Identification of SLEEPLESS, a sleep-promoting factor

Abstract

Sleep is an essential process conserved from flies to humans. The importance of sleep is underscored by its tight homeostatic control. Through a forward genetic screen, we identified a gene, sleepless, required for sleep in Drosophila. The sleepless gene encodes a brain-enriched, glycosylphosphatidylinositol-anchored protein. Loss of SLEEPLESS protein caused an extreme (>80%) reduction in sleep; a moderate reduction in SLEEPLESS had minimal effects on baseline sleep but markedly reduced the amount of recovery sleep after sleep deprivation. Genetic and molecular analyses revealed that quiver, a mutation that impairs Shaker-dependent potassium current, is an allele of sleepless. Consistent with this finding, Shaker protein levels were reduced in sleepless mutants. We propose that SLEEPLESS is a signaling molecule that connects sleep drive to lowered membrane excitability.

Fig. 1

Sleep phenotype of sss mutants. (A) Histogram showing the distribution of daily sleep for ∼3,500 mutant lines (∼8 female flies per line). For each line, daily sleep is shown as the difference from the mean of a group of about 100−250 lines tested simultaneously. The arrow indicates the sss mutant line. (B) Sleep profile in 30-min intervals for sss flies (open diamonds) versus background controls (ctrl, closed diamonds). Data for male (M) and female (F) flies are shown. The bar below the x-axis indicates light (white) and dark (black) periods. (C) Daily sleep amount for ctrl (162 males and 148 females), ctrl/sss (111 males and 113 females), and sss flies (146 males and 148 females). Data from the same animals are shown in (C-F). (D-F) Activity counts/minute awake (D), sleep bout duration (E), and daily number of sleep bouts (F) for male and female ctrl, ctrl/sss, and sss flies. In this and subsequent figures, error bars represent SEM. *P < 0.05; **P < 0.0001. For (C), (E), and (F), significance level is shown for sss mutants compared to both ctrl and ctrl/sss flies. For (D), significance level is shown for pairwise comparisons as indicated by lines. In (E), sleep bout duration, which is not normally distributed, is presented as simplified box plots. The line inside each box indicates the median, and the top and bottom represent 75^th and 25^th percentiles, respectively. Approximately 9% of animals exhibiting zero sleep were excluded from calculation of sleep bout duration.

Fig. 2

sss encodes a novel brain-enriched, GPI-anchored protein. (A) Schematic of the genomic structure of the sss locus. Non-coding regions of the cDNA are shaded, while coding regions are shown in white. (B) Schematic of structural features of the SSS protein. The primary sequence contains a predicted signal peptide, a N-type glycosylation site (ψ), and a potential GPI attachment site (*). (C) Amino acid sequence of SSS. Amino acids 1−32 comprise the predicted signal peptide (shown boxed), and the predicted N-type glycosylation site is underlined. * denotes the predicted GPI attachment site. (D) Glycosylation of the SSS protein. Western blot analysis with anti-SSS antibody reveals two bands detected in head extracts from wild-type (ctrl) but not sss flies. Deglycosylation of head extracts by treatment with PNGase F results in detection of a single band. Because our antibody to SSS does not recognize glycosylated SSS well, Western blots were treated with PNGase F before being probed with antibody to SSS. In this and subsequent Western blots, antibody to MAP kinase (MAPK) is used to control for loading. (E) Surface expression of SSS in cultured Drosophila cells. S2R+ cells were transfected with a pIZ-sss construct, and stained with or without permeabilization to assay for total or surface expression, respectively. Transfection with the pIZ vector alone shows specificity of our SSS antibody. (F) Reduced surface expression of SSS after PI-PLC treatment. S2R+ cells transfected with a pIZ-sss construct were stained without permeabilization after PI-PLC(+) or mock(−) treatment. (G) Release of SSS into the culture medium by PI-PLC. Western blot analysis of S2R+ cells transfected with pIZ-sss was performed after PI-PLC(+) or mock(−) treatment. (H) Enrichment of SSS expression in brain and head compared to body. An equal amount of total protein (∼40 ug) was loaded per lane. The experiments in (D) through (H) were performed 3−4 times with similar results.

Fig. 3

Genetic analysis of sss. (A) Daily sleep amount for precise excision (Pr, n=26), sss^Δ40 imprecise excision (Im, n=15), precise/sss^P1 (Pr/sss^P1, n=24), and imprecise/sss^P1 (Im/sss^P1, n=35) female flies. (B) Western blot analysis of SSS protein levels. Similar levels of SSS protein are seen in head extracts from background control (ctrl) and precise excision (Pr) flies. SSS protein is undetectable in sss^P1 and sss^Δ40 imprecise excision (Im) flies. Similar results were obtained in 2 additional experiments. (C) Daily sleep amount for female sss^P1 mutant flies with (sss^P1;TG1−3, n=15,8,16, respectively) or without (sss^P1, n=16) a genomic sss transgene. TG1−3 refer to three independent transgene insertions, and either 1 or 2 copies of the transgene were present in the flies tested. (D) Daily sleep amount for sss^P2 (n=110) versus background control (ctrl, n=80), as well as ctrl/sss^P1 (n=80) versus sss^P2/sss^P1 (n=112) female flies. (E) Reduced levels of SSS protein in sss^P2 and transheterozygous sss^P2/sss^P1 flies. Similar results were obtained in 3 additional experiments. Data from male flies of the genotypes shown in (A), (C), and (D) are available in fig S2. *P < 0.05; **P < 0.0001.

Fig. 4

Reduced homeostatic response to sleep deprivation in female sss mutants. (A) Amount of sleep lost during 6 or 12 hours of deprivation by the end of the dark period for background control (ctrl), sss^P2, ctrl/sss^P1, and sss^P2/sss^P1 flies. Data from 13−56 female flies are presented. (B) Amount of sleep gained during 6 hours of recovery following deprivation as in (A). (C) Change in sleep latency following deprivation, compared to undisturbed controls as in (A). Data from male files are shown in fig. S3. *P < 0.05; **P < 0.001.

Fig. 5

Circadian rhythm and longevity phenotypes of sss mutants. (A) Average activity records for background control (ctrl, n=64) and sss^P1 male flies (n=81) assayed in DD. The activity records are double plotted so that each horizontal line represents data for 2 days. The gray and black bars above each activity record indicate subjective day and night, respectively. (B) Activity records showing average activity in DD for ctrl/sss^P1 and ctrl/sss^P2 (n=76) versus sss^P2/sss^P1 (n=65) male flies. Circadian data for ctrl/sss^P1 and ctrl/sss^P2 were statistically similar and thus pooled. (C) Cycling of PER protein in large ventral lateral neurons in control (ctrl) and sss^P1 mutants. Ventral lateral neurons for ctrl and sss^P1 animals are stained for PER at indicated Zeitgeber times (ZT). PER protein level is elevated at ZT2 and ZT20, and low at ZT8 and ZT14. (D) Survivorship curves of background control (ctrl, closed diamonds) and sss^P1 (open diamonds) flies. Female sss flies (n=187) show a significantly shorter lifespan (P < 0.0001) than controls (n=198). Data from male flies are shown in fig. S5.

Fig. 6

sss is allelic to qvr and affects Sh expression. (A) Daily sleep amount for qvr (qvr, n=31), versus background control (ctrl, n=32), as well as ctrl/sss^P1 (n=30) versus qvr/sss^P1 (n=32) female flies. **P < 0.0001. (B) Altered sss transcripts in qvr mutants. RT-PCR products obtained with qvr and background control (ctrl) RNA and with water used as a negative control (neg). (C) Schematic representation of sss transcripts in qvr mutants. qvr 1, 2, and 3 correspond to the top, middle, and bottom bands, respectively. In background control (ctrl) transcripts, 163 nucleotides of Intron 6 are spliced out. In contrast, the entire intron is present in qvr 1 transcripts. In qvr 2 and 3 transcripts, splice donor sites differ from the one used in wild-type control transcripts, as indicated by the nucleotide numbers for splice sites. (D) Sequence change in qvr genomic DNA in Intron 6 of sss. The fifth nucleotide in Intron 6 has a G to A transition. (E) Altered expression of SSS in qvr mutants. Fly head extracts from background control (ctrl), qvr, and sss^P1 flies were analyzed by Western blotting with anti-SSS antibody. (F) Reduced expression of Sh in sss mutants. Western blot analysis of head extracts with anti-Sh antibody reveals a Sh-specific band that is substantially reduced in sss^P1 mutants compared with background control (ctrl) flies. Sh¹⁴ flies were used to identify a Sh-specific band. Non-specific bands (*) may have obscured additional Sh bands. The experiments in (E) and (F) were performed 3 times with similar results.