| Project/Area Number |
21K15004
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 42040:Laboratory animal science-related
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| Research Institution | University of Tsukuba |
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Principal Investigator |
KIM Staci・Jakyong 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (80865246)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2022)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2023: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2022: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
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| Keywords | Sleep / Intracellular signaling / Sleep depth / Sik3 / Gene-modified mice |
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| Outline of Research at the Start |
Sleep is universal behavior found in all complex animals. However, the molecular mechanism that control sleep remains largely unknown. Our research group has identified SIK3 as a key regulator involved in sleep homeostasis, but it is still unclear how SIK3 controls sleep. This study will focus on HDAC5, a known SIK3 target, with Cre-Flox system in mice to examine specific neuronal population linked to sleep behavior. We will also examine transcriptomic changes in sleep homeostasis within the identified cell population.
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| Outline of Annual Research Achievements |
There has been increased interest in understanding how sleep/wake behavior is controlled as the emphasis on the well-maintained sleep has grown. However, the regulatory mechanism of sleep and wake at the molecular level remains unclear to date. This study is based on our previous finding of SIK3 kinase as a key regulator of sleep homeostasis. We identified several SIK3 substrate molecules where SIK3-induced phosphorylation of these proteins localizes them to the cytoplasm and result in desuppression of target gene expression in the nucleus. We observed abnormal sleep and wake behavior in the loss-of-function mutants and phosphodeficient SIK3-substrate mice. The loss-of-function mutants showed increased sleep time and sleep need, consistent with the Sik3-Sleepy mutant phenotype. On the other hand, the phosphodeficient mutants showed increased wakefulness with decrease in sleep need marker. These results suggest that the SIK3 cascade may constitute an important molecular pathway in sleep need regulation. To investigate responsible neural population in sleep regulation, we established floxed mice to induce conditional knockout of the target gene when mated with Cre-driver mice. It is plausible that sleep time and sleep need regulation may be controlled by distinct population of neurons and cell types. Several Cre-driver strains were selected to mate with the floxed mice and examined for changes in any sleep parameters. We are proceeding with investigation of the role of SIK3 substrates in brain region- and cell type-specific manner using these newly established mouse line.
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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
As stated above, we have identified SIK3 substrate molecules as the intracellular signaling components in sleep homeostasis regulation. These findings have been published peer-reviewed journal (S. J. Kim et al., Nature 2022). We have completed the sleep recording and analysis of the target conditional knockout mice. Mice lacking the substrate in Vglut2-positive glutamatergic neurons and Vgat-positive GABAergic neurons show altered sleep architecture. In addition, the preparation for further analyses for identifying brain regions and cell types has been completed and the study is being progressed.
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| Strategy for Future Research Activity |
Based on the sleep/wake behavioral analysis and identified target regions in the brain, we will investigate the changes in transcriptome by single-nucleus (sn) RNA sequencing. We have identified phosphodeficient mutation of the SIK3-targeted site results in decreased sleep time and brain activity in mice. This phosphorylation site is conserved among the Class IIa HDACs, including Hdac4. This finding, in addition to our observation of sleep disruption in Hdac5 conditional knockout mice, suggest that SIK3 may modulate its substrates differently in distinct population of neurons. Using the brain samples from Hdac4 and Hdac5 mutant mice, we will perform the snRNA-seq to identify the molecular marker for sleep homeostasis, as well as the neuronal markers of this key signaling pathway.
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