Identification of Gene Expression Changes in Sleep Mutants Associated With Reduced Longevity in Drosophila
Sleep deprivation has become a common problem in modern society, yet the physiological consequences of sleep deprivation remain poorly understood. Sleep disruption has been shown to shorten lifespan in multiple animal species. In my thesis study, I investigated the relationship between sleep and longevity using Drosophila melanogaster. By analyzing the sleep and longevity profiles from a panel of sleep mutants, I discovered a robust positive correlation between daily sleep time and median longevity. This discovery led to my hypothesis that sleep disruption as a result of genetic mutation would elicit certain gene expression changes that ultimately would lead to shortened lifespan. To identify the genes that exhibited altered expression, I profiled the transcriptomes of wild type flies, aged wild type flies, and two sleep mutants insomniac1 (inc1) and wide awakeD2 (wakeD2) with total RNA samples collected every 4 hours for 2 days. Circadian analysis was performed to detect oscillating transcripts in each group, and the results revealed substantial variations in number of oscillating genes. By comparing the experimental groups to the control wild type group, I discovered large scale rhythmicity changes in the experimental groups. Aged wild type flies predominantly showed a gain of rhythmicity, while loss of rhythmicity was more evident in sleep mutants. Furthermore, with the transcriptome data as input, I developed a differential gene expression (DGE) analysis pipeline to select candidate genes that might serve as the connection between sleep disruption and longevity reduction. In the DGE analysis, each experimental group was compared with the control wild type group. Results from each comparison were subsequently intersected to pinpoint genes that were significantly changed in all experimental groups. Ultimately, 15 candidate genes stood out from the analysis. For the scope of this study, I focused on candidate gene Neuropeptide-like precursor 3 (Nplp3), a putative neuropeptide precursor. RNA sequencing results revealed that expression levels of Nplp3 were reduced in sleep mutants and aged wild type animals, compared to control flies. These results were validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). More importantly, I found that Nplp3 expression was reduced in several sleep mutants in addition to inc1 and wakeD2. Decreased Nplp3 expression resulted in significant shortening in lifespan but did not affect sleep amount. Transgenic fly strains were generated to selectively overexpress either wild type Nplp3 or Nplp3 without signal peptide using Nplp3 specific driver. Overexpression of normal Nplp3 reduced sleep. Longevity results indicated that signal peptide might be important for Nplp3 function. These findings expanded our understandings of the relationship between sleep and longevity and suggested a potential neuropeptide signaling pathway for regulation of longevity by sleep.
sleep deprivation, longevity, drosophila, neuropeptides, circadian rhythm, gene expression, Life Sciences
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