An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster

Abstract

Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice. The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population. The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals. Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions. Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80%. The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction. Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay. We used this assay as a screen to identify drugs that extend lifespan in flies. Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions. We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan.

Fig. 1.

Death curves of two different biomarkers of aging coupled to tetanus toxin expression. Flies were kept at a density of 25 flies per sex and vial on lightly yeasted standard cornmeal food and passed every other day. Dead flies were counted daily. (A) DJ651-driven toxin expression leads to average survival times of 8 days for both males and females. Toxin expression is delayed in DJ634-driven flies, and therefore, average survival times increased to 18 days. (B) Compared with survivorship curves of Canton-S flies, DTT females live 80% shorter.

Fig. 2.

Environmental interactions that increase lifespan also extend survival times of DTT flies. Both male (Left) and female (Right) flies live longer in the DTT assay when subjected to environmental interventions that are known to extend lifespan of D. melanogaster. Lower ambient temperature (A), reduced caloric intake (B), and virginity (C) extend survival times up to 74%.

Fig. 3.

Long-lived mutants extend survival times of DTT flies. Homozygous DTT flies were crossed to the indicated stocks, and death curves of offspring were generated. (A) When crossed to freshly isogenized Indy mutants, males lived 50% longer than genetically matched control. (B) Male offspring of a cross to the hypomorph rpd3^P-UTR mutant lived significantly longer than genetically matched control. Males lived 40% longer.

Fig. 4.

The DTT system can be used as a drug-discovery tool. (A) Culturing DTT flies on food containing 0.005% lipoic acid increases the average survival times of males (Left) by 4% and of females (Right) by 12%. No yeast was added because the yeast/lipoic acid combination at any concentration was toxic. (B) Food containing 200 μM polyphenol resveratrol increased the average survival times of males (Left) by 9% and of females (Right) by 8%.

Fig. 5.

Drugs extending survival times of DTT flies also extend lifespan of Canton-S flies. Canton-S flies were kept on food without yeast according to the conditions established in Fig. 4, and survivorship curves were generated. (A) The 0.005% lipoic acid increased average and median lifespan of males (Upper) by 4% and 6% (P = 0.5498), respectively, and average, median, and maximum lifespan of females (Lower) by 12%, 15%, and 5% (P = 0.0025), respectively, which were the same as increases seen in DTT flies. (B) Average, median, and maximum lifespan extension by 16%, 13%, and 15%, respectively, in females (Lower)(P < 0.0001) and 10%, 10%, and 18%, respectively, in males (Upper)(P < 0.0001) is observed when flies are fed 200 μM resveratrol.