A sleep-inducing gene, nemuri, links sleep and immune function in Drosophila

Abstract

Sleep remains a major mystery of biology. In particular, little is known about the mechanisms that account for the drive to sleep. In an unbiased screen of more than 12,000 Drosophila lines, we identified a single gene, nemuri, that induces sleep. The NEMURI protein is an antimicrobial peptide that can be secreted ectopically to drive prolonged sleep (with resistance to arousal) and to promote survival after infection. Loss of nemuri increased arousability during daily sleep and attenuated the acute increase in sleep induced by sleep deprivation or bacterial infection. Conditions that increase sleep drive induced expression of nemuri in a small number of fly brain neurons and targeted it to the sleep-promoting, dorsal fan-shaped body. We propose that NEMURI is a bona fide sleep homeostasis factor that is particularly important under conditions of high sleep need; because these conditions include sickness, our findings provide a link between sleep and immune function.

Fig. 1.. nemuri (nur) overexpression promotes sleep.

(A) Summary of the gain-of-function screen. A UAS insertion in each line was driven by elav-GS, which was induced by adding RU486 to the food. Average sleep time (minutes per 24 hours; x axis) is shown for males (top) and females (bottom). Lines within one standard deviation (SD) of the average sleep time are indicated in black, one to two SDs in yellow, and two or more SDs in red. An arrow indicates the line termed nemuri. (B) Left: Sleep profile of elav-GS/UAS-nemuri flies +RU486 (black) or −RU486 (blue) obtained by video tracking. Red arrows indicate longer sleep in the presence of RU486 (black line) in nur-overexpressing flies. Right: Median ± interquartile minutes of sleep during daytime, night, and total 24-hour period in +RU486 flies versus vehicle controls; n = 24 females for each group. **P < 0.01, ****P < 0.0001 (Mann-Whitney U test). (C) Sleep architecture assayed through video tracking. Median ± interquartile number of sleep bouts and bout duration is shown for the daytime (left) and night (right) in +RU486 flies and vehicle controls; n = 24 females for each group. *P < 0.05, ****P < 0.0001 (Mann-Whitney U test); n.s., not significant. (D) Top: Sleep/wake patterns in the Drosophila activity monitoring system of flies subjected to a mechanical stimulus at ZT20 (red arrow). Sleep (left) and activity (right) are shown for nur overexpressors (black) and control (blue) flies (n = 32 females per group). Bottom: Bar graphs indicate the percentage of RU486-treated (+) or control (−) flies in sleep (red) versus wake (blue) states, before (left) and after (right) the stimulus.

Fig. 2.. NUR is a secreted protein and its ectopic expression induces sleep.

(A) SEAP assay. Left: Culture medium from S2R+ cells expressing alkaline phosphatase (AP), NUR::AP, or a fusion of AP with the immunoglobulin binding protein (BiP), BiP::AP, used as a secreted control, with (+) or without (−) induction by Cu²⁺ in a 96-well plate. The arrow indicates NUR expression in the media. Right: Quantification of the SEAP assay by measuring absorbance at 595 nm for each sample (A.U., absorbance units). ****P < 0.0001 (Student t test). Error bars denote SEM. (B) Protein immunoblot of S2R+ cells (left) or conditioned medium (right) using antibodies to GFP. Cells expressed myr::GFP (negative control), NUR::GFP, or BiP::GFP (positive control); with (+) or without (−) induction by Cu²⁺.The arrow indicates the detection of NUR::GFP in the medium. (C) Sleep profiles and expression patterns of Gal4 lines ectopically expressing nur in various subsets of neurons in the brain. Upper panels show the sleep pattern of each genotype: Gal4/UAS-nur (red), Gal4 control (blue), and UAS-nur control (green). The average number of minutes of total sleep in eight female flies is indicated. Lower panels show the expression pattern of each Gal4 line, as determined by expression of UAS-mCD8::GFP (green) and costaining with nc82 (neuropile marker; magenta) (images from Brainbase: https://braingazer.org/).

Fig. 3.. nur encodes an antimicrobial peptide that promotes survival upon expression in neurons.

(A) Alignment between NUR and a fish cathelicidin. The C-terminal domain of NUR shows sequence similarity with a Greenland cod (Gadus ogac) cathelicidin. Note the overlap of glycine and arginine residues, indicated by asterisks. Amino acid abbreviations: A, Ala; D, Asp; E, Glu; G, Gly; I, Ile; K, Lys; L, Leu; R, Arg; S, Ser; T, Thr; V, Val. (B) Top: Alamar Blue cell viability assay; S. marcescens in PBS alone (center), with kanamycin (left), or with NUR protein (right). Blue color indicates lack of bacteria. Bottom: The assay was quantified by measuring absorbance at 595 nm (n = 3). ***P < 0.001 (Student t test). Error bars denote SEM. (C) Kaplan-Meier plot depicting survival of infection with S. marcescens in nur-overexpressing flies (+RU486) and controls (−RU486); n = 68 and 74 females, respectively. *P < 0.02 (log rank test). (D) Daytime sleep in nur-overexpressing flies (black) and controls (blue) after infection at ZT18 the previous night; n = 49 control and 62 +RU486-fed female flies, respectively. Median ± interquartile is shown. ****P < 0.0001 (Mann-Whitney U test). Note that daytime sleep values are lower than in other figures (fig. S1B). This effect was also observed in uninfected controls and may be caused by the CO₂ anesthesia necessary for infecting flies. (E) Bacterial load in control flies (−) and nur overexpressors (+) at indicated times after infection; n = 9 for each. Median ± interquartile is shown; cfu, colony-forming units. *P < 0.05 (Mann-Whitney U test).

Fig. 4.. Requirement of nur for sleep depth and for acute sleep induction after sleep deprivation or infection.

(A) Gene targeting to knock out nur using CRISPR/Cas9. Arrows indicate regions targeted by guide RNA. Homology arms (~1 kbp; blue) are flanked by attp-Loxp-3xP3 (eyeless promoter)–DsRed-Loxp sequences. After the targeting, hs-cre was crossed in to flip out the Loxp-3xP3-DsRed-LoxP sequence. (B) Sleep pattern of nur² mutants or wild-type controls (iso31) in males and females. Horizontal white and black bars correspond to light and dark cycles. (C) Day, night, and total sleep in the genotypes indicated; n = 15, 16, and 16 females for nur²/+, nur³/+, and nur²/nur³, respectively. Median ± interquartile is shown in all cases. P > 0.05 (Mann-Whitney U test). (D) Arousal threshold assay using a light pulse of 10 s in the middle of the night. Shown are proportions of flies awakened by the stimulus; n = 44, 54, and 73 females for nur²/+, nur³/+, and nur²/nur³, respectively. *P < 0.05 (Fisher exact test). (E and F) Speed after awakening (E) and latency to sleep (F) of flies awakened by light stimuli in (D). Speed in (E) was measured by video tracking after awakening until they fell asleep again. Median ± interquartile is shown; n = 12, 27, and 24 females for nur²/+, nur³/+, and nur²/nur³, respectively. ***P < 0.001, **P < 0.01 (Kruskal-Wallis test with Dunn post hoc comparison). (G) Recovery sleep after 6 hours of sleep deprivation. Rebound was calculated as the mean ± SEM net change in sleep relative to the baseline day before deprivation; n = 64 females for each genotype. (H) Infection-induced sleep, calculated as the mean ± SEM net change in sleep in the morning after infection relative to the corresponding baseline; n = 45 and 37 females for W¹¹¹⁸ control and nur² homozygous mutants, respectively. In (G) and (H), sleep values are plotted against zeitgeber time. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way analysis of variance followed by Bonferroni multiple comparison test).

Fig. 5.. Increased nur mRNA expression after sleep deprivation.

(A) Expression of nur mRNA in adult fly heads; n = 3. qPCR for nur expression used actin as a normalization control. ***P < 0.001 (Student t test). Error bars denote SEM. (B) Brains of nur^Gal4/UAS-CD4::tdGFP flies stained with anti-GFP (green) (top), nc82 (neuropil marker; magenta) (middle), or merged (bottom) in control (left), sleep-deprived (SD) (center), or caffeine-fed (right) animals. Arrows indicate GFP-positive projections; arrowheads indicate GFP-positive cell bodies. Scale bars, 50 μm.

Fig. 6.. NUR is induced by sleep loss and is localized to the dFSB area of the brain.

(A) Expression of endogenous NUR after sleep deprivation. Staining is for anti-NUR (green) and nc82 (magenta). The insets in the upper images are magnified below. Red arrows indicate expression of NUR in the dFSB. Scale bars, 50 μm. (B) Ectopic expression of NUR by different Gal4 drivers. Each Gal4 line was crossed with flies carrying UAS-syt::GFP and UAS-NUR::HA. Staining is with anti-GFP (top), anti-HA (middle), or merge (bottom). Note NUR expression in the dFSB area. Scale bars, 20 μm.