Honda Takato

本多 隆利

Tsukuba Repository

Investigation of Sleep/Wake Regulatory
Mechanisms Through the Sik3 Gene Identified by
Forward Genetics
著者 Honda Takato
year 2018
その他のタイトル フォワード・ジェネティクスにより同定した新規睡
眠覚醒制御遺伝子Sik3 の解析による睡眠覚醒制御
機構の解明
学位授与大学 筑波大学 (University of Tsukuba)
学位授与年度 2017
報告番号 12102甲第8749号
URL http://hdl.handle.net/2241/00152850
    
 
 
 
 
論文の要旨 
Abstract of thesis 
The mechanism for homeostatic sleep/wakefulness regulation, as well as the neural 
substrate for “sleepiness,” remains as the biggest mystery in sleep biology. To make a 
breakthrough in this issue, the candidate's laboratory has initiated a large-scale forward 
genetic screen of sleep/wake abnormalities in mice based on somnographic (EEG/EMG) 
measurements, which are the gold standard in mammalian sleep/wake assessment. The laboratory 
including the candidate has so far screened > 8,000 heterozygous ENU-mutagenized mice and 
established multiple pedigrees exhibiting heritable and specific sleep/wake abnormalities. 
By combining linkage analysis and whole-exome sequencing, the applicant identified two 
dominant mutations that strongly affect sleep/wakefulness (Funato H, Honda T, Yanagisawa M 
et al., Nature 2016). A splicing mutation in the Sik3 protein kinase gene (termed Sleepy 
mutation) causes marked hypersomnia (i.e., increased non-REM sleep), owing to an increase in 
inherent sleep need. A missense mutation in the sodium leak channel NALCN (termed Dreamless 
mutation) reduces the total amount and episode duration of REM sleep, apparently by 
increasing the excitability of REM sleep-inhibiting neurons. Since these dominant mutations 
cause severe and specific sleep abnormalities, the applicant expects that the mutated genes 
play central roles for regulating sleep/wake amounts.  
To elucidate the molecular basis how Sik3 gene is involved in sleep/wake regulation, 
the applicant has performed the genetic and biochemical analysis. SIK3 has a serine-
threonine kinase domain at the N terminus and a protein kinase A (PKA) recognition site 
(Ser551) in the middle portion. The skipping of exon 13 resulted in an in-frame deletion of 
52 amino acids, encompassing the PKA site. From this point, it is hypothesized that, since 
Sleepy mutant mice lack the exon 13 including Ser551 (PKA recognition site), phosphorylation 
state could be constitutively defective form in vivo, which cause the prolonged NREM sleep. 
氏     名 本多 隆利 
A学 位 の 種 類       E 博士（ 人間生物学  ） 
A学 位 記 番 号       E 博甲第  ８７４９  号 
A学位授与年月       E 平成 ３０年 ３月 ２３日 
A学位授与の要件       E 学位規則 第４条第１項該当（昭和２８年４月１日文部省令第９号） 
審 査 組 織 グローバル教育院 
学位論文題目  Investigat ion of Sleep/Wake Regulatory Mechanisms 
Through the Sik3  Gene Identif ied by Forward Genetics 
（フォワード・ジェネティクスにより同定した新規睡眠覚醒
制御遺伝子 Sik3  の解析による睡眠覚醒制御機構の解明）  
 （職名） （学位） （氏名） 
主   査 筑波大学准教授 博士（理学） Lazarus Michael 
副   査 筑波大学教授(ｸﾞﾛｰﾊﾞﾙ教育院） Ph.D. Joseph S. Takahashi 
副   査 筑波大学教授 医学博士 柳沢 正史 
副   査 筑波大学助教 博士(医学） 水野 聖哉 
 To examine this hypothesis, the candidate generated the phosphorylation-defective SIK3 
(Ser551Ala, termed S551A mutant) and constitutive phosphorylation-active SIK3 (Ser551Asp, 
termed S551D mutant) by CRISPR/Cas9 gene editing.  
As a result, both S551A and S551D mutant mice exhibited the longer NREM sleep and 
decreased wakefulness similar to the phenotype of the exon skipped original Sleepy (ΔEx13) 
mutant. To elucidate the mechanism of their sleep/wake phenotypes, the candidate performed 
the biochemical analysis by using the FLAG/HA-tagged mice and the series of expression 
plasmids (WT, Sleepy (ΔEx13), S551A, S551D) to examine how the phosphorylation state on 
Ser551 effects on the signaling pathway and binding partners of SIK3. Through 
immunoprecipitation (IP) and western-blotting, the applicant confirmed that Sleepy (ΔEx13), 
S551A, S551D showed the similar binding patterns toward PKA and 14-3-3 which correspond to 
their similar sleep phenotypes. These results suggest that the phosphorylation state of 
Ser551 in Sik3 gene and its signaling pathway including PKA and 14-3-3 have a key role on 
sleep/wake regulation under normal physiological conditions. Notably, the exon 13-encoding 
region of Sik3, including Ser551, is highly conserved among vertebrate animals, which 
suggests the biological importance of the phosphorylation pathway PKA -> SIK3 (determined by 
Ser551) -> 14-3-3 in sleep/wake regulation. These findings provide the landmark information 
about the novel sleep/wake regulatory mechanisms connecting from the intracellular signaling 
pathway to the dynamic in vivo sleep/wake behaviors. 
 
 
審査の要旨 
 Abstract of assessment result 
 
【批評 Review】 
 
The experimental performance of the applicant is outstanding. The candidate generated 
phosphorylation-defective SIK3 and constitutive phosphorylation-active SIK3 by CRISPR/Cas9 
gene editing for behavioral and biochemical analysis. In the final examination, the 
applicant presented clear data on the sleep/wake behavior of the mutants and the 
intracellular signaling pathway of the mouse SIK3 phosphorylation site S551. His findings 
reveal a vital role of SIK3 in sleep/wake regulation.  
The applicant will continue to study forward-genetic mutant mice with sleep 
abnormalities and learning and memory deficits as postdoctoral work and already secured a 
position at MIT and 2-year funding from the Japan Society for the Promotion of Science. 
Therefore, the PhD committee is confident to say that the applicant is an exceptional 
student and fulfills all necessities to graduate from the Human Biology PhD program. 
 
【最終試験の結果 Result】 
The final examination committee conducted a meeting as a final examination on 26 
January, 2018. The applicant provided an overview of dissertation, addressed questions and 
comments raised during Q&A session. All of the committee members reached a final decision 
that the applicant has passed the final examination. 
 
【結論 Conclusion】 
Therefore, the final examination committee approved that the applicant is qualified to 
be awarded Doctor of Philosophy in Human Biology. 
 
 
 


フォワード・ジェネティクスにより同定した新規睡眠覚醒制御遺伝子Sik3 の解析による睡眠覚醒制御機構の解明

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フォワード・ジェネティクスにより同定した新規睡眠覚醒制御遺伝子Sik3 の解析による睡眠覚醒制御機構の解明

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