xenograft survival with no toxicity. Acetyltransferase inhibitor CPI-1612 was brain penetrant and extended xenograft survival with no toxicity. Our data support the hypothesis that G3 MBs exploit a transient mechanism of telomere repair mediated by ZSCAN4. Targeting ZSCAN4 or downstream histone acetylation is therapeutically viable. Pan-cancer whole-genome sequencing data showed that MBs have lower telomeric content than most other cancers. Thus, MBs may be particularly vulnerable to therapeutic strategies that inhibit telomere repair.

CSP7 Protects Alveolar Epithelial Cells by Targeting p53-Fibrinolytic Pathways During Lung Injuries.

Impaired alveolar epithelial regeneration in patients with idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) is attributed to telomere dysfunction in type II alveolar epithelial cells (A2Cs). Genetic susceptibility, aging, and toxicant exposures, including tobacco smoke (TS), contribute to telomere dysfunction in A2Cs. Here we investigated whether improvement of telomere function plays a role in CSP7-mediated protection of A2Cs against ongoing senescence and apoptosis during bleomycin (BLM)-induced pulmonary fibrosis (PF) as well as alveolar injury caused by chronic TS exposure. We found a significant telomere shortening in A2Cs isolated from IPF and COPD lungs in line with other studies. These cells showed increased p53 in addition to its post-translational modification with induction of activated caspase-3 and β-galactosidase, suggesting a p53-mediated loss of A2C renewal. Further, we found increased expression of SIAH-1, a p53-inducible E3 ubiquitin ligase known to down-regulate telomere repeats binding factor 2 (TRF2). Consistent with the loss of TRF2 and upregulation of TRF1, telomerase reverse transcriptase (TERT) was downregulated in A2Cs. A2Cs from fibrotic lungs of mice either repeatedly instilled with BLM or isolated from chronic TS exposure-induced lung injury model showed reduced telomere length along with induction of p53, PAI-1, SIAH1 and TRF1 as well as loss of TRF2 and TERT, which were reversed in wild-type mice after treatment with CSP7. Interestingly, PAI-1-/- mice, or those lacking microRNA-34a expression in A2Cs, resisted telomere dysfunction, while uPA-/- mice failed to respond to CSP7 treatment, suggesting p53-microRNA-34a feed-forward induction and p53-uPA pathway contributes to telomere dysfunction.

Association of hTERT expression, Her2Neu, estrogen receptors, progesterone receptors, with telomere length before and at the end of treatment in breast cancer patients.

Breast cancer shows significant clinical, morphologic, and molecular variation. Telomeres are nucleoprotein complexes composed of hexanucleotide repeat DNA sequence, TTAGGG, and numerous telomere-associated proteins. The maintenance of telomere length is carried out by a ribonucleoprotein called telomerase, which consists of two main components: a catalytic subunit called hTERT (human telomerase reverse transcriptase) and an RNA template called hTR (human telomerase RNA). The importance of evaluating hTERT expression lies in its potential therapeutic application, being an attractive target due to its almost non-existent expression in normal somatic cells. It is also expected that the anti-neoplastic effect would appear earlier in neoplastic cells with shorter telomeres. Additionally, a significant relationship has been observed between Her2-Neu overexpression and Her2-Neu positivity, which could suggest new combined therapies.The aim of this study was to detect the expression of hTERT, estrogen receptor (ER), progesterone receptor (PR), and HER2-Neu in neoplastic breast tissue embedded in paraffin before treatment and to investigate the relationship between them and with baseline and post-treatment telomere length, as well as with various clinicopathological parameters. A cross-sectional-correlational, 21 women diagnosed with breast cancer at the Oncology Service of the High Specialty Medical Unit No. 1 of Bajio of the Mexican Institute of Social Security. The study complies with the Helsinki Declaration and was approved by the Institutional Ethical Committee of the Mexican Institute of Social Security (R-2019-1001-127). A peripheral blood sample was obtained before oncological treatment and at the end of oncological treatment for the measurement of telomere length by extracting DNA from leukocytes, was performed by the quantitative polymerase chain reaction (PCR) method described by Cawthon. Tumor samples were collected from each patient at the oncology department for immunohistochemical determination of biomarker expression (ER, PR, Her2/neu) and hTERT. Of the 21 cases included in the study, the median age was 57.57 years. Eighteen cases were classified as invasive ductal carcinoma NOS (85.71%), 10 were histologic grade 2 (47.61%), 16 cases were hormone receptor positive (76.19%), 7 were Her2Neu positive (33.33%), and only 2 cases were triple negative (9.52%). Positive hTERT expression was detected in 11 cases (52.38%) and was negative in the remaining cases. A significant association was identified between hTERT-positive cases and Her2-Neu positive cases (p = 0.04). Baseline and post-treatment telomere lengths showed a significant difference using the non-parametric Wilcoxon t-test (p = 0.002). In hTERT-positive cases, there was significant telomere shortening at the end of oncological treatment (6.14 ± 1.54 vs. 4.75 ± 1.96 Kb, p = 0.007). Positive hTERT immunostaining cases were associated with poor prognostic factors, such as Her2-Neu overexpression and post-treatment telomere shortening. In the future, hTERT immunostaining could be used to select patients for therapies with antagonistic effects on hTERT, as well as in the selection of more appropriate chemotherapy regimens for patients who express it.

Telomere Maintenance Is Associated with Type 2 Diabetes Remission in Response to a Long-Term Dietary Intervention without Non-Weight Loss in Patients with Coronary Heart Disease: From the CORDIOPREV Randomized Controlled Trial.

In order to evaluate whether telomere maintenance is associated with type 2 diabetes remission, newly diagnosed type 2 diabetes patients without glucose-lowering treatment (183 out of 1002) from the CORDIOPREV study (NCT00924937) were randomized to consume a Mediterranean or low-fat diet. Patients were classified as Responders, those who reverted from type 2 diabetes during the 5 years of dietary intervention (n = 69), and Non-Responders, who did not achieve diabetes remission by the end of the follow-up period (n = 104). We found no differences in diabetes remission between the two diets, and we determined telomere length (TL) by measuring qPCR, telomerase activity using the TRAP assay, and direct redox balance based on the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSH) via colorimetric assay. Responders exhibited higher baseline TL in comparison with Non-Responders (p = 0.040), and a higher TL at baseline significantly predicted a higher probability of type 2 diabetes remission (OR 2.13; 95% CI, 1.03 to 4.41). After the dietary intervention, Non-Responders showed significant telomere shortening (-0.19, 95% CI -0.32 to 0.57; p = 0.005). Telomere shortening was significantly pronounced in type 2 diabetes patients with a worse profile of insulin resistance and/or beta-cell functionality: high hepatic insulin resistance fasting, a high disposition index (-0.35; 95% CI, -0.54 to -0.16; p < 0.001), and a low disposition index (-0.25; 95% CI, -0.47 to -0.01; p = 0.037). In addition, changes in TL were correlated to the GSH/GSSG ratio. Responders also showed increased telomerase activity compared with baseline (p = 0.048), from 0.16 (95% CI, 0.08 to 0.23) to 0.28 (95% CI, 0.15 to 0.40), with a more marked increase after the dietary intervention compared with Non-Responders (+0.07; 95% CI, -0.06-0.20; p = 0.049). To conclude, telomere maintenance may play a key role in the molecular mechanisms underlying type 2 diabetes remission in newly diagnosed patients. However, further larger-scale prospective studies are necessary to corroborate our findings.

Functional and transcriptional sequelae of cardiomyocyte telomere shortening and their pathologic relevance in dilated cardiomyopathy and ischemic heart failure

Abstract Background Heart failure (HF) is associated with cardiomyocyte (CM) telomere shortening. Sequelae have been described with the telomere-p53-mitochondrion model. P53 deficiency only partially ameliorates mitochondrial defects in telomerase deficient mice hinting at additional pathways. Purpose We assessed the cardiac phenotype and transcriptome of early/advanced telomere shortening and evaluated the pathological relevance by correlation to published HF datasets. Methods Male (6-8w) telomerase RNA-component deficient mice, early (generation 2, mTRG2) and advanced (mTRG5) were phenotyped vs age-matched C57BL/6 mice (B6). Adult CMs from mTRG2/G5 vs B6 mice were characterized (live imaging, quantitative fluorescence in situ hybridization (Q-FISH) and transcriptome analysis (microarray Mouse MTA 1.0)). Gene expression analysis was performed (Ingenuity Pathway Analysis, Qiagen) and correlated to comparable datasets from &amp;gt; 120 000 curated datasets. Results CMs exhibited significant telomere shortening in mTR(-/-) mice increasing with every generation (mTRG2/G5 p&amp;lt;0.0001 vs B6). In parallel, echocardiography revealed increasing defects of left ventricular (LV) systolic function (LVEF, mTRG5 &amp;lt;0.01 vs B6), LV global longitudinal strain mTRG2/G5 p&amp;lt;0.001 vs B6) and LV contractile reserve (mTRG2/G5 p&amp;lt;0.05 vs B6). Live imaging showed increasing impairment of mitochondrial polarization with all energy substrates (mTRG2/G5 p&amp;lt;0.0001 vs B6). Transcriptome analysis revealed 357/2906 differentially expressed genes (DEGs) in mTRG2/G5-CMs vs B6-CMs (fold change &amp;lt;-1.5 or &amp;gt;1.5 and p-value &amp;lt;0.05). Upstream Regulator (UR) analysis revealed significant overrepresentation of numerous regulators including angiotensinogen (z-score 7.5, p-value 1.1E-32), transforming growth factor (TGF)-b1 (z-score 7.5, p-value 2.2E-60), interleukin (IL)-1 (z-score 5.6, p-value 1.1E-26), IL-6 (expression fold change 11.3, z-score 6.9 p-value 2.65E-32), IL-17A (z-score 4.8, p-value 4.4E-27) and NFkB (z-score 6.0 p-value 4.9E-19). Activation of TP53 (z-score 5.8, p-value 5.3E-31) and inhibition of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) (z-score -4.4, p-value 6.3E-19) confirmed the relevance of the p53-mitochondrion-axis. Analysis matching revealed a significant overlap of gene expression patterns in the same direction (positive z-score) between mTR-/--mice and all comparable datasets of dilated (DCM) and ischemic cardiomyopathy (ICM) (human/mouse), the overlap increasing with increasing telomere shortening (e.g. DCM vs mTRG2: z-score 17.7, UR p-value 1.5E-16; DCM vs mTRG5: z-score 56.8, UR-value 1.5E-62). Matching to datasets after cardiac regeneration (e.g. after left ventricular assist device implantation) revealed a reversal of gene expression patterns (negative z-score). Conclusions Our results reveal the large extent of transcriptional alterations upon cardiac telomere shortening and support their central role in the pathogenesis of HF.

Long telomeres at baseline and male sex are main determinants of telomere loss following chemotherapy exposure in lymphoma patients.

Although chemotherapy (CHT) exposure is an established cause of telomere attrition, determinants of telomere length (TL) dynamics after chemotherapy are poorly defined. In this study, we analyzed granulocyte telomere dynamics in 34 adult lymphoma patients undergoing first-line CHT. TL was measured by southern blot at each CHT cycle and after 1year from CHT completion. Median age was 59yrs (range 22-77). Median number of CHT cycles was 6 (range 3-6). The majority of patients (79%, n=27) experienced TL shortening following CHT exposure. Mean telomere loss was 673 base pairs (bp) by cycle 6. Telomere shortening was an early event as 87% of the total telomere loss (mean 586 bp) occurred by the end of cycle 3, with no significant recovery after 1year. A significant correlation was observed between baseline TL and total or fractional telomere loss (p<0.001), with telomere shortening by cycle 3 observed predominantly in male patients with long telomeres at pre-treatment evaluation. Stratifying the analysis by gender and age only young women (<51years of age) did not show significant telomere shortening following chemotherapy exposure. These findings indicate that gender and baseline TL are major determinants of TL dynamics following chemotherapy exposure in lymphoma patients.

Alternative telomere maintenance mechanism in Alligator sinensis provides insights into aging evolution

Lifespan is a life-history trait that undergoes natural selection. Telomeres are hallmarks of aging, and shortening rate predicts species lifespan, making telomere maintenance mechanisms throughout different lifespans a worthy topic for study. Alligators are suitable for the exploration of anti-aging molecular mechanisms, because they exhibit low or even negligible mortality in adults and no significant telomere shortening. Telomerase reverse transcriptase (TERT) expression is absent in the adult Alligator sinensis, as in humans. Selection analyses on telomere maintenance genes indicated that ATM, FANCE, SAMHD1, HMBOX1, NAT10, and MAP3K4 experienced positive selection on A.sinensis. Repressed pleiotropic ATM kinase in A.sinensis suggests their fitness optimum shift. In ATM downstream, Alternative Lengthening of Telomeres (ALT)-related genes were clustered in a higher expression pattern in A.sinensis, which covers 10-15% of human cancers showing no telomerase activities. In summary, we demonstrated how telomere shortening, telomerase activities, and ALT contributed to anti-aging strategies.

Telomere shortening in hypertensive heart disease depends on NOX2-mediated loss of PRDX1 and oxidative DNA damage

Abstract Background Heart failure (HF) coincides with cardiomyocyte telomere shortening. Arterial hypertension is the most prominent risk factor for HF. Both HF and arterial hypertension are associated with dysregulation of the neurohormonal axis and increased ROS. However, how neurohormonal activation is linked to telomere shortening in the pathogenesis of HF is incompletely understood. Further, recent findings suggest that peroxiredoxin 1 (PRDX1) can act as a telomere specific antioxidant. Methods To induce hypertensive HF, male C57BL/6J mice were subjected to AngII-infusion, uninephrectomy and high-salt (AngII++) for 5 weeks. Cardiac function was assessed by ultrasound. Mouse adult cardiomocytes (CMs) isolated from C57BL/6J mice and mice lacking the NADPH-oxidase (NOX) 2/gp91phox as well as rat ventricular CM-derived cells (H9C2) were stimulated with AngII. Telomere length was quantified by Q-FISH after staining with a C-rich telomere probe (TelC). DNA/RNA-damage was evaluated after staining for Oxo-8-Gua (8-oxo-7,8-dihydroguanine) and Oxo-8-G (8-oxo-7,8-dihydroguanosine). Superoxide (O2-) was quantified by 2-hydroxyethidium (2-HE) using HPLC analysis. Colocalization of Oxo-8-Gua and TelC was quantified by automated image analysis. (Sub)cellular and tissue expression of gp91phox/Nox2 and PRDX1 was evaluated by ICC/IHC. All image quantification was conducted semiautomatically. Results In mice subjected to hypertensive HF, CM telomere shortening correlated significantly with both left ventricular (LV) dilatation and impairment of LV systolic function, paralleled by a significant loss of myocardial PRDX1 and significantly increased myocardial DNA/RNA-damage. Similarly, CMs stimulated with AngII exhibited significant telomere shortening, significant loss of PRDX1 and significantly increased DNA/RNA-damage, together with significantly increased CM superoxide production and significantly increased expression of the superoxide generating enzyme gp91phox/NOX2. Correspondingly, deficiency of gp91phox/NOX2 significantly prevented AngII-induced CM telomere shortening, DNA/RNA-damage and PRDX1-depletion. A similar effect could be observed upon stimulation of CMs with the specific histone deacetylase (HDAC) 6-inhibitor tubastatin, which prevents deacetylation of PRDX1. Conclusion We could provide first evidence that in heart failure, ROS originating from increased NOX2-activity leads to depletion of the telomere-targeted antioxidant and repair-protein PRDX1, which results in damage to the telomeric DNA. Both depletion of NOX2 as well as harnessing the intrinsic antioxidant defense by stabilizing PRDX1 via HDAC6-inhibition prevented CM telomere shortening and could be further evaluated as potential therapeutic targets to address heart failure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Federal Ministry for Education and Research

Abstract 2316: Malignant potential of colorectal adenoma based on the telomere length

Abstract Telomere attrition is a well-known hallmark of malignancy in cancer tissue. Our study analyzed the relative telomere length in adenomas, precursor lesions of colorectal adenocarcinoma. We investigated if the accelerated telomere length shortening occurs before potential malignant transformation to adenocarcinoma and corresponds with clinicopathological characteristics. Relative telomere length was measured in 145 patients using multiplex qPCR. We have recorded significant shortening of telomeres in the adenoma tissue compared to adjacent mucosa (Wilcoxon Signed Rank test, p &amp;lt; 0.0001). Further, we have observed a weak inverse relationship between both adenoma and mucosa telomere length and age (Spearman's and Pearson's correlation, p = 0.021 and p = 0.027, respectively), contrary to our previous study of colorectal cancer patients (n = 696), where the inverse correlation of telomere length was detected only in the non-malignant mucosa (Pearson's correlation, p = 0.029). However, the telomere length ratio of adenoma to adjacent mucosa did not correspond with the degree of dysplasia according to the Vienna classification (Kruskal-Wallis test, p = 0.572). Also, there was no difference in the telomere length ratio among histological types of adenomas and no association with the adenoma localization in proximal and distal colon or rectum (Kruskal-Wallis test, p = 0.648 and p = 0.459, respectively). Preliminary results indicate that telomere shortening occurs indeed as early as in a stage of adenomatous polyps, and it is less pronounced in adenomas compared to cancerous cells. However, the relative telomere length does not seem to correspond with adenoma progression towards malignancy. In planned experiments, we intend to assess mitochondrial DNA damage caused by 8-oxoguanine DNA adducts. These major oxidative base lesions can arise from endogenous reactive oxygen species and impact, among other things, the integrity of telomeres through double-strand breaks to DNA. Mitochondrial DNA is more prone to accumulation of oxidative damage than nuclear DNA and thus might be useful as an early, additional indicator of the precancerous character of adenoma. The study will reveal whether increased mitochondrial DNA damage and telomere loss are commonly present and linked together in adenomas. This study was supported by the Charles University (project GA UK No. 120, UNCE/MED/006) and the Grant Agency of the Czech Republic (21-04607X, 21-27902S). Citation Format: Kristyna Tomasova, Michal Kroupa, Anna Siskova, Josef Horak, Ondrej Vycital, Vaclav Liska, Ludmila Vodickova, Pavel Vodicka. Malignant potential of colorectal adenoma based on the telomere length [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2316.

Multiwalled Carbon Nanotubes Induce Fibrosis and Telomere Length Alterations.

Telomere shortening can result in cellular senescence and in increased level of genome instability, which are key events in numerous of cancer types. Despite this, few studies have focused on the effect of nanomaterial exposure on telomere length as a possible mechanism involved in nanomaterial-induced carcinogenesis. In this study, effects of exposure to multiwalled carbon nanotubes (MWCNT) on telomere length were investigated in mice exposed by intrapleural injection, as well as in human lung epithelial and mesothelial cell lines. In addition, cell cycle, apoptosis, and regulation of genes involved in DNA damage repair were assessed. Exposure to MWCNT led to severe fibrosis, infiltration of inflammatory cells in pleura, and mesothelial cell hyperplasia. These histological alterations were accompanied by deregulation of genes involved in fibrosis and immune cell recruitment, as well as a significant shortening of telomeres in the pleura and the lung. Assessment of key carcinogenic mechanisms in vitro confirmed that long-term exposure to the long MWCNT led to a prominent telomere shortening in epithelial cells, which coincided with G1-phase arrest and enhanced apoptosis. Altogether, our data show that telomere shortening resulting in cell cycle arrest and apoptosis may be an important mechanism in long MWCNT-induced inflammation and fibrosis.

Reduced telomere length in amniocytes: an early biomarker of abnormal fetal development?

Telomeres protect chromosome ends and control cell division and senescence. During organogenesis, telomeres need to be long enough to ensure the cell proliferation necessary at this stage of development. Previous studies have shown that telomere shortening is associated with growth retardation and congenital malformations. However, these studies were performed in newborns or postnatally, and data on telomere length (TL) during the prenatal period are still very limited. We measured TL using quantitative PCR in amniotic fluid (AF) and chorionic villi (CV) samples from 69 control fetuses with normal ultrasound (52 AF and 17 CV) and 213 fetuses (165 AF and 48 CV) with intrauterine growth retardation (IUGR) or congenital malformations diagnosed by ultrasound. The samples were collected by amniocentesis at the gestational age (GA) of 25.0 ± 5.4weeks and by CV biopsy at 18.1 ± 6.3weeks. In neither sample type was TL influenced by GA or fetal sex. In AF, a comparison of abnormal versus normal fetuses showed a significant telomere shortening in cases of IUGR (reduction of 34%, P < 10-6), single (29%, P < 10-6) and multiple (44%, P < 10-6) malformations. Similar TL shortening was also observed in CV from abnormal fetuses but to a lesser extent (25%, P = 0.0002; 18%, P = 0.016; 20%, P = 0.004, respectively). Telomere shortening was more pronounced in cases of multiple congenital anomalies than in fetuses with a single malformation, suggesting a correlation between TL and the severity of fetal phenotype. Thus, TL measurement in fetal samples during pregnancy could provide a novel predictive marker of pathological development.

Telomere shortening in heart failure due to excess neurohormonal activation is associated with loss of nuclear TRF-2 and increased telomeric DNA damage and predicts cardiac recovery

Abstract The mechanisms and clinical significance of telomere shortening in heart failure remain elusive. Mammalian cardiomyocyte (CM) regeneration is limited and CM cell division cannot account for telomere shortening. Whether telomere shortening in turn affects cardiac recovery remains unexplored. We induced heart failure by excess neurohormonal activation (NHA), a universal dysregulation in the failing heart. B6 mice were subjected to AngII-infusion, uninephrectomy and high-salt (AngII++) to induce excess NHA for 5 weeks. Cardiac function was assessed by ultrasound. Human iPSC-derived CMs, mouse CMs and rat ventricular CMs (H9C2) were stimulated with AngII. Human cardiac biopsies from patients with heart failure with reduced ejection fraction (HFrEF) were grouped depending on cardiac recovery. Telomere length was quantified by Q-FISH. ROS-induced DNA/RNA-damage was evaluated after staining for 8-oxo-7,8-dihydro-2'-deoxyguanosine, -guanine and – guanosine-residues (8-oxo-dG). H9C2-cells were co-stained for telomeres, telomere repeat binding factor 2 (TRF-2) and 8-oxo-dG, telomere-specific DNA-damage was quantified by automated colocalization analysis. Superoxide (O2-) was quantified by 2-hydroxyethidium (2-HE) using HPLC analysis. Fibrosis was quantified by Sirius red staining. AngII++-mice exhibited left ventricular (LV) hypertrophy (p&amp;lt;0.0001 vs B6) along with reduced LV ejection fraction (p=0.03 vs B6), reduced global longitudinal strain (p&amp;lt;0.001 vs B6) along with significant telomere shortening in CMs isolated from AngII++-mice (p=0.0082 vs B6). Telomere length showed a significant correlation with LV systolic function (r2=0.64, p=0.006). While 8-oxo-dG-staining revealed significantly increased ROS-induced DNA/RNA damage in AngII++-myocardial sections overall (p&amp;lt;0.0001 vs B6), DNA/RNA-damage was highest in areas of fibrotic adverse remodeling congruent with significant telomere shortening (p&amp;lt;0.0122 fibrotic vs non-fibrotic AngII++-myocardium, p&amp;lt;0.0001 vs B6). In all cells tested, NHA resulted in significant telomere shortening. As a potential explanation, CMs stimulated with AngII revealed significantly increased, dose-dependent O2–production (AngII 10 nM p=0.03, AngII 50 nm p=0.0004 vs H9C2) along with significant loss of nucelar TRF2 (AngII nM p=0.0049, Ang II 50 nM p&amp;lt;0.0001 vs H9C2), while colocalization analysis revealed a significantly increased ROS-induced damage to the telomeric DNA (AngII 10/50 nm p&amp;lt;0.05 vs H9C2). HFrEF-patients that had recovered cardiac function (ΔLVEF +5 – 30%) showed significantly longer myocardial telomeres than patients with no recovery/worsening (ΔLVEF −10 – 0%) (p=0.008), overall the telomere signal correlated significantly with ΔLVEF (r2=0.41, p=0.03). Our data provides insides into how excess NHA via increased ROS, loss of nuclear TRF-2 and increased telomeric DNA-damage causes myocardial telomere shortening in heart failure and further that the extent of telomere shortening determines cardiac recovery. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BMBF 01EO1003

Biallelic variants in YRDC cause a developmental disorder with progeroid features

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient’s dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.

Significant Shortening of Leukocyte Telomere Length in Korean Patients with Bipolar Disorder 1

ObjectiveTelomere shortening has been seen in major psychiatric disorders, including major depressive disorder. However, only a few small studies have examined this in bipolar disorder (BD). We compared the telomere length in patients with BD1 or BD2 with that in matched healthy controls.MethodsWe included 215 patients with BD (128 BD1, 87 BD2) and 204 age- and sex-matched healthy controls. Relative telomere length was determined by quantitative polymerase chain reaction. The patients and controls were compared separately for age groups, sex, and BD subgroups (BD1 and BD2).ResultsWe found significant telomere shortening in patients with BD1 (p < 0.001), but not in patients with BD2. In male patients with BD1, the 30−39 year age group had significant shortening of telomere length than controls (p = 0.01). Female patients with BD1 in the 19−29-year age group had significantly shortened telomeres compared to the controls (p < 0.01).ConclusionOur results suggest a significant reduction in telomere length in BD1. Telomere shortening would be a potential biomarker for BD.

Evidence for Biological Age Acceleration and Telomere Shortening in COVID-19 Survivors.

The SARS-CoV-2 infection determines the COVID-19 syndrome characterized, in the worst cases, by severe respiratory distress, pulmonary and cardiac fibrosis, inflammatory cytokine release, and immunosuppression. This condition has led to the death of about 2.15% of the total infected world population so far. Among survivors, the presence of the so-called persistent post-COVID-19 syndrome (PPCS) is a common finding. In COVID-19 survivors, PPCS presents one or more symptoms: fatigue, dyspnea, memory loss, sleep disorders, and difficulty concentrating. In this study, a cohort of 117 COVID-19 survivors (post-COVID-19) and 144 non-infected volunteers (COVID-19-free) was analyzed using pyrosequencing of defined CpG islands previously identified as suitable for biological age determination. The results show a consistent biological age increase in the post-COVID-19 population, determining a DeltaAge acceleration of 10.45 ± 7.29 years (+5.25 years above the range of normality) compared with 3.68 ± 8.17 years for the COVID-19-free population (p < 0.0001). A significant telomere shortening parallels this finding in the post-COVID-19 cohort compared with COVID-19-free subjects (p < 0.0001). Additionally, ACE2 expression was decreased in post-COVID-19 patients, compared with the COVID-19-free population, while DPP-4 did not change. In light of these observations, we hypothesize that some epigenetic alterations are associated with the post-COVID-19 condition, particularly in younger patients (< 60 years).

Unbalanced translocation der(5;17) resulting in a TP53 loss as recurrent aberration in myelodysplastic syndrome and acute myeloid leukemia with complex karyotype.

A complex karyotype, detected in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML), is associated with a reduced median survival. The most frequent chromosomal aberrations in complex karyotypes are deletions of 5q and 17p harboring the tumor suppressor gene TP53. The unbalanced translocation der(5;17) involving chromosome 5q and 17p is a recurrent aberration in MDS/AML, resulting in TP53 loss. We analyzed the karyotypes of 178 patients with an unbalanced translocation der(5;17) using fluorescence R-/G-banding analysis. Whenever possible, fluorescence in situ hybridization (FISH) (n = 138/141), multicolor FISH (n = 8), telomere length measurement (n = 9), targeted DNA sequencing (n = 13), array-CGH (n = 7) and targeted RNA sequencing (n = 2) were conducted. The der(5;17) aberration was accompanied with loss of genetic material in 7q (53%), -7 (27%), gain of 21q (29%), +8 (17%) and - 18 (16%) and all analyzed patients (n = 13) showed a (likely) pathogenic variant inTP53. The der(5;17) cohort showed significantly shortened telomeres in comparison to the healthy age-matched controls (P < .05), but there was no significant telomere shortening in comparison to MDS/AML patients with a complex karyotype without der(5;17). No fusion genes resulted from the unbalanced translocation. This study demonstrates that the unbalanced translocation der(5;17) is associated with a biallelic inactivation of TP53 due to a deletion of TP53 in one allele and a pathogenic variant of the second TP53 allele. Since the breakpoints are located within (near-) heterochromatic regions, alterations of DNA methylation or histone modifications may be involved in the generation of der(5;17).

Relationship between telomere shortening and age in Korean individuals with mild cognitive impairment and Alzheimer's disease compared to that in healthy controls.

Although telomere length (TL) is highly variable, a shorter TL indicate increased biological age. This multicenter study was conducted to identify the overall correlation between age and TL in Koreans and investigate the associations between age and TL in healthy individuals and patients with mild cognitive impairment (MCI) and Alzheimer’s disease (AD). TL was measured in peripheral leukocyte DNA. MCI and AD were diagnosed based on clinical examinations and amyloid deposition on positron emission tomography. This study enrolled 437 individuals. Multivariable linear analysis showed an overall approximate TL decrease of 37 bp per 1-year increase in age in all individuals (B=-0.037; P=0.002). There was no significant difference in the mean TL between healthy individuals and individuals with AD. Multivariable linear regression analysis showed that the mean rate of telomere shortening was 60 bp per year in individuals with AD (B=-0.060; P=0.006). There was a negative association between age and TL in our study. Our study results showed more significant telomere shortening per year in women than that in men. In addition, individuals with AD had greater telomere shortening every year than healthy individuals and individuals with MCI.

Swc4 positively regulates telomere length independently of its roles in NuA4 and SWR1 complexes.

Telomeres at the ends of eukaryotic chromosomes are essential for genome integrality and stability. In order to identify genes that sustain telomere maintenance independently of telomerase recruitment, we have exploited the phenotype of over-long telomeres in the cells that express Cdc13-Est2 fusion protein, and examined 195 strains, in which individual non-essential gene deletion causes telomere shortening. We have identified 24 genes whose deletion results in dramatic failure of Cdc13-Est2 function, including those encoding components of telomerase, Yku, KEOPS and NMD complexes, as well as quite a few whose functions are not obvious in telomerase activity regulation. We have characterized Swc4, a shared subunit of histone acetyltransferase NuA4 and chromatin remodeling SWR1 (SWR1-C) complexes, in telomere length regulation. Deletion of SWC4, but not other non-essential subunits of either NuA4 or SWR1-C, causes significant telomere shortening. Consistently, simultaneous disassembly of NuA4 and SWR1-C does not affect telomere length. Interestingly, inactivation of Swc4 in telomerase null cells accelerates both telomere shortening and senescence rates. Swc4 associates with telomeric DNA in vivo, suggesting a direct role of Swc4 at telomeres. Taken together, our work reveals a distinct role of Swc4 in telomere length regulation, separable from its canonical roles in both NuA4 and SWR1-C.

Context-Sensitive Cleavage of Folded DNAs by Loop-Targeting bPNAs.

Herein, we demonstrate context-dependent molecular recognition of DNA by synthetic bPNA iron and copper complexes, using oxidative backbone cleavage as a chemical readout for binding. Oligoethylenimine bPNAs displaying iron·EDTA or copper·phenanthroline sites were found to be efficient chemical nucleases for designed and native structured DNAs with T-rich single-stranded domains. Cleavage reactivity depends strongly on structural context, as strikingly demonstrated with DNA substrates of the form (GGGTTA)n. This repeat sequence from the human telomere is known to switch between parallel and antiparallel G-quadruplex (G4) topologies with a change from potassium to sodium buffer: notably, bPNA-copper complexes efficiently cleave long repeat sequences into ∼22-nucleotide portions in sodium, but not potassium, buffer. We hypothesize preferential cleavage of the antiparallel topology (Na+) over the parallel topology (K+) due to the greater accessibility of the TTA loop to bPNA in the antiparallel (Na+) form. Similar ion-sensitive telomere shortening upon treatment with bPNA nucleases can be observed in both isolated and intracellular DNA from PC3 cells by quantitative polymerase chain reaction. Live cell treatment was accompanied by accelerated cellular senescence, as expected for significant telomere shortening. Taken together, the loop-targeting approach of bPNA chemical nucleases complements prior intercalation strategies targeting duplex and quadruplex DNA. Structurally sensitive loop targeting enables discrimination between similar target sequences, thus expanding bPNA targeting beyond simple oligo-T sequences. In addition, bPNA nucleases are cell membrane permeable and therefore may be used to target native intracellular substrates. In addition, these data indicate that bPNA scaffolds can be a platform for new synthetic binders to particular nucleic acid structural motifs.

Aging and biomarkers: Transcriptional levels evaluation of Osteopontin/miRNA-181a axis in hepatic tissue of rats in different age ranges.

Osteopontin (OPN), a novel hepatic damage marker, as well as several non-coding RNA seem to be associated with liver aging, a little studied and not yet defined process. The aim of the study was to evaluate the expression variations of OPN and miRNA-181a, the transcriptional profiling of long non coding (lnc) RNA GAS-5/miRNA-222 axis and lncRNA NEAT-1 in liver tissue of rats. In addition, to monitor the senescence process, the telomere shortening and TERT/TERC gene expression were also measured. Three groups of male Wistar rats were studied: A (n=6, young); B (n=13, adult); C (n=10, old). Total RNA, including miRNAs, was extracted from liver and analysed by Real-Time PCR. Ultrasound and biochemical evaluation were performed in all rats as well as the histological analysis. OPN mRNA resulted lower in C with respect to A and B while miRNA-181a expression was significantly increased as a function of age. An increasing of both NEAT-1 and miRNA-222 expression as a function of age in parallel with a decreasing of GAS-5 expression in young and old rats, but not in the adults, was observed. A positive correlation was detected between miRNA-181a and miRNA-222. The hepatic ultrasound analysis revealed areas of hyperechogenicity distributed as a function of age. A significant telomere shortening was measured as a function of age while the two subunits TERT and TERC expressions showed an opposite trend. This work could provide a valid starting point to better understand the physiopathological changes during aging, pinpointing in the OPN/miRNA-181a axis significant predictors of successful aging.