Identification of genes associated with resilience/vulnerability to sleep deprivation and starvation in Drosophila

Abstract

Background and study objectives: Flies mutant for the canonical clock protein cycle (cyc(01)) exhibit a sleep rebound that is ∼10 times larger than wild-type flies and die after only 10 h of sleep deprivation. Surprisingly, when starved, cyc(01) mutants can remain awake for 28 h without demonstrating negative outcomes. Thus, we hypothesized that identifying transcripts that are differentially regulated between waking induced by sleep deprivation and waking induced by starvation would identify genes that underlie the deleterious effects of sleep deprivation and/or protect flies from the negative consequences of waking.

Design: We used partial complementary DNA microarrays to identify transcripts that are differentially expressed between cyc(01) mutants that had been sleep deprived or starved for 7 h. We then used genetics to determine whether disrupting genes involved in lipid metabolism would exhibit alterations in their response to sleep deprivation.

Setting: Laboratory.

Patients or participants: Drosophila melanogaster.

Interventions: Sleep deprivation and starvation.

Measurements and results: We identified 84 genes with transcript levels that were differentially modulated by 7 h of sleep deprivation and starvation in cyc(01) mutants and were confirmed in independent samples using quantitative polymerase chain reaction. Several of these genes were predicted to be lipid metabolism genes, including bubblegum, cueball, and CG4500, which based on our data we have renamed heimdall (hll). Using lipidomics we confirmed that knockdown of hll using RNA interference significantly decreased lipid stores. Importantly, genetically modifying bubblegum, cueball, or hll resulted in sleep rebound alterations following sleep deprivation compared to genetic background controls.

Conclusions: We have identified a set of genes that may confer resilience/vulnerability to sleep deprivation and demonstrate that genes involved in lipid metabolism modulate sleep homeostasis.

Keywords: Drosophila melanogaster; lipid metabolism; lipid storage; microarray; sleep homeostasis; transcriptional changes.

Figure 1

Gene expression profiles in sleep deprived and starved cyc⁰¹, per⁰, and Wild-type Canton-S (CS) flies. (A) Relative fold changes versus untreated genetic controls in representative genes derived from complementary DNA arrays (6 samples/condition) or quantitative polymerase chain reaction (qPCR, 1 sample/group, n = 20 flies). All flies were maintained in dark-dark cycle (DD). cyc⁰¹ and per⁰¹ were sleep deprived (SlDn) or starved (Stv) concurrently for 7 h while CS flies were deprived for 12 h during their primary sleep period. (B) Percent change versus untreated genetic controls expressed as mean ± standard error of the mean. Fold changes between sleep deprived and starved flies are highlighted in gray.

Figure 2

Genetic validation of microarray. (A) Sleep homeostasis is increased in bgm¹ mutants (n = 21) compared to its background genetic control, bgm^rev (n = 24). * P = 0.0045, Student t-test. (B) bgm messenger RNA (mRNA) from flies in Actin-GAL4/bgm^EY03176 compared to bgm^EY01376/+. mRNA is expressed as a percentage of bgm^EY01376/+ (n = 5 flies/group). *P < 0.05 by Student t-test. (C) bgm^EY03176/+ flies (n = 45) also show an increased homeostatic response in comparison to Act-GAL4/bgm^EY03176 (n = 27). *P = 0.00065 by Student t-test. (D) cue mRNA levels are decreased in cue² homozygotes compared with the background control with the P-element precisely excised (cue^rev). Levels are presented as a percentage of cue^rev. *P < 0.05 by Student t-test. (E) Flies homozygous for cue² (n = 129), or hemizygous cue²/Df(3L)Ar14-8 (n = 32) have a significantly reduced sleep rebound compared to genetic background controls in which the P-element has been excised, cue^rev (n = 39). One-way analysis of variance F2,197 = 5.80; *P < 0.001 modified Bonferroni test. (F) Total triglyceride levels were significantly decreased in cue²/cue² mutants compared to its background control cue^rev/cue^rev. *P = 0.017 using Student t-test with an n = 5 groups of 10 flies per genotype.

Figure 3

Triglyceride levels are decreased with ubiquitous hll knockdown. (A) Representative triglyceride profiles from flies with ubiquitous knockdown of hll (tubGS- > UAS-hll^RNAi induced by RU486, left tracing) and the uninduced, genetically identical siblings on the vehicle control (veh, right tracing). We chose to use the GeneSwitch system to minimize the number of groups analyzed and to better control for genetic background. Tracings were generated using positive ion electrospray ionization (ESI) mass spectrometric (MS) analyses of lipid Li⁺ adducts from whole flies. Peaks are labeled by their mass to charge (m/z) ratios, and the boxed m/z values correspond to the peaks quantified in (B). The arrow denotes internal standard (m/z 684), and the most intense peak is normalized to 100. Although there is a decrease in overall triglyceride abundance, there is no change in the distribution of triglyceride molecular species. (B) Quantification of triglyceride peaks from the mass spectra of hll knockdown and the uninduced siblings (four samples per group; n = 5 flies/sample). Numbers above graph represent the m/z value from (A) and the smaller numbers are the lipid species that could correspond to the given m/z value. The intensity of the peak in question was divided by that of the internal standard (I.S.), and the resultant ratio was then normalized to protein content to determine the fold-increase of the triglyceride species represented by the peak. *P < 0.05) or **P < 0.01 as calculated by Student t-test with a Bonferroni correction.

Figure 4

Identification of the constituents that constitute the peak at m/z 783. Mass spectrometric MS/MS spectra obtained from ions with the m/z value of 783 in the total ion current tracing (Figure 4) corresponds to the Li⁺ adduct of triacylglycerol (TAG) species from the RU-treated flies in which the total number of carbon atoms in the fatty acyl chains is 46 and the total number of double bonds is 1 (denoted 46:1-TAG). The tandem spectrum obtained from collisionally activated dissociation (CAD) of m/z 783 is displayed in the figure, and it indicates that the predominant molecular species represented by that peak is 14:0/16:1/16:0-TAG. Features of the spectrum that establish that assignment are the presence of ions that represent neutral losses of each of the substituents as a free fatty acid at m/z 527 (loss of 16:0), 529 (loss of 16:1), and 555 (loss of 14:0), respectively. There are also ions reflecting loss of each substituent as a Li⁺ salt at m/z 521, 523, and 549, respectively. The ions representing loss of the sn-2 substituent of TAG-Li⁺ species are less abundant than the ions reflecting loss of the sn-1 or sn-3 substituent, and the sn-1 and sn-3 positions of TAG molecules are not distinguishable by mass spectrometry. Other ions consistent with this assignment are the acylium ion of 14:0 (m/z 211) and ions representing Li⁺ adducts of 14:0 (m/z 235) and 16:0 (m/z 263). There are also ions representing combined losses of 16:1 and 16:0 (m/z 275) or 14:0 (m/z 303) as an α,β-unsaturated fatty acid. Such combined losses always include the sn-2 substituent, indicating that 16:1 is the sn-2 substituent in the major TAG isomer contributing ion current to the m/z 783 peak. That there are less abundant isomers with the overall composition 46:1-TAG, e.g., 12:0/18:1/16:0-TAG, is reflected by relatively low abundance ions at m/z 501 and m/z 583 that represent loss of 18:1 and 12:0 as free fatty acids, respectively. MS and MS/MS analyses of lipid extracts of vehicle-treated flies revealed the same major TAG molecules observed for the RU-treated flies.

Figure 5

hll mutants exhibit reduced sleep homeostasis. (A) Levels of hll messenger RNA (mRNA) from tubGS > hll^RNAi induced with RU486 are decreased compared to sibling flies uninduced using vehicle control. mRNA is expressed as a percentage of vehicle control (n = 5 flies/group). *P < 0.05 by Student t-test. (B) Sleep homeostasis is reduced when tubGS > hll^RNAi are induced with the drug mifepristone (RU486, n = 28) compared to when the flies are treated with the vehicle control (veh, n = 28). (C) mRNA for hll was reduced in whole heads in Actin-Gal4/UAS-hll^RNAi flies compared to parental lines (Actin-GAL4/+ and UAS-hll^RNAi/+). mRNA levels were normalized to UAS-hll^RNAi/+; n = 20 heads/group. *P < 0.05 by planned comparison Student t-test in indicated comparison. (D) Sleep homeostasis is reduced in Actin-Gal4/UAS-hll^RNAi (n = 66) compared to Act-GAL4/+ (n = 39) and UAS-hll^RNAi/+ (n = 66) parental lines. One-way analysis of variance F2,168 = 7.51; *P < 0.01 modified Bonferroni Test. *P < 0.01 by Student t-test.