Wang, T. V. | bioRxiv Science

bioRxiv2023

Biochemical and chemical biological approaches to mammalian sleep: roles of calcineurin in site-specific dephosphorylation and sleep regulation

Understanding of sleep mechanisms traditionally rely on electrophysiology and genetics but here we have initiated biochemical and chemical biological studies. Sleep was increased in mouse mutants with an alanine replacing threonine at residue 469 (T469A) of the salt inducible kinase 3 (SIK3). We searched for T469 phosphatases by classic purification with HEK293 cells and by a new photo-crosslinking method with mouse brains. Both led to PPP3CA, a catalytic subunit of calcium/calmodulin activated phosphatase (calcineurin). It dephosphorylated T469 and serine (S) 551 but not T221 in SIK3 in vitro. PPP3CA knockdown increased phosphorylation of T469 and S551 but not T221 in mouse brains. Knockdown of its regulatory subunit PPP3R1 significantly reduced daily sleep by more than 5 hours, exceeding other known mouse mutants. Our results have uncovered in vitro and in vivo evidence for site-specific SIK3 dephosphorylation by calcineurin, demonstrated a physiological role for calcineurin in sleep, and suggested sleep control by calcium dependent dephosphorylation.

neuroscience↗

bioRxiv2022

ppGpp is Present in and Functions to Regulate Sleep in Drosophila

Sleep is essential for animals, and receives inputs from circadian, homeostasis, and environment, yet the mechanisms of sleep regulation remain elusive. Discovery of molecules in living systems and demonstration of their functional roles are pivotal in furthering our understanding of the molecular basis of biology. Here we report that ppGpp (guanosine-5-diphosphate, 3-diphosphate), a molecule that has been detected in prokaryotes for more than five decades, is present in Drosophila, and plays an important role in regulation of sleep and SISL (starvation induced sleep loss). ppGpp is detected in germ-free Drosophila and hydrolyzed by an enzyme encoded by the mesh1 gene in Drosophila. Nighttime sleep and SISL were defected in mesh1 mutant flies, and rescued by expression of wildtype Mesh1, but not the enzymatically defective mutant Mesh1E66A. Ectopic expression of RelA, the E. coli synthetase for ppGpp, phenocopied mesh1 knockout mutants, whereas overexpression of Mesh1 resulted in the opposite phenotypes, supporting that ppGpp is both necessary and sufficient in sleep regulation. A chemoconnectomic screen followed by genetic intersection experiments implicate the Dilp2 neurons in the pars intercerebralis (PI) brain region as the site of ppGpp function. Our results have thus supported that ppGpp is present in animals after long lag since its discovery in bacteria, and revealed a physiological role of ppGpp in sleep regulation for the first time.

genetics↗