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, 29 (22), 7148-57

Identifying Sleep Regulatory Genes Using a Drosophila Model of Insomnia

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Identifying Sleep Regulatory Genes Using a Drosophila Model of Insomnia

Laurent Seugnet et al. J Neurosci.

Abstract

Although it is widely accepted that sleep must serve an essential biological function, little is known about molecules that underlie sleep regulation. Given that insomnia is a common sleep disorder that disrupts the ability to initiate and maintain restorative sleep, a better understanding of its molecular underpinning may provide crucial insights into sleep regulatory processes. Thus, we created a line of flies using laboratory selection that share traits with human insomnia. After 60 generations, insomnia-like (ins-l) flies sleep 60 min a day, exhibit difficulty initiating sleep, difficulty maintaining sleep, and show evidence of daytime cognitive impairment. ins-l flies are also hyperactive and hyperresponsive to environmental perturbations. In addition, they have difficulty maintaining their balance, have elevated levels of dopamine, are short-lived, and show increased levels of triglycerides, cholesterol, and free fatty acids. Although their core molecular clock remains intact, ins-l flies lose their ability to sleep when placed into constant darkness. Whole-genome profiling identified genes that are modified in ins-l flies. Among those differentially expressed transcripts, genes involved in metabolism, neuronal activity, and sensory perception constituted over-represented categories. We demonstrate that two of these genes are upregulated in human subjects after acute sleep deprivation. Together, these data indicate that the ins-l flies are a useful tool that can be used to identify molecules important for sleep regulation and may provide insights into both the causes and long-term consequences of insomnia.

Figures

Figure 1.
Figure 1.
Sleep patterns of ins-l flies over generations. A, Frequency distribution of total sleep time in 60 min bins in wild-type Cs flies (n = 1000) and in ins-l flies at generation 65 (n = 364). B, Total sleep time in males (n = 40) and females (n = 40) for successive generations of ins-l flies. C, Daily sleep patterns (min/h) for progressive generations of ins-l flies (n ≈ 32/generation). D, Sleep in minutes per hour for 24 h in Cs (n = 32) and ins-l (generation 65; n = 32) flies. E, Daily total sleep time is shown for 37 d in one Cs and three ins-l flies. F, Daily total sleep time is decreased in ins-l/ins-l as well as in ins-l/Cs flies compared with Cs (ANOVA, F (1,89) = 5.50E + 06, p = < 0.0005). Error bars represent SEM.
Figure 2.
Figure 2.
Characterization of insomnia-like traits. A, Sleep latency is increased in ins-l flies (n = 28) versus Cs flies (n = 33; *p = 8.78 × 10−8, one-tailed t test). B, Average sleep bout duration is reduced during the dark period in ins-l (n = 32) versus Cs (n = 32) flies; ANOVA F (1,122) = 4.34 × 104, p < 0.0005, *p < 0.05 planned comparison with Tukey correction). C, Cumulative sleep lost or gained during 12 h of sleep deprivation and subsequent recovery in ins-l (n = 32) and Cs (n = 32) flies; striped bar indicates sleep deprivation. For each hour, the amount of sleep obtained during baseline is subtracted from the respective amount of sleep time obtained during the corresponding hour of the sleep deprivation and recovery days; the difference scores are then summed across each hour to create the cumulated gained-lost plot. A negative slope indicates sleep lost, a positive slope indicates sleep gained; when the slope is zero, recovery is complete. D, Amylase mRNA levels are elevated in ins-l flies (percentage of Cs expression) at ZT0–1 (p = 0.0002, one-sample t test). E, Intensity of locomotor activity as measured by the counts/waking minutes for 24 h in Cs flies (n = 57) and ins-l flies (n = 61; *p = 1.67 × 10−10, two-tailed t test). F, Geotaxic response to a sudden shock is greater in ins-l versus Cs flies. Percentage of flies present in the top half of a 50 ml tube (responders). Three groups of 10 flies were tested for each genotype (genotype × time interaction, F (6,24) = 4.301, p = 0.0006). Error bars represent SEM.
Figure 3.
Figure 3.
Physiological changes associated with the insomnia-like phenotype. A, Learning in Cs flies, longer-sleeping ins-l flies (average daily sleep time, 347 ± 55 min), and short-sleeping ins-l flies (average daily sleep, 26 ± 7 min; n = 10 for each group; *ANOVA, F (1,27) = 10.26, p = 0.0005, *p < 0.05, planned comparison with Tukey correction). B, PI and QSI in Cs flies (n = 5) and short-sleeping ins-l flies (n = 5). C, Number of falls during 30 min in Cs (n = 20) and ins-l flies (n = 18; *p < 0.0001, two-tailed t test). D–F, Head neurotransmitter levels in Cs and ins-l flies, as measured by HPLC (n = 4 replicates of 20 heads; *p = 0.02, two-tailed t test). G–I, Whole-body lipid content in Cs and ins-l flies (n = 3 replicates of 10 flies): triglycerides (G), free fatty acids (H), and cholesterol (I) [*p = 0.0001 (G), p = 0.002 (H), p = 0.01 (I), two-tailed t test]. J, Representative survival curve of aging ins-l compared with Cs flies (30 flies/group). K, L, Representative survival curves of ins-l and Cs flies after exposure to desiccation (K) or starvation (L) (30 flies/group). D, Error bars represent SEM.
Figure 4.
Figure 4.
Sleep in the absence of zeitgebers. A, Baseline sleep in minutes per hour in Cs and ins-l flies maintained under LD and after placement into DD (arrow). Black rectangle indicates lights off. B, Top graph, Sleep in minutes per hour in ins-l flies maintained in LD or in DD. Bottom graph, Circadian oscillations in temperature used to entrain the flies in DD. C, Sleep was evaluated in Cs and ins-l flies placed into DD for 24 h (black bars) and then into LD for recovery (white bars). Sleep is expressed as a percentage of baseline sleep in LD. D, Amylase levels in ins-llong flies under LD (n = 20) and after 24 h in DD (n = 20); data are presented as percentage change from age-matched ins-lshort and ins-llong flies, respectively (*p < 0.05, two-tailed t test). E, Learning in ins-llong flies in LD (average daily sleep 331 ± 19 min) and after 5 d in DD (average daily sleep at DD5, 72 ± 14 min; n = 10/group; *p = 0.036, one-tailed t test). F, Representative actograms for single female flies from DD1 to DD5 are shown. Similar results were obtained with ins-l males. G, Immunolocalization of PER protein in the sLNvs at DD5 (single confocal sections are shown). Ten brains were evaluated for each time point; a representative example is shown. Error bars represent SEM.
Figure 5.
Figure 5.
Gene expression in ins-l flies. A, GO categories identified using GOToolBox software schematic. Note the overrepresentation of genes involved in sensory perception. Nb in set, Number of genes differentially expressed for the corresponding the GO category; Freq, number of genes with the corresponding GO annotation divided by the total number of GO-annotated genes differentially expressed. B, QPCR confirmation of gene expression changes in short-sleeping ins-l flies expressed as fold change from normal-sleeping Cs controls. RNA was collected from all flies after 3 h of spontaneous waking at ZT3. Genes were rank ordered by p value, and confirmation was conducted on the top 5% (black), 5–50% (gray), and bottom 50% (white) of all genes. Thick arrow indicates confirmation of Amylase expression. Error bars represent SEM.
Figure 6.
Figure 6.
Genes differentially regulated ins-l flies are modulated by sleep loss in humans. A, Filamin-A (Cheerio) and Malic enzyme (Men) are both upregulated in ins-lw flies compared with nsw flies. QPCR data from 20 fly heads collected at ZT0. Levels are expressed as percentage of nsw expression. B, Filamin-A and Malic enzyme expressions are elevated after 28 h of waking in humans (n = 8; Wilcoxon signed rank test, p = 0.02 and p = 0.059, respectively). QPCR data obtained from saliva mRNA extracts. Each saliva sample collected after sleep deprivation was compared with a circadian matched baseline sample from the same subject. Levels are expressed as percentage of baseline expression. Error bars represent SEM.

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