Regulation of sleep amount by CRTC1 via transcription ofCrhin mice

The cAMP-response element binding protein (CREB) is required for regulation of daily sleep amount, whereas gain-of-function of CREB-regulated transcription coactivator 1 (CRTC1) causes severe insomnia in mice. However, the physiological functions of CRTCs and their downstream target genes in the regulation of sleep amount remain unclear. Here, we use adult brain chimeric (ABC)-expression/knockout platform for somatic genetics analysis of sleep in adult male mice. ABC-expression of constitutively active mutant CRTC1/2 CA in the mouse brain neurons significantly reduces the amount of non-rapid eye movement sleep (NREMS) and/or REMS. Consistent with that SIK3 phosphorylates and inhibits CRTCs, ABC-expression of CRTC1/2/3 CA rescues the hypersomnia phenotype of Sleepy ( Sik3 Slp ) mice. While ABC- Crtc2 KO or Crtc3 KO causes no sleep phenotype, ABC- Crtc1 KO or ABC-expression of dominant-negative CRTC (dnCRTC) results in modest reduction of NREMS amount accompanied with elevated NREMS delta power. Moreover, ABC-expression of CRTC1 CA or dnCRTC in the excitatory neurons causes bidirectional changes of NREMS/REMS amount and/or NREMS delta power, whereas expression of CRTC1 CA in the inhibitory neurons decreases REMS amount and increases NREMS delta power. The ability of CRTC1 CA to regulate sleep requires its transactivation domain and CREB-binding domain and is dependent on CREB. Furthermore, we showed that inducible ABC-expression of corticotropin releasing hormone ( Crh ) and brain-derived neurotrophic factor ( Bdnf )–two target genes of CRTCs–significantly reduces daily sleep amount. Notably, ABC- Crh KO , but not Bdnf KO , rescues the insomnia phenotype of ABC-CRTC1 CA mice. Taken together, these results indicate that CREB-CRTC1 complex regulates daily sleep amount by modulating the transcription of Crh in the mouse brain neurons. Significance Statement CRTCs function as coactivators for CREB, a transcription factor required for the regulation of daily sleep amount in mice. Here, we found that CRTC1/2/3 function similarly to suppress NREMS and REMS in a CREB-dependent manner. Consistent with that CRTCs are substrates of SIK3 kinase, expression of constitutive active mutant CRTCs CA rescue the hypersomnia phenotype of Sleepy ( Sik3 Slp ) mice. Furthermore, we showed that CRTC1 reduces NREMS amount by upregulating the expression of neuropeptide CRH. These results elucidate the mechanism by which CRTCs regulates sleep amount and suggest a potential transcriptional mechanism in the stress-induced sleep/wake regulation.