Cross-translational studies in human and Drosophila identify markers of sleep loss

Inadequate sleep has become endemic, which imposes a substantial burden for public health and safety. At present, there are no objective tests to determine if an individual has gone without sleep for an extended period of time. Here we describe a novel approach that takes advantage of the evolutionary conservation of sleep to identify markers of sleep loss. To begin, we demonstrate that IL-6 is increased in rats following chronic total sleep deprivation and in humans following 30 h of waking. Discovery experiments were then conducted on saliva taken from sleep-deprived human subjects to identify candidate markers. Given the relationship between sleep and immunity, we used Human Inflammation Low Density Arrays to screen saliva for novel markers of sleep deprivation. Integrin αM (ITGAM) and Anaxin A3 (AnxA3) were significantly elevated following 30 h of sleep loss. To confirm these results, we used QPCR to evaluate ITGAM and AnxA3 in independent samples collected after 24 h of waking; both transcripts were increased. The behavior of these markers was then evaluated further using the power of Drosophila genetics as a cost-effective means to determine whether the marker is associated with vulnerability to sleep loss or other confounding factors (e.g., stress). Transcript profiling in flies indicated that the Drosophila homologues of ITGAM were not predictive of sleep loss. Thus, we examined transcript levels of additional members of the integrin family in flies. Only transcript levels of scab, the Drosophila homologue of Integrin α5 (ITGA5), were associated with vulnerability to extended waking. Since ITGA5 was not included on the Low Density Array, we returned to human samples and found that ITGA5 transcript levels were increased following sleep deprivation. These cross-translational data indicate that fly and human discovery experiments are mutually reinforcing and can be used interchangeably to identify candidate biomarkers of sleep loss

Cross-Translational Studies in Human and <i>Drosophila</i> Identify Markers of Sleep Loss

Inadequate sleep has become endemic, which imposes a substantial burden for public health and safety. At present, there are no objective tests to determine if an individual has gone without sleep for an extended period of time. Here we describe a novel approach that takes advantage of the evolutionary conservation of sleep to identify markers of sleep loss. To begin, we demonstrate that IL-6 is increased in rats following chronic total sleep deprivation and in humans following 30 h of waking. Discovery experiments were then conducted on saliva taken from sleep-deprived human subjects to identify candidate markers. Given the relationship between sleep and immunity, we used Human Inflammation Low Density Arrays to screen saliva for novel markers of sleep deprivation. Integrin αM (ITGAM) and Anaxin A3 (AnxA3) were significantly elevated following 30 h of sleep loss. To confirm these results, we used QPCR to evaluate ITGAM and AnxA3 in independent samples collected after 24 h of waking; both transcripts were increased. The behavior of these markers was then evaluated further using the power of Drosophila genetics as a cost-effective means to determine whether the marker is associated with vulnerability to sleep loss or other confounding factors (e.g., stress). Transcript profiling in flies indicated that the Drosophila homologues of ITGAM were not predictive of sleep loss. Thus, we examined transcript levels of additional members of the integrin family in flies. Only transcript levels of scab, the Drosophila homologue of Integrin α5 (ITGA5), were associated with vulnerability to extended waking. Since ITGA5 was not included on the Low Density Array, we returned to human samples and found that ITGA5 transcript levels were increased following sleep deprivation. These cross-translational data indicate that fly and human discovery experiments are mutually reinforcing and can be used interchangeably to identify candidate biomarkers of sleep loss.</p

Overall strategy to exploit the evolutionary conservation of sleep to identify and validate biomarkers of sleepiness.

<p>Overall strategy to exploit the evolutionary conservation of sleep to identify and validate biomarkers of sleepiness.</p

Human subjects show an increase in salivary IL-6.

<p>(<b>A</b>) Subjective sleepiness as assessed using the Stanford Sleepiness Scale was increased following 30 h of wakefulness. Each subject served as their untreated circadian-matched control; t-test p = 0.0008. (<b>B</b>–<b>D</b>) Cognitive performance, assessed using the psychomotor vigilance task (PVT), was impaired following 30 h of wakefulness: (<b>B</b>) Lapses increased during waking compared to controls; t-test, p = 0.03. (<b>C</b>) Mean reaction time, expressed as 1/RT) slowed; t-test p = 0.001. (<b>D</b>) Reaction times in the slowest 10% also slowed t-test p = 0002. (<b>E</b>) Levels of IL-6 protein from human saliva after 30 hours of waking. Levels in sleep deprived subjects (n = 16) are expressed as a % of that their own untreated circadian-matched sample (con); t-test p = 0.042.</p

Immune-related transcripts are associated with increased sleep drive in <i>Drosophila</i> clock mutants.

<p>(<b>A</b>) Immune related transcripts are substantially increased in <i>cyc⁰¹</i> mutants following 7 h of sleep deprivation but are not consistently activated when 7 h of waking is induced by starvation; data are expressed as % change from untreated siblings. Experiments were conducted in constant darkness (DD). (<b>B</b>–<b>E</b>) Transcript levels for <i>AttB</i>, <i>Dro</i>, <i>Mtk</i> and <i>Drs</i> in <i>tim⁰¹</i> mutants following 3, 6, 9 and 12 h of sleep deprivation. Immune related transcripts are consistently elevated following 9 and 12 hour sleep deprivation (Light Green) but not after 3 or 6 h of sleep deprivation when no sleep rebound is observed (dark Green).</p

The <i>Drosophila</i> integrin <i>scb</i> and Human <i>ITGA5</i> are modified by sleep loss.

<p>(<b>A</b>) <i>scb</i> is elevated following caffeine administration but not after waking induced by methamphetamine. (<b>B</b>) <i>scb</i> is elevated in sleep deprived <i>cyc⁰¹</i> mutants but is not increased when waking is induced by starvation. (<b>C</b>) <i>scb</i> mRNA levels remain low following deprivations that do not activate homeostatic mechanisms (3 and 6 h SD), but are elevated following deprivations that activate homeostatic mechanisms (9 and 12 h SD). (<b>D</b>–<b>E</b>) Salivary <i>ITGA5</i> transcripts are increased in humans following 30 h of wakefulness while 24 h of wakefulness do not quite reach significance; t-test and p = 0.04 and p = 0.056 respectively. Each subject serves as their own circadian matched untreated control; data are expressed as a percent change from control.</p

IL-6 is elevated in serum of chronic total sleep deprived rats.

<p>(<b>A</b>) Energy Expenditure expressed as % of home cage controls (HCC, n = 7) is significantly increased in total sleep deprived (TSD, n = 7) rats compared to yoked controls (TSC, n = 6); One way ANOVA F_(2,11) = 6.79, p = .012, * modified Bonferroni test p<0.05 (<b>B</b>) Serum concentrations of IL-6 protein from individual TSD, TSC, and HCC rats. Arrows denote which animals tested positive for bacteria. (<b>C</b>) Mean concentration of IL-6 from individual rats in (B). One way ANOVA F_(2,17) = 5.22, p = .017 * p<0.05 modified Bonferroni test <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061016#pone.0061016-Keppel1" target="_blank">[58]</a>. Data are presented as mean±SEM.</p

Transcripts for immune-related genes are elevated in flies under conditions of increased sleepiness.

<p>(<b>A</b>) Immune-related transcripts are significantly elevated in <i>Cs</i> flies following a night of sleep deprivation; data are expressed as % of untreated circadian-matched siblings. (<b>B</b>) <i>Cs</i> flies with normal total sleep time but spontaneously fragmented sleep (average night sleep bouts <30 min) exhibit an increase in immune-related transcripts expressed as % change from siblings with consolidated sleep and matched by total sleep time; error bars are present but not visible given the magnitude of the changes. Abbreviations: <i>Drosocin</i> (<i>Dro</i>), <i>Attacin-B</i> (<i>AttB</i>), <i>Drosomycin</i> (<i>Drs</i>), <i>Metchnikowin</i> (<i>Mtk</i>), <i>Immune induced molecule 23</i> (<i>IM23</i>), and <i>Defensin</i> (<i>Def</i>).</p