Regulation of dietary polyamine uptake in Drosophila melanogaster
Final Report Abstract
In the present project, I have investigated the uptake and the impact of dietary polyamines on the model organism Drosophila melanogaster. To this end, polyamine-free holidic diets were employed to study the effect of supplemented polyamines under standardized dietary conditions. Since it turned out that the existing holidic diets did not promote a proper egg to adult development of D. melanogaster, a modified optimized holidic diet formula was generated that supported egg to adult development of Drosophila even under gnotobiotic conditions similar to standard complex media. Moreover, I show that the polyamine levels of fruit flies dynamically changed within the first days after eclosion and led to higher spermidine levels in female flies compared to males. Beyond day 6 after eclosion, the polyamine concentrations remain constant in both sexes during the aging process. By using the spermidine analogue norspermidine as a tool to study the uptake of polyamines, I found that male flies accumulated less polyamines from the diet than female flies and that the impact of dietary polyamines on the polyamine level of flies declined during the aging process, again more prominent in males than in females. In line with these data, supplementation of the Drosophila diet with 2.5 mM norspermidine reduced the lifespan in female but not in male flies. Similarly, the lifespan extending effect of dietary spermidine was found solely in female flies. However, this longevity effect of dietary spermidine was contextspecific with respect to the amino acid geometry and amino acid concentration (control versus amino acid restriction) of the diet, despite the fact that dietary spermidine elevated the spermidine level of fruit flies under all conditions tested. My data also indicate that lifespan extension in fruit flies by spermidine supplementation cannot be explained by a compensatory effect owing to an age-dependent decline of polyamine levels. To examine the function of antizyme, a putative regulator of the polyamine metabolism, I generated a antizyme(+1-frameshift):GFP Drosophila transgene by replacing the endogenous antizyme via Crispr/Cas9 methodology. According to the specific physiological regulation of antizyme expression, a +1 ribosomal frameshift within the antizyme part of the mRNA is necessary to obtain an in-frame GFP expression. Although the Crispr construct has been successfully integrated into the antizyme gene locus of D. melanogaster and is transcribed to an mRNA with a correct sequence, up to now, I could not identify a physiological condition that led to a GFP expression in the fly indicative for a successful +1 frameshift during translation. In addition, I generated a D. melanogaster transgene with an antizyme gene copy that lacks the +1 frameshift site and that is under the control of the GAL4/UAS system. Overexpression of this in-frame antizyme copy by the intestine-specific driver NP1 had no effect on the accumulation of dietary polyamines, however, caused an increased sensitivity towards oral infection with the pathogenic bacterium Pseudomonas entomophila. In many organisms, antizyme is a regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, by accelerating the ODC protein degradation process. In cell culture studies, I found that the two Drosophila ODC proteins had a short half-life of below 1 h comparable to their mammalian and yeast counterparts. However, both Drosophila amino acid sequences lack a degron domain that is responsible for the rapid decay of mammalian and yeast ODC proteins. Moreover, in contrast to other ODC proteins, the half-life of the Drosophila ODC was not further affected by increased spermidine levels in the culture medium or by the knock down of antizyme expression via RNAi.
Publications
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(2018) Drosophila melanogaster as a versatile model organism in food and nutrition research J Agric Food Chem. 66(15):3737-3753
Staats S, Lüersen K, Wagner AE, Rimbach G
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(2019) Drosophila melanogaster in nutrition research - The importance of standardizing experimental diets. Genes Nutr.14
Lüersen K, Röder T, Rimbach G
