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生物の適応能を飛躍的に高めたリズム制御分子と高次中枢システムの世界
1S3-1
Design of circadian clock by two ATPases in cyanobacterial clock protein KaiC
Kondo Takao
Division of Biological Science, Nagoya University, Furocho 1, Chikusa, Nagoya, Aichi, 464-8602, Japan

We reconstituted the self-sustainable circadian oscillation of the KaiC phosphorylation state by incubating purified cyanobacterial clock proteins, KaiC with KaiA, KaiB, and ATP. This in vitro oscillation is the primary pacemaker of the cyanobacterial circadian clock, and revealed a novel function of proteins as timing devices that govern cellular metabolism. We further found that the ATPase activity of KaiC defines the period length and its temperature compensation. KaiC possesses extremely weak but stable ATPase activity(15 molecules of ATP per day). As the ATPase activity of KaiC is inherently temperature-invariant, suggesting that temperature compensation of the circadian period could be attained by simple ATPase reaction. Interestingly, the activities of five period-mutant proteins are directly proportional to their in vivo circadian frequencies, indicating that the ATPase activity defines the circadian period. We propose that KaiC ATPase activity constitutes the most fundamental reaction underlying circadian periodicity in cyanobacteria.Based on these observations, we propose a model of the protein circadian clock, in which the clock is composed from two units, pacemaker and driver that respectively take charge of C1 and CII domain of KaiC ATPase. Functionally, this design is important to mix two basically different processes function together to achieve precision of period length and robustness of the oscillation. The first unit can be achived by intramolecular feedback that generate mechanical tension inside the protein and the second unit could be energy-dependent phosphorylation cycle that is basis for robustness of the oscillation. These two processes could be combined by a uynique mechanism similar to escapement mechanism of the pendulum clock.
1S3-2
Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9-based Rett syndrome model mouse
Yagita Kazuhiro
Dept. of Physiology and Systems Bioscience, Kyoto Pref. Univ. Med.

Methyl-CpG-binding protein 2(Mecp2)is an X-linked gene encoding a methylated DNA binding nuclear protein which regulates transcriptional activity. The mutation of MECP2 in humans is associated with Rett syndrome(RTT), a neurodevelopmental disorder. RTT patients frequently exhibit abnormal sleep patterns and sleep-associated problems, in addition to autistic symptoms, raising the possibility of circadian clock dysfunction in RTT. In this study, we investigated circadian clock function in Mecp2-deficient mice. We successfully generated both male and female Mecp2-deficient mice on the wild-type C57BL/6 background and PER2Luciferase(PER2Luc)knock-in background by utilizing the clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 system. Generated Mecp2-deficient mice recapitulated reduced activity in mouse models of RTT, and their activity rhythms were diminished in constant dark conditions. Bioluminescence rhythms were analyzed using photomultiplier tubes(PMT)and high-sensitivity EMCCD camera-based microscopy in order to evaluate the molecular clockwork in the master pacemaker suprachiasmatic nucleus(SCN)with or without lacking Mecp2, PER2Luc. Real-time bioluminescence imaging revealed that the amplitude of PER2Luc driven circadian oscillation was significantly attenuated in Mecp2 deficient SCN neurons. On the other hands, in vitro circadian rhythm development assay using Mecp2 deficient mouse embryonic stem cells(ESCs)showed slight period-length changes of PER2Luc bioluminescence rhythms without apparent dampening. Together, these results demonstrate that Mecp2 deficiency abrogates the circadian pacemaking ability of the SCN, which may be a therapeutic target to treat the sleep problems of RTT patients.
1S3-3
Understanding the mechanism of seasonal time measurement
Yoshimura Takashi1,2,3
1Institute of Transformative Bio-Molecules(wpi-itbm), nagoya Univ.,2Grad. Sch. of Bioagr. Sci., Nagoya Univ.,3Div. of Seasonal Biol., Natl. Inst. of Basic Biol.

Animals living outside the tropics use changes in day length to adapt to seasonal changes in environment, but mechanisms underlying seasonal time measurement are not fully understood. Japanese quail is an excellent model for the study of these mechanisms because of its rapid and dramatic response. We have demonstrated that local thyroid hormone catabolism within the mediobasal hypothalamus(MBH)by thyroid hormone-activating enzyme(type 2 deiodinase:DIO2)regulates photoperiodism. Functional genomics analysis demonstrated that long day stimulus induces thyrotropin(thyroid stimulating hormone:TSH)production in the pars tuberalis(PT)of the pituitary gland, which triggers DIO2 expression in the ependymal cells of the MBH. In mammals, nocturnal melatonin secretion provides an endocrine signal of the photoperiod to the PT that contains melatonin receptors in high density. We have also demonstrated the involvement of TSH signaling pathway in mammals by using the TSH receptor null mice. Well known function of TSH derived from pars distalis(PD)of the pituitary gland is stimulation of thyroid gland. However, it was unclear how these two TSHs avoid functional crosstalk. We demonstrated that tissue-specific glycosylation is central to this mechanism. Although fish also exhibit clear seasonal responses, they do not possess an anatomically distinct PT. We found expression of TSH, DIO2, and rhodopsin family genes in the coronet cell of the saccus vasculosus(SV), suggesting the existence of a photoperiodic signaling pathway from light input to neuroendocrine output. Functional analysis suggested that the SV acts as a seasonal sensor in fish. We are currently trying to develop transformative bio-molecules that improve animal production and human health.
1S3-4
Regulation of memory retrieval by forebrain circadian clock
Kida Satoshi
Department of Bioscience, Tokyo University of Agriculture

Cognitive performance in people varies according to time-of-day, with memory retrieval declining in the late afternoon-early evening. Here we show that mice exhibit a similar time-of-day retrieval profile following weak hippocampus-dependent learning, with reduced retrieval efficiency correlating with low forebrain activity of circadian transcription factor, BMAL1. To test whether BMAL1 activity regulates retrieval efficiency, we inducibly expressed a dominant negative BMAL1(dnBMAL1)in mouse forebrain. dnBMAL1 expression had no effect on memory encoding but disrupted retrieval at Zeitgeber Time 8-12, and not at other time. Importantly, these effects were observed across multiple hippocampal memories and were independent of retention delay, time of encoding and Zeitgeber entrainment cue. Additionally, cAMP signals were suggested to mediate regulation of retrieval efficiency by BMAL1. Furthermore, forebrain dnBMAL1 expression did not affect locomotor rhythm or BMAL1-mediated transcription in the SCN. Thus forebrain clock regulates the efficiency of hippocampus-dependent memory retrieval independent of core time-keeping cells.