Project description:<p>Chronic sleep loss profoundly impacts metabolic health and shortens lifespan, but studies of the mechanisms involved have focused largely on acute sleep deprivation. To identify metabolic consequences of chronically reduced sleep, we conducted unbiased metabolomics on heads of three adult Drosophila short-sleeping mutants with very different mechanisms of sleep loss: fumin (fmn), redeye (rye), and sleepless (sss). Common features included elevated ornithine and polyamines, with lipid, acyl-carnitine, and TCA cycle changes suggesting mitochondrial dysfunction. Studies of excretion demonstrate inefficient nitrogen elimination in adult sleep mutants, likely contributing to their polyamine accumulation. Increasing levels of polyamines, particularly putrescine, promote sleep in control flies but poison sleep mutants. This parallels the broadly enhanced toxicity of high dietary nitrogen load from protein in chronically sleep-restricted Drosophila, including both sleep mutants and flies with hyper-activated wake-promoting neurons. Together, our results implicate nitrogen stress as a novel mechanism linking chronic sleep loss to adverse health outcomes-and perhaps for linking food and sleep homeostasis at the cellular level in healthy organisms.</p>
Project description:A method for the long-term maintenance of germ-free flies was established using aseptic isolators. The methodology effectively and reliably yields large numbers of germ-free flies in homogeneous cultures. Germ-free flies exhibited increased lifespan (only female flies) and decreased egg production, markedly reduced fat storage, less midday sleep, and enhanced aggressiveness (male flies). Fructilactobacillus—a species of fly intestinal microbes—was re-colonized in germ-free flies, and these gnotobiotic flies were successfully maintained for numerous generations. The proteome of those flies were analyzed.
Project description:Genome-wide profiling of rhythmic gene expression has offered new avenues for studying the contribution of circadian clock to diverse biological processes. Sleep has been considered one of the most important physiological processes that are regulated by the circadian clock, however, the effects of chronic sleep loss on rhythmic gene expression remain poorly understood. In the present study, we exploited Drosophila sleep mutants insomniac1 (inc1) and wide awakeD2 (wakeD2) as models for chronic sleep loss. We profiled the transcriptomes of heads collected from 4-week-old wild type flies, inc1 and wakeD2 at timepoints around the clock. Analysis of gene oscillation revealed a substantial loss of rhythmicity in inc1 and wakeD2 compared to wild type flies, with most of the affected genes common to both mutants. The disruption of gene oscillation was not due to changes in average gene expression levels. We also identified a subset of genes whose loss of rhythmicity was shared among animals with chronic sleep loss and old flies, suggesting a contribution of aging to chronic, sleep-loss-induced disruption of gene oscillation.
Project description:Because pink1-mutant flies exhibit a global shutdown of protein synthesis, we decided to measure the levels of individual proteins in adult flies through quantitative proteomics.
Project description:We used a microarray platform to survey the daily levels of 78 D. melanogaster miRNAs in adult heads of wildtype control flies and the arrhythmic clock mutant cyc01. At least 2 miRNAs were identified that showed robust daily abundance changes in control flies but not the cyc01 mutant. Keywords: microRNA, Circadian regulation yw and yw;cyc01 flies were maintained in standard media. For collections, approximately 50-100 young (2-5 day old) flies were placed in vials that were incubated at 25oC for four days in standard 12hr light-12hr dark cycles [LD; where zeitgeber time 0 (ZT0) is defined as lights-on]. Flies were collected by freezing in dry ice during the fourth LD cycle at the following times; ZT1, 7, 13 and 19. Subsequently low molecular RNAs were extracted for microarray analysis using a mirMAX microarray.
Project description:Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. To identify the genes responsible for the protective effects of starvation we conducted transcription profiling of sleep deprived flies that accrue sleep debt compared to starved siblings that do not. Genes involved in lipid metabolism were highly enriched in our dataset of 84 differentially regulated transcripts. Follow up genetic studies established that 6 genes involved in lipid metabolism strongly influence sleep homeostasis. Two of these genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), are in the same lipolysis pathway but exert antagonistic effects on lipid storage. bmm mutant flies have excess fat stores and display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking. Two-condition experiments: sleep deprived vs starved. RNA from 8 biological replicates for each condition was pooled in groups of 2 to create 4 samples. Each of the 4 samples is run in duplicate with untreated circadian matched controls.
Project description:Long noncoding RNAs (lncRNAs) are a diverse class of RNAs that are critical for gene regulation, DNA repair and splicing, and have been implicated in cancer, stress response, and development. However, the function of many lncRNAs remains unknown. In Drosophila melanogaster, U snoRNA host gene 4 (Uhg4) encodes an antisense long noncoding RNA that is host to seven small nucleolar RNAs (snoRNAs). Uhg4 is expressed ubiquitously during development and in all adult fly tissues with maximal expression in ovaries; however, it has no annotated function(s). Here, we used CRISPR-Cas9 germline gene editing to generate multiple deletions spanning the promoter region and first exon of Uhg4. Mutant flies were sterile, showed delayed development and decreased viability, and changes in sleep and responses to stress. Whole genome RNA sequencing of Uhg4 deletion flies and their controls identified coregulated genes and genetic interaction networks. Gene ontology analyses highlighted a broad spectrum of biological processes, including morphogenesis, stress response, and regulation of transcription and translation. Thus, Uhg4 is a lncRNA essential for reproduction with pleiotropic effects on multiple fitness traits.