Common Mechanisms for Calorie Restriction and AC5 Knockout Models of Longevity

Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared to WT mice. CR applied to AC5 KO result in rapid decrease in body weight, metabolic deterioration and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene Ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD and olfactory receptors in both young and intermediate age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

The phenotypic impact of naturally occurring genetic and molecular mitochondrial variation in a Drosophila Genetic Reference Population

Roel Paulus Josephus Bevers

Genome-wide association (GWA) studies aim to dissect the relationship between genotype and phenotype. So far, the focus has been on nuclear genetic determinants as variation in the mitochondrial genome is often low in the study cohorts. Despite this observation, mitochondrial genomic variants are increasingly linked to metabolic and neurological disorders. Thus, there is much to gain from studying how mitochondrial variation interacts with nuclear variation and/or environment, and how this subsequently affects phenotypes. In my thesis I address these topics using Drosophila melanogaster as a model system, and specifically the Drosophila Genetic Reference Panel (DGRP). The DGRP consists of 200 sequenced and genetically diverse Drosophila populations and has been extensively used to study a wide-range of quantitative traits. However, in all of these studies, researchers have focussed on the influence of nuclear genetic variants in the absence of a high-resolution map of mitochondrial variants. To address this problem, we have developed a high-throughput method to specifically sequence mitochondrial DNA that enabled us to characterize mitochondrial genomic variation to an unprecedented depth. On average, each DGRP line had 25 mitochondrial variants (1 in 600 bp) and that most of the variants detected were unique for each line. However, we also found that some variants are more prevalent and show they can be used to construct mitochondrial haplotypes. We then used these haplotypes to test whether mitochondrial variation affected previously published phenotypes. We detected significant associations with 12 out of 259 publicly accessible phenotypes and found that these were mostly related to metabolism and responses to environmental cues. We selected one of the significant phenotypes, food intake in males, to verify our findings. We demonstrated the effect of mitochondrial haplotypes on the phenotype by swapping mitochondria from ¿high food intake¿ lines with mitochondria from ¿low food intake¿ lines and vice versa. We observed that, largely independent of the nuclear background, DGRP lines with a ¿high food intake mitochondria¿ consumed more food and concomitantly, DGRP lines with the ¿low food intake mitochondria¿ consumed less food. Furthermore, we investigated the influence of natural genetic variation was on the response to a high-fat diet. We designed a control and a high-fat diet and fed this to >100 DGRP lines and measured the lifespan on each diet. On average, lifespan was decreased by 48% on a high-fat diet, however this ranged from an increase of 2% to a decrease of 60% accentuating the complexity of the underlying genetic architecture. Our dataset was relatively small and limited our genome-wide and mitochondrial haplotype association analyses. Nonetheless, our GWA analysis detected variants related to genes involved in lipid metabolism and mitochondria but we did not detect significant associations with mitochondrial haplotypes. This suggests that while mitochondrial function may be important for the response to a high-fat diet, it is not dependent on the mitochondrial haplotype. Taken together, the work described in my thesis demonstrates the significance of accounting for mitochondrial haplotypes in phenotype association analyses. By doing so, it can open up new insights into genotype-to-phenotype relationships and gene regulation.

EPFL2018

Unraveling Patterns of Site-to-Site Synonymous Rates Variation and Associated Gene Properties of Protein Domains and Families

Slavica Dimitrieva Janeva

In protein-coding genes, synonymous mutations are often thought not to affect fitness and therefore are not subject to natural selection. Yet increasingly, cases of non-neutral evolution at certain synonymous sites were reported over the last decade. To evaluate the extent and the nature of site-specific selection on synonymous codons, we computed the site-to-site synonymous rate variation (SRV) and identified gene properties that make SRV more likely in a large database of protein-coding gene families and protein domains. To our knowledge, this is the first study that explores the determinants and patterns of the SRV in real data. We show that the SRV is widespread in the evolution of protein-coding sequences, putting in doubt the validity of the synonymous rate as a standard neutral proxy. While protein domains rarely undergo adaptive evolution, the SRV appears to play important role in optimizing the domain function at the level of DNA. In contrast, protein families are more likely to evolve by positive selection, but are less likely to exhibit SRV. Stronger SRV was detected in genes with stronger codon bias and tRNA reusage, those coding for proteins with larger number of interactions or forming larger number of structures, located in intracellular components and those involved in typically conserved complex processes and functions. Genes with extreme SRV show higher expression levels in nearly all tissues. This indicates that codon bias in a gene, which often correlates with gene expression, may often be a site-specific phenomenon regulating the speed of translation along the sequence, consistent with the co-translational folding hypothesis. Strikingly, genes with SRV were strongly overrepresented for metabolic pathways and those associated with several genetic diseases, particularly cancers and diabetes.

Public Library of Science2014

Detection of environmental glucocorticoids and investigation of their effects using the zebrafish embryo as a model

Anita Orsolya Hidasi

Synthetic glucocorticoids (GCs) are widely used anti-inflammatory drugs. They mimic cortisol, the natural stress hormone in humans and in fish. Cortisol binds and activates the glucocorticoid receptor (GR), which regulates genes governing development, immune response, osmoregulation, and behavior. Therefore, the presence of GCs in the aquatic environment may cause endocrine disruption through impairing these essential physiological processes in fish. The aim of this thesis was to assess the effects of environmentally relevant concentrations of GCs in teleost fish, focusing on the inflammatory response and molecular mechanisms of GC action on different levels, from mRNA to proteins to metabolites. To confirm the environmental presence of GCs, GC activity was determined along a Swiss freshwater stream impacted by a wastewater treatment plant, using effect-directed analysis. The effluent samples showed the highest potency to activate the GR, as determined by the GR-CALUX® assay. The chemical analysis found clobetasol propionate (CP), a highly potent GC, to be responsible for 63% of the total GR activity in the sample. CP was selected as a model chemical to study GC effects in fish. Embryos of zebrafish (Danio rerio) aged until 5 days post fertilization (dpf) were used as a model organism; working with embryonic stages is a refinement of animal experiments. We demonstrated that CP is readily taken up by the embryos. Bacterial lipopolysaccharides (LPS) induce one of the main inflammatory cascades. As observed in LPS challenge assays, the inflammatory response of the embryos was significantly reduced after exposure to ¿0.1 nM CP. We showed that mRNA expression of several LPS mechanism- and GC action-related genes was regulated by CP. Among them, Annexin a1b (anxa1b) was significantly down-regulated after exposure to ¿0.05 nM CP. Sensitive regulation of anxa1b by the non-steroidal anti-inflammatory drug diclofenac (DCF) suggested that this gene product is a potential biomarker of steroidal and non-steroidal immunosuppressive effects. An LC-MS/MS-based targeted proteomics technique was developed to measure proteins, as it is these molecules that actually carry out cellular functions. 12 out of 40 GC action-related protein targets were detectable in control embryo digests and were subsequently monitored after CP, DCF and grab water sample exposures. We found that the muscle protein Myhz2 was regulated only after DCF exposure, while I¿B¿, an anti-inflammatory protein, was regulated only by CP, in agreement with mRNA level data. Global and targeted metabolomics analyses were performed in zebrafish embryos exposed to CP from 4 to 5 dpf, in collaboration with M. Lamoree (VU Amsterdam). Several of the detected compounds showed significant response to CP: lysine, nicotinamide, choline, hypoxanthine, tyrosine and tryptophan. To conclude, this thesis demonstrates that GCs are present in the aquatic environment and may suppress the inflammatory response of fish at environmentally relevant concentrations.

EPFL2016