Increased telomerase improves motor function and alpha-synuclein pathology in a transgenic mouse model of Parkinson's disease associated with enhanced autophagy

Protective effects of the telomerase protein TERT have been shown in neurons and brain. We previously demonstrated that TERT protein can accumulate in mitochondria of Alzheimer’s disease (AD) brains and protect from pathological tau in primary mouse neurons. This prompted us to employ telomerase activators in order to boost telomerase expression in a mouse model of Parkinson’s disease (PD) overexpressing human wild type α-synuclein. Our aim was to test whether increased Tert expression levels were able to ameliorate PD symptoms and to activate protein degradation. We found increased Tert expression in brain for both activators which correlated with a substantial improvement of motor functions such as gait and motor coordination while telomere length in the analysed region was not changed. Interestingly, only one activator (TA-65) resulted in a decrease of reactive oxygen species from brain mitochondria. Importantly, we demonstrate that total, phosphorylated and aggregated α-synuclein were significantly decreased in the hippocampus and neocortex of activator-treated mice corresponding to enhanced markers of autophagy suggesting an improved degradation of toxic alpha-synuclein. We conclude that increased Tert expression caused by telomerase activators is associated with decreased α-synuclein protein levels either by activating autophagy or by preventing or delaying impairment of degradation mechanisms during disease progression. This encouraging preclinical data could be translated into novel therapeutic options for neurodegenerative disorders such as PD

Feedback between p21 and reactive oxygen production is necessary for cell senescence

The sustained activation of CDKN1A (p21/Waf1/Cip1) by a DNA damage response induces mitochondrial dysfunction and reactive oxygen species (ROS) production via signalling through CDKN1A-GADD45A-MAPK14- GRB2-TGFBR2-TGFbeta in senescing primary human and mouse cells in vitro and in vivo.Enhanced ROS production in senescing cells generates additional DNA damage. Although this damage is repairable and transient, it elevates the average levels of DNA damage response permanently, thus forming a positive feedback loop.This loop is necessary and sufficient to maintain the stability of growth arrest until a ‘point of no return' is reached during establishment of senescence

A Stochastic Step Model of Replicative Senescence Explains ROS Production Rate in Ageing Cell Populations

Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells

Preterm infants have significantly longer telomeres than their term born counterparts

There are well-established morbidities associated with preterm birth including respiratory, neurocognitive and developmental disorders. However several others have recently emerged that characterise an `aged' phenotype in the preterm infant by term-equivalent age. These include hypertension, insulin resistance and altered body fat distribution. Evidence shows that these morbidities persist into adult life, posing a significant public health concern. In this study, we measured relative telomere length in leukocytes as an indicator of biological ageing in 25 preterm infants at term equivalent age. Comparing our measurements with those from 22 preterm infants sampled at birth and from 31 term-born infants, we tested the hypothesis that by term equivalent age, preterm infants have significantly shorter telomeres (thus suggesting that they are prematurely aged). Our results demonstrate that relative telomere length is highly variable in newborn infants and is significantly negatively correlated with gestational age and birth weight in preterm infants. Further, longitudinal assessment in preterm infants who had telomere length measurements available at both birth and term age (n = 5) suggests that telomere attrition rate is negatively correlated with increasing gestational age. Contrary to our initial hypothesis however, relative telomere length was significantly shortest in the term born control group compared to both preterm groups and longest in the preterm at birth group. In addition, telomere lengths were not significantly different between preterm infants sampled at birth and those sampled at term equivalent age. These results indicate that other, as yet undetermined, factors may influence telomere length in the preterm born infant and raise the intriguing hypothesis that as preterm gestation declines, telomere attrition rate increases

Acute exercise leads to regulation of Telomere-Associated genes and MicroRNA expression in immune Cells

Telomeres are specialized nucleoprotein structures that protect chromosomal ends from degradation. These structures progressively shorten during cellular division and can signal replicative senescence below a critical length. Telomere length is predominantly maintained by the enzyme telomerase. Significant decreases in telomere length and telomerase activity are associated with a host of chronic diseases; conversely their maintenance underpins the optimal function of the adaptive immune system. Habitual physical activity is associated with longer leukocyte telomere length; however, the precise mechanisms are unclear. Potential hypotheses include regulation of telomeric gene transcription and/or microRNAs (miRNAs). We investigated the acute exercise-induced response of telomeric genes and miRNAs in twenty-two healthy males (mean age = 24.1±1.55 years). Participants undertook 30 minutes of treadmill running at 80% of peak oxygen uptake. Blood samples were taken before exercise, immediately post-exercise and 60 minutes post-exercise. Total RNA from white blood cells was submitted to miRNA arrays and telomere extension mRNA array. Results were individually validated in white blood cells and sorted T cell lymphocyte subsets using quantitative real-time PCR (qPCR). Telomerase reverse transcriptase (TERT) mRNA (P = 0.001) and sirtuin-6 (SIRT6) (P<0.05) mRNA expression were upregulated in white blood cells after exercise. Fifty-six miRNAs were also differentially regulated post-exercise (FDR <0.05). In silico analysis identified four miRNAs (miR-186, miR-181, miR-15a and miR-96) that potentially targeted telomeric gene mRNA. The four miRNAs exhibited significant upregulation 60 minutes post-exercise (P<0.001). Telomeric repeat binding factor 2, interacting protein (TERF2IP) was identified as a potential binding target for miR-186 and miR-96 and demonstrated concomitant downregulation (P<0.01) at the corresponding time point. Intense cardiorespiratory exercise was sufficient to differentially regulate key telomeric genes and miRNAs in white blood cells. These results may provide a mechanistic insight into telomere homeostasis and improved immune function and physical health. Funding NHMR

Aletris aurea

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells

Inorganic nitrate supplementation in young and old obese adults does not affect acute glucose and insulin responses but lowers oxidative stress

© 2016 American Society for Nutrition. Background: Aging and obesity are associated with raised oxidative stress and a reduction of nitric oxide (NO) bioavailability, with subsequent decline in insulin sensitivity and endothelial function. Inorganic nitrate is converted into NO via a 2-step reduction process and may be an effective nutritional intervention to modify vascular and metabolic functions. Objectives: This study tested whether inorganic nitrate supplementation improved glucose disposal and attenuated the acute effects of hyperglycemia on oxidative stress, inflammation, and vascular function in young and old obese participants. Methods: Ten young (aged 18-44 y) and 10 old (aged 55-70 y) obese participants consumed 75 g glucose followed by either potassium nitrate (7 mg/kg body weight) or potassium chloride (placebo) in a randomized, double-blind crossover design. Resting blood pressure (BP), endothelial function, and blood biomarkers were measured for 3 h postintervention. Biomarkers included plasma nitrate/nitrite (NOx), glucose, insulin, cyclic GMP, interleukin 6, 3-nitrotyrosine, E- and P-selectins, intercellular adhesion molecule 3 (ICAM-3), and thrombomodulin, as well as superoxide in freshly isolated peripheral blood mononuclear cells (PBMCs). Results: Inorganic nitrate supplementation did not affect plasma glucose (P = 0.18) or insulin (P = 0.26) responses. The increase in plasma NOx concentrations 3 h after the administration of inorganic nitrate was significantly higher in young than in old participants (234% increase compared with 149% increase, respectively, P < 0.001). Plasma 3-nitrotyrosine concentrations declined significantly after inorganic nitrate supplementation compared with placebo (3 h postdose, 46% decrease compared with 27% increase, respectively, P = 0.04), and a similar nonsignificant trend was observed for superoxide concentrations (3 h postdose, 16% decrease compared with 23% increase, respectively, P = 0.06). Plasma cyclic GMP, ICAM-3, and thrombomodulin concentrations differed between young and old participants (P < 0.01). Inorganic nitrate supplementation did not improve BP or endothelial function. Conclusions: Oral supplementation with inorganic nitrate did not improve glucose and insulin responses but reduced oxidative stress in old individuals during acute hyperglycemia

Mitochondrial TERT protects from apoptosis induction after H₂O₂ treatment and X-irradiation compared to cells transfected with nuclear TERT.

<p>Representative images of activated caspase 3 (shown in red) in <b>A</b>: Hela, <b>B</b>: MRC/SV40, <b>C</b>: U87 cells transfected with mito TERT and nuclear TERT (myc-tag, shown in green) after 400 µM H₂O₂ treatment for 3 h or irradiation with 20 Gy. <b>D</b>: Quantification of the percentage of apoptotic cells of the 3 cell lines after H₂O₂ treatment, E: Quantification of the percentage of apoptotic cells of the 3 cell lines after X-irradiation. Bars present mean and standard error from around 45 transfected cells per condition and cell line. * p<0.05.</p

Nuclear TERT localization correlates with high DNA damage levels after treatment with H₂O₂

<p><b>while mitochondrial telomerase prevents it. A–C:</b> Representative images of TERT localization (green), and γH2A.X staining (red). Blue: DAPI nuclear counterstain <b>A:</b> HeLa <b>B:</b> MCF7 <b>C:</b> MRC-5/hTERT cells. Cells were treated for 3 h with 400 µM H₂O₂. TERT localization was determined as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052989#pone-0052989-g001" target="_blank">Figure 1B</a> and grouped into 3 categories: nuclear TERT (N) TERT (C) and intermediary TERT (I) localization. Examples for the 3 different localizations are indicated with arrows. <b>D:</b> Correlation between subcellular TERT localization and nuclear DNA damage levels (number of γH2A.X foci). Cytoplasmic TERT localization correlates with low nuclear DNA damage in all 3 cell lines while nuclear TERT localization results in high nuclear damage after 3 h of treatment with 400 µM H₂O₂. Intermediary TERT localization results in intermediate DNA damage levels. Black bars: HeLa, red bars: MCF7, green bars: MRC-5/hTERT. Bars are mean ± SE from at least 40–100 cells per cell line in repeated experiments. * P<0.05.</p

Mitochondrially localized TERT protects against mitochondrial ROS generation after H₂O₂ treatment and irradiation in 4 different cell lines. A:

<p>Upper panel: Representative images of ROS staining (red, mitosox) and TERT localization (myc-tag, green) after organelle specific TERT transfection and 100 µM H₂O₂ treatment for 3 h in HeLa cells. Upper row: mito- TERT, lower row: nuclear TERT. Arrows indicate transfected cells. Lower panel: Quantification of ROS levels measured as percentage of mitosox positive area from whole cytoplasm using ImageJ in transfected and un-transfected cells. <b>B:</b> MCF7 cells, panels as described for A. <b>C:</b> Quantification of ROS in U87 cells after 3 h of 100 µM H₂O₂ treatment. <b>D–F:</b> Quantification of ROS levels after X-irradiation. <b>D:</b> MCF7 after 20 Gy X-irradiation. <b>E:</b> U87 after 20 Gy X-irradiation <b>F:</b> MRC-5/SV40 after 10 Gy X-irradiation. Bars represent mean ± SE from 3 independent experiments. * P<0.05.</p