2018 Fiscal Year Research-status Report
| Project/Area Number |
17K15592
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| Research Institution | University of Tsukuba |
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Principal Investigator |
Ma Jing 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (70793222)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | sleep / immunoprecipitation / mass spectrometric / Sleepy |
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| Outline of Annual Research Achievements |
In research period, we performed quantitative phosphoproteomic studies of whole mouse brains from two models of sleep/wake perturbation. A combined proteome and phosphoproteome data for 9,410 mouse proteins and 62,384 phosphopeptides were examined. Comparison of two models identifies 80 mostly synaptic Sleep-Need-Index-PhosphoProteins (SNIPPs), whose phosphorylation states closely parallel changes of sleep need. To examine whether SNIPPs are substrates of SLEEPY kinase, we compared the interactomes of SLEEPY and SIK3 by immunoprecipitation (IP) and mass spectrometric analysis using whole-brain lysates from the Flag-HA-Sik3+ and Flag-HA-Sik3Slp knock-in mice. Accordingly, SLEEPY preferentially associated with synaptic proteins, including 28 of 80 SNIPPs. We applied the “AMPK Motif Analyzer27” to predict 2,943 phosphopeptides as potential AMPK-like substrates in the Slp/WT phosphoproteome dataset. The 28 SLEEPY-interacting SNIPPs contain 47 putative AMPK sites showing significant changes, of which 40 (85%) become hyper-phosphorylated, in Sleepy brains. Taken together, these observations suggest that SLEEPY may increase phosphorylation of SNIPPs by enhancing kinase-substrate association. Inhibition of SIK3 activity reduces phosphorylation state of SNIPPs and slow wave activity (SWA) during non-rapid-eye-movement sleep (NREMS), in both Sleepy and sleep-deprived wild-type mice.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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. These results have been published on Nature.
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| Strategy for Future Research Activity |
The intracerebroventricular (i.c.v.) injection-based and AAV overexpression-based EEG/EMG sleep recording system have been established, we will investigate the function of several SNIPPs through pharmacological and AAV overexpression approaches, and then generate conditional knockout mice and specific phosphorylation site knock-in mice for SNIPPs genes to elucidate their roles in sleep regulation and function.
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| Causes of Carryover |
The bioinformatics analysis of the phosphoproteome data generated new insights and more interesting Sleepy substrates. Two sleep study system, i.c.v. and AAV overexpression, have been established to test the functions of those substrates.
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