Nils Hartmann

TELOMERE LENGTH OF PATIENTS WITH MAJOR DEPRESSION IS SHORTENED BUT INDEPENDENT FROM THERAPY AND SEVERITY OF THE DISEASE

Background: Shortened telomere length has been observed in a variety of diseases. Our objective was to analyze mean telomere length of patients with major depressive disorder. A key question was whether telomere length varies in different groups of depressive patients. Methods: We obtained blood samples from patients with major depressive disorder (n=54) and healthy subjects (n=20). We isolated genomic DNA and measured mean telomere length using telomere restriction fragments and Southern blotting. We grouped patients according to the therapy they received including total antidepressant dose. Results: Mean telomere length of the entire patient group (7.20±0.61 kb) was significantly shorter than in the control group (7.55±0.54 kb). We observed no significant difference in telomere length among the different patient groups, but each of these patient groups had significantly shorter telomeres than the healthy subjects. Further analysis revealed no significant association between telomere length and illness duration and between telomere length and the severity of depression, as determined by the Hamilton score. Conclusion: These results provide further evidence that major depressive disorder is associated with shortened telomeres. However, differences in the applied therapy, the duration of illness, or the severity of depression do not seem to have any influence on telomere length. Depression and Anxiety, 2010.  © 2010 Wiley‐Liss, Inc.

Measuring telomere length and telomere dynamics in evolutionary biology and ecology

Telomeres play a fundamental role in the protection of chromosomal DNA and in the regulation of cellular senescence. Recent work in human epidemiology and evolutionary ecology suggests adult telomere length (TL) may reflect past physiological stress and predict subsequent morbidity and mortality, independent of chronological age. Several different methods have been developed to measure TL, each offering its own technical challenges. The aim of this review is to provide an overview of the advantages and drawbacks of each method for researchers, with a particular focus on issues that are likely to face ecologists and evolutionary biologists collecting samples in the field or in organisms that may never have been studied in this context before. We discuss the key issues to consider and wherever possible try to provide current consensus view regarding best practice with regard to sample collection and storage, DNA extraction and storage, and the five main methods currently available to measure TL. Decisions regarding which tissues to sample, how to store them, how to extract DNA, and which TL measurement method to use cannot be prescribed, and are dependent on the biological question addressed and the constraints imposed by the study system. What is essential for future studies of telomere dynamics in evolution and ecology is that researchers publish full details of their methods and the quality control thresholds they employ.

Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri

Age research in vertebrates is often limited by the longevity of available models. The teleost fish Nothobranchius furzeri has an exceptionally short lifespan with 3.5 months for the laboratory strain GRZ and about 6 months for the wild-derived strain MZM-0403. Here we have investigated telomere length in muscle and skin tissue of young and old fish of both strains using different methods. We found age-dependent telomere shortening in the MZM-0403 strain with the longer lifespan, whereas the short-lived GRZ strain showed no significant telomere shortening with advanced age. Sequencing of the two main telomerase genes Tert and Terc revealed that both genes are highly conserved between the N. furzeri strains while there is little conservation to other fish species and humans. Both genes are ubiquitously expressed in N. furzeri and expression levels of Tert and Terc correlate with telomerase activity in a tissue-specific manner. Unexpectedly, the expression level of Tert is increased in aged muscle and skin tissue of MZM-0403 suggesting that telomeres shorten upon ageing despite increased Tert expression and hence high telomerase activity. We further conclude that the extremely short lifespan of the GRZ strain is not caused by diminished telomerase activity or accelerated telomere shortening.

Effects of dietary restriction on mortality and age-related phenotypes in the short-lived fish Nothobranchius furzeri

The short‐lived annual fish Nothobranchius furzeri shows extremely short captive life span and accelerated expression of age markers, making it an interesting model system to investigate the effects of experimental manipulations on longevity and age‐related pathologies. Here, we tested the effects of dietary restriction (DR) on mortality and age‐related markers in N. furzeri. DR was induced by every other day feeding and the treatment was performed both in an inbred laboratory line and a longer‐lived wild‐derived line. In the inbred laboratory line, DR reduced age‐related risk and prolonged maximum life span. In the wild‐derived line, DR induced early mortality, did not reduce general age‐related risk and caused a small but significant extension of maximum life span. Analysis of age‐dependent mortality revealed that DR reduced demographic rate of aging, but increased baseline mortality in the wild‐derived strain. In both inbred‐ and wild‐derived lines, DR prevented the expression of the age markers lipofuscin in the liver and Fluoro‐Jade B (neurodegeneration) in the brain. DR also improved performance in a learning test based on conditioning (active avoidance in a shuttle box). Finally, DR induced a paradoxical up‐regulation of glial fibrillary acidic protein in the brain.

Mitochondrial DNA copy number and function decrease with age in the short-lived fish Nothobranchius furzeri.

Among vertebrates that can be kept in captivity, the annual fish Nothobranchius furzeri possesses the shortest known lifespan. It also shows typical signs of aging and is therefore an ideal model to assess the role of different physiological and environmental parameters on aging and lifespan determination. Here, we used Nothobranchius furzeri to study whether aging is associated with mitochondrial DNA (mtDNA) alterations and changes in mitochondrial function. We sequenced the complete mitochondrial genome of N. furzeri and found an extended control region. Large‐scale mtDNA deletions have been frequently described to accumulate in other organisms with age, but there was no evidence for the presence of detectable age‐related mtDNA deletions in N. furzeri. However, mtDNA copy number significantly decreased with age in skeletal muscle, brain, liver, skin and dorsal fin. Consistent with this finding, expression of Pgc‐1α that encodes a transcriptional coactivator of mitochondrial biogenesis and expression of Tfam and mtSsbp both encoding mtDNA binding factors was downregulated with age. The investigation of possible changes in mitochondrial function revealed that the content of respiratory chain complexes III and IV was reduced in skeletal muscle with age. In addition, ADP‐stimulated and succinate‐dependent respiration was decreased in mitochondria of old fish. These findings suggest that despite the short lifespan, aging in N. furzeri is associated with a decline in mtDNA copy number, the downregulation of mtDNA‐associated genes and an impairment of mitochondrial function.

High tandem repeat content in the genome of the short-lived annual fish Nothobranchius furzeri: a new vertebrate model for aging research

BackgroundThe annual fish Nothobranchius furzeri is the vertebrate with the shortest known life span in captivity. Fish of the GRZ strain live only three to four months under optimal laboratory conditions, show explosive growth, early sexual maturation and age-dependent physiological and behavioral decline, and express aging related biomarkers. Treatment with resveratrol and low temperature significantly extends the maximum life span. These features make N. furzeri a promising new vertebrate model for age research.ResultsTo contribute to establishing N. furzeri as a new model organism, we provide a first insight into its genome and a comparison to medaka, stickleback, tetraodon and zebrafish. The N. furzeri genome contains 19 chromosomes (2n = 38). Its genome of between 1.6 and 1.9 Gb is the largest among the analyzed fish species and has, at 45%, the highest repeat content. Remarkably, tandem repeats comprise 21%, which is 4-12 times more than in the other four fish species. In addition, G+C-rich tandem repeats preferentially localize to centromeric regions. Phylogenetic analysis based on coding sequences identifies medaka as the closest relative. Genotyping of an initial set of 27 markers and multi-locus fingerprinting of one microsatellite provides the first molecular evidence that the GRZ strain is highly inbred.ConclusionsOur work presents a first basis for systematic genomic and genetic analyses aimed at understanding the mechanisms of life span determination in N. furzeri.

Mapping of quantitative trait loci controlling lifespan in the short‐lived fish Nothobranchius furzeri– a new vertebrate model for age research

The African annual fish Nothobranchius furzeri emerged as a new model for age research over recent years. Nothobranchius furzeri show an exceptionally short lifespan, age‐dependent cognitive/behavioral decline, expression of age‐related biomarkers, and susceptibility to lifespan manipulation. In addition, laboratory strains differ largely in lifespan. Here, we set out to study the genetics of lifespan determination. We crossed a short‐ to a long‐lived strain, recorded lifespan, and established polymorphic markers. On the basis of genotypes of 411 marker loci in 404 F2 progeny, we built a genetic map comprising 355 markers at an average spacing of 5.5 cM, 22 linkage groups (LGs) and 1965 cM. By combining marker data with lifespan values, we identified one genome‐wide highly significant quantitative trait locus (QTL) on LG 9 (P < 0.01), which explained 11.3% of the F2 lifespan variance, and three suggestive QTLs on LG 11, 14, and 17. We characterized the highly significant QTL by synteny analysis, because a genome sequence of N. furzeri was not available. We located the syntenic region on medaka chromosome 5, identified candidate genes, and performed fine mapping, resulting in a c. 40% reduction of the initial 95% confidence interval. We show both that lifespan determination in N. furzeri is polygenic, and that candidate gene detection is easily feasible by cross‐species analysis. Our work provides first results on the way to identify loci controlling lifespan in N. furzeri and illustrates the potential of this vertebrate species as a genetic model for age research.

The transcript catalogue of the short-lived fish Nothobranchius furzeri provides insights into age-dependent changes of mRNA levels

BackgroundThe African annual fish Nothobranchius furzeri has over recent years been established as a model species for ageing-related studies. This is mainly based on its exceptionally short lifespan and the presence of typical characteristics of vertebrate ageing. To substantiate its role as an alternative vertebrate ageing model, a transcript catalogue is needed, which can serve e.g. as basis for identifying ageing-related genes.ResultsTo build the N. furzeri transcript catalogue, thirteen cDNA libraries were sequenced using Sanger, 454/Roche and Solexa/Illumina technologies yielding about 39 Gb. In total, 19,875 protein-coding genes were identified and annotated. Of these, 71% are represented by at least one transcript contig with a complete coding sequence. Further, transcript levels of young and old fish of the strains GRZ and MZM-0403, which differ in lifespan by twofold, were studied by RNA-seq. In skin and brain, 85 differentially expressed genes were detected; these have a role in cell cycle control and proliferation, inflammation and tissue maintenance. An RNA-seq experiment for zebrafish skin confirmed the ageing-related relevance of the findings in N. furzeri. Notably, analyses of transcript levels between zebrafish and N. furzeri but also between N. furzeri strains differed largely, suggesting that ageing is accelerated in the short-lived N. furzeri strain GRZ compared to the longer-lived strain MZM-0403.ConclusionsWe provide a comprehensive, annotated N. furzeri transcript catalogue and a first transcriptome-wide insight into N. furzeri ageing. This data will serve as a basis for future functional studies of ageing-related genes.

Branched-chain amino acid catabolism is a conserved regulator of physiological ageing

Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFβ, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan.

A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri)

Background: A challenge in age research is the absence of short‐lived vertebrate model organisms. The turquoise killifish Nothobranchius furzeri has an exceptionally short lifespan of 4–10 months depending on the strain. Thus, it possesses the shortest known maximum lifespan of a vertebrate species that can be bred in captivity. Results: Here we show the successful introduction of DNA and RNA molecules into the one‐cell embryo of N. furzeri. For this purpose, we adapted existing microinjection protocols to inject through the remarkably thick and robust chorion of N. furzeris eggs. The injected DNA transgene was integrated into the genome and transmitted to subsequent generations as indicated by the expression of the fluorophore enhanced green fluorescent protein (EGFP). Furthermore, we could confirm a special phase during embryonic development in which embryogenesis occurs within a re‐aggregated mass of previously dispersed cells as it has been described for other related cyprinodont fish species. Conclusions: The transgenesis protocol described here provides a basis for a variety of genetic manipulations including overexpression of genes and determining their effects on lifespan and longevity. The feasibility to perform transgenesis is an important step to establish N. furzeri as a new model in age research. Developmental Dynamics 241:1133–1141, 2012.