Morphine treatment is associated with diminished telomere length together with down-regulated TERT and TERF2 mRNA levels

ABSTRACT Telomeres, the chromosome ends, are maintained by a balance of activities that erode and replace the terminal DNA sequences. Furthermore, telomere-proximal genes are often silenced in an epigenetic manner. In Saccharomyces cerevisiae, average telomere length and telomeric silencing are reduced by loss of function of UPF genes required in the nonsense-mediated mRNA decay (NMD) pathway. Because NMD controls the mRNA levels of several hundred wild-type genes, we tested the hypothesis that NMD affects the expression of genes important for telomere functions. In upf mutants, high-density oligonucleotide microarrays and Northern blots revealed that the levels of mRNAs were increased for genes encoding the telomerase catalytic subunit (Est2p), in vivo regulators of telomerase (Est1p, Est3p, Stn1p, and Ten1p), and proteins that affect telomeric chromatin structure (Sas2p and Orc5p). We investigated whether overexpressing these genes could mimic the telomere length and telomeric silencing phenotypes seen previously in upf mutant strains. Increased dosage of STN1, especially in combination with increased dosage of TEN1, resulted in reduced telomere length that was indistinguishable from that in upf mutants. Increased levels of STN1 together with EST2 resulted in reduced telomeric silencing like that of upf mutants. The half-life of STN1 mRNA was not altered in upf mutant strains, suggesting that an NMD-controlled transcription factor regulates the levels of STN1 mRNA. Together, these results suggest that NMD maintains the balance of gene products that control telomere length and telomeric silencing primarily by maintaining appropriate levels of STN1, TEN1, and EST2 mRNA.

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Telomere dynamics during aging in polygenic left ventricular hypertrophy

Physiological Genomics ◽

10.1152/physiolgenomics.00083.2015 ◽

2016 ◽

Vol 48 (1) ◽

pp. 42-49 ◽

Cited By ~ 11

Author(s):

Francine Z. Marques ◽

Scott A. Booth ◽

Priscilla R. Prestes ◽

Claire L. Curl ◽

Lea M. D. Delbridge ◽

...

Keyword(s):

Heart Failure ◽

Left Ventricular Hypertrophy ◽

Cardiac Hypertrophy ◽

Telomere Length ◽

Ventricular Hypertrophy ◽

Telomerase Activity ◽

Left Ventricular ◽

Mrna Levels ◽

Control Strain ◽

Increased Risk

Short telomeres are associated with increased risk of cardiovascular disease. Here we studied cardiomyocyte telomere length at key ages during the ontogeny of cardiac hypertrophy and failure in the hypertrophic heart rat (HHR) and compared these with the normal heart rat (NHR) control strain. Key ages corresponded with the pathophysiological sequence beginning with fewer cardiomyocytes (2 days), leading to left ventricular hypertrophy (LVH) (13 wk) and subsequently progression to heart failure (38 wk). We measured telomere length, tissue activity of telomerase, mRNA levels of telomerase reverse transcriptase ( Tert) and telomerase RNA component ( Terc), and expression of the telomeric regulator microRNA miR-34a. Cardiac telomere length was longer in the HHR compared with the control strain at 2 days and 38 wk, but shorter at 13 wk. Neonatal HHR had higher cardiac telomerase activity and expression of Tert and miR-34a. Telomerase activity was not different at 13 or 38 wk. Tert mRNA and Terc RNA were overexpressed at 38 wk, while miR-34a was overexpressed at 13 wk but downregulated at 38 wk. Circulating leukocytes were strongly correlated with cardiac telomere length in the HHR only. The longer neonatal telomeres in HHR are likely to reflect fewer fetal and early postnatal cardiomyocyte cell divisions and explain the reduced total cardiomyocyte complement that predisposes to later hypertrophy and failure. Although shorter telomeres were a feature of cardiac hypertrophy at 13 wk, they were not present at the progression to heart failure at 38 wk.

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Genetic Variants of the TERT Gene, Telomere Length, and Circulating TERT as Prognostic Markers in Rectal Cancer Patients

Cancers ◽

10.3390/cancers12113115 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3115

Author(s):

Enrica Rampazzo ◽

Erika Cecchin ◽

Paola Del Bianco ◽

Chiara Menin ◽

Gaya Spolverato ◽

...

Keyword(s):

Rectal Cancer ◽

Telomere Length ◽

Cancer Patients ◽

Neoadjuvant Chemoradiotherapy ◽

Response To Therapy ◽

Disease Outcome ◽

Mrna Levels ◽

Tert Gene ◽

Low Levels ◽

Telomere Erosion

Single-nucleotide polymorphisms (SNPs) in the TERT gene can affect telomere length and TERT expression and have been associated with risk and/or outcome for several tumors, but very few data are available about their impact on rectal cancer. Eight SNPs (rs2736108, rs2735940, rs2736098, rs2736100, rs35241335, rs11742908, rs2736122 and rs2853690), mapping in regulatory and coding regions of the TERT gene, were studied in 194 rectal cancer patients to evaluate their association with constitutive telomere length, circulating TERT mRNA levels, response to neoadjuvant chemoradiotherapy (CRT) and disease outcome. At diagnosis, the rs2736100CC genotype was associated with longer telomeres measured pre-CRT, while the rs2736100CC, rs2736108TT and rs2735940AA were associated with greater telomere erosion evaluated post-CRT. The rs2736108CC and rs2853690AA/GG genotypes, respectively associated with lower telomere erosion and lower levels of circulating TERT post-CRT, were also independently associated with a better response to therapy [OR 4.6(1.1–19.1) and 3.0(1.3–6.9)]. Overall, post-CRT, low levels (≤ median value) of circulating TERT and its stable/decreasing levels compared to those pre-CRT, were independently associated with a better response to therapy [OR 5.8(1.9–17.8) and 5.3(1.4–19.4), respectively]. Furthermore, post-CRT, patients with long telomeres (>median value) and low levels of circulating TERT had a significantly lower risk of disease progression [HR 0.4(0.1–0.9) and 0.3(0.1–0.8), respectively]. These findings suggest that TERT SNPs could be a useful tool for improving the selection of patients who could benefit from CRT and support the role of telomere length and circulating TERT mRNA levels as useful markers for monitoring the response to therapy and disease outcome in rectal cancer patients.

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Habenula kisspeptin retrieves morphine impaired fear memory in zebrafish

Scientific Reports ◽

10.1038/s41598-020-76287-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Mageswary Sivalingam ◽

Satoshi Ogawa ◽

Ishwar S. Parhar

Keyword(s):

Fear Memory ◽

Treated Group ◽

Mrna Levels ◽

Morphine Treatment ◽

Conditioned Place Avoidance ◽

Serotonin System ◽

Total Brain ◽

Evolutionarily Conserved ◽

Place Avoidance

Abstract The habenula is an evolutionarily conserved brain structure, which has recently been implicated in fear memory. In the zebrafish, kisspeptin (Kiss1) is predominantly expressed in the habenula, which has been implicated as a modulator of fear response. Hence, in the present study, we questioned whether Kiss1 has a role in fear memory and morphine-induced fear memory impairment using an odorant cue (alarm substances, AS)-induced fear avoidance paradigm in adult zebrafish, whereby the fear-conditioned memory can be assessed by a change of basal place preference (= avoidance) of fish due to AS-induced fear experience. Subsequently, to examine the possible role of Kiss1 neurons-serotonergic pathway, kiss1 mRNA and serotonin levels were measured. AS exposure triggered fear episodes and fear-conditioned place avoidance. Morphine treatment followed by AS exposure, significantly impaired fear memory with increased time-spent in AS-paired compartment. However, fish administered with Kiss1 (10–21 mol/fish) after morphine treatment had significantly lower kiss1 mRNA levels but retained fear memory. In addition, the total brain serotonin levels were significantly increased in AS- and Kiss1-treated groups as compared to control and morphine treated group. These results suggest that habenular Kiss1 might be involved in consolidation or retrieval of fear memory through the serotonin system.

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103 ECTOPIC EXPRESSION OF TELOMERASE IN BOVINE PREIMPLANTATION EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab103 ◽

2008 ◽

Vol 20 (1) ◽

pp. 132

Author(s):

K. Iqbal ◽

W. A. Kues ◽

H. Niemann

Keyword(s):

Telomere Length ◽

Fluorescent Protein ◽

Ectopic Expression ◽

Blastocyst Stage ◽

Mrna Levels ◽

Bovine Embryos ◽

Cell Stage ◽

Fluid Medium ◽

Gfp Fluorescence ◽

Morula Stage

Recently, we have demonstrated a stage-specific increase of telomere length at morula-blastocyst transition in bovine and murine embryos (Schaetzlein et al. 2004 PNAS 101, 8034–8038). Telomeres are composed of repetitive hexanucleotide sequences (TTAGGG) encompassing several kilobasepairs and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage up to the morula stage, telomerase is not active or only found at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) was selected as the target molecule due to its 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from local abattoir followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. In preliminary tests, it was shown that co-injection of green fluorescent protein (GFP) and red fluorescent protein (dsRed) constructs resulted in the simultaneous expression of the 2 proteins. The onset of fluorescent protein expression was recorded 30 to 40 hours after injection by fluorescence microscopy. Because all cDNA were driven by the cytomegalovirus (CMV) promoter, it was assumed that hTERT is expressed in parallel. In the main experiment, 2 constructs encoding human TERT and GFP were co-injected to allow live separation of embryos. A total of 400 bovine embryos were injected, 209 (53%) of the treated embryos showed specific GFP fluorescence; out of a total of 104 blastocysts (26%), 55 showed GFP fluorescence (53%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic human TERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. Surprisingly, expression pattern of the endogenous TERT revealed a transient increase at the 2–4 cell stage and a major increase at the blastocyst stage. The mRNA level of the ectopic hTERT started to rise from 4 to 8 cell stage and remained high up to the morula stage. Currently, qFISH and TRAP techniques are being employed to assess enzymatic activity of hTERT and to quantitatively determine telomere length. In conclusion, this study demonstrates the ectopic expression of TERT and fluorescent proteins in early embryos; overexpression of TERT may facilitate the derivation of bovine ES cells. Supported by DFG and Goyaike S.A.A.C.I. Y.F.

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277 PLASMID MEDIATED EXPERIMENTAL TELOMERE EXTENSION IN BOVINE EMBRYOS BY ECTOPIC EXPRESSION OF HUMAN TERT

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab277 ◽

2009 ◽

Vol 21 (1) ◽

pp. 235 ◽

Cited By ~ 1

Author(s):

K. Iqbal ◽

W. A. Kues ◽

H. Niemann

Keyword(s):

Telomere Length ◽

Ectopic Expression ◽

Embryonic Stem ◽

Blastocyst Stage ◽

Human Embryos ◽

Mrna Levels ◽

Bovine Embryos ◽

Fluid Medium ◽

Gfp Fluorescence ◽

Morula Stage

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situ hybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells. Supported by DFG and Goyaike SAACIYF.

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Cholecystokinin (CCK)-like material and CCK mRNA levels in the rat brain and spinal cord after acute or repeated morphine treatment

Neuropeptides ◽

10.1016/0143-4179(92)90044-w ◽

1992 ◽

Vol 21 (3) ◽

pp. 193-200 ◽

Cited By ~ 14

Author(s):

M. Pohl ◽

E. Collin ◽

J.J. Benoliel ◽

S. Bourgoin ◽

F. Cesselin ◽

...

Keyword(s):

Spinal Cord ◽

Rat Brain ◽

Mrna Levels ◽

Morphine Treatment ◽

Brain And Spinal Cord

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Differential effects of acute and repeated morphine treatment on κ-opioid receptor mRNA levels in mesocorticolimbic system

Pharmacological Reports ◽

10.1016/s1734-1140(12)70786-7 ◽

2012 ◽

Vol 64 (2) ◽

pp. 445-448 ◽

Cited By ~ 4

Author(s):

Yu Gang ◽

Yan Hui ◽

Gong Ze-Hui

Keyword(s):

Opioid Receptor ◽

Mrna Levels ◽

Morphine Treatment ◽

Differential Effects ◽

Mesocorticolimbic System ◽

Receptor Mrna

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Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event

Journal of Applied Physiology ◽

10.1152/japplphysiol.00997.2011 ◽

2012 ◽

Vol 112 (5) ◽

pp. 773-781 ◽

Cited By ~ 28

Author(s):

Matthew J. Laye ◽

Thomas P. J. Solomon ◽

Kristian Karstoft ◽

Karin K. Pedersen ◽

Susanne D. Nielsen ◽

...

Keyword(s):

Skeletal Muscle ◽

Telomere Length ◽

Mononuclear Cells ◽

Physiological Stress ◽

Telomerase Activity ◽

Mrna Levels ◽

Htert Mrna ◽

Peripheral Blood Mononuclear ◽

Shelterin Complex ◽

Blood Mononuclear Cells

Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects ( n = 8, mean age = 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 ( P < 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P < 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4+ or CD8+ cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle.

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