記述言語：英語   出版者・発行元：Nature Communications  

Entrainment is characterized by phase response curves (PRCs), which provide a summary of responses to perturbations at each circadian phase. The synchronization of mammalian circadian clocks is accomplished through the receipt of a variety of inputs from both internal and external time cues. A comprehensive comparison of PRCs for various stimuli in each tissue is required. Herein, we demonstrate that PRCs in mammalian cells can be characterized using a recently developed estimation method based on singularity response (SR), which represents the response of desynchronized cellular clocks. We confirmed that PRCs can be reconstructed using single SR measurements and quantified response properties for various stimuli in several cell lines. SR analysis reveals that the phase and amplitude after resetting are distinguishable among stimuli. SRs in tissue slice cultures reveal tissue-specific entrainment properties. These results demonstrate that SRs can be employed to unveil entrainment mechanisms with diverse stimuli in multiscale mammalian clocks.

DOI： 10.1038/s41467-023-38392-x

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PubMed

記述言語：英語   出版者・発行元：Science Advances  

The mammalian central circadian clock, located in the suprachiasmatic nucleus (SCN), coordinates the timing of physiology and behavior to local time cues. In the SCN, second messengers, such as cAMP and Ca2+, are suggested to be involved in the input and/or output of the molecular circadian clock. However, the functional roles of second messengers and their dynamics in the SCN remain largely unclear. In the present study, we visualized the spatiotemporal patterns of circadian rhythms of second messengers and neurotransmitter release in the SCN. Here, we show that neuronal activity regulates the rhythmic release of vasoactive intestinal peptides from the SCN, which drives the circadian rhythms of intracellular cAMP in the SCN. Furthermore, optical manipulation of intracellular cAMP levels in the SCN shifts molecular and behavioral circadian rhythms. Together, our study demonstrates that intracellular cAMP is a key molecule in the organization of the SCN circadian neuronal network.

DOI： 10.1126/sciadv.abq7032

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記述言語：英語   出版者・発行元：Scientific Reports  

Real-time monitoring of cellular temperature responses is an important technique in thermal biology and drug development. Recent study identified that Na+/Ca2+ exchanger (NCX)-dependent Ca2+ influx transduces cold signals to circadian clock in mammalian cultured cells. The finding raised an idea that cellular responses to the cold signals can be analyzed by monitoring of clock gene expression. We found that Per1 and Per2 were up-regulated after culture at 27 °C compared to 37 °C in Rat-1 fibroblasts. In order to monitor cold-Ca2+-dependent transcription in living cells, we developed a luciferase-based real-time reporting system by using Per1 promoter, Per2 promoter, Ca2+/cAMP-response elements (CRE) or NFAT-binding elements. We found that benzyloxyphenyl NCX inhibitor KB-R7943 and SN-6, but not SEA-0400 or YM-244769 inhibited the cold induction of Per2. Our study established a real-time monitoring system for cold Ca2+ signaling which can be applied to evaluation of drugs.

DOI： 10.1038/s41598-022-22166-4

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記述言語：日本語   出版者・発行元：公益社団法人 日本薬理学会  

<p>Circadian clock generates many physiological rhythms. In mammals, most of the rhythms are generated by transcriptional rhythms based on transcriptional and translational feedback loops (TTFL). Importantly, period lengths of the rhythms are almost constant in the range of physiological temperatures, and the property is called as temperature compensation.</p><p>In order to understand the mechanism of the temperature compensation, we screened small-molecule inhibitors by using Rat-1 fibroblasts expressing luciferase reporter of the transcriptional rhythms. The temperature compensation was compromised in the presence of an inhibitor of NCX (KB-R7943, SEA0400) or CaMKII (KN-93). Further analysis revealed that temperature lowering enhances NCX-dependent Ca²⁺ influx to activate CaMKII. CaMKII facilitates heterodimerization of CLOCK and BMAL1, bHLH transcription factors, thereby activating gene expression through E-box DNA elements. Thus, NCX-dependent cold Ca²⁺-CaMKII signaling compensates for deceleration of TTFL at lower temperatures. In addition to the temperature compensation, activities of NCX and CaMKII are essential for cell-autonomous oscillation of the rhythms. These results demonstrate that NCX is a new target for drug discovery based on the circadian clock function. (Kon <i>et al</i>., <b><i>Genes and Development</i></b>, 2014; Kon <i>et al</i>. <b><i>Science Advances</i></b>, 2021).</p>

DOI： 10.1254/jpssuppl.95.0_3-s41-4

CiNii Research

記述言語：英語   出版者・発行元：Scientific Reports  

Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.

DOI： 10.1038/s41598-021-00499-w

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担当区分：筆頭著者,　最終著者,　責任著者   記述言語：日本語   掲載種別：記事・総説・解説・論説等（学術雑誌）