Characterisation of telomere length dynamics in dairy cattle and association with productive lifespan

Telomeres form protective caps at the ends of linear chromosomes. They consist of repetitive DNA nucleotides and associated proteins of the shelterin complex. In vitro telomeres become shorter during cell division and when a critical shortness is reached they trigger a DNA damage response that leads to replicative senescence or apoptosis. Telomere shortening is a recognised hallmark of cellular ageing and seems to be also associated with organismal ageing. Telomere length (TL) and the rate of shortening vary across individuals and several studies have found that short telomeres and fast telomere depletion are associated with poor survival and early onset of age related diseases. However, longitudinal studies are needed to better understand the relationship of TL and TL dynamics with longevity measures. Relevant studies on livestock species are largely missing from the literature. In the dairy industry, farmers are forced to cull a considerable percentage of their heifers and cows at a young age due to fertility problems or diseases. As a consequence many replacement heifers have to be reared to maintain a specific herd size. This results in increased costs, consumption of resources, and damage to the environment. Breeding for an improved productive lifespan is difficult because longevity measures are recorded at the end of life and are known to have a low heritability. Therefore, the expected genetic improvement is generally slow, but could be considerably accelerated if an early life heritable biomarker was identified that is predictive of productive lifespan and could be used for animal selection. The question is if TL could be used as such a biomarker. The objectives of this thesis were to 1) develop robust methods to measure average relative leukocyte TL (RLTL) in cattle, 2) examine RLTL dynamics with age at a population as well as at an individual level, 3) estimate genetic parameters and 4) assess the association of RLTL and RLTL dynamics with productive lifespan. A quantitative polymerase chain reaction (qPCR) based assay developed for human studies was adapted to cattle and delivered robust results (repeatability > 80%, coefficient of variation=0.05). Different DNA extraction methods were tested for their effect on RLTL measurements and it was demonstrated that fast silica based DNA extraction methods are suitable for telomere projects which can improve the sample throughput and enable large-scale projects. Subsequently, RLTL in 1328 whole blood samples of 308 Holstein Friesian dairy cows and additionally in 284 whole blood samples of 38 female calves was measured. Repeatability and random regression models were used for the statistical analysis of telomere data. RLTL decreased considerably within the first year of life, but remained relatively stable afterwards at population level. Animals varied significantly in their amount and direction of telomere change. The genetic correlation between consecutive measurements in the same individual weakened with increasing sample interval from r=1 to r=0.69 which indicates that TL in the beginning of life might be under a different genetic control than TL later in life. For the first time in a livestock species we calculated heritability estimates for RLTL which were high (0.32-0.38) and remained constant over life. Long telomeres at birth were not predictive of better productive lifespan. However, animals with long RLTL at the ages of one and five years had a survival advantage. Also, animals that showed less average RLTL attrition over their lives remained in production for longer. TL dynamics differed among individuals and a considerable subset of individuals demonstrated telomere lengthening between consecutive measurements. On average, telomeres tend to shorten early in life and then remain relatively constant. While TL is a heritable trait throughout lifetime, telomere change is not heritable. Short TL at specific ages and telomere attrition over life were associated with poorer productive lifespan

Consequences of measurement error in qPCR telomere data:A simulation study

The qPCR method provides an inexpensive, rapid method for estimating relative telomere length across a set of biological samples. Like all laboratory methods, it involves some degree of measurement error. The estimation of relative telomere length is done subjecting the actual measurements made (the Cq values for telomere and a control gene) to non-linear transformations and combining them into a ratio (the TS ratio). Here, we use computer simulations, supported by mathematical analysis, to explore how errors in measurement affect qPCR estimates of relative telomere length, both in cross-sectional and longitudinal data. We show that errors introduced at the level of Cq values are magnified when the TS ratio is calculated. If the errors at the Cq level are normally distributed and independent of true telomere length, those in the TS ratio are positively skewed and proportional to true telomere length. The repeatability of the TS ratio declines sharply with increasing error in measurement of the Cq values for telomere and/or control gene. In simulated longitudinal data, measurement error alone can produce a pattern of low correlation between successive measures of relative telomere length, coupled with a strong negative dependency of the rate of change on initial relative telomere length. Our results illustrate the importance of reducing measurement error: a small increase in error in Cq values can have large consequences for the power and interpretability of qPCR estimates of relative telomere length. The findings also illustrate the importance of characterising the measurement error in each dataset-coefficients of variation are generally unhelpful, and researchers should report standard deviations of Cq values and/or repeatabilities of TS ratios-and allowing for the known effects of measurement error when interpreting patterns of TS ratio change over time

Heritable variation in telomere length predicts mortality in soay sheep

Telomere length (TL) is considered an important biomarker of whole-organism health and aging. Across humans and other vertebrates, short telomeres are associated with increased subsequent mortality risk, but the processes responsible for this correlation remain uncertain. A key unanswered question is whether TL–mortality associations arise due to positive effects of genes or early-life environment on both an individual’s average lifetime TL and their longevity, or due to more immediate effects of environmental stressors on within-individual TL loss and increased mortality risk. Addressing this question requires longitudinal TL and life history data across the entire lifetimes of many individuals, which are difficult to obtain for long-lived species like humans. Using longitudinal data and samples collected over nearly two decades, as part of a long-term study of wild Soay sheep, we dissected an observed positive association between TL and subsequent survival using multivariate quantitative genetic models. We found no evidence that telomere attrition was associated with increased mortality risk, suggesting that TL is not an important marker of biological aging or exposure to environmental stress in our study system. Instead, we find that among-individual differences in average TL are associated with increased lifespan. Our analyses suggest that this correlation between an individual’s average TL and lifespan has a genetic basis. This demonstrates that TL has the potential to evolve under natural conditions, and suggests an important role of genetics underlying the widespread observation that short telomeres predict mortality

Brain matters: unveiling the distinct contributions of region, age, and sex to glia diversity and CNS function

The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are often differentially affected in human neurodegenerative diseases across CNS region, age and sex. We hypothesize that this selective vulnerability is underpinned by physiological variation in white matter glia. Using single nucleus RNA sequencing of human post-mortem white matter samples from the brain, cerebellum and spinal cord and subsequent tissue-based validation we found substantial glial heterogeneity with tissue region: we identified region-specific oligodendrocyte precursor cells (OPCs) that retain developmental origin markers into adulthood, distinguishing them from mouse OPCs. Region-specific OPCs give rise to similar oligodendrocyte populations, however spinal cord oligodendrocytes exhibit markers such as SKAP2 which are associated with increased myelin production and we found a spinal cord selective population particularly equipped for producing long and thick myelin sheaths based on the expression of genes/proteins such as HCN2. Spinal cord microglia exhibit a more activated phenotype compared to brain microglia, suggesting that the spinal cord is a more pro-inflammatory environment, a difference that intensifies with age. Astrocyte gene expression correlates strongly with CNS region, however, astrocytes do not show a more activated state with region or age. Across all glia, sex differences are subtle but the consistent increased expression of protein-folding genes in male donors hints at pathways that may contribute to sex differences in disease susceptibility. These findings are essential to consider for understanding selective CNS pathologies and developing tailored therapeutic strategies

Method specific calibration corrects for DNA extraction method effects on relative telomere length measurements by quantitative PCR

Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study