Telomeric Repeat Binding Factor Research Articles

ÜRİNER SİSTEM ENFEKSİYONLARINDA SHELTERİN PROTEİNLERİ VE AGMATİN İLİŞKİSİ

Abstract: The presence of microorganisms in the kidney, collecting system, or bladder with pyuria and clinical symptoms is called Urinary Tract Infection (UTI). UTI is one of the most common diseases in both nosocomial and community-acquired infections and affects millions of people every year. The clinical types of UTI range from cystitis to sepsis. In this study, we aimed to contribute to the literature by comparing the levels of shelterin proteins and agmatine in the serum of patients diagnosed with cystitis, one of the types of UTI, and healthy volunteers without any known medical disorder, and to bring new approaches to issues such as clinical severity, response to treatment, and pathogenesis of the disease.
 Methods: In this study, 30 individuals diagnosed with cystitis from UTIs and 30 individuals without any systemic disease participated. Serum agmatine levels were measured by a fluorescence detector using the ultra-high-performance liquid chromatography method (UHPLC). Serum shelterin proteins levels were measured using the ELISA method.
 Results: When the patients and healthy controls were compared, serum Telomeric Repeat Binding Factor2 (TRF2) levels of the patients were found to be lower than the controls; serum agmatine levels were found to be higher than the controls and statistically significant (p0.05).
 Conclusion: In conclusion, the statistically significant difference in serum TRF2 and agmatine levels between the groups may be associated with oxidative stress. It is thought that the shortening of telomere lengths may be associated with decreased TRF2 levels.

Lactoferrin improves symptoms of dextran sulfate sodium-induced colitis in mice through modulation of cellular senescence

The multifaceted effects of lactoferrin (LF) on the digestive and immune systems make it an attractive therapeutic option in inflammatory bowel diseases. In this study, we aimed to explore the anti-inflammatory effects of LF in colitis, particularly in relation to cellular senescence. We hypothesize that LF has the potential to modulate the senescence process. The effects of LF on senescence were tested in vitro using HCT116 and SW480 cell lines, and in vivo, the dextran sulfate sodium–induced mouse model of colitis. LF (500 mg/kg) alleviated symptoms of colitis in mice with a significant decrease in colon damage (P < .0001 vs. control) and microscopic (P < .05 vs. control) scores. Cellular senescence markers p16 and p21 were significantly upregulated in the mouse colon during inflammation (both P < .01 vs. control), and LF at 500 mg/kg decreased these markers (both P < .05 vs. dextran sulfate sodium–treated mice). In vitro, LF significantly affected the expression of p16 and p21 (P < .05–P < .0001 vs. control), senescence associated secretory phenotype (P < .01–P < .0001 vs. control), and telomere-specific proteins: telomeric repeat binding factor 1 and 2 (P < .05–P < .0001 vs. control) in a concentration-dependent manner. LF modulates the expression of cellular senescence markers and shows hallmarks of senolytic and pro-senescent activity, depending on dose. Further studies are needed to fully understand the anti-inflammatory effect of LF in the context of senescence and safe utilization in patients with inflammatory bowel diseases.

Proteomic Profiles and Protein Network Analysis of Primary Human Leukocytes Revealed Possible Clearance Biomarkers for Staphylococcus aureus Infection.

Staphylococcus aureus is a serious pathogen that can survive within host cells after a typical course of treatment completion, leading to chronic infection. Knowledge of host proteomic patterns after clearance of this pathogen from cells is limited. Here, we looked for S. aureus clearance biomarkers produced by in vitro-infected leukocytes. Extracellular proteins from primary human leukocytes infected with S. aureus ATCC 25923 were investigated as possible treatment-monitoring clearance biomarkers by applying a proteomics approach combining liquid chromatography with tandem mass spectrometry (LC-MS/MS) and protein interaction network analysis. It was found that the expression patterns of proteins secreted by S. aureus-infected leukocytes differed among stages of infection. Proteomic profiles showed that an ATPase, aminophospholipid transporter-like, Class I, type 8A, member 2 (ATP8A2) was expressed in the clearance stage and was not detected at any earlier stage or in uninfected controls. Protein network analysis showed that TERF2 (telomeric repeat-binding factor 2), ZNF440 (zinc finger protein 440), and PPP1R14A (phosphatase 1 regulatory subunit 14A) were up-regulated, while GLE1, an essential RNA-export mediator, was suppressed in both infection and clearance stages, suggesting their potential roles in S. aureus infection and clearance. These findings are the first to report that the ATP8A2 has potential as a clearance biomarker for S. aureus infection.

Endothelial senescence alleviates cognitive impairment in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is among the most prevalent age-related neurodegenerative diseases. Endothelial cell (EC) senescence was discovered in the AD brain, but its function in AD pathogenesis was unidentified. Here we created an AD mouse model with EC senescence (APP/PS1;TERF2DN mice) by intercrossing APP/PS1 mice with Tie2 promoter-driven dominant negative telomeric repeat-binding factor 2 transgenic mice (TERF2DN-Tg mice). We evaluated cognitive functions and AD brain pathology in APP/PS1;TERF2DN mice. Surprisingly, compared with the control APP/PS1 mice, APP/PS1;TERF2DN mice demonstrated the attenuation of cognitive impairment and amyloid-β (Aβ) pathology, accompanied by the compaction of Aβ plaques with increased microglial coverage and reduced neurite dystrophy. Moreover, we evaluated whether EC senescence could affect microglial morphology and phagocytosis of Aβ. Compared with wild-type mice, microglia in TERF2DN-Tg mice display increased numbers of endpoints (a morphometric parameter to quantify the number of processes) and Aβ phagocytosis and related gene expression. Single-cell RNA-sequencing analysis showed that compared with APP/PS1 mouse microglia, APP/PS1;TERF2DN mouse microglia displayed a modest decline in disease-associated microglia, accompanied by an altered direction of biological process branching from antigen synthesis and arrangement to ribonucleoprotein complex biogenesis. Our outcomes indicate that EC senescence alters microglia toward a protective phenotype with a rise in phagocytic and barrier roles, and may offer a clue to create a novel preventive/therapeutic method to treat AD.

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments.

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.

Non-canonical telomere protection role of FOXO3a of human skeletal muscle cells regulated by the TRF2-redox axis

Telomeric repeat binding factor 2 (TRF2) binds to telomeres and protects chromosome ends against the DNA damage response and senescence. Although the expression of TRF2 is downregulated upon cellular senescence and in various aging tissues, including skeletal muscle tissues, very little is known about the contribution of this decline to aging. We previously showed that TRF2 loss in myofibers does not trigger telomere deprotection but mitochondrial dysfunction leading to an increased level of reactive oxygen species. We show here that this oxidative stress triggers the binding of FOXO3a to telomeres where it protects against ATM activation, revealing a previously unrecognized telomere protective function of FOXO3a, to the best of our knowledge. We further showed in transformed fibroblasts and myotubes that the telomere properties of FOXO3a are dependent on the C-terminal segment of its CR2 domain (CR2C) but independent of its Forkhead DNA binding domain and of its CR3 transactivation domain. We propose that these non-canonical properties of FOXO3a at telomeres play a role downstream of the mitochondrial signaling induced by TRF2 downregulation to regulate skeletal muscle homeostasis and aging.

The neuro-protective role of telomerase via TERT/TERF-2 in the acute phase of spinal cord injury.

To investigate the interaction of telomerase activity and telomere length on neuro-protection or neuro-degeneration effects after spinal cord injury (SCI). A contusive SCI model was developed using 56 Sprague-Dawley rats. Seven rats were allocated into acute injury phase groups (1, 3, 8, 24, and 48h), and sub-acute and chronic injury phase groups (1, 2, and 4weeks). Telomerase activity was assessed by telomerase reverse transcriptase (TERT) and telomeric repeat binding factor-2 (TERF-2). Differentiation of activated neural stem cells was investigated by co-expression of neuronal/glial cell markers. Apoptosis expression was also investigated by caspase-3, 8, and 9 using terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Immunofluorescence staining and western blotting were performed for quantitative analyses. Expression of TERT increased gradually until 24h post-injury, and was decreased following SCI (P < 0.05). TERF-2 also was increased following SCI until 24h post-injury and then decreased with time (P < 0.05). Co-localization of TERT and TERF-2 was higher at 24h post-injury. High expression of TERT was seen in neurons (Neu N Ab), however, expression of TERT was relatively lower in astrocytes and oligodendrocytes. Apoptosis analysis showed persistent high expression of caspases-3, -9, and -8 during the observation period. Increased TERT and TERF-2 activity were noted 24h post-injury in the acute phase of SCI with TERF-2 maintaining telomeric-repeat length. Our results suggest that increased activity of telomere maintenance may be related to neuro-protective mechanisms against subsequent apoptosis resulting from DNA damage after acute SCI.

Telomere Targeting Chimera Enables Targeted Destruction of Telomeric Repeat-Binding Factor Proteins.

Telomeres are naturally shortened after each round of cell division in noncancerous normal cells, while the activation of telomerase activity to extend telomere in the cancer cell is essential for cell transformation. Therefore, telomeres are regarded as a potential anticancer target. In this study, we report the development of a nucleotide-based proteolysis-targeting chimera (PROTAC) designed to degrade TRF1/2 (telomeric repeat-binding factor 1/2), which are the key components of the shelterin complex (telosome) that regulates the telomere length by directly interacting with telomere DNA repeats. The prototype telomere-targeting chimeras (TeloTACs) efficiently degrade TRF1/2 in a VHL- and proteosome-dependent manner, resulting in the shortening of telomeres and suppressed cancer cell proliferation. Compared to the traditional receptor-based off-target therapy, TeloTACs have potential application in a broad spectrum of cancer cell lines due to their ability to selectively kill cancer cells that overexpress TRF1/2. In summary, TeloTACs provide a nucleotide-based degradation approach for shortening the telomere and inhibiting tumor cell growth, representing a promising avenue for cancer treatment.

Inhibition of TRF2 Leads to Ferroptosis, Autophagic Death, and Apoptosis by Causing Telomere Dysfunction.

Gastric cancer (GC) is an aggressive malignancy with a high mortality rate and poor prognosis. Telomeric repeat-binding factor 2 (TRF2) is a critical telomere protection protein. Emerging evidence indicates that TRF2 may be an essential treatment option for GC; however, the exact mechanism remains largely unknown. We aimed to explore the role of TRF2 in GC cells. The function and molecular mechanisms of TRF2 in the pathogenesis of GC were mainly discussed in this study. Relevant data from GEPIA and TCGA databases regarding TRF2 gene expression and its prognostic significance in GC samples were analyzed. Analysis of 53BP1 foci at telomeres by immunofluorescence, metaphase spreads, and telomere-specific FISH analysis was carried out to explore telomere damage and dysfunction after TRF2 depletion. CCK8 cell proliferation, trypan blue staining, and colony formation assay were performed to evaluate cell survival. Apoptosis and cell migration were determined with flow cytometry and scratch-wound healing assay, respectively. qRT-PCR and Western blotting were carried out to analyze the mRNA and protein expression levels after TRF2 depletion on apoptosis, autophagic death, and ferroptosis. By searching with GEPIA and TCGA databases, the results showed that the expression levels of TRF2 were obviously elevated in the samples of GC patients, which was associated with adverse prognosis. Knockdown of TRF2 suppressed the cell growth, proliferation, and migration in GC cells, causing significant telomere dysfunction. Apoptosis, autophagic death, and ferroptosis were also triggered in this process. The pretreatment of chloroquine (autophagy inhibitor) and ferrostatin-1 (ferroptosis inhibitor) improved the survival phenotypes of GC cells. Our data suggest that TRF2 depletion can inhibit cell growth, proliferation, and migration through the combined action of ferroptosis, autophagic death, and apoptosis in GC cells. The results indicate that TRF2 might be used as a potential target to develop therapeutic strategies for treating GC.

Arabidopsis TRB proteins function in H3K4me3 demethylation by recruiting JMJ14

Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.

TERRA G-quadruplex stabilization as a new therapeutic strategy for multiple myeloma

BackgroundMultiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability, and telomere dysfunction is an important cause of acquired genomic alterations. Telomeric repeat-containing RNA (TERRA) transcripts are long non-coding RNAs involved in telomere stability through the interaction with shelterin complex. Dysregulation of TERRAs has been reported across several cancer types. We recently identified a small molecule, hit 17, which stabilizes the secondary structure of TERRA. In this study, we investigated in vitro and in vivo anti-MM activities of hit 17.MethodsAnti-proliferative activity of hit 17 was evaluated in different MM cell lines by cell proliferation assay, and the apoptotic process was analyzed by flow cytometry. Gene and protein expressions were detected by RT-qPCR and western blotting, respectively. Microarray analysis was used to analyze the transcriptome profile. The effect of hit 17 on telomeric structure was evaluated by chromatin immunoprecipitation. Further evaluation in vivo was proceeded upon NCI-H929 and AMO-1 xenograft models.ResultsTERRA G4 stabilization induced in vitro dissociation of telomeric repeat‐binding factor 2 (TRF2) from telomeres leading to the activation of ATM-dependent DNA damage response, cell cycle arrest, proliferation block, and apoptotic death in MM cell lines. In addition, up-regulation of TERRA transcription was observed upon DNA damage and TRF2 loss. Transcriptome analysis followed by gene set enrichment analysis (GSEA) confirmed the involvement of the above-mentioned processes and other pathways such as E2F, MYC, oxidative phosphorylation, and DNA repair genes as early events following hit 17-induced TERRA stabilization. Moreover, hit 17 exerted anti-tumor activity against MM xenograft models.ConclusionOur findings provide evidence that targeting TERRA by hit 17 could represent a promising strategy for a novel therapeutic approach to MM.

In silico exploration and molecular dynamics of deleterious SNPs on the human TERF1 protein triggering male infertility

By limiting chromosome erosion and end-to-end fusions, telomere integrity is critical for chromosome stability and cell survival. During mitotic cycles or due to environmental stresses, telomeres become progressively shorter and dysfunctional, thus triggering cellular senescence, genomic instability and cell death. To avoid such consequences, the telomerase action, as well as the Shelterin and CST complexes, assure the telomere’s protection. Telomeric repeat binding factor 1 (TERF1), which is one of the primary components of the Shelterin complex, binds directly to the telomere and controls its length and function by regulating the telomerase activity. Several reports about TERF1 gene variations have been associated with different diseases, and some of them have linked these variations to male infertility. Hence, this paper can be advantageous to investigate the association between the missense variants of the TERF1 gene and the susceptibility to male infertility. The stepwise prediction of SNPs pathogenicity followed in this study was based on stability and conservation analysis, post-translational modification, secondary structure, functional interaction prediction, binding energy evaluation and finally molecular dynamic simulation. Prediction matching among the tools revealed that out of 18 SNPs, only four (rs1486407144, rs1259659354, rs1257022048 and rs1320180267) were predicted as the most damaging and highly deleterious SNPs affecting the TERF1 protein and its molecular dynamics when interacting with the TERB1 protein by influencing the function, structural stability, flexibility and compaction of the overall complex. Interestingly, these polymorphisms should be considered during genetic screening so they can be used effectively as genetic biomarkers for male infertility diagnosis. Communicated by Ramaswamy H. Sarma

Generation of telomeric repeat binding factor 1 (TRF1)-knockout human embryonic stem cell lines, KRIBBe010-A-95, KRIBBe010-A-96, and KRIBBe010-A-97, using CRISPR/Cas9 technology

Telomeric repeat binding factor 1 (TRF1) plays an essential role in maintaining telomere length. Here, we established TRF1-knockout human pluripotent stem cells (hPSCs; hTRF1-KO) using the CRISPR/Cas9 technology. The hTRF1-KO cell lines expressed pluripotency markers and demonstrated a normal karyotype (46, XX) and DNA profile. In addition, hTRF1-KOcells spontaneously differentiated into all three germ layers in vitro. Thus, these cell lines could be useful models in various research fields.

TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived fromhuman iPSCs.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of DMD cardiomyocytes. Here, we test whether prevention of telomere attrition is possible in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSC-CMs) and if preventing telomere shortening impacts cardiomyocyte function. We observe reduced cell size, nuclear size, and sarcomere density in DMD iPSC-CMs compared with healthy isogenic controls. We find that expression of just one telomere-binding protein, telomeric repeat-binding factor 2 (TRF2), a core component of the shelterin complex, prevents telomere attrition and rescues deficiencies in cell size as well as sarcomere density. We employ a bioengineered platform to micropattern cardiomyocytes for calcium imaging and perform Southern blots of telomere restriction fragments, the gold standard for telomere length assessments. Importantly, preservation of telomere lengths in DMD cardiomyocytes improves their viability. These data provide evidence that preventing telomere attrition ameliorates deficits in cell morphology, activation of the DNA damage response, and premature cell death, suggesting that TRF2 is a key player in DMD-associated cardiac failure.

Identification of new telomere- and telomerase-associated autoantigens in systemic sclerosis

PurposeIn up to 20% of patients with systemic sclerosis (SSc) no known autoantibody specificity can be identified. Recently discovered autoantigens, such as telomeric repeat binding factor 1 (TERF1), as well as established autoantigens, like RuvBL1/2, are associated with telomere and telomerase biology. We aimed to identify new telomere- and telomerase-associated autoantigens in patients with SSc without known autoantibody specificity. MethodsUnlabelled protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS) was performed with sera of 106 patients with SSc from two tertiary referral centres that had a nuclear pattern on HEp-2 indirect immunofluorescence without previously identified autoantibody. Telomere- or telomerase-associated proteins or protein complexes precipitated by individual sera were identified. Candidate autoantigens were confirmed through immunoprecipitation-western blot (IP-WB). A custom Luminex xMAP assay for 5 proteins was evaluated with sera from persons with SSc (n = 467), other systemic autoimmune rheumatic diseases (n = 923), non-rheumatic disease controls (n = 187) and healthy controls (n = 199). ResultsEight telomere- and telomerase-associated autoantigens were identified in a total of 11 index patients, including the THO complex (n = 3, all with interstitial lung disease and two with cardiac involvement), telomeric repeat-binding factor 2 (TERF2, n = 1), homeobox-containing protein 1 (HMBOX1, n = 2), regulator of chromosome condensation 1 (RCC1, n = 1), nucleolar and coiled-body phosphoprotein 1 (NOLC1, n = 1), dyskerin (DKC1, n = 1), probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2, n = 1) and nuclear valosin-containing protein-like (NVL, n = 2). A Luminex xMAP assay for THO complex subunit 1 (THOC1), TERF2, NOLC1, NOP2 and NVL revealed high reactivity in all index patients, but also in other patients with SSc and disease controls. However, the reactivity by xMAP assay in these other patients was not confirmed by IP-WB. ConclusionIP-MS revealed key telomere- and telomerase-associated proteins and protein complexes as autoantigens in patients with SSc.

ZNF281 drives hepatocyte senescence in alcoholic liver disease by reducing HK2-stabilizedPINK1/Parkin-mediated mitophagy.

We investigated the role of zinc-finger protein 281 (ZNF281), a novel molecule, in ethanol-induced hepatocyte senescence and uncovered the potential mechanism. Real-time PCR, Western blot, immunofluorescence staining, and enzyme-linked immunosorbent assay were performed to explore the role of ZNF281 in hepatocyte senescence. ZNF281 expression was upregulated in both alcohol-fed mice livers and ethanol-treated hepatocytes. Silence of ZNF281 in hepatocytes using siRNA mitigated ethanol-caused decrease in cell viability and increased release of aspartate aminotransferase, alanine transaminase, and lactate dehydrogenase. ZNF281 siRNA reduced senescence-associated β-galactosidase-positive cells under ethanol exposure, abolished cell cycle arrest at G0/G1 phase, and diminished senescence-associated secretory phenotype and proinflammatory cytokines (IL-1β and IL-6) release. At molecular level, ZNF281 deficiency altered the expression profile of senescence-associated proteins including p53, p21, p16, high mobility group AT-hook 1, and phospho-histone H2A.X and telomerase-associated regulatory factors including telomerase reverse transcriptase, telomeric repeat binding factor 1 (TRF1), and TRF2. ZNF281 knockdown promoted hepatocyte recovery from ethanol-induced mitochondrial dysfunction and ROS production, which was correlated with rescuing HK2-PINK1/Parkin signalling-mediated mitophagy. Mechanistically, ZNF281 directly bound to 5'-GGCGGCGGGCGG-3' motif within HK2 promoter region and transcriptionally repressed HK2 expression. Systematic ZNF281 knockdown by adeno-associated virus encoding ZNF281 shRNA protected mice from alcohol feeding-caused hepatocyte injury and senescence. This study provides a novel factor ZNF281 as a driver of hepatocyte senescence during alcoholic liver disease.

Midazolam alleviates cellular senescence in SH-SY5Y neuronal cells in Alzheimer's disease.

Alzheimer's disease (AD) impacts the daily life of aging people. Oligomerized amyloid β (Aβ)-associated neuronal senescence is involved in the pathological mechanism of AD. Blockage of neuronal senescence has been considered an important strategy for the treatment of AD. Midazolam is a hypnotic-sedative drug with pleiotropic properties. However, the effects of Midazolam in oligomerized Aβ1.42 -induced neurotoxicity have not been reported previously. Here, we investigate whether Midazolam possesses a beneficial effect against oligomerized Aβ1.42 in SH-SY5Y neuronal cells. Cellular senescence was assessed using senescence-associated β-galactosidase staining. Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA. First, the lactate dehydrogenase release assay demonstrates that 10 and 20 µM are the optimal concentrations of Midazolam used for cell cultures. Senescence-associated β-galactosidase staining results indicate that exposure to oligomerized Aβ1.42 significantly increased cellular senescence of SH-SY5Y cells, but it was significantly alleviated by Midazolam. Additionally, Midazolam restored the oligomerized Aβ1.42 -induced reduction of telomerase activity. Interestingly, we found that oligomerized Aβ1.42 remarkably reduced human telomerase reverse transcriptase (hTERT) gene expression but increased the telomeric repeat-binding factor 2 (TERF2) expression. However, treatment with Midazolam reversed the effects of oligomerized Aβ1.42 on the hTERT and TERF2 gene expressions. Importantly, the presence of Midazolam attenuated Aβ1.42 -induced p53 and p21 expressions. Mechanistically, Midazolam repressed the level of cyclooxygenase-2 (COX-2) and the release of prostaglandin E2. Importantly, overexpression of COX-2 abolished the impact of Midazolam against oligomerized Aβ1.42 in neuronal senescence. We conclude that the usage of Midazolam is a potential treatment strategy for AD.

MiR-182-3p targets TRF2 and impairs tumor growth of triple-negative breast cancer.

The telomeric repeat-binding factor 2 (TRF2) is a telomere-capping protein that plays a key role in the maintenance of telomere structure and function. It is highly expressed in different cancer types, and it contributes to cancer progression. To date, anti-cancer strategies to target TRF2 remain a challenge. Here, we developed a miRNA-based approach to reduce TRF2 expression. By performing a high-throughput luciferase screening of 54 candidate miRNAs, we identified miR-182-3p as a specific and efficient post-transcriptional regulator of TRF2. Ectopic expression of miR-182-3p drastically reduced TRF2 protein levels in a panel of telomerase- or alternative lengthening of telomeres (ALT)-positive cancer cell lines. Moreover, miR-182-3p induced DNA damage at telomeric and pericentromeric sites, eventually leading to strong apoptosis activation. We also observed that treatment with lipid nanoparticles (LNPs) containing miR-182-3p impaired tumor growth in triple-negative breast cancer (TNBC) models, including patient-derived tumor xenografts (PDTXs), without affecting mouse survival or tissue function. Finally, LNPs-miR-182-3p were able to cross the blood-brain barrier and reduce intracranial tumors representing a possible therapeutic option for metastatic brain lesions.

Abstract 15649: Terf2ip K240 Sumoylation Drives Endothelial Senescence and Atherogenesis via Activating Parp1

The goal of this study: Telomeric repeat-binding factor 2 (TRF2) interacting protein 1 (TERF2IP) not only associates with TRF2 to protect telomeres but also associates with RSK to promote senescence. The later has been detected in endothelial cells (ECs) under disturbed flow (DF), which involved TERF2IP S205 phosphorylation. Besides, it has been suggested that TERF2IP K240 can be SUMOylated; however, its role in senescence and atherogenesis remains unknown. Methods: We investigated the functional role of TERF2IP K240 SUMOylation utilizing a model of DF-induced atherogenesis in TERF2IP K237R knock-in (KI) (mouse K237 is equivalent to human K240) mouse carotid artery by partial ligation and RNA isolated from mouse lung ECs. Results: TERF2IP K240 SUMOylation was increased in ECs under DF and provoked telomere shortening-associated senescence. TERF2IP K240 SUMOylation occurred independently of TERF2IP S205 phosphorylation although both events were blunted by the RSK specific inhibitor (FMK-MEA), and EC senescence as well as related events were abolished by both depletion and point mutation of TERF2IP at S205 or K240 residue. Whereas point mutation of TERF2IP at S205 prevented RSK-associated NF-kB activation, that of K240 exhibited no effect, suggesting that K240 SUMOylation did not involve in EC inflammation. By inducing persistent telomere DNA damage, TERF2IP K240 SUMOylation distinctive provoked telomere shortening-associated EC senescence via inducing persistent DNA damage response and subsequent PARP1 activation. Finally, a mouse model of DF-induced atherogenesis showed that atherogenesis was inhibited in TER2IP K237R KI mice although body weight and cholesterol level were unaltered Discussions: These data have revealed that EC senescence plays a critical role in atherogenesis and that TERF2IP K240 SUMOylation can be a novel therapeutic target for atherogenesis.

Telomere Length and Development of Systemic Lupus Erythematosus: A Mendelian Randomization Study.

Previous observational studies demonstrated that a subset of patients with systemic lupus erythematosus (SLE) have markedly short telomere length in leukocytes. This study was undertaken to test whether leukocyte telomere length is causally associated with risk of SLE. A 2-sample Mendelian randomization (MR) analysis was conducted to estimate causality of telomere length on SLE in European populations. A replication 2-sample MR study using Asian genetic data was also conducted. A reverse MR analysis was then performed to test the effects of SLE on telomere length. The autoantibodies targeting telomere-associated protein (telomeric repeat-binding factor 1 [TERF1] autoantibodies) were detected in patients with SLE, healthy controls, and patients with rheumatoid arthritis. The results of the inverse variance-weighted method (odds ratio [OR] 2.96 [95% confidence interval (95% CI) 1.58-5.55], P < 0.001) showed strong evidence for a causal relationship between longer telomere length and risk of SLE in people with European ancestry. The outcomes of MR-Egger regression analysis (OR 29.46 [95% CI 3.02-287.60], P=0.033) and MR pleiotropy residual sum and outlier analysis (OR 3.62 [95% CI 2.03-6.46], P=0.002) also showed that longer telomere length was significantly associated with increased risk of SLE in a European population. Sensitivity analyses using different methods and summary data sets showed that the results were still broadly consistent. A replication MR study using Asian genetic data yielded similar findings. However, the reverse MR analysis showed that genetically predicted SLE was not causally associated with telomere length. In addition, we found that TERF1 autoantibodies were present in 2 of 40 SLE patients (5.0%). In contrast with previous observational studies, MR analyses show that longer telomere length is significantly associated with increased risk of SLE.