Identification of Redeye, a new sleep-regulating protein whose expression is modulated by sleep amount

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Identification of Redeye, a new sleep-regulating protein whose expression is modulated by sleep amount

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DOI: http://dx.doi.org/10.7554/eLife.01473Published February 4, 2014 Cite as eLife 2014;3:e01473

Abstract

In this study, we report a new protein involved in the homeostatic regulation of sleep in Drosophila. We conducted a forward genetic screen of chemically mutagenized flies to identify short-sleeping mutants and found one, redeye (rye) that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor α subunit required for Drosophila sleep. Levels of RYE oscillate in light–dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. We propose that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep.

DOI: http://dx.doi.org/10.7554/eLife.01473.001

eLife digest

Almost all animals need to sleep, including most insects. In experiments in the 1980s, a group of rats that were completely deprived of sleep died within only a few weeks. Sleep has been implicated in processes including tissue repair, memory consolidation and, more recently, the removal of waste materials from the brain. However, a full understanding of why we sleep is still lacking.

As anyone who has experienced jetlag can testify, the timing of the sleep/wake cycle is governed by the circadian clock, which leads us to feel sleepy at certain points of the day–night cycle and alert at others. The duration of sleep is regulated by a second process called sleep/wake homeostasis. The longer we remain awake, the more the body’s need for sleep—or ‘sleep drive’—increases, until it becomes almost impossible to stay awake any longer. Whereas many components of the circadian clock have been identified, relatively little is known about the molecular basis of this second process.

Now, Shi et al. have identified a key component of the sleep/wake homeostatic system using the fruit fly and genetic model organism, Drosophila. Flies with a mutation in one particular gene, subsequently named redeye, were found to sleep only half as long as normal flies. While the insects were able to fall asleep, they would wake again only a few minutes later.

Redeye encodes a subunit of a receptor that has previously been implicated in the control of wakefulness, known as the nicotinic acetylcholine receptor. Mutant flies had normal circadian rhythms, suggesting that their sleep problems were the result of disrupted sleep/wake homeostasis. Consistent with this, levels of redeye showed two daily peaks, one corresponding to night-time sleep and the second to the time at which flies would normally take an afternoon siesta. This suggests that redeye signals an acute need for sleep, and then helps to maintain sleep once it is underway.

While redeye is not thought to be the factor that triggers sleep per se, it is directly under control of the sleep homeostat, and may be a useful biomarker for sleep deprivation. The fact that redeye was identified in fruit flies, a species whose genome has been fully sequenced, opens up the possibility of further studies to identify the genetic basis of sleep regulation.

DOI: http://dx.doi.org/10.7554/eLife.01473.002

Main text

Introduction

Sleep is a common and prominent behavior in almost all vertebrate animals and also in most invertebrates (Cirelli, 2009; Sehgal and Mignot, 2011). The function of sleep is a mystery, but it is surely of great importance to animals, as prolonged sleep deprivation can lead to death. Anatomical studies in mammals and birds have revealed brain structures and neurotransmitters that regulate sleep and wakefulness (Saper et al., 2010). However, our understanding of the molecular mechanisms that drive the need to sleep is still in its infancy, partially due to the challenge of performing genetic experiments with mammalian models. In the past decade, several premier genetic organisms have been introduced into the sleep field, including fruit flies, worms and zebra fish. Mammalian counterparts of some sleep components identified in these model animals also regulate sleep (Joho et al., 2006), which argues that (i) behavioral genetics in lower organisms provides an efficient tool to identify sleep components, (ii) at least some of the mechanisms underlying sleep are conserved through evolution.

A two-process model for sleep regulation has been widely accepted by the sleep field. Process C (the circadian clock) controls the timing, in other words the onset and offset of sleep, whereas process S (the sleep homeostat) regulates sleep duration based on the sleep pressure built up during prior wakefulness (Borbely, 1982). This simple model explains sleep related phenomena, including sleep rebound after sleep deprivation. Molecular mechanisms of circadian control have been well characterized (Zheng and Sehgal, 2012), but, as noted above, relatively little is known about process S. Forward genetic screens in Drosophila have led to the identification of several mutants with altered sleep length, but while the genes implicated by these mutants are required for implementation of sleep drive, they have not yet been directly linked to this drive (Sehgal and Mignot, 2011).

Using a forward genetic screen, we identified a new sleep mutant we termed redeye (rye), which is directly controlled by the homeostatic drive to sleep. The rye mutation maps to a nicotinic acetylcholine receptor (nAChR), which interacts with a previously identified sleep-regulating protein, SLEEPLESS (SSS). Levels of RYE are expressed cyclically, in conjunction with the sleep state, and reflect sleep need such that they are up-regulated after sleep deprivation and in short-sleeping mutants. We conclude that homeostatic sleep drive promotes sleep at least in part by increasing expression of RYE.

Results

Identification of redeye, a short-sleeping mutant

As previous screens have identified sleep components on the X chromosome and the second chromosome (Cirelli et al., 2005; Koh et al., 2008; Stavropoulos and Young, 2011), we sought to identify sleep-altering mutations on the third chromosome in Drosophila. To this end, we generated a stock of iso31 (Ryder et al., 2004) flies carrying a newly isogenized third chromosome and treated males of this stock with 10–25 mM ethyl-methanesulfonate (EMS). Individual male progeny were bred to achieve homozygosity of the third chromosome, and females of the F3 generation were tested for daily sleep patterns in the presence of light:dark cycles (Koh et al., 2008). As seen in previous screens (Koh et al., 2008), sleep amounts were normally distributed in the 1857 lines assayed (Figure 1A, Figure 1—source data 1). Two homozygous mutant lines showed a severe reduction of sleep length, and we focused on one of these that we named redeye (rye). rye homozygotes show a >50% reduction in both daytime and night-time sleep (Figure 1C). The activity (average number of beam crossings during periods of activity) of rye mutants is comparable with that of wild type controls, suggesting it is not a hyperactive mutant (Figure 1B). rye heterozygotes have slightly less sleep than wild type, suggesting that the rye mutation is partially dominant (Figure 1B,C). The dramatic reduction of baseline sleep results largely from a shortening of the average sleep episode duration (Figure 1D), which is indicative of a defect in sleep maintenance. The average number of sleep episodes at night is actually increased significantly in rye mutants, perhaps because the sleep homeostat senses sleep loss and compensates by initiating more bouts. We also assayed rye mutants in sleep deprivation assays and found that they did not lose much additional sleep in response to mechanical stimulation (data not shown). We surmise that an inability to sustain sleep bouts leads to the buildup of high sleep need in rye mutants, which makes them resist any further sleep loss.

Figure 1.
Download figureOpen in new tabDownload powerpointFigure 1. Identification of a short sleep mutant, redeye (rye), through a chemical mutagenesis (EMS) screen.

(A) Histogram depicting average sleep levels in females from homozygous EMS-mutagenized lines (n = 1857). The mean sleep value for each mutant was calculated by assaying sleep in 4–8 individual flies in the presence of 12 hr L-12 hr D cycles. Average sleep for all lines is indicated on the X axis in increments of 25 min. The Y axis depicts the number of mutant lines within each group. The dashed lines mark the sleep values that correspond to plus/minus 3× standard deviation of the mean. The arrowhead indicates the redeye mutant. (B) Top: average activity pattern of females from control lines and from rye mutants recorded in 12hr L-D cycles (n = 14–16 in 5 days). Bottom: sleep profiles with standard error bars. Black: lines isogenized on the 3rd chromosome for chemical mutagenesis; Blue: flies heterozygous for rye and the isogenized control chromosome; Orange: rye homozygotes. (C) Mean values of total sleep, daytime sleep and night-time sleep for rye mutants (Figure 1—source data 1). For each genotype 14–16 flies were assayed over a 5 day period. Bars represent standard error. One-way ANOVA was performed followed by Tukey post hoc analysis. * represents p<0.05, **p<0.01 and ***p<0.001. (D) Sleep quality of rye mutants: Daytime and night-time sleep episode length and episode number are plotted in the box-and-whisker diagram (Figure 1—source data 1). The middle line represents the median value; Bottom and top line of each box represent 25% and 75% respectively; Bottom bar and top bar represent 5% and 95% respectively. ns: not significant, ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.01473.003

Figure 1—Source data 1. Measurement of sleep duration for all EMS mutants and sleep analysis of rye mutants.

DOI: http://dx.doi.org/10.7554/eLife.01473.004

Download source data [figure-1—source-data-1.media-1.xlsx]

Normal circadian rhythms in rye homozygotes

As the circadian clock regulates the timing of sleep and some clock mutants show reduced sleep (Hendricks et al., 2003), we also assayed rye flies for defects in circadian rhythms. These endogenously generated rhythms are best monitored under constant conditions, in the absence of cyclic environmental cues, so we monitored behavior in constant darkness (DD). rye mutants display robust rest:activity rhythms in DD (∼62% rhythmicity) despite showing prolonged duration of activity (Figure 2A; Table 1). Of the ‘arrhythmic’ rye homozygotes detected, half (6/11) actually displayed a rhythm of ∼12 hr period length, which likely resulted from persistence of bimodal behavior (typically seen in light:dark) in DD. Thus, most rye homozygotes are rhythmic. Consistent with the intact circadian behavior, circadian cycling of the clock protein, PERIOD (PER), is normal in central clock cells of rye mutants. The peaks and troughs of expression, as well as the subcellular localization, at different times of day are similar to those in iso controls (Figure 2B). Thus, PER is largely nuclear at ZT20, entirely nuclear at ZT2, expressed at low levels at ZT8 and undetectable at ZT14. We conclude that only the homeostatic regulation of sleep is affected in rye mutants.

Figure 2.
Download figureOpen in new tabDownload powerpointFigure 2. rye homozygotes display normal circadian rhythms and clock protein cycling.

(A) Double plotted activity records of iso controls and rye homozygotes in DD. Rhythmic, but prolonged cross-beam activity was recorded in rye homozygotes. (B) Immunostaining of PERIOD in ventral lateral neurons (LNvs) of iso controls and rye homozygotes at CT time points on the second day of DD following LD entrainment. PER oscillates in these clock neurons in iso as well as in rye homozygotes. PDF labels the small and large LNvs.

DOI: http://dx.doi.org/10.7554/eLife.01473.005

View this table:
Table 1.

Rhythmic behavior of rye homozygotes in constant darkness

DOI: http://dx.doi.org/10.7554/eLife.01473.006

Identification of rye as an α subunit of the nicotinic acetylcholine receptor

We identified the sleep-altering lesion in rye mutants through a combination of recombination mapping and deep-sequencing. Despite starting with a newly isogenized line, deep-sequencing identified many nucleotide changes in the rye background relative to the parental control and so considerable mapping was required to pinpoint the relevant mutation. Meiotic recombination analysis positioned rye between two genetic markers, thread and curled, in the centromeric region of the 3rd chromosome. Further mapping studies, using self identified single nucleotide polymorphism (SNP) markers, narrowed it down to ∼11 mega-bases (Figure 3A, Figure 3—source data 1). For finer localization, we utilized the genome-wide sequence data, which provided 40× and 10× genomes worth of nucleotide sequence, corresponding to 95% and 88% coverage, for the rye and iso31 genomes respectively. Following alignment with the published Drosophila genome, unique nucleotide polymorphisms present in rye mutants were pooled together as potential EMS-induced mutations (n = 26,224). Within the 11 Mb region identified through recombination mapping, there were 1457 potential mutations, but only nine that produced an amino-acid change in coding regions. Sanger sequencing allowed us to exclude seven of these. Two turned out to be deep-sequencing errors and five polymorphisms were also present in the wild type iso stock but not detected due to the low coverage of deep-sequencing in this stock. One polymorphism, in a gene annotated CG7320, was at a site that was also polymorphic in iso, although the amino acid change was different in the two strains. Because of the change in iso, which does not produce a sleep phenotype, we considered CG7320 a less likely candidate for rye. The last of the nine changes identified by deep-sequencing was a C-T transition (typical for EMS-induced mutations) that leads to a threonine to methionine change in CG12414 and is present only in rye mutants (Figure 3B). CG12414 encodes an α subunit of a nicotinic acetylcholine receptor (nAchR) (Lansdell and Millar, 2000). Five alternatively spliced variants produced by this gene are predicted to generate three possible open reading frames with one (pC/pG) containing an intact ligand bind domain (LBD) (Figure 3C). The predicted protein is homologous to several nAChR subunits, including α3, α2, α6, α4 and α7 in order of similarity. Alignment of CG12414 with α3 and α7 using the ClustalW2 algorithm shows that the rye mutation is at the junction between the LBD and the trans-membrane domain (TM) (Figure 3D,E). Interestingly, this junction region is highly conserved across species and the specific threonine mutated in rye is conserved in all α7 subunits (Figure 3D).

Figure 3.
Download figureOpen in new tabDownload powerpointFigure 3. Genetic mapping and genome-wide deep-sequencing reveal a missense mutation in a nicotinic acetylcholine receptor α subunit gene in rye mutants.

(A) Through genetic mapping, using classical phenotypic markers and newly identified SNP markers, rye was mapped between two SNP markers (SNP_L and SNP_R) near the centromere on the 3rd chromosome. (B) Paired-end genomic deep-sequencing of rye mutants identified a missense mutation in CG12414. We confirmed this through Sanger sequencing. This C-T/G-A transition that generated the ryeT227M allele is typical of EMS-induced mutations. (C) Schematic representation of the rye candidate gene, a nicotinic acetylcholine receptor (CG12414: nAcRα-80B). The gene spans ∼100 kb with alternative splicing predicted to produce five transcripts (RC-RG). The spliced forms are predicted to translate into three proteins isoforms (pC-pG). pC(pG) produces the largest protein with the longest ligand binding domain (LBD). (D) Alignment of the partial sequence of Drosophila RYE with nAChR α3 and α7 subunits in other animals using ClustalW2. The region shown is at the boundary of the ligand binding domain (LBD) and transmembrane domain (TM), and is evolutionarily conserved. T227 in RYE is marked in bold form. ‘*’ identity; ‘:’ high similarity; ‘.’ similarity. (E) Protein sequence analysis predicts four transmembrane domains (TM) in RYE, which is typical of most nAChR proteins. RYE appears to contain a single ligand binding domain (LBD) in the extracellular region, and four TMs with three loop regions (L1-L3). The mutated threonine227 is in the LBD, close to the beginning of the TM.

DOI: http://dx.doi.org/10.7554/eLife.01473.007

Figure 3—Source data 1. Recombination mapping of the rye mutation using a chromosome marked with visible markers h,th,cu,sr,e, and using SNP markers.

DOI: http://dx.doi.org/10.7554/eLife.01473.008

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Functional analysis of rye

The genetic mapping and deep-sequencing data combined implicated CG12414 as a candidate gene responsible for the rye sleep phenotype. To verify this function for CG12414, we attempted to rescue rye mutants with a CG12414 transgene. To drive expression in areas that normally express this gene, we cloned the CG12414 promoter region (1.8 kb) and generated a GAL4 construct. This GAL4 is expressed broadly in the Drosophila brain (Figure 4A). We also cloned the CG12414 cDNA into a UAS construct and crossed the GAL4 and UAS transgenes into an iso31 stock to ensure a homogenous genetic background. These transgenes were then introduced into rye mutants. While either transgene alone did not alter the rye short sleep phenotype, the rye promoter driving RYE expression (ryeP > uas-rye) partially rescued the mutant phenotype and restored total sleep to levels seen in rye heterozygotes (Figure 4B). This is expected, given that ryeT227M is partially dominant over wild type. In a wild type background, ryeP > uas-rye does not increase sleep length (data not shown), suggesting that the increase in sleep is specific to the mutant. Thus, we identified the gene responsible for the short sleep phenotype in rye mutants, and henceforth refer to this gene CG12414, previously called nAChR-80B, as redeye.

Figure 4.
Download figureOpen in new tabDownload powerpointFigure 4. An alpha subunit of the nicotinic acetylcholine receptor accounts for the rye mutant phenotype.

(A) Expression pattern of ryeP-GAL4. Rye-GAL4 was used to express nGFP, which is visualized with an anti-GFP antibody. The anti-nc82 staining marks the neuropil. (B) Expression of the alpha subunit, as described in Figure 3, increases sleep duration in rye mutants (Figure 4—source data 1). Left: sleep profiles, with standard error, of rye mutants and mutants expressing a UAS construct of the putative rye cDNA under control of its own promoter (ryeP-Gal4) in a 12:12 LD cycle. Right: quantification of sleep length. **p<0.01, ***p<0.001. (C) Reduction of rye expression in rye neurons through the expression of an RNAi construct, together with Dicer2, diminishes sleep length (Figure 4—source data 1). Left: sleep profile in a 12:12 LD cycle. Right: quantification of sleep length. *p<0.05. (D) Western blot analysis shows reduced expression of RYE when actin-GAL4 is used to drive rye RNAi (VDRC#11392) with Uas-Dicer2 in female and male flies.

DOI: http://dx.doi.org/10.7554/eLife.01473.009

Figure 4—Source data 1. Sleep behaviour of transgenically rescued rye mutants and rye RNAi lines.

DOI: http://dx.doi.org/10.7554/eLife.01473.010

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To further characterize rye function, we knocked down RYE expression through RNA interference. A rye RNAi line (#11392) from the VDRC stock center in Vienna was crossed into the ryeP-GAL4 line along with a UAS-Dicer2 transgene, included to improve efficacy of the RNAi. Levels of RYE were reduced (Figure 4D), as was the total sleep length (Figure 4C). Since ryeT227M reduces sleep as well (Figure 1), these data suggest that the T227M mutant allele causes loss of RYE function. The sleep phenotype produced by rye knock-down is rather mild in comparison with the phenotype of the homozygous rye mutant, possibly due to inefficient knockdown in relevant cells or because ryeT227M is a dominant-negative mutation. The nicotinic acetylcholine receptor forms a pentameric structure, consisting of five homo-oligomeric or hetero-oligomeric subunits (Miwa et al., 2011), so a dominant negative mutation in any one subunit may well interfere with activity of the complex.

RYE interacts with SSS

We next addressed if rye interacts with other known sleep mutants, in particular with a mutant we identified previously, sleepless (sss), that has an extreme short-sleeping phenotype (Koh et al., 2008). While transheterozygotes of rye and sss (sss/+; rye/+) showed sleep duration comparable to that of rye/+ alone (data not shown), rye heterozygotes carrying a genomic sss transgene, which increases SSS expression above that of wild type controls, showed a further reduction of sleep (Figure 5A). The effect was observed with three independent insertions of the sss transgene, indicating that it was not due to insertion site effects (Figure 5B). We note that this sss transgene does not have a significant effect on sleep in wild type flies although it completely rescues the sleep phenotype of sss mutants (Koh et al., 2008). Thus, the effect is specific to rye mutants.

Figure 5.
Download figureOpen in new tabDownload powerpointFigure 5. Overexpression of sss exacerbates the rye phenotype by repressing RYE activity.

(A) An extra copy of SSS reduces sleep in rye heterozygotes. Sleep profile of rye heterozygotes with or without a genomic sss transgenic line inserted on the third chromosome (sssTG2) and of sss transgenics alone. (B) Quantification of sleep in the genotypes as indicated (Figure 5—source data 1). TG1 and TG3 are additional independent genomic sss transgenes inserted on the 3rd and X chromosomes respectively. Sleep reduction was observed in rye transheterozygotes with all three sss transgenic lines. *p<0.05. (C) Heterologous expression of SSS reduces current following ACh application in Xenopus oocytes expressing Drosophila RYE and human nAChR β2 (Figure 5—source data 1). *p<0.05. (D) Heterologous expression of SSS reduces current following ACh application in Xenopus oocytes expressing human nAChR α4β2 (Figure 5—source data 1). ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.01473.011

Figure 5—Source data 1. Sleep analysis of rye mutants overexpressing sss, and whole-cell membrane current recording of Xenopus oocytes.

DOI: http://dx.doi.org/10.7554/eLife.01473.012

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SSS is a GPI-anchored protein that regulates the Shaker potassium channel and its predicted structure resembles that of a specific class of toxins (Wu et al., 2010; Dean et al., 2011). It is most similar to the mammalian Lynx-1 protein, which is known to inhibit activity of the nicotinic acetylcholine receptor (Miwa et al., 2011). The fact that SSS exacerbated the phenotype of flies heterozygous for rye, which have reduced activity of nAChR, suggested that SSS also regulates nAChRs. We tested a possible Lynx-1-like function of SSS in regulating nAChR activity by using a heterologous expression system, specifically Xenopus laveis oocytes. For functional expression of wild type RYE, we expressed it with a human β2 subunit, as insect β subunits typically do not work in heterologous systems (Millar and Lansdell, 2010). cRNAs encoding sss, wild type rye and human β2 were injected into oocytes, and 4–7 days later whole cell currents were measured following application of ACh (Kuryatov and Lindstrom, 2011). Heterologous expression of insect nAChRs has, in general, been challenging (Millar, 2009), which accounts for the low current values observed (Figure 5C). However, we still detected a significant reduction of nAChR current upon co-expression with SSS (Figure 5C). We also co-expressed in vitro transcribed sss and human α4 and β2 subunits and found that SSS also inhibits the robust current produced by the human receptor complex (Figure 5D). Thus, in addition to its sleep-promoting role reported previously, SSS may also promote wake by repressing activity of nAChRs. As discussed below, loss of the sleep-promoting role has a dominant effect in sss mutants.

RYE is expressed cyclically in association with the sleep state

We next asked if levels of rye vary over the course of the day. Groups of wild type flies were housed in a 12 hr-light and 12 hr-dark incubator and harvested at different Zeitgeber times (ZT). Zeitgeber time defines time based on the environmental stimulus, so ZT0 corresponds to light-on and ZT12 to light-off. RNA was extracted and subjected to quantitative PCR analyses. While period (per), a well-known circadian clock component, oscillates with a circadian rhythm (Zheng and Sehgal, 2012), rye mRNA levels were found to be constant (Figure 6A).

To examine protein levels of RYE, we generated an antibody against the cytoplasmic L3 domain of RYE (Figure 3E), which is the most variable region across all nAChR subunits (Lindstrom, 2003). On western blots, the antibody recognizes a band (or sometimes a doublet) of ∼60 Kd, which is the size predicted for the full-length isoform. Surprisingly, in contrast to the flat mRNA levels, levels of RYE protein show a robust rhythm with two peaks each day, one at around ZT6-10 and the other at ZT18-22 (Figure 6B, Figure 6—figure supplement 1). Interestingly, these correspond to the two daily times of sleep, the afternoon siesta and night-time sleep (Figure 1B), suggesting that RYE is expressed in phase with sleep. The cycling of RYE persists in constant darkness, as do rhythms of sleep (data not shown). However, RYE does not appear to be regulated like other cycling clock components, such as PER, which usually show only one peak (Figure 6B). Furthermore, we found that RYE continues to cycle in Clkjrk flies that lack a circadian clock, although the phase of RYE expression tends to be more variable in these flies (Figure 6C, Figure 6—figure supplement 2). These data suggest that the RYE oscillation per se does not require process C of the two process model, but may depend upon process S (sleep homeostasis). However, the circadian clock may function to provide more precise timing of RYE expression, perhaps through its control of sleep onset and offset.

Expression of RYE is under control of the sleep homeostat

Increases in RYE expression could occur as a consequence of sleep, and thereby reflect the sleep state, but in that case levels should remain high throughout the sleep state. Given that RYE levels do not remain high throughout the night or through the afternoon siesta, another possibility is that elevations in RYE correspond to sleep drive and so occur at the time of sleep onset. To test this idea, we assayed RYE expression in short-sleeping mutants. These mutants have low sleep levels, but probably have high sleep drive that cannot be implemented. As shown in Figure 7, for the most part RYE expression exhibits two peaks in these mutants, but the phase is more variable (Figure 7—figure supplements 1–3), indicating that defects in sleep behavior affect the RYE expression pattern. Interestingly, overall RYE values are higher in fumin (Kume et al., 2005; Ueno et al., 2012), sss (Koh et al., 2008) and insomniac (Stavropoulos and Young, 2011) mutants than in wild type controls. As noted above, the sss mutation affects channel activity, fumin is a mutation in the dopamine transporter (short sleep is thought to result from the high levels of dopamine in the synaptic cleft) and insomniac is thought to affect protein turnover. The one feature these mutants have in common is short sleep, yet they all have elevated RYE expression during an LD cycle (Figure 7A–C). Based upon these data, we suggest that RYE reflects sleep need. It is elevated at the time of sleep onset and is further increased in short-sleeping mutants because they have high sleep need.

If the expression of RYE changes in response to the accumulation of sleep need, it should also be affected by acute sleep deprivation. We mechanically deprived flies of sleep in the second half night (6 hr) in an LD cycle, and confirmed, through the presence of a substantial sleep rebound the following morning, that the flies were successfully deprived (Figure 8A; Koh et al., 2008). RYE levels were assayed during the 5 hr sleep rebound window. In the non-deprived control, RYE levels were low at ZT0 and high at ZT5, as noted above. Sleep-deprived flies had high levels of RYE immediately following deprivation (ZT0), but levels were low at ZT5 (Figure 8A, Figure 8—figure supplement 1). This fits the profile for sleep drive, which is expected to be high at the end of deprivation, but then dissipated over the course of rebound sleep.

Discussion

The molecular mechanism of sleep homeostasis is a mystery and a subject of intense research in the sleep field (Suzuki et al., 2013). In addition to the investigation of mechanisms underlying sleep drive, considerable effort is being put into identifying biomarkers of sleep need. Based on what we know about the so-called sleep homeostat, which consists of increasing sleep pressure during wakefulness and dissipation of such pressure following sleep, we suggest that a component or direct output of the homeostat should satisfy three criteria: (i) the gene product should regulate the sleep:wake cycle (i.e., genetic alleles of this gene should have some sleep phenotype); (ii) expression levels or activity of the gene product should go up during wakefulness or during sleep deprivation and (iii) expression levels or activity should decrease after sleep. The function of RYE and the molecular kinetics of the RYE protein largely satisfy these criteria. However, while RYE builds up during sleep deprivation, it does not accumulate gradually over the wake period in a daily cycle. Rather, it displays a marked increase close to the time of sleep onset, suggesting that it is not a central component of the homeostat, but responds to an upstream homeostatic signal, perhaps when that signal reaches a certain threshold. The fact that over-expression of RYE does not promote sleep also supports the idea that it is not the sleep-inducing homeostatic signal (further discussed below). Nevertheless, RYE is not simply a sleep output gene or sleep biomarker. It is required for implementation of signals from the homeostat and it functions to maintain sleep. Thus, we propose that rye is a sleep-regulating gene immediately downstream of the homeostat.

RYE expression reflects sleep need

We suggest that RYE represents a molecular correlate of delta power, a characteristic of an electroencephalogram (EEG) that reflects sleep drive. Recently, a few other molecules were reported to change with sleep drive, but the effects were at the level of the mRNA, the magnitude of the increase was less than we report here for RYE and loss of the molecules did not affect baseline sleep duration (Seugnet et al., 2006; Maret et al., 2007; Naidoo et al., 2007). In addition, only one is expressed cyclically (Nelson et al., 2004), indicating that others reflect sleep drive only under pathological conditions of sleep deprivation. RYE levels oscillate robustly in a daily cycle, although the phase is not as coherent as seen for circadian clock proteins. The timing of the peak varies within a temporal range, such that there is almost always a daytime peak and a night-time peak but not necessarily at the exact same time (Figure 6—figure supplement 1). We suggest that RYE cycles under control of the sleep homeostat, which may not time behavior as precisely as the circadian clock, perhaps because sleep can be influenced by many factors. The variability in RYE cycling is particularly pronounced in short-sleeping mutants and in the ClkJrk circadian clock mutant (Figure 6—figure supplement 2, Figure 7—figure supplements 1–3), suggesting that the clock does influence RYE expression although it is not required for its cycling in an LD cycle. Interestingly, RYE cycles exclusively at the level of the protein, indicating translational or post-translational mechanisms. It is worth noting that a recently identified sleep regulator, Insomniac, is a component of specific protein degradation pathways in the cell (Stavropoulos and Young, 2011). Although our study indicates that RYE cycling does not require Insomniac (Figure 7C), it is possible that it is regulated by other protein turn-over machinery. Thus, translational/posttranslational regulation appears to be part of the mechanism of sleep homeostasis.

RYE is required to maintain the sleep state

We show that RYE not only reflects sleep drive, but is also required for sleep maintenance (Figures 1, 4 and 8). Given that RYE is induced by sleep deprivation and it promotes sleep, one might expect over-expression of the protein to increase sleep. However, transgenic expression of rye in a wild type background does not increase sleep, suggesting that while rye is necessary, it is not sufficient for sleep onset. We cannot exclude the possibility that RYE functions together with other signals as part of the sleep-inducing homeostatic drive. On the other hand, it is also possible that transgenic expression does not produce adequate amounts of RYE protein in relevant cells. This might be the case if RYE is tightly regulated at the level of protein stability. For the moment, though, we prefer the parsimonious explanation noted above, that RYE is not part of the homeostat, but immediately downstream of it.

Acetylcholine signaling has been long proposed as an arousal factor, as the nAChR complex is a cation channel that normally promotes neuronal activity and ACh is released during wakefulness in mammals (Platt and Riedel, 2011). In contrast, our study indicates that at least one nAChR subunit (RYE) promotes sleep in the fly. There are more than 10 paralogs of nAChR subunits in the fly genome. One possibility is that RYE is expressed specifically in sleep promoting neurons, while other subunits of AChRs are in wake-promoting cells. An increase in ACh during wakefulness may contribute to the accumulation of sleep drive and to the increase in RYE (Lindstrom, 2003). Alternatively, sleep drive may increase RYE independently of ACh, but in either scenario, RYE then promotes sleep. The precise site of RYE action is currently not known. rye-gal4 driven GFP is expressed widely in the brain (Figure 4A), but we cannot be sure that endogenous RYE is as widespread, as our antibody was not effective in immunohistochemistry experiments, and we find that GAL4 drivers are often quite promiscuous (unpublished observations).

SSS inhibits RYE activity and promotes arousal in a sensitized background

SSS was previously identified as a sleep promoting factor, essential for maintaining baseline sleep and for homeostatic rebound (Koh et al., 2008). An interaction between rye and sss is therefore not surprising. What is surprising is that overexpression of SSS promotes wakefulness in ryeT227M heterozygotes (Figure 5A,B). SSS is a GPI-anchored protein that functions as a neuronal modulator. Previous studies indicate that SSS promotes activity of the voltage-gated potassium channel, Shaker (Wu et al., 2010). In this study, we report that SSS acts like a brake on nAChR (RYE) activity (Figure 5C,D), as does Lynx-1, a SSS-like molecule, in mammals (Miwa et al., 2011). Although the data we show for Drosophila receptors used only the RYE α subunit, it is likely that SSS also inhibits activity of other Drosophila nAChR receptors. As both sssP1 (a null mutation) and sssP2 (a hypomorphic allele) are short-sleeping mutants (Koh et al., 2008), we propose that the overall effect of SSS is to promote sleep. The reduced sleep in sss mutants probably results from an increase of neuronal excitability, through inactivation of potassium channels (Shaker) (Dean et al., 2011), or from hyperactivity of nAChR channels in wake-promoting neurons. Thus, typically the sleep-inhibiting effect of SSS, mediated through RYE, is masked by these other more dominant influences. However, in a sensitized background (i.e., rye/+), this effect is evident. RYE promotes sleep, and so loss of RYE results in a decrease in sleep, which is further impacted by SSS overexpression (Figure 8B).

We note that there are some caveats to these data. For instance, the ryeT227M allele could confer a neomorphic function that accounts for the interaction with sss. Likewise, the effects in oocytes could be non-physiological, not necessarily reflecting what happens in the fly brain. However, given that we observe interactions in these two very different types of assays, and both assays indicate repression of nAchR function by SSS, which is the effect predicted from the role of the mammalian SSS-like protein, Lynx1, we believe SSS does indeed regulate nAchRs such as RYE. Interactions between SSS and nicotinic acetylcholine receptors are also reported by recent work from another laboratory (W Joiner, personal communication).

It is interesting that genes identified through independent genetic screens in Drosophila are turning out to interact with one another. SSS and Shaker were isolated independently as sleep-regulating genes (Cirelli et al., 2005; Koh et al., 2008), and subsequently shown to interact, and now we find that RYE interacts with SSS. Given that each of these genes represents a relatively infrequent hit in an unbiased screen, the interactions suggest that genetic approaches are converging upon specific sleep-regulating pathways. Interestingly, a recent GWAS study for sleep-altering loci in humans identified significant effects of SNPs in an nAchR subunit as well as in a regulatory subunit of Shaker, suggesting that these mechanisms are also conserved across species (Allebrandt et al., 2013).

Materials and methods

Generation of an isogenic 3rd chromosome stock

Wild type iso31 (Ryder et al., 2004) was crossed with TM2/TM6C, Sb (Bloomington stock #5906), and a single male progeny was selected and crossed with #5906 to balance the 3rd chromosome and generate a line isogenic for this chromosome.

Behavioral analysis

Flies were housed in Percival incubators (Perry, IA) and beam–break activity was recorded with the Trikinetics DAM system (http://www.trikinetics.com/). Pysolo (Gilestro and Cirelli, 2009) software was used to analyze and plot sleep patterns. Sleep deprivation was achieved through repeated mechanical shaking (2 s randomized shaking in every 20-s interval).

Mapping analysis

A 3rd chromosome marker line h, th, cu, sr, e (Bloomington stock #576) was crossed to rye/TM6C, Sb, and heterozygote female progeny (h, th, cu, sr, e/rye) were further crossed to TM2/TM6C, Sb (#5906). A single recombinant male offspring was back-crossed to #576 to score the genotype of recessive markers and also back-crossed to rye for behavioral analysis to score the rye genotype. Overlap in the genotypes of recombinants narrowed down the location of the rye mutation to the region between markers th and cu.

Genomic DNA of homozygous recombinants was subject to SNP analysis. The primer set 5′TGTTTAGTGGTGTTGTGTGAGC3′ and 5′GCCGAGTGTCATCGCCTTTG3′ for SNP_L and the primer set 5′AAAGGTCATCTTGCTTCGGAGTTG3′ and 5′GGAGTGGCTTCCTCGTCATC3′ for SNP_R were used for PCR amplification. Nucleotide polymorphisms were identified between iso and #576. Thus, those recombinants were scored accordingly and rye was further narrowed down to a region between those two SNP markers.

Sequencing and cloning

Fragmented genomic DNA (∼400 bp in size) obtained through the Covaris instrument (Woburn, MA) was subjected to Illumina paired-end DNA library preparation. The libraries were amplified for 10 PCR cycles prior to Illumina Hi-Seq analyses. SNP calling analyses identified nucleotide polymorphisms in both rye mutants and iso flies.

The primer set 5′GGCTCGATGTGCTTTCAAGAGTTC3′ and 5′CATGCCAGATGAGTGCGTTTC3′ was used to clone the rye promoter region from genomic DNA derived from the iso stock. The primer set 5′TTTAGGCTTAGTCCGCTACC 3′ and 5′AATGTCGTGGTTTGAAGTGC 3′ was used to clone the full length rye cDNA. The PhiC31 integration system was used for injection. The rye promoter construct was specifically inserted onto the 2nd chromosome and the Uas-rye construct was integrated on the 3rd chromosome.

Generation of RYE antibodies

The primer set 5′ATGCATCATCACCATCACCATAGTATCTGCGTGACGGTTGTTG3′ and 5′GTGGTGGTGCACAACTGCCAACGTGAATATCC 3′ was used to clone the L3 epitope of RYE. A GST-RYEL3 fusion protein was expressed in Escherichia coli, excised from a PAGE gel and injected into rats for antibody generation.

Extractions and measurements of DNA, RNA and protein

Drosophila genomic DNA: flies (3–15) were frozen and homogenized in DNA extraction buffer. After LiCl/KAc precipitation, supernatants were subject to isopropanol precipitation. DNA pellets were dissolved in TE for sequencing analyses and PCR amplification.

RNA: adult flies were collected at indicated time points and fly heads (10∼20) were subject to Trizol extraction (Ambion, Life Technologies, Grand Island, NY). cDNA libraries were made through high-capacity cDNA reverse transcription kits (Applied Biosystems, Life Technologies, Grand Island, NY). Quantitative PCR analysis was performed using SYBR green reagents in the Applied Biosystems 7000 sequence detection system. The primer set 5′AGTTGAATGGAAGCCACCAGC3′ and 5′TGTTCATCCATGTGCCTCAG3′ was used for qPCR analysis of rye expression.

Protein: flies were collected at indicated time points and fly heads (∼5) were prepared for protein extraction and for Western analyses as previously described (Luo and Sehgal, 2012).

Immunocytochemistry assays

Adult fly brains were dissected in cold PBS buffers at indicated CT time points. Brains were fixed in 4% PFA for 30 m and incubated with 5% Normal Donkey Serum (NDS) for 1 hr at the blocking step. Incubation with primary rabbit anti-PER (1:1000), mouse anti-PDF (1:1000) and rabbit anti-GFP, mouse anti nc82 was performed overnight in the cold room. After extensive washing, brains were incubated with the donkey anti-rabbit or anti-mouse secondary antibody (1:1000) for 2 hr. Images were taken under a Leica confocal microscope.

Electrophysiological current recording in Xenopus oocytes

Oocytes were removed surgically from Xenopus laevis and placed in an OR-2 solution containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, and pH 7.5. They were defolliculated in this buffer containing 2 mg/ml collagenase type IA (Sigma, St. Louis, MO) for 1.5 hr. After defolliculation, oocytes were incubated at 18°C in 50% L15 (Invitrogen, Carlsbad, CA) 10 mM HEPES, pH 7.5, 10 units/ml penicillin, and 10 g/ml streptomycin at 18°C. 3–6 days after injection, whole-cell membrane currents evoked by acetylcholine were recorded in oocytes at room temperature with a standard two-electrode voltage-clamp amplifier (Oocyte Clamp OC-725; Warner Instrument, Hamden, CT). Recordings were performed at a holding potential of −50 mV. All perfusion solutions contained 0.5M atropine to block responses of endogenous muscarinic AChRs that might be present in oocytes. Acetylcholine was applied by means of a set of 2-mm glass tubes directed to the animal pole of the oocytes. Application was achieved by manual unclamping/clamping of a flexible tube connected to the glass tubes and to reservoirs with the test solutions. The recording chamber was perfused at a flow rate of 15–20 ml/min. cRNAs for human AChR subunits 4 and 2 and Drosophila sss were synthesized in vitro using SP6 RNA polymerase (mMESSAGEmMACHINETM, Ambion). Oocytes were injected with 5 ng of cRNA of each of the subunits.

References

Acknowledgements

We are grateful to Christine Quake for technical support of this project, Matt Kayser, Dan Cavanaugh, Katarina Moravcevic and Christine Dubowy for critical reading of the manuscript and other members of the laboratory for useful discussions. We thank William Joiner for communicating results prior to publication. We also thank Natania Field for suggesting the name, redeye. AS is an Investigator of the HHMI.

Decision letter

Leslie C Griffith, Reviewing editor, Brandeis University, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Identification of Redeye, a new sleep-regulating protein whose expression is modulated by sleep amount” for consideration at eLife. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors, and one of whom, Subhabrata Sanyal (Reviewer 3), has agreed to reveal his identity.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

The consensus of the reviewers was that the identification of rye is an important and novel contribution. To our knowledge there are no other protein markers that have been reported that are independent of the clock and external conditions and respond only to sleep drive. RYE levels therefore potentially represent an important new biomarker for sleep deprivation. The reviewers also felt that the data on the interaction of rye and sss were the weakest part of the paper and that without deeper investigation the precise interpretation of those experiments was difficult. While it would be satisfying to have mechanism, this study is still quite important without a full understanding of the interaction of rye and sss. We would therefore recommend publication of a suitably revised paper. The full reviews below contain a number of minor things that will help the authors to make the paper stronger, but the main concerns/suggestions are enumerated as follows:

1) Eliminate Figure 5 or cut back on the mechanistic claims and give a much fuller discussion of the caveats surrounding the data (see Reviewer 1 comments).

2) More balanced discussion of the issue of phase and whether rye is downstream of S or actually part of the homeostat (see Reviewer 2 comments). The overexpression data suggest the former.

3) Discussion of the issues surrounding the variability of the western blots (see comment from Reviewers 1 and 3).

4) More information on rye expression (or GAL4 pattern) and localization of requirement would strengthen the paper, especially if Figure 5 is eliminated. If such data exist it would be good to include them, but we would not require new experiments to be done.

Reviewer 1 comments:

This is a very interesting study. The authors used the power of genetics to identify a novel sleep regulator: RYE. The fact that RYE levels are apparently controlled by sleep drive and not the clock make this a very novel and important discovery. I believe this may be the first molecule of this class and for that reason I am very enthusiastic about this paper.

Unfortunately, I think that the data that go beyond the characterization of rye are not very strong and are in many cases not interpretable in the simple way that the authors try to suggest. I almost wonder if the sss connection should not just be taken out of the paper until the authors have more data and can present a believable story. The isolation of the rye gene could stand alone as a brief communication. As it is the SSS stuff just muddies the waters. Perhaps if they added more detail about how the mutants are resistant to SD to the basic story?

I am not convinced that there is a specific interaction between SSS and RYE that regulates sleep in WT animals. First, the authors claim that the sssTg lines have no effects on sleep, but they show data that the sleep levels in these lines are equivalent to rye/+, which has reduced sleep. The fact that now combining them results in even less sleep suggests a simple additive and independent effect of each manipulation on sleep. Second, there are no data on RNAi of RYE + OE of SSS. It is possible that the SSS effect is specific to the mutant allele because it has neomorphic function and that SSS may not regulate WT RYE.

The oocyte data are not deep enough to ameliorate these concerns. The fact that SSS can decrease current from both human and fly alpha containing complexes makes me suspicious that the SSS effect is indirect via some effect on trafficking/sorting in general or perhaps increased K channel expression. No data on RMP etc. are presented. Many more controls and experiments would be needed to make this cleanly interpretable (e.g., does SSS block current from other unrelated ionotropic receptors? Is there physical interaction of RYE and SSS? Co-expression of the point mutant RYE – is it really DN? Etc.).

It is also interesting that SSS seems to promote SHAKER expression yet suppress RYE. How do the authors fit this into a global model? They casually say its sleep promoting role is dominant but how would this really work? One thing we are never shown is where RYE is expressed in the brain. What is the pattern of rye-GAL4? Does this shed any light on the issue of SSS function? Since SHAKER is basically all over the brain perhaps RYE is more tightly localized and this might allow dissection of differential local effects of SSS on activity?

Reviewer 2 comments:

This paper presents studies that are truly a break-through for the sleep field – the identification of a mutant (rye) in which the affected protein is clearly part of the sleep homeostat (process S), which responds to increasing periods of wakefulness by producing a proportional increase in sleep drive. While the existence of such a homeostat is well supported by sleep behavior, the molecular basis has remained mysterious, and therefore this manuscript presents an important entry point for such a molecular analysis. The experiments are thorough, well controlled and convincing. The mutation affects a subunit for the nicotinic acetyl choline receptor, and the fact that a protein identified as mutated in a previously isolated short-sleeping mutant (sss) is shown to negatively regulate RYE is important because it suggests that the genetic analysis of sleep in Drosophila may be identifying some mutants in a single genetic pathway – something that has not been clear thus far. One perhaps could have hoped that this manuscript would also reveal the signals that lead to upregulation of RYE (i.e., more about the pathway for the sleep homeostat), but this will undoubtedly require more years of work by multiple labs.

The data supporting a role for RYE in the sleep homeostat are very compelling, but unless I'm missing something it seems to me that RYE expression is more likely to be triggered by an upstream S process than to be a direct part of the actual S process. For one thing, the phase for RYE expression is not right. A factor which directly responds to S should build up during wakefulness and dissipate during sleep, but RYE accumulates when the flies are asleep and only dissipates towards the end of sleep. While this feature of RYE expression alone might suggest that RYE accumulates during sleep to measure sleep recovery and promote waking, the short-sleep phenotype of the mutant and the elevated levels of RYE in various short-sleep mutants and under conditions of sleep deprivation are not consistent with this alternative (so it does seem that RYE is part of the process S). Another interesting feature is that RYE overexpression in a wild type genetic background does not alter sleep, which is inconsistent with a factor that directly drives the S process (this should elevate or phase advance sleep). The authors note this paradox and postulate that RYE overexpression may not be sufficient to induce sleep, or may not produce higher levels of RYE in the relevant cells (perhaps overexpressed proteins is not stable in these cells). Along these lines, perhaps RYE can only accumulate when the S process reaches a threshold that triggers sleep, and then RYE remains high until sleep debt is dissipated. Consideration of these points needs to be added to the Discussion; if nothing else, there needs to be more discussion of the phase of RYE expression and its relationship to its postulated role in the sleep homeostat.

Reviewer 3 comments:

This manuscript by Shi et al. describes sleep phenotypes of a very interesting mutation in Drosophila named redeye (rye). The authors do a very commendable job of isolating, identifying, characterizing and defining this mutation and in so doing further establish Drosophila as a powerful platform to study the biochemical and genetic basis of sleep. The authors convincingly show that loss of Rye leads to a short-sleeping phenotype that can be rescued partially by adding back Rye in a domain circumscribed by a newly generated Rye-GAL4 driver line and weakly phenocopied using RNAi. What is most interesting though is that the Rye protein (assayed using newly generated Rye antibodies) cycles during the day and is most prominently present just before sleep onset, is elevated by sleep deprivation and in several short-sleeping mutants such as sleepless, insomniac and fumin, but is not controlled by the clock machinery. These are hallmarks of a true “somnogen”; however, Rye fails in one test, i.e., artificially increased expression of Rye does not promote sleep. Even so, it seems clear that as the authors suggest, Rye is part of the sleep homeostat and is necessary but not sufficient for sleep drive. This is a very interesting study and is suitable for publication in eLife. One area that could have been investigated a bit more deeply is that of the tissue-specificity of Rye. The authors use the Rye-Gal4 and yet suggest that the expression pattern of this GAL4 line may not reflect that of endogenous Rye expression. Given the large number of extant GAL4 lines and some understanding of the sleep circuitry in Drosophila, it might have been instructive to test a few of these (e.g., pan neuronal, dopaminergic neurons, fan shaped body etc) to define the tissue-restricted requirement of Rye, if any. This could be most simply achieved using RNAi (though the phenotypes are arguably weaker than Rye mutants), selective rescue of Rye phenotypes using the UAS-Rye transgene, or perhaps even attempting to test if strong Rye over-expression in neuronal subsets might induce sleep (due to either stronger over-expression than Rye-GAL4 or a circuit-specific effect of Rye elevation). A minor point is the difference in the timing of the first peak of Rye between wild type and clock mutants (Figure 6 B and C). Do the authors think this is relevant or is the peak of Rye temporally more loosely defined than, say, clock proteins? Overall, this is an excellent manuscript and one of the few that describes a protein that is clearly correlated with sleep drive. Mechanistic associations with sleepless and channel activity further highlight the importance of this protein in sleep regulation.

DOI: http://dx.doi.org/10.7554/eLife.01473.025

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