A gain-of-function screen to identify genes that reduce lifespan in the adult of Drosophila melanogaster

Abstract

Background: Several lines of evidence associate misregulated genetic expression with risk factors for diabetes, Alzheimer's, and other diseases that sporadically develop in healthy adults with no background of hereditary disorders. Thus, we are interested in genes that may be expressed normally through parts of an individual's life, but can cause physiological defects and disease when misexpressed in adulthood.

Results: We attempted to identify these genes in a model organism by arbitrarily misexpressing specific genes in adult Drosophila melanogaster, using 14,133 Gene Search lines. We identified 39 "reduced-lifespan genes" that, when misexpressed in adulthood, shortened the flies' lifespan to less than 30% of that of control flies. About half of these genes have human orthologs that are known to be involved in human diseases. For about one-fourth of the reduced-lifespan genes, suppressing apoptosis restored the lifespan shortened by their misexpression. We determined the organs responsible for reduced lifespan when these genes were misexpressed specifically in adulthood, and found that while some genes induced reduced lifespan only when misexpressed in specific adult organs, others could induce reduced lifespan when misexpressed in various organs. This finding suggests that tissue-specific dysfunction may be involved in reduced lifespan related to gene misexpression. Gene ontology analysis showed that reduced-lifespan genes are biased toward genes related to development.

Conclusions: We identified 39 genes that, when misexpressed in adulthood, shortened the lifespan of adult flies. Suppressing apoptosis rescued this shortened lifespan for only a subset of the reduced-lifespan genes. The adult tissues in which gene misexpression caused early death differed among the reduced-lifespan genes. These results suggest that the cause of reduced lifespan upon misexpression differed among the genes.

Figure 1

A gain-of-function screen to identify reduced-lifespan genes. (A) Crosses for the primary screen: GS lines were crossed with hs-GAL4, and their F1 progeny were raised, at 18°. Typically, 10 to 20 F1 adult males carrying each GS vector and hs-GAL4 were collected for 5 days after eclosion, reared for another 2 days at 18°, and then heat-shocked at 37° for 20 min. The flies were reared for another 5 days at 25°, and then dead flies were counted. Lethality was calculated as the percentage of total heat-shocked flies that were dead flies. (B) A dot plot showing the lethality (determined by the percentage of survivors 5 days after heat shock) of individual lines; the shaded area in the graph corresponds to 80-100% lethality. GS lines with lethality greater than 80% (227 lines, indicated as 1^st positive lines) were used for a secondary screen.

Figure 2

Comparison of the mean longevity of F1 progeny with or without heat shock. (A and B) The mean longevity of F1 progeny carrying each GS vector and hs-GAL4 was measured with or without heat shock. The numbers of GS lines with the indicated mean longevity are shown for each 2-day period. White and black bars correspond to the number of GS lines treated without (−) or with (+) heat shock, respectively. (A) Mean longevity averages for 122 randomly selected GS lines: the average mean longevity of flies treated without or with heat-shock was 56.6 and 55.2 days, respectively. (B) GS lines misexpressing reduced-lifespan genes, identified from the screen. The average of the mean longevities of flies that were or were not heat-shocked was 3.7 and 38.5 days, respectively. (C and D) Box-and-whisker plots of the mean longevity averages for 122 randomly selected GS lines (C) and lines with reduced-lifespan genes (D), with (+) or without (−) heat shock (hs). Whiskers and outliers were determined by Tukey’s method. Significance indicated (**) is P < 0.001 (unpaired t-test). NS: not significant.

Figure 3

Inhibiting apoptosis suppressed the reduced lifespan associated with misexpressed reduced-lifespan genes. Survival curves of adult flies expressing UAS-p35 (magenta), which encodes an apoptosis inhibitor, or UAS-GFP (green). Survivor rates are shown for 2-day intervals. (A) Control (Canton-S) flies; (B-D) flies coexpressing the reduced-lifespan gene (B) UAS-rpr, (C) atlastin (misexpression driven by GS12903), or (D) dream (misexpression driven by GS16231).

Figure 4

Misexpression of reduced-lifespan genes in specific tissues was responsible for reduced lifespan in adult flies. Misexpression of reduced-lifespan genes in specific tissues was responsible for reduced lifespan in adult flies. (A-D) Survival curves of adult flies misexpressing reduced-lifespan genes. Tissue-specific expression of reduced-lifespan genes in adult flies was induced with the TARGET system. Survivor rates were scored every three days. (A, B) Two representative reduced-lifespan genes (A) GS5065 (CG11819) and (B) GS10665 (High mobility group protein D) were misexpressed using various GAL4 drivers, indicated in the upper right. (C, D) Survival curves for all identified reduced-lifespan genes when driven by the GAL4 driver (C) 24B-GAL4 or (D) P{GawB}l(3)31-1^31–1. Blue lines: survival curve of a wild-type (Canton-S) control. Magenta lines: survival curves showing a lifespan less than 1/3 that of wild-type control flies; the corresponding reduced-lifespan genes are shown in the upper right. Survival curves showing a lifespan greater than 1/3 of that of wild-type control flies are shown in black; the corresponding gene names are not shown.