Summary Sleep is a behavior conserved from invertebrates to vertebrates, and tightly regulated in a homeostatic manner. The molecular and cellular mechanism determining the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remains unknown. Here we identified two dominant mutations affecting sleep/wakefulness through an electroencephalogram/electromyogram-based screening of randomly mutagenized mice. A splicing mutation of the Sik3 protein kinase gene causes a profound decrease in total wake time, due to an increase in inherent sleep need. Sleep deprivation affects regulatory-site phosphorylation of the kinase. Sik3 orthologues regulate sleep also in fruit flies and roundworms. A missense mutation of the leak cation channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the utility of forward genetic approach for sleep behaviors in mice, demonstrating the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

Sleep and wake have global effects on brain physiology, from molecular changes and neuronal activities to synaptic plasticity. Sleep-wake homeostasis is maintained by the generation of a sleep need that accumulates during waking and dissipates during sleep. Here we investigate the molecular basis of sleep need using quantitative phosphoproteomic analysis of the sleep-deprived and Sleepy mouse models of increased sleep need. Sleep deprivation induces cumulative phosphorylation of the brain proteome, which dissipates during sleep. Sleepy mice, owing to a gain-of-function mutation in the Sik3 gene , have a constitutively high sleep need despite increased sleep amount. The brain proteome of these mice exhibits hyperphosphorylation, similar to that seen in the brain of sleep-deprived mice. Comparison of the two models identifies 80 mostly synaptic sleep-need-index phosphoproteins (SNIPPs), in which phosphorylation states closely parallel changes of sleep need. SLEEPY, the mutant SIK3 protein, preferentially associates with and phosphorylates SNIPPs. Inhibition of SIK3 activity reduces phosphorylation of SNIPPs and slow wave activity during non-rapid-eye-movement sleep, the best known measurable index of sleep need, in both Sleepy mice and sleep-deprived wild-type mice. Our results suggest that phosphorylation of SNIPPs accumulates and dissipates in relation to sleep need, and therefore SNIPP phosphorylation is a molecular signature of sleep need. Whereas waking encodes memories by potentiating synapses, sleep consolidates memories and restores synaptic homeostasis by globally downscaling excitatory synapses. Thus, the phosphorylation-dephosphorylation cycle of SNIPPs may represent a major regulatory mechanism that underlies both synaptic homeostasis and sleep-wake homeostasis.

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Sleep is an evolutionally conserved behavior from vertebrates to invertebrates. The molecular mechanisms that determine daily sleep amounts and the neuronal substrates for homeostatic sleep need remain unknown. Through a large-scale forward genetic screen of sleep behaviors in mice, we previously demonstrated that the Sleepy mutant allele of the Sik3 protein kinase gene markedly increases daily nonrapid-eye movement sleep (NREMS) amounts and sleep need. The Sleepy mutation deletes the in-frame exon 13 encoding a peptide stretch encompassing S551, a known PKA recognition site in SIK3. Here, we demonstrate that single amino acid changes at SIK3 S551 (S551A and S551D) reproduce the hypersomnia phenotype of the Sleepy mutant mice. These mice exhibit increased NREMS amounts and inherently increased sleep need, the latter demonstrated by increased duration of individual NREMS episodes and higher EEG slow-wave activity during NREMS. At the molecular level, deletion or mutation at SIK3 S551 reduces PKA recognition and abolishes 14-3-3 binding. Our results suggest that the evolutionally conserved S551 of SIK3 mediates, together with PKA and 14-3-3, the intracellular signaling crucial for the regulation of daily sleep amounts and sleep need at the organismal level.

Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1−/− mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.

The hypoxia-inducible transcription factors (HIF)-1␣ and -2␣ mediate responses to hypoxia, such as tumor neovascularization. To determine the function of HIF-2␣ in vascular endothelial cells (ECs), we examined vascular formation in HIF-2␣ knockdown (kd/kd) mice transplanted with tumors. We observed that both the tumor size and the number of large vessels growing within transplanted melanomas were significantly reduced in kd/kd recipients compared with wild-type (WT) mice. In contrast, we observed a similar extent of vascular formation within fibrosarcomas transplanted from either kd/kd or WT mice into WT recipients. Thus, HIF-2␣ expression in host animal ECs, but not in the tumor cells, is crucial for tumor neovascularization. HIF-2␣ may function through ephrin A1 as the expression of ephrin A1 and related genes was markedly reduced in kd/kd ECs, and HIF-2␣ specifically bound a hypoxiaresponse element sequence in the ephrin A1 promoter. Treatment of WT ECs with an ephrin A1 inhibitor (ephrin A1-Fc) also impaired neovascularization. We conclude that in ECs, HIF-2␣ plays an essential role in vascular remodeling during tumor vascularization through activation of at least ephrin A1.

A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity.

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