NAGANO Mamoru

    Department of Medicine Associate Professor
Last Updated :2024/03/24

Researcher Information

Degree

  • (BLANK)(Kinki University)

J-Global ID

Research Interests

  • 神経解剖学   Neuroanatomy   

Research Areas

  • Life sciences / Neuroanatomy and physiology

Education

  •        - 1982  Kindai University  Faculty of Pharmacy  Department of Pharmacy
  •        - 1982  Kinki University  Faculty of Pharmaceutical Science

Association Memberships

  • 日本時計生物学会   日本薬理学会   日本神経科学学会   日本組織細胞化学会   日本電子顕微鏡学会   日本解剖学会   

Published Papers

  • Makito Hirano; Motoi Kuwahara; Yuko Yamagishi; Makoto Samukawa; Kanako Fujii; Shoko Yamashita; Masahiro Ando; Nobuyuki Oka; Mamoru Nagano; Taro Matsui; Toshihide Takeuchi; Kazumasa Saigoh; Susumu Kusunoki; Hiroshi Takashima; Yoshitaka Nagai
    Scientific reports 13 (1) 17801 - 17801 2023/10 
    Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) has recently been attributed to biallelic repeat expansions in RFC1. More recently, the disease entity has expanded to atypical phenotypes, including chronic neuropathy without cerebellar ataxia or vestibular areflexia. Very recently, RFC1 expansions were found in patients with Sjögren syndrome who had neuropathy that did not respond to immunotherapy. In this study RFC1 was examined in 240 patients with acute or chronic neuropathies, including 105 with Guillain-Barré syndrome or Miller Fisher syndrome, 76 with chronic inflammatory demyelinating polyneuropathy, and 59 with other types of chronic neuropathy. Biallelic RFC1 mutations were found in three patients with immune-mediated neuropathies, including Guillain-Barré syndrome, idiopathic sensory ataxic neuropathy, or anti-myelin-associated glycoprotein (MAG) neuropathy, who responded to immunotherapies. In addition, a patient with chronic sensory autonomic neuropathy had biallelic mutations, and subclinical changes in Schwann cells on nerve biopsy. In summary, we found CANVAS-related RFC1 mutations in patients with treatable immune-mediated neuropathy or demyelinating neuropathy.
  • Yuji Kanazawa; Yuri Ikeda-Matsuo; Hiaki Sato; Mamoru Nagano; Satoshi Koinuma; Tatsuo Takahashi; Hirokazu Suzuki; Ryo Miyachi; Yasufumi Shigeyoshi
    International Journal of Molecular Sciences MDPI AG 24 (11) 9209 - 9209 2023/05 
    Obesity and aging are known to affect the skeletal muscles. Obesity in old age may result in a poor basement membrane (BM) construction response, which serves to protect the skeletal muscle, thus making the skeletal muscle more vulnerable. In this study, older and young male C57BL/6J mice were divided into two groups, each fed a high-fat or regular diet for eight weeks. A high-fat diet decreased the relative gastrocnemius muscle weight in both age groups, and obesity and aging individually result in a decline in muscle function. Immunoreactivity of collagen IV, the main component of BM, BM width, and BM-synthetic factor expression in young mice on a high-fat diet were higher than that in young mice on a regular diet, whereas such changes were minimal in obese older mice. Furthermore, the number of central nuclei fibers in obese older mice was higher than in old mice fed a regular diet and young mice fed a high-fat diet. These results suggest that obesity at a young age promotes skeletal muscle BM formation in response to weight gain. In contrast, this response is less pronounced in old age, suggesting that obesity in old age may lead to muscle fragility.
  • Atsuko Fujioka; Mamoru Nagano; Keisuke Ikegami; Koh-hei Masumoto; Tomoko Yoshikawa; Satoshi Koinuma; Ken-ichi Nakahama; Yasufumi Shigeyoshi
    Brain Research Elsevier BV 1798 148129 - 148129 0006-8993 2023/01 
    The localization and function of synaptotagmin (syt)17 in the suprachiasmatic nucleus (SCN) of the brain, which is the master circadian oscillator, were investigated. The Syt17 mRNA-containing neurons were mainly situated in the shell region while SYT17 immunoreactive cell bodies and neural fibers were detected in the core and shell of the SCN and the subparaventricular zone (SPZ). Further, electron microscopy analysis revealed SYT17 in the rough endoplasmic reticulum (rER), Golgi apparatus (G), and large and small vesicles of neurons. Syt17 mRNA expression in the SCN showed a circadian rhythm, and light exposure at night suppressed its expression. In addition, the free running period of locomotor activity rhythm was shortened in Syt17-deletion mutant mice. These findings suggest that SYT17 is involved in the regulation of circadian rhythms.
  • Tadamitsu Morimoto; Tomoko Yoshikawa; Mamoru Nagano; Yasufumi Shigeyoshi
    PLOS ONE Public Library of Science (PLoS) 17 (10) e0276372 - e0276372 2022/10 
    In mammals, the center of the circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Many studies have suggested that there are multiple regions generating different circadian periods within the SCN, but the exact localization of the regions has not been elucidated. In this study, using a transgenic rat carrying a destabilized luciferase reporter gene driven by a regulatory element of Per2 gene (Per2::dLuc), we investigated the regional variation of period lengths in horizontal slices of the SCN. We revealed a distinct caudal medial region (short period region, SPR) and a rostro-lateral region (long period region, LPR) that generate circadian rhythms with periods shorter than and longer than 24 hours, respectively. We also found that the core region of the SCN marked by dense VIP (vasoactive intestinal peptide) mRNA-expressing neurons covered a part of LPR, and that the shell region of the SCN contains both SPR and the rest of the LPR. Furthermore, we observed how synchronization is achieved between regions generating distinct circadian periods in the SCN. We found that the longer circadian rhythm of the rostral region appears to entrain the circadian rhythm in the caudal region. Our findings clarify the localization of regionality of circadian periods and the mechanism by which the integrated circadian rhythm is formed in the SCN.
  • Yuji Kanazawa; Tatsuo Takahashi; Takashi Higuchi; Ryo Miyachi; Mamoru Nagano; Satoshi Koinuma; Yasufumi Shigeyoshi
    Medical Molecular Morphology Springer Science and Business Media LLC 1860-1480 2022/09
  • 継続的なストレッチングがヒラメ筋の基底板に与える影響
    金澤 佑治; 樋口 隆志; 宮地 諒; 長野 護; 鯉沼 聡; 重吉 康史
    日本筋学会学術集会プログラム・抄録集 (一社)日本筋学会 8回 136 - 136 2433-975X 2022/08
  • Yuji Kanazawa; Mamoru Nagano; Satoshi Koinuma; Shinichi Sugiyo; Yasufumi Shigeyoshi
    Microscopy (Oxford, England) 71 (4) 245 - 248 2022/03 [Refereed]
     
    We investigated the effect of aging on the basement membrane during post-injury muscle recovery. Using a rat model, we found that aging delayed muscle fiber and basement membrane recovery. In addition, expression of basement membrane-related factors peaked 7 days after muscle injury among both young and older rats. Peak expression of collagen IV synthetic factors decreased with age, whereas expression of the degradative factor was unaffected by age. These results suggest that age-related delays in post-injury muscle fiber and basement membrane recovery may be related to suppression of collagen IV synthetic factors.
  • Yuji Kanazawa; Mamoru Nagano; Satoshi Koinuma; Shinichi Sugiyo; Yasufumi Shigeyoshi
    Acta histochemica et cytochemica 54 (5) 167 - 175 2021/10 
    The basement membrane (BM)-related factors, including collagen IV, are important for the maintenance and recovery of skeletal muscles. Aging impairs the expression of BM-related factors during recovery after disuse atrophy. Muscle activity facilitates collagen synthesis that constitutes the BM. However, the effect of endurance exercise on the BM of aged muscles is unclear. Thus, to understand the effect of endurance exercise on the BM of the skeletal muscle in aged rats, we prescribed treadmill running in aged rats and compared the differences in the expression of BM-related factors between the aged rats with and without exercise habits. Aged rats were subjected to endurance exercise via treadmill running. Exercise increased the mRNA expression levels of the BM-related factors, the area and intensity of collagen IV-immunoreactivity and the width of lamina densa in the soleus muscle of aged rats. These finding suggests that endurance exercise promotes BM construction in aged rats.
  • Yoichi Minami; Tomoko Yoshikawa; Mamoru Nagano; Satoshi Koinuma; Tadamitsu Morimoto; Atsuko Fujioka; Keiichi Furukawa; Keisuke Ikegami; Atsuhiro Tatemizo; Kentaro Egawa; Teruya Tamaru; Taizo Taniguchi; Yasufumi Shigeyoshi
    The European journal of neuroscience 53 (6) 1783 - 1793 2021/03 
    The circadian rhythms are endogenous rhythms of about 24 h, and are driven by the circadian clock. The clock centre locates in the suprachiasmatic nucleus. Light signals from the retina shift the circadian rhythm in the suprachiasmatic nucleus, but there is a robust part of the suprachiasmatic nucleus that causes jet lag after an abrupt shift of the environmental lighting condition. To examine the effect of attenuated circadian rhythm on the duration of jet lag, we established a transgenic rat expressing BMAL1 dominant negative form under control by mouse Prnp-based transcriptional regulation cassette [BMAL1 DN (+)]. The transgenic rats became active earlier than controls, just after light offset. Compared to control rats, BMAL1 DN (+) rats showed smaller circadian rhythm amplitudes in both behavioural and Per2 promoter driven luciferase activity rhythms. A light pulse during the night resulted in a larger phase shift of behavioural rhythm. Furthermore, at an abrupt shift of the light-dark cycle, BMAL1 DN (+) rat showed faster entrainment to the new light-dark cycle compared to controls. The circadian rhythm has been regarded as a limit cycle phenomenon, and our results support the hypothesis that modification of the amplitude of the circadian limit cycle leads to alteration in the length of the phase shift.
  • Yuji Kanazawa; Mamoru Nagano; Satoshi Koinuma; Shinichi Sugiyo; Yasufumi Shigeyoshi
    Biomedical research (Tokyo, Japan) 42 (3) 115 - 119 2021 
    The basement membrane (BM), with collagen IV as a major component, plays an important role in the maintenance of muscle structure and its robustness. To investigate the effects of aging on factors related to BM construction, we compared the expression status of these factors in 3- and 20-month-old male Wistar rats. The expression levels of Col4a1 and Col4a2 (encoding collagen IV), Sparc (involved in collagen IV functionalization), and Mmp14 (a collagen IV degradation factor) were decreased. These results suggest that aging suppresses collagen IV synthetic and degradative factors and affects BM-related factors in the steady state.
  • 筋損傷後の基底板再構築における加齢の影響
    金澤 佑治; 長野 護; 鯉沼 聡; 筋野 貢; 南 陽一; 杉生 真一; 武田 功; 重吉 康史
    日本筋学会学術集会プログラム・抄録集 日本筋学会 6回 75 - 75 2433-975X 2020/12
  • Keisuke Ikegami; Masato Nakajima; Yoichi Minami; Mamoru Nagano; Satoru Masubuchi; Yasufumi Shigeyoshi
    Biochemical and Biophysical Research Communications Elsevier BV 0006-291X 2020/08 [Refereed]
  • Yuji Kanazawa; Mamoru Nagano; Satoshi Koinuma; Mitsugu Sujino; Yoichi Minami; Shinichi Sugiyo; Isao Takeda; Yasufumi Shigeyoshi
    Connective tissue research 62 (5) 1 - 12 2020/07 [Refereed]
     
    PURPOSE: Collagen IV is a component of the basement membrane (BM) that provides mechanical support for muscle fibers. In addition, transcription factor 4 (TCF4) is highly expressed in muscle connective tissue fibroblasts and regulates muscle regeneration. However, the expression of collagen IV and TCF4 (+) cells in response to exercise-induced muscle injury is not well-known. Here, we investigated the expression and localization of collagen IV and TCF4 (+) cells during the recovery process after muscle injury induced by different exercise loads. MATERIALS AND METHODS: Muscle injury was observed in the soleus muscle of young Wistar rats after 12 or 18 sets - downhill running (DR) on a treadmill. After running, the rats were permitted to recover for a period of 0.5 days, 2 days, or 7 days. RESULTS: Ectopic localization of collagen IV in injured muscle fibers was observed after DR, and the number increased at 0.5 days after 18 sets DR and at 2 days after 12 or 18 sets - DR as compared to the number observed at baseline. BM disruption was observed after DR. TCF4 (+) cells appeared in the inside and around injured muscle fibers at 0.5 day of recovery. After 18 sets DR, TCF4 (+) cells were more abundant for a longer period than that observed after 12 sets DR. CONCLUSIONS: DR induces BM disruption accompanied by muscle fiber damage. It is possible that BM destruction may be accompanied by muscle damage and that TCF4 (+) cells contribute to muscle fiber and BM recovery.
  • Shoko Takemura; Mamoru Nagano; Ayami Isonishi; Tatsuhide Tanaka; Kouko Tatsumi; Mariko Yamano; Yoichi Minami; Yasufumi Shigeyoshi; Akio Wanaka
    Neuroscience letters 727 134897 - 134897 2020/05 [Refereed]
     
    Entrainment of mammalian circadian rhythms requires receptor-mediated signaling in the hypothalamic suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker. Receptor-mediated signaling is regulated by endocytosis, indicating that endocytosis-related proteins contribute to SCN pacemaking. Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. In this study, we showed that Snx25 mRNA and SNX25 protein are highly expressed and exhibit remarkable circadian rhythms in the SCN of adult mice. Expression was maximal at about zeitgeber time (ZT) 16 in the subjective night and minimal at ZT8 in the subjective day. Prominent SNX25 immunoreactivity was found in the arginine vasopressin-positive neurons of the SCN. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the circadian pacemaking system.
  • Keisuke Ikegami; Kazumasa Saigoh; Atsuko Fujioka; Mamoru Nagano; Ken Kitajima; Chihiro Sato; Satoru Masubuchi; Susumu Kusunoki; Yasufumi Shigeyoshi
    Scientific reports 9 (1) 13634 - 13634 2019/09 [Refereed]
     
    ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.
  • 筋損傷後に生じる骨格筋内の線維芽細胞による基底板構築
    金澤 佑治; 長野 護; 鯉沼 聡; 筋野 貢; 南 陽一; 杉生 真一; 武田 功; 重吉 康史
    日本筋学会学術集会プログラム・抄録集 日本筋学会 5回 124 - 124 2433-975X 2019/08
  • Mamoru Nagano; Keisuke Ikegami; Yoichi Minami; Yuji Kanazawa; Satoshi Koinuma; Mitsugu Sujino; Yasufumi Shigeyoshi
    Brain research 1714 73 - 80 0006-8993 2019/07 [Refereed]
     
    The suprachiasmatic nucleus (SCN) is the center of the mammalian circadian system. Environmental photic signals shifts the phase of the circadian rhythm in the SCN except during the dead zone, when the photic signal is gated somewhere on the way from the retina to the neurons in the SCN. Here we examined the phase of the dead zone after an abrupt delay of the LD cycles for several days by observing the mc-Fos induction in the SCN by light pulses. After an abrupt shift of the LD cycles, the dead zone showed a slow phase shift, about two hours per day, which was well corresponded with the slow phase shift of the rest-activity cycles. In our previous study we demonstrated that, after an abrupt shift of the LD cycles, the SCN showed transient endogenous desynchronization between shell and core regions that showed a slow and a rapid shift of the circadian rhythms, respectively. Therefore, the present findings on the phase shift of the dead zone after the LD cycles shift suggest that the phase of the dead zone is under the control of the timing signals from the shell region of the SCN.
  • Yasutaka Niwa; Genki N Kanda; Rikuhiro G Yamada; Shoi Shi; Genshiro A Sunagawa; Maki Ukai-Tadenuma; Hiroshi Fujishima; Naomi Matsumoto; Koh-Hei Masumoto; Mamoru Nagano; Takeya Kasukawa; James Galloway; Dimitri Perrin; Yasufumi Shigeyoshi; Hideki Ukai; Hiroshi Kiyonari; Kenta Sumiyama; Hiroki R Ueda
    Cell reports 24 (9) 2231 - 2247 2018/08 [Refereed]
     
    Sleep regulation involves interdependent signaling among specialized neurons in distributed brain regions. Although acetylcholine promotes wakefulness and rapid eye movement (REM) sleep, it is unclear whether the cholinergic pathway is essential (i.e., absolutely required) for REM sleep because of redundancy from neural circuits to molecules. First, we demonstrate that synaptic inhibition of TrkA+ cholinergic neurons causes a severe short-sleep phenotype and that sleep reduction is mostly attributable to a shortened sleep duration in the dark phase. Subsequent comprehensive knockout of acetylcholine receptor genes by the triple-target CRISPR method reveals that a similar short-sleep phenotype appears in the knockout of two Gq-type acetylcholine receptors Chrm1 and Chrm3. Strikingly, Chrm1 and Chrm3 double knockout chronically diminishes REM sleep to an almost undetectable level. These results suggest that muscarinic acetylcholine receptors, Chrm1 and Chrm3, are essential for REM sleep.
  • Mitsugu Sujino; Takeshi Asakawa; Mamoru Nagano; Satoshi Koinuma; Koh-Hei Masumoto; Yasufumi Shigeyoshi
    Scientific reports 8 (1) 854 - 854 2018/01 [Refereed]
     
    In mammals, the principal circadian oscillator exists in the hypothalamic suprachiasmatic nucleus (SCN). In the SCN, CLOCK works as an essential component of molecular circadian oscillation, and ClockΔ19 mutant mice show unique characteristics of circadian rhythms such as extended free running periods, amplitude attenuation, and high-magnitude phase-resetting responses. Here we investigated what modifications occur in the spatiotemporal organization of clock gene expression in the SCN of ClockΔ19 mutants. The cultured SCN, sampled from neonatal homozygous ClockΔ19 mice on an ICR strain comprising PERIOD2::LUCIFERASE, demonstrated that the Clock gene mutation not only extends the circadian period, but also affects the spatial phase and period distribution of circadian oscillations in the SCN. In addition, disruption of the synchronization among neurons markedly attenuated the amplitude of the circadian rhythm of individual oscillating neurons in the mutant SCN. Further, with numerical simulations based on the present studies, the findings suggested that, in the SCN of the ClockΔ19 mutant mice, stable oscillation was preserved by the interaction among oscillating neurons, and that the orderly phase and period distribution that makes a phase wave are dependent on the functionality of CLOCK.
  • Yuji Kanazawa; Keisuke Ikegami; Mitsugu Sujino; Satoshi Koinuma; Mamoru Nagano; Yuki Oi; Tomoya Onishi; Shinichi Sugiyo; Isao Takeda; Hiroshi Kaji; Yasufumi Shigeyoshi
    EXPERIMENTAL GERONTOLOGY PERGAMON-ELSEVIER SCIENCE LTD 98 153 - 161 0531-5565 2017/11 [Refereed]
     
    Aging is known to lead to the impaired recovery of muscle after disuse as well as the increased susceptibility of the muscle to damage. Here, we show that, in the older rats, reloading after disuse atrophy, causes the damage of the muscle fibers and the basement membrane (BM) that structurally support the muscle fibers. Male Wistar rats of 3-(young) and 20-(older) months of age were subjected to hindlimb-unloading for 2 weeks followed by reloading for a week. In the older rats, the soleus muscles showed necrosis and central nuclei fiber indicating the regeneration of muscle fibers. Furthermore, ectopic immunoreactivity of collagen IV, a major component of the BM, remained mostly associated with the necrotic appearance, suggesting that the older rats were impaired with the ability of repairing the damaged BM. Further, after unloading and reloading, the older rats did not show a significant alteration, although the young rats showed clear response of Col4a1 and Col4a2 genes, both coding for collagen IV. In addition, during the recovery phase, the young rats showed increase in the amount of Hsp47 and Sparc mRNA, which are protein folding-related factor genes, while the older rats did not show any significant variation. Taken together, our findings suggest that the atrophic muscle fibers of the older rats induced by unloading were vulnerable to the weight loading, and that attenuated reactivity of the BM-synthesizing fibroblast to gravity contributes to the fragility of muscle fibers in the older animals.
  • Atsuko Kubo; Mitsugu Sujino; Koh-hei Masumoto; Atsuko Fujioka; Toshio Terashima; Yasufumi Shigeyoshi; Mamoru Nagano
    ACTA HISTOCHEMICA ET CYTOCHEMICA JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY 50 (2) 95 - 104 0044-5991 2017 [Refereed]
     
    Both prokineticin receptor 2 (pkr2) and prokineticin 2 (pk2) gene-deficient mice have hypoplasia of the main olfactory bulb (MOB). This hypoplasia has been attributed to disruption of the glomerulus that is caused by loss of afferent projection from olfactory sensory neurons (OSN), and to the impaired migration of granule cells, a type of interneuron. In the present study, we examined whether migration of the second type of interneuron, periglomerular cells (PGC), is dependent on the pkr2 expression by observing the localization of distinct subpopulations of PGC: calretinin (CR)-, calbindin (CB)- and tyrosine hydroxylase (TH)-expressing neurons. In the Pkr2(-/-) mice, the construction of the layered structure of the MOB was partially preserved, with the exception of the internal plexiform layer (IPL) and the glomerular layer (GL). In the outermost layer of the MOB, abundant CR- and CB-immunopositive neurons were observed in the hypoplastic olfactory bulb. In addition, although markedly decreased, TH-immunopositive neurons were also observed in the outermost cell-dense region in the Pkr2(-/-). The findings suggest that the migration of PGC to the MOB, as well as the migration from the core to the surface region of the MOB, is not driven by the PK2-PKR2 system.
  • Akihito A Adachi; Atsuko Fujioka; Mamoru Nagano; Koh-hei Masumoto; Toru Takumi; Takashi Yoshimura; Shizufumi Ebihara; Kentaro Mori; Yoshifumi Yokota; Yasufumi Shigeyoshi
    Zoological science 30 (12) 1011 - 8 0289-0003 2013/12 [Refereed]
     
    The mammalian circadian oscillator is composed of interacting positive and negative transcription events. The clock proteins PER1 and PER2 play essential roles in a negative limb of the feedback loop that generates the circadian rhythm in mammals. In addition, the proteins CLOCK and BMAL1 (also known as ARNTL) form a heterodimer that drives the Per genes via the E-box consensus sequences within their promoter regions. In the present study, we demonstrate that Id2 is involved in stabilization of the amplitudes of the circadian oscillations by suppressing transcriptional activation of clock genes Clock and Bmal1. Id2 shows dynamic oscillation in the SCN, with a peak in the late subjective night. Under constant dark conditions (DD), Id2(-/-) mice showed no apparent difference in locomotor activity, however, under constant light conditions (LL), Id2(-/-) mice exhibit aberrant locomotor activity, with lower circadian oscillation amplitudes, although the free running periods in Id2(-/-) mice show no differences from those in either wild type or heterozygous mice. Id2(-/-) animals also exhibit upregulation of Per1 in constant light, during both the subjective night and day. In wild type mice, Id2 is upregulated by constant light exposure during the subjective night. We propose that Id2 expression in the SCN contributes to maintenance of dynamic circadian oscillations.
  • Satoshi Koinuma; Takeshi Asakawa; Mamoru Nagano; Keiichi Furukawa; Mitsugu Sujino; Koh-Hei Masumoto; Yoshihiro Nakajima; Seiichi Hashimoto; Kazuhiro Yagita; Yasufumi Shigeyoshi
    EUROPEAN JOURNAL OF NEUROSCIENCE WILEY-BLACKWELL 38 (6) 2832 - 2841 0953-816X 2013/09 [Refereed]
     
    The suprachiasmatic nucleus (SCN) is the mammalian circadian rhythm center. Individual oscillating neurons have different endogenous circadian periods, but they are usually synchronized by an intercellular coupling mechanism. The differences in the period of each oscillating neuron have been extensively studied; however, the clustering of oscillators with similar periods has not been reported. In the present study, we artificially disrupted the intercellular coupling among oscillating neurons in the SCN and observed regional differences in the periods of the oscillating small-latticed regions of the SCN using a transgenic rat carrying a luciferase reporter gene driven by regulatory elements from a per2 clock gene (Per2::dluc rat). The analysis divided the SCN into two regions - a region with periods shorter than 24h (short-period region, SPR) and another with periods longer than 24h (long-period region, LPR). The SPR was located in the smaller medial region of the dorsal SCN, whereas the LPR occupied the remaining larger region. We also found that slices containing the medial region of the SCN generated shorter circadian periods than slices that contained the lateral region of the SCN. Interestingly, the SPR corresponded well with the region where the SCN phase wave is generated. We numerically simulated the relationship between the SPR and a large LPR. A mathematical model of the SCN based on our findings faithfully reproduced the kinetics of the oscillators in the SCN in synchronized conditions, assuming the existence of clustered short-period oscillators.
  • Kaori Tsujino; Ryohei Narumi; Koh-hei Masumoto; Etsuo A. Susaki; Yuta Shinohara; Takaya Abe; Masayuki Iigo; Atsushi Wada; Mamoru Nagano; Yasufumi Shigeyoshi; Hiroki R. Ueda
    GENES TO CELLS WILEY-BLACKWELL 18 (7) 575 - 588 1356-9597 2013/07 [Refereed]
     
    Organisms have seasonal physiological changes in response to day length. Long-day stimulation induces thyroid-stimulating hormone beta subunit (TSH beta) in the pars tuberalis (PT), which mediates photoperiodic reactions like day-length measurement and physiological adaptation. However, the mechanism of TSH beta induction for day-length measurement is largely unknown. To screen candidate upstream molecules of TSH beta, which convey light information to the PT, we generated Luciferase knock-in mice, which quantitatively report the dynamics of TSH beta expression. We cultured brain slices containing the PT region from adult and neonatal mice and measured the bioluminescence activities from each slice over several days. A decrease in the bioluminescence activities was observed after melatonin treatment in adult and neonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSH beta expression to melatonin. Thus, we concluded that our experimental system monitors TSH beta expression dynamics in response to external stimuli.
  • Mark J. McCabe; Carles Gaston-Massuet; Louise C. Gregory; Kyriaki S. Alatzoglou; Vaitsa Tziaferi; Oualid Sbai; Philippe Rondard; Koh-Hei Masumoto; Mamoru Nagano; Yasufumi Shigeyoshi; Marija Pfeifer; Tony Hulse; Charles R. Buchanan; Nelly Pitteloud; Juan-Pedro Martinez-Barbera; Mehul T. Dattani
    Journal of Clinical Endocrinology and Metabolism 3 98 (3) E547 - E557 0021-972X 2013/03 [Refereed]
     
    Context: Loss-of-function mutations in PROK2 and PROKR2 have been implicated in Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia. Recent data suggest overlapping phenotypes/genotypes between KS and congenital hypopituitarism (CH), including septo-optic dysplasia (SOD). Objective: We screened a cohort of patients with complex forms of CH (n=422) for mutations in PROK2 and PROKR2. Results:Wedetected 5 PROKR2 variants in 11 patients with SOD/CH: novel p.G371R and previously reported p.A51T, p.R85L, p.L173R, and p.R268C-the latter 3 being known functionally deleterious variants. Surprisingly, 1 patient with SOD was heterozygous for the p.L173R variant, whereas his phenotypically unaffected mother was homozygous for the variant. We sought to clarify the role of PROKR2 in hypothalamopituitary development through analysis of Prokr2-/- mice. Interestingly, these revealed predominantly normal hypothalamopituitary development and terminal cell differentiation, with the exception of reduced LH this was inconsistent with patient phenotypes and more analogous to the healthy mother, although she did not have KS, unlike the Prokr2-/- mice. Conclusions: The role of PROKR2 in the etiology of CH, SOD, and KS is uncertain, as demonstrated by no clear phenotype-genotype correlation loss-of-function variants in heterozygosity or homozygosity can be associated with these disorders. However, we report a phenotypically normal parent, homozygous for p.L173R. Our data suggest that the variants identified herein are unlikely to be implicated in isolation in these disorders other genetic or environmental modifiers may also impact on the etiology. Given the phenotypic variability, genetic counseling may presently be inappropriate. Copyright © 2013 by The Endocrine Society.
  • Yukihiro Hamada; Kazumasa Saigoh; Koh-hei Masumoto; Mamoru Nagano; Susumu Kusunoki; Yasufumi Shigeyoshi
    NEUROSCIENCE LETTERS ELSEVIER IRELAND LTD 535 12 - 17 0304-3940 2013/02 [Refereed]
     
    Polysialic acids are implicated in various biological processes such as neural cell migration, axonal growth, synaptogenesis and resetting of the circadian rhythm. Recently, polysialation has been reported to be involved in the formation and resetting of the circadian clock. However, the genes that control the circadian rhythm of polysialation have not been elucidated. In the present study, we investigated the expression profile of ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 (ST8Sia VI) in the suprachiasmatic nucleus (SCN), which is one of the modification transferases that add sialic acids to type O carbohydrate chains. ST8Sia VI mRNA showed strong expression in the SCN with dynamic circadian rhythm. Further, the amount of ST8Sia VI mRNA in the SCN was increased by brief light exposure. Interestingly, the localization of ST8Sia VI mRNA in the SCN differs from those of arginine vasopressin and vasoactive intestinal peptide mRNAs, which are typical SCN subregion markers showing shell and core, dorsomedial and ventrolateral, or light-responsive and unresponsive regions, respectively. The present findings suggest that ST8siVI is involved in rhythmic polysialation in the SCN and that ST8siVI expression provides a novel compartmentation of the mammalian circadian center. (C) 2013 Elsevier Ireland Ltd. All rights reserved.
  • Mitsugu Sujino; Keiichi Furukawa; Satoshi Koinuma; Atsuko Fujioka; Mamoru Nagano; Masayuki Iigo; Yasufumi Shigeyoshi
    ENDOCRINOLOGY ENDOCRINE SOC 153 (5) 2277 - 2286 0013-7227 2012/05 
    The suprachiasmatic nucleus is the master circadian clock and resets the peripheral clocks via various pathways. Glucocorticoids and daily feeding are major time cues for entraining most peripheral clocks. However, recent studies have suggested that the dominant timing factor differs among organs and tissues. In our current study, we reveal differences in the entrainment properties of the peripheral clocks in the liver, kidney, and lung through restricted feeding (RF) and antiphasic corticosterone (CORT) injections in adrenalectomized rats. The peripheral clocks in the kidney and lung were found to be entrained by a daily stimulus from CORT administration, irrespective of the meal time. In contrast, the liver clock was observed to be entrained by an RF regimen, even if daily CORT injections were given at antiphase. These results indicate that glucocorticoids are a strong zeitgeber that overcomes other entrainment factors regulating the peripheral oscillators in the kidney and lung and that RF is a dominant mediator of the entrainment ability of the circadian clock in the liver. (Endocrinology 153: 2277-2286, 2012)
  • Mitsugu Sujino; Keiichi Furukawa; Satoshi Koinuma; Atsuko Fujioka; Mamoru Nagano; Masayuki Iigo; Yasufumi Shigeyoshi
    ENDOCRINOLOGY ENDOCRINE SOC 153 (5) 2277 - 2286 0013-7227 2012/05 [Refereed]
     
    The suprachiasmatic nucleus is the master circadian clock and resets the peripheral clocks via various pathways. Glucocorticoids and daily feeding are major time cues for entraining most peripheral clocks. However, recent studies have suggested that the dominant timing factor differs among organs and tissues. In our current study, we reveal differences in the entrainment properties of the peripheral clocks in the liver, kidney, and lung through restricted feeding (RF) and antiphasic corticosterone (CORT) injections in adrenalectomized rats. The peripheral clocks in the kidney and lung were found to be entrained by a daily stimulus from CORT administration, irrespective of the meal time. In contrast, the liver clock was observed to be entrained by an RF regimen, even if daily CORT injections were given at antiphase. These results indicate that glucocorticoids are a strong zeitgeber that overcomes other entrainment factors regulating the peripheral oscillators in the kidney and lung and that RF is a dominant mediator of the entrainment ability of the circadian clock in the liver. (Endocrinology 153: 2277-2286, 2012)
  • Application of layer-specific markers in the evaluation of abnormal cytoarchitecture in the olfactory bulb of prokineticin receptor 2 deficient mice
    重吉 康史; 山崎千尋; 久保厚子; 長野 護; 筋野 貢
    Acta Med Kinki Univ 37 (1) 35 - 43 2012
  • Profile of tyrosine hydroxylase-expressing neurons in the olfactory bulb of prokineticin type 2 receptor-deficient mice during embryonic development.
    重吉 康史; 久保厚子; 山崎千尋; 長野 護; 筋野 貢; 升本 宏平
    Acta Med Kinki Univ 37 (1) 25 - 33 2012
  • Takeya Kasukawa; Koh-hei Masumoto; Itoshi Nikaido; Mamoru Nagano; Kenichiro D. Uno; Kaori Tsujino; Carina Hanashima; Yasufumi Shigeyoshi; Hiroki R. Ueda
    PLOS ONE PUBLIC LIBRARY SCIENCE 6 (8) e23228  1932-6203 2011/08 [Refereed]
     
    The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of similar to 50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems.
  • Masashi Kitazawa; Mamoru Nagano; Koh-hei Masumoto; Yasufumi Shigeyoshi; Tohru Natsume; Seiichi Hashimoto
    ENDOCRINOLOGY ENDOCRINE SOC 152 (7) 2558 - 2567 0013-7227 2011/07 
    Angiopoietin-like (Angptl) 2, a member of the Angptl protein family, is predominantly secreted from adipose tissue and the heart. Here, we demonstrate that the expression of Angptl2 in epididymal adipose tissue of C57BL/6J mice shows pulsatility and circadian rhythmicity and that the rhythmicity was disrupted in high-fat-fed and leptin receptor-deficient diabetic db/db mice with insulin resistance. To investigate whether the reduction in Angptl2 expression was related to the progression of diabetes, wetreated db/db mice with recombinant Angptl2 for 4wk during the peak period of Angptl2 expression in C57BL/6J mice. Angptl2-treated mice showed decreases in plasma glucose, insulin, triglyceride, and fatty acid levels and an increase in plasma adiponectin, a therapeutic regulator of insulin resistance, leading to improvements in glucose tolerance. In cultured adipocytes, recombinant Angptl2 increased adiponectin expression and stimulated insulin sensitivity partially by reducing the levels of tribbles homolog 3, a specific Akt kinase inhibitory protein. Conversely, Angptl2 small interfering RNA reduced adiponectin expression, resulting in insulin resistance. In preadipocytes, treatment with Angptl2 small interfering RNA inhibited differentiation to adipocytes and reduced adiponectin expression. Taken together, our results suggest that replenishment of Angptl2 stimulates insulin sensitivity and improves the type 2 diabetic state. (Endocrinology 152: 2558-2567, 2011)
  • Masashi Kitazawa; Mamoru Nagano; Koh-hei Masumoto; Yasufumi Shigeyoshi; Tohru Natsume; Seiichi Hashimoto
    ENDOCRINOLOGY ENDOCRINE SOC 152 (7) 2558 - 2567 0013-7227 2011/07 [Refereed]
     
    Angiopoietin-like (Angptl) 2, a member of the Angptl protein family, is predominantly secreted from adipose tissue and the heart. Here, we demonstrate that the expression of Angptl2 in epididymal adipose tissue of C57BL/6J mice shows pulsatility and circadian rhythmicity and that the rhythmicity was disrupted in high-fat-fed and leptin receptor-deficient diabetic db/db mice with insulin resistance. To investigate whether the reduction in Angptl2 expression was related to the progression of diabetes, wetreated db/db mice with recombinant Angptl2 for 4wk during the peak period of Angptl2 expression in C57BL/6J mice. Angptl2-treated mice showed decreases in plasma glucose, insulin, triglyceride, and fatty acid levels and an increase in plasma adiponectin, a therapeutic regulator of insulin resistance, leading to improvements in glucose tolerance. In cultured adipocytes, recombinant Angptl2 increased adiponectin expression and stimulated insulin sensitivity partially by reducing the levels of tribbles homolog 3, a specific Akt kinase inhibitory protein. Conversely, Angptl2 small interfering RNA reduced adiponectin expression, resulting in insulin resistance. In preadipocytes, treatment with Angptl2 small interfering RNA inhibited differentiation to adipocytes and reduced adiponectin expression. Taken together, our results suggest that replenishment of Angptl2 stimulates insulin sensitivity and improves the type 2 diabetic state. (Endocrinology 152: 2558-2567, 2011)
  • T. Svingen; K. S. McClelland; K. Masumoto; M. Sujino; M. Nagano; Y. Shigeyoshi; P. Koopman
    SEXUAL DEVELOPMENT KARGER 5 (6) 294 - 303 1661-5425 2011 
    Kallmann syndrome is a form of hypogonadotropic hypogonadism also associated with the loss of smell. It is a phenotypically and genetically heterogeneous disorder, with mutations in several known causative genes now accounting for approximately 30% of cases. The prevalence for the disease is also much higher in males than in females, a phenomenon that remains to be fully explained. Here, we show that loss of Prokr2, which is linked to autosomal recessive Kallmann syndrome type 3 ( KAL3; OMIM 244200), affects fetal testis differentiation in mice. We find that Prokr2 is specifically expressed in the XY gonads during sex determination and fetal sexual differentiation, and knockout mice display a variable degree of compromised vasculature in the fetal testes. This phenotype offers potential insight into the clinical heterogeneity observed within familial cases, and may contribute to the gender bias in Kallmann syndrome patients. Copyright (C) 2012 S. Karger AG, Basel
  • Koh-hei Masumoto; Maki Ukai-Tadenuma; Takeya Kasukawa; Mamoru Nagano; Kenichiro D. Uno; Kaori Tsujino; Kazumasa Horikawa; Yasufumi Shigeyoshi; Hiroki R. Ueda
    NEUROSCIENCE RESEARCH ELSEVIER IRELAND LTD 71 E172 - E172 0168-0102 2011 
    多くの生物は日長の変化に応じて季節変化を感じ取り、体内の生理機能を調節して環境変化に適応する。この日長の変化に伴う現象のことを光周性と呼び、動物では生殖腺の発達や冬眠などが光周性に起因していることが知られている。
    これまでにウズラの下垂体正中隆起部で、日長が長くなると甲状腺刺激ホルモンβサブユニット(TSHβ)が誘導され、これがいわば春ホルモンであることが報告されていた。環境の明暗情報は、光の情報によって下垂体正中隆起部に伝えられると考えられている。しかし、光の情報がどのように伝わり春ホルモンTSHβを誘導するのか、その誘導制御機構はいまだに明らかにされていなかった。これまで、一般的な研究用マウスは光周性を示さないと考えられていた。しかし近年、CBA/Nマウスはウズラと同様に、日長が長くなると下垂体正中隆起部でTSHβを誘導することが報告された。そこで、TSHβの誘導制御機構を解明するために、DNAマイクロアレイを
  • Koh-hei Masumoto; Maki Ukai-Tadenuma; Takeya Kasukawa; Mamoru Nagano; Kenichiro D. Uno; Kaori Tsujino; Kazumasa Horikawa; Yasufumi Shigeyoshi; Hiroki R. Ueda
    CURRENT BIOLOGY CELL PRESS 20 (24) 2199 - 2206 0960-9822 2010/12 [Refereed]
     
    Living organisms detect seasonal changes in day length (photoperiod) [1-3] and alter their physiological functions accordingly to fit seasonal environmental changes. TSH beta, induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism [4, 5]. However, the upstream inducers of TSH beta expression remain unknown. Here we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSH beta expression response is preserved [6]. This analysis identified "short-day" and "long-day" genes, including TSH beta, and further predicted the acute induction of long-day genes by late-night light stimulation. We verified this by advancing and extending the light period by 8 hr, which induced TSH beta expression within one day. In the following genome-wide expression analysis under this acute long-day condition, we searched for candidate upstream genes by looking for expression that preceded TSH beta's, and we identified the Eya3 gene. We demonstrated that Eya3 and its partner Six/synergistically activate TSH beta expression and that this activation is further enhanced by Tef and Hlf. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of TSH beta expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system.
  • Mamoru Nagano; Akihito Adachi; Koh-hei Masumoto; Elizabeth Meyer-Bemstein; Yasufumi Shigeyoshi
    BRAIN RESEARCH ELSEVIER SCIENCE BV 1289 37 - 48 0006-8993 2009/09 
    Photic resetting of a biological clock is one of the fundamental characteristics of circadian systems and allows living organisms to adjust to a particular environment. Nocturnal light induces the Per1 and Per2 genes, which leads to a resetting of the circadian clock in the suprachiasmatic nucleus (SCN), the mammalian circadian center. In our present study, we investigated whether light differentially induces the rat Per1 (rPer1) and Per2 (rPer2) genes to enable resetting of their circadian clocks. In a 24-hour LD cycle (12 h light:12 h dark), which is shorter than the normal free-running period for rats, Per1 alone showed strong induction in the ventrolateral region of the SCN (VLSCN) during the early day. In contrast, in a 2S hour LD cycle (12.5 h light:12.5 h dark), which is longer than the free running period for these animals, rPer2 alone was strongly induced in the VLSCN, at the end of the light phase and during the early dark periods. our current findings therefore suggest that Per1 and Per2 are differentially regulated for daily entrainment to the LD cycle. (C) 2009 Elsevier B.V. All rights reserved.
  • Yuichi Kumaki; Maki Ukai-Tadenuma; Ken-ichiro D. Uno; Junko Nishio; Koh-hei Masumoto; Mamoru Nagano; Takashi Komori; Yasufumi Shigeyoshi; John B. Hogenesch; Hiroki R. Ueda
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 105 (39) 14946 - 14951 0027-8424 2008/09 
    Mammalian circadian clocks consist of regulatory loops mediated by Clock/Bmal1-binding elements, DBP/E4BP4 binding elements, and RevErbA/ROR binding elements. As a step toward system-level understanding of the dynamic transcriptional regulation of the oscillator, we constructed and used a mammalian promoter/enhancer database (http://promoter.cdb.riken.jp/) with computational models of the Clock/Bmal1-binding elements, DBP/E4BP4 binding elements, and RevErbA/ROR binding elements to predict new targets of the clock and subsequently validated these targets at the level of the cell and organism. We further demonstrated the predictive nature of these models by generating and testing synthetic regulatory elements that do not occur in nature and showed that these elements produced high-amplitude circadian gene regulation. Biochemical experiments to characterize these synthetic elements revealed the importance of the affinity balance between transactivators and transrepressors in generating high-amplitude circadian transcriptional output. These results highlight the power of comparative genomics approaches for system-level identification and knowledge-based design of dynamic regulatory circuits.
  • Mitsugu Sujino; Mamoru Nagano; Atsuko Fujioka; Yasufumi Shigeyoshi; Shin-Ichi T. Inouye
    EUROPEAN JOURNAL OF NEUROSCIENCE BLACKWELL PUBLISHING 26 (10) 2731 - 2738 0953-816X 2007/11 
    The mammalian hypothalamic suprachiasmatic nucleus (SCN) is the master oscillator that regulates the circadian rhythms of the peripheral oscillators. Previous studies have demonstrated that the transplantation of embryonic SCN tissues into SCN-lesioned arrhythmic mice restores the behavioral circadian rhythms of these animals. In our present study, we examined the clock gene expression profiles in a transplanted SCN and peripheral tissues, and also analysed the circadian rhythm of the locomotor activity in SCN-grafted mice. These experiments were undertaken to elucidate whether the transplanted SCN generates a dynamic circadian oscillation and maintains the phase relationships that can be detected in intact mice. The grafted SCN indeed showed dynamic circadian expression rhythms of clock genes such as mPeriod1 (mPer1) and mPeriod2 (mPer2). Furthermore, the phase differences between the expression rhythms of these genes in the grafted SCN and the locomotor activity rhythms of the transplanted animals were found to be very similar to those in intact animals. Moreover, in the liver, kidney and skeletal muscles of the transplanted animals, the phase angles between the circadian rhythm of the grafted SCN and that of the peripheral tissues were maintained as in intact animals. However, in the SCN-grafted animals, the amplitudes of the mPer1 and mPer2 rhythms were attenuated in the peripheral tissues. Our current findings therefore indicate that a transplanted SCN has the capacity to generate a dynamic intrinsic circadian oscillation, and can also lock the normal phase angles among the SCN, locomotor activity and peripheral oscillators in a similar manner as in intact control animals.
  • Hideki Ukai; Tetsuya J. Kobayashi; Mamoru Nagano; Koh-hei Masumoto; Mitsugu Sujino; Takao Kondo; Kazuhiro Yagita; Yasufumi Shigeyoshi; Hiroki R. Ueda
    NATURE CELL BIOLOGY NATURE PUBLISHING GROUP 9 (11) 1327 - U280 1465-7392 2007/11 
    Singularity behaviour in circadian clocks(1,2) - the loss of robust circadian rhythms following exposure to a stimulus such as a pulse of bright light - is one of the fundamental but mysterious properties of clocks. To quantitatively perturb and accurately measure the dynamics of cellular clocks(3,4), we synthetically produced photo-responsiveness within mammalian cells by exogenously introducing the photoreceptor melanopsin(5-8) and continuously monitoring the effect of photo-perturbation on the state of cellular clocks. Here we report that a critical light pulse drives cellular clocks into singularity behaviour. Our theoretical analysis consistently predicts and subsequent single-cell level observation directly proves that desynchronization of individual cellular clocks underlies singularity behaviour. Our theoretical framework also explains why singularity behaviours have been experimentally observed in various organisms, and it suggests that desynchronization is a plausible mechanism for the observable singularity of circadian clocks. Importantly, these in vitro and in silico findings are further supported by in vivo observations that desynchronization underlies the multicell-level amplitude decrease in the rat suprachiasmatic nucleus induced by critical light pulses.
  • 長野 護; 重吉 康史
    Clinical Neuroscience Clinical Neuroscience 中外医学社 25 (10) 1120 - 1123 0289-0585 2007/10 
    哺乳類において、視交叉上核は体内時計の中枢と考えられている。この視交叉上核における各ペプチド含有細胞の分布、出力系、体内時計中枢のリセット(光同調)について形態学的な視点から解説した。また、我々の研究において解明した時差症候群(時差ボケ)の機序について紹介した。
  • Ryutaro Fujinaga; Akie Yanai; Hirokazu Nakatsuka; Kumiko Yoshida; Yukio Takeshita; Kanako Uozumi; Changjiu Zhao; Kazuko Hirata; Keiji Kokubu; Mamoru Nagano; Koh Shinoda
    HISTOCHEMISTRY AND CELL BIOLOGY SPRINGER 128 (4) 335 - 348 0948-6143 2007/10 
    The anti-serum against an unknown human placental antigen complex X-P2 (hPAX-P2) immunohistochemically recognizes three putative molecules (hPAX-P2S, hPAX-P2N, and hPAX-P2R), each of which is associated with the stigmoid bodies (STBs), necklace olfactory glomeruli (NOGs), or reticulo-filamentous structures (RFs) in the rat brain. The STBs also contain huntingtin-associated protein 1 (HAP1), and the HAP1-cDNA transfection induces STB-like inclusions in cultured cells. In order to clarify the relationship between hPAX-P2S and HAP1 isoforms (A/B), we performed Western blotting, immuno-histo/cytochemistry for light- and electron-microscopy and pre-adsorption tests with HAP1 deletion fragments. The results showed that the anti-hPAX-P2 anti-serum recognizes HAP1(474-577) of HAP1A/B in Western blotting and strongly immunostains HAP1A-induced STB-like inclusions but far weakly detects HAP1B-induced diffuse structures in HAP1-transfected HEK 293 cells. In the rat brain, immunoreactivity of the anti-hPAX-P2 anti-serum for the STBs was eliminated by pre-adsorption with HAP1(474-577), whereas no pre-adsorption with any different HAP1 fragments can suppress immunoreactivity for the NOGs and RFs, which were not immunoreactive to anti-HAP1 anti-serum. These findings indicate that hPAX-P2S, which is distinct from hPAX-P2N and hPAX-P2R, is identical with STB-constituted HAP1 and that the HAP1-induced/immunoreactive inclusions correspond to the hPAX-P2-immunoreactive STBs previously identified in the brain.
  • 古河 惠一; 重吉 康史; 長野 護
    近畿大学医学雑誌 Med J Kinki Univ 近畿大学医学会 32 (3) 171 - 174 0385-8367 2007/09 
    哺乳類における体内時計の場所である視交叉上核とその中に存在する小領域と、体内時計はどうして変位するか。すなわち、日々の時刻合わせがどのようになっているかについて説明し、時差症候群の成り立ちについて説明をする。
  • Shihoko Kojima; Ken Matsumoto; Matsumi Hirose; Miyuki Shimada; Mamoru Nagano; Yasufumi Shigeyoshi; Shin-ichi Hoshino; Kumiko Ui-Tei; Kaoru Saigo; Carla B. Green; Yoshiyuki Sakaki; Hajime Tei
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 104 (6) 1859 - 1864 0027-8424 2007/02 
    The mammalian molecular clock is composed of feedback loops to keep circadian 24-h rhythms. Although much focus has been on transcriptional regulation, it is clear that posttranscriptional controls also play important roles in molecular circadian clocks. In this study, we found that mouse LARK (mLARK), an RNA binding protein, activates the posttranscriptional expression of the mouse Period1 (mPer1) mRNA. A strong circadian cycling of the mLARK protein is observed in the suprachiasmatic nuclei with a phase similar to that of mPER1, although the level of the Lark transcripts are not rhythmic. We demonstrate that LARK causes increased mPER1 protein levels, most likely through translational regulation and that the LARK1 protein binds directly to a cis element in the 3' UTR of the mPer1 mRNA. Alterations of mLark expression in cycling cells caused significant changes in circadian period, with mLark knockdown by siRNA resulting in a shorter circadian period, and the overexpression of mLARK1 resulting in a lengthened period. These data indicate that mLARKs are novel posttranscriptional regulators of mammalian circadian clocks.
  • Koh-hei Masumoto; Mamoru Nagano; Naoyuki Takashima; Naoto Hayasaka; Hideki Hiyama; Shun-ichiro Matsumoto; Shin-Ichi T. Inouye; Yasufumi Shigeyoshi
    EUROPEAN JOURNAL OF NEUROSCIENCE BLACKWELL PUBLISHING 23 (11) 2959 - 2970 0953-816X 2006/06 
    The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA-positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double-labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA-containing neurons co-expressed either vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA-containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2-expressing neurons in the SCN.
  • S Matsumoto; C Yamazaki; KH Masumoto; M Nagano; M Naito; T Soga; H Hiyama; M Matsumoto; J Takasaki; M Kamohara; A Matsuo; H Ishii; M Kobori; M Katoh; H Matsushime; K Furuichi; Y Shigeyoshi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 103 (11) 4140 - 4145 0027-8424 2006/03 
    Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-) but Pkr2(-/-) mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.
  • A Matsuo; S Matsumoto; M Nagano; K Masumoto; J Takasaki; M Matsumoto; M Kobori; M Katoh; Y Shigeyoshi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 331 (1) 363 - 369 0006-291X 2005/05 
    G-protein-coupled receptors (GPCRs) are important mediators of signal transduction and are therefore potential targets for pharmacological therapeutics. Here, we report the identification and characterization of an orphan GPCR, termed GPRg1, which was found in the GenBank database following searches with GPCR query sequences. Quantitative PCR analysis revealed that GPRg1 transcripts are expressed almost exclusively in the brain. Moreover, in situ hybridization experiments in brain demonstrated that GPRg1 is abundantly expressed in the ventrolateral region of caudate putamen, the habenular nucleus, the zona incerta, and the medial mammillary nucleus. In addition, overexpression of GPRg1 in 293-EBNA cells activates serum response factor mediated transcription, which was completely inhibited by the Gq/11 selective inhibitor YM-254890, indicating the coupling of GPRg1 with Gq/11. These findings suggest that GPRg1 is a candidate receptor for novel physiologically bioactive substrates and that it plays important roles in the central nervous system. © 2005 Elsevier Inc. All rights reserved.
  • H Ito; H Ito; M Nagano; S Nakano; Y Shigeyoshi; H Kusaka
    NEUROLOGY LIPPINCOTT WILLIAMS & WILKINS 64 (6) 1073 - 1075 0028-3878 2005/03 
    The authors examined skeletal muscle specimens from four patients with myositis and hepatitis C virus (HCV) infection. PCR analysis identified HCV RNA in muscle homogenates from two patients. In situ hybridization signals for HCV RNA were detected within muscle fibers as well as in infiltrating lymphocytes from the same patients. The results may relate to the pathomechanism of myositis in patients with HCV infection.
  • R Fujinaga; J Kawano; Y Matsuzaki; K Kamei; A Yanai; ZJ Sheng; M Tanaka; KI Nakahama; M Nagano; K Shinoda
    JOURNAL OF COMPARATIVE NEUROLOGY WILEY-LISS 478 (1) 88 - 109 0021-9967 2004/10 
    Huntingtin-associated protein 1 (HAP1) was identified as an interactor of the gene product (Huntingtin) responsible for Huntington's disease and found to be a core component of the stigmoid body. Even though HAP1 is highly expressed in the brain, detailed information on HAP1 distribution has not been fully described. Focusing on the neuroanatomical analysis of HAP1-mRNA expression using in situ hybridization histochemistry, the present study clarified its detailed regional distribution in the entire mouse brain. Mouse HAP1 (Hap1)-mRNAs were abundantly expressed in the limbic-related forebrain regions and midline/periventricular brainstem regions including the olfactory bulb, limbic-associated cortices, hippocampus, septum, amygdala, bed nucleus of the stria terminalis, preoptico-hypothalamic regions, central gray, raphe nuclei, locus coeruleus, parabrachial nuclei, nucleus of the solitary tract, and area postrema. In contrast, little expression was detected in the striatum and thalamus, implying that Hap1 is associated with neurodegeneration-sparing regions rather than target lesions in Huntington's disease. The distribution pattern, resembling that of the stigmoid body, suggests that HAP1 and the stigmoid body are implicated in protection from neuronal death rather than induction of neurodegeneration in Huntington's disease, and that they play an important role in integrating instinct behaviors and underlying autonomic, visceral, arousal, drive, memory, and neuroendocrinergic functions, particularly during extensive homeostatic or emotional processes. These data will provide an important morphological base for a future understanding of functions of HAP1 and the stigmoid body in the brain. (C) 2004 Wiley-Liss, Inc.
  • T Iwasaki; K Nakahama; M Nagano; A Fujioka; H Ohyanagi; Y Shigeyoshi
    LIFE SCIENCES PERGAMON-ELSEVIER SCIENCE LTD 74 (25) 3093 - 3102 0024-3205 2004/05 
    The liver is among the peripheral organs that display a clear circadian rhythmicity. To investigate whether specific pathological conditions affect circadian rhythms in the liver, we examined the expression profiles of the clock-related and glyceraldehyde 3-phosphate dehydrogenase (GADPH) genes following a partial hepatectomy in the mouse. This surgical procedure causes dynamic proliferation of residual hepatocytes and within one day of the operation the hepatectomized mice demonstrated higher expression of both mPer1 and mPer2 genes in the remaining liver tissue when compared to control mice that had undergone a Sham-operation. In contrast, the mCry1 gene in hepatectomized mice displayed a circadian gene expression profile that was similar to the control group. In addition, GAPDH levels, that demonstrated no oscillations in Sham-hepatectomized mice, underwent daily alterations following a partial hepatectomy. These findings suggest that the regenerative state of the liver affects the expression not only of clock-related genes but also of genes that are constitutively expressed under steady state conditions. (C) 2004 Elsevier Inc. All rights reserved.
  • K Kawamoto; M Nagano; F Kanda; K Chihara; Y Shigeyoshi; H Okamura
    JOURNAL OF NEUROSCIENCE RESEARCH WILEY-LISS 74 (6) 852 - 857 0360-4012 2003/12 
    Vasoactive intestinal peptide (VIP) neurons constitute a large group in the suprachiasmatic nucleus (SCN) and it is thought that they are involved in the generation and entrainment of circadian rhythm. We have characterized these VIP-expressing neurons in rat SCN by their ability to induce the mammalian Period1 (Per1) gene in response to light exposure, innervation of retinal afferents, day-night variations in VIP mRNA, and coexpression of gastrin releasing peptide (GRP). VIP neurons in the ventrolateral SCN (SCNVL) were subdivided into two groups, light-evoked Per1-inducible main SCNVL (SCNVLmain) and non-Per1-inducible medially located SCNVL (SCNVLmed). Retinal innervation was abundant in the SCNVLmain but nearly absent in the SCNVLmed. Day-night variation in VIP mRNA expression level was observed in the SCNVLmain but not in the SCNVLmed. GRP mRNA was seen in rarely SCNVLmed but abundant in SCNVLmain, where some neurons coexpressed VIP mRNA. These findings indicate that VIP neurons in the SCN can be divided into two topographically and functionally distinct groups. (C) 2003 Wiley-Liss, Inc.
  • M Nagano; A Adachi; K Nakahama; T Nakamura; M Tamada; E Meyer-Bernstein; A Sehgal; Y Shigeyoshi
    JOURNAL OF NEUROSCIENCE SOC NEUROSCIENCE 23 (14) 6141 - 6151 0270-6474 2003/07 
    The suprachiasmatic nucleus (SCN) is the neuroanatomical locus of the mammalian circadian pacemaker. Here we demonstrate that an abrupt shift in the light/dark (LD) cycle disrupts the synchronous oscillation of circadian components in the rat SCN. The phases of the RNA cycles of the period genes Per1 and Per2 and the cryptochrome gene Cry1 shifted rapidly in the ventrolateral, photoreceptive region of the SCN, but were relatively slow to shift in the dorsomedial region. During the period of desynchrony, the animals displayed increased nighttime rest, the timing of which was inversely correlated with the expression of Per1 mRNA in the dorsomedial SCN. Molecular resynchrony required similar to6 d after a 10 hr delay and 9 similar to 13 d after a 6 hr advance of the LD cycle and was accompanied by the reemergence of normal rest - activity patterns. This dissociation and slow resynchronization of endogenous oscillators within the SCN after an LD cycle shift suggests a mechanism for the physiological symptoms that constitute jet lag.
  • M Nagano; A Adachi; K Nakahama; T Nakamura; M Tamada; E Meyer-Bernstein; A Sehgal; Y Shigeyoshi
    JOURNAL OF NEUROSCIENCE SOC NEUROSCIENCE 23 (14) 6141 - 6151 0270-6474 2003/07 
    The suprachiasmatic nucleus (SCN) is the neuroanatomical locus of the mammalian circadian pacemaker. Here we demonstrate that an abrupt shift in the light/dark (LD) cycle disrupts the synchronous oscillation of circadian components in the rat SCN. The phases of the RNA cycles of the period genes Per1 and Per2 and the cryptochrome gene Cry1 shifted rapidly in the ventrolateral, photoreceptive region of the SCN, but were relatively slow to shift in the dorsomedial region. During the period of desynchrony, the animals displayed increased nighttime rest, the timing of which was inversely correlated with the expression of Per1 mRNA in the dorsomedial SCN. Molecular resynchrony required similar to6 d after a 10 hr delay and 9 similar to 13 d after a 6 hr advance of the LD cycle and was accompanied by the reemergence of normal rest - activity patterns. This dissociation and slow resynchronization of endogenous oscillators within the SCN after an LD cycle shift suggests a mechanism for the physiological symptoms that constitute jet lag.
  • HR Ueda; WB Chen; A Adachi; H Wakamatsu; S Hayashi; T Takasugi; M Nagano; K Nakahama; Y Suzuki; S Sugano; M Iino; Y Shigeyoshi; S Hashimoto
    NATURE NATURE PUBLISHING GROUP 418 (6897) 534 - 539 0028-0836 2002/08 
    Mammalian circadian clocks consist of complex integrated feedback loops(1-10) that cannot be elucidated without comprehensive measurement of system dynamics and determination of network structures(11). To dissect such a complicated system, we took a systems-biological approach based on genomic, molecular and cell biological techniques. We profiled suprachiasmatic nuclei and liver genome-wide expression patterns under light/dark cycles and constant darkness. We determined transcription start sites of human orthologues for newly identified cycling genes and then performed bioinformatical searches for relationships between time-of-day specific expression and transcription factor response elements around transcription start sites. Here we demonstrate the role of the Rev-ErbA/ROR response element in gene expression during circadian night, which is in phase with Bmal1 and in antiphase to Per2 oscillations. This role was verified using an in vitro validation system, in which cultured fibroblasts transiently transfected with clock-controlled reporter vectors exhibited robust circadian bioluminescence(12).
  • K Nakahama; A Fujioka; M Nagano; S Satoh; K Furukawa; H Sasaki; Y Shigeyoshi
    GENES TO CELLS BLACKWELL PUBLISHING LTD 7 (7) 731 - 741 1356-9597 2002/07 
    Background: Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, which is localized in the astrocyte plasma membrane. Membrane targeting of AQP4 is essential to perform its function. The mechanism(s) of membrane targeting is not clear in astrocytes. Results: We investigated the role of the C-terminus of AQP4 (short isoform) in its membrane targeting by an expression study of C-terminal mutants of AQP4 in cultured astrocytes. The deletion of 26 C-terminal residues of AQP4 (AQP4(Delta276-301aa) ) results in the intracellular localization of the protein. However, smaller deletions than 21 C-terminal residues did not alter its plasma membrane localization. These results suggest that C-terminal residues between Val(276) and Ile(280) play an important role in the expression of AQP4 in the plasma membrane. However, the plasma membrane localization of the AQP4(A(276) AAAA(280) ) mutant (alanine substitution of Val(276) -Ile(280) of AQP4) suggests that another signal for membrane targeting exists in the C-terminus of AQP4. The deletion or point mutations of the PDZ binding motif of the AQP4(A(276) AAAA(280) ) mutant resulted in the intracellular localization of the proteins. These results suggest that the PDZ binding motif may also be involved in the membrane targeting of AQP4. Conclusions: We found that the C-terminal sequence of AQP4 contains two important signals for membrane expression of AQP4 in cultured astrocytes. One is a hydrophobic domain and the other is a PDZ binding motif that exists in the C-terminus.
  • Yoshiki Ishida; Kazuhiro Yagita; Tsuyoshi Fukuyama; Masataka Nishimura; Mamoru Nagano; Yasufumi Shigeyoshi; Shun Yamaguchi; Takahide Komori; Hitoshi Okamura
    Journal of Neuroscience Research 64 (6) 612 - 616 0360-4012 2001/06 
    Casein kinase Iε (CKIε) and casein kinase Iδ (CKIδ) phosphorylate clock oscillating mPER proteins, and play a key role in the transcription (post)translation feedback loop that generates circadian rhythm. In the present study, the expression profiles of CKIε and CKIδ mRNAs were examined in the mice clock center, suprachiasmatic nucleus (SCN). Moderate levels of CKIε and CKIδ mRNAs were constantly expressed in the SCN in both light:dark and constant dark conditions. This finding supports the hypothesis that CKI may form a constant threshold to the nuclear entry of mPER proteins as in the Drosophila homologue, double-time. Further, we demonstrated that the light exposure at subjective night induced a delayed increase in CKIε and CKIδ mRNAs in the SCN. CKIε and CKIδ proteins may play a role on light-induced phase-shift. © 2001 Wiley-Liss, Inc.
  • Y Ishida; K Yagita; T Fukuyama; M Nishimura; M Nagano; Y Shigeyoshi; S Yamaguchi; T Komori; H Okamura
    JOURNAL OF NEUROSCIENCE RESEARCH WILEY-LISS 64 (6) 612 - 616 0360-4012 2001/06 
    Casein kinase I epsilon (CKI epsilon) and casein kinase I delta (CKI delta) phosphorylate clock oscillating mPER proteins, and play a key role in the transcription (post)translation feedback loop that generates circadian rhythm. In the present study, the expression profiles of CKI epsilon of and CKI delta mRNAs were examined in the mice clock center, suprachiasmatic nucleus (SCN), Moderate levels of CKI epsilon and CKI delta mRNAs were constantly expressed in the SCN in both light, dark and constant dark conditions. This finding supports the hypothesis that CKI may form a constant threshold to the nuclear entry of mPER proteins as in the Drosophila homologue, double-time. Further, we demonstrated that the light exposure at subjective night induced a delayed increase in CKI epsilon and CKI delta mRNAs in the SCN, CKI epsilon and CKI delta proteins may play a role on light-induced phase-shift. (C) 2001 Wiley-Liss, Inc.
  • K Nakahama; M Nagano; A Fujioka; K Shinoda; H Sasaki
    GLIA WILEY-LISS 25 (3) 240 - 246 0894-1491 1999/02 
    Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, localized in the astrocyte plasma membrane. The regulation of AQP4 is believed to be important for the homeostasis of water in the brain, but the AQP4 regulatory mechanisms are not yet known. In this study, we investigated the effect of a protein kinase C (PKC) activator on the expression of AQP4 mRNA in cultured rat astrocytes. Cultured rat astrocytes constitutively expressed AQPQ mRNA. Treatment of the cells with 0.1 mM of phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, caused a rapid decrease in AQP4 mRNA. This effect was time- and dose-dependent. The TPA-induced decrease in AQP4 mRNA was inhibited by a relatively specific PKC inhibitor, l-(5-isoquinoline sulfonyl)-2-methylpiperazine (H7) in a dose-dependent manner. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decrease in AQP4 mRNA by TPA. These results strongly suggest that the TPA-induced decrease in AQP4 mRNA is mediated by PKC activation. To test whether the effect of TPA requires protein synthesis, astrocytes were pretreated with cycloheximide, an inhibitor of protein synthesis. Pretreatment of the cells with cycloheximide did not inhibit the decrease in AQP4 mRNA induced by TPA. To test whether the TPA-induced decrease in AQP4 was due to a decrease in the mRNA stability we examined the effect of actinomycin D, an inhibitor of transcription, on TPA-treated cells. The stability of AQP4 mRNA was not decreased by the pretreatment of the cells with actinomycin D. The results suggest that AQP4 mRNA is inhibited by TPA via PKC activation without de novo protein synthesis, and that the inhibition of AQP4 mRNA could be at the transcriptional level. (C) 1999 Wiley-Liss, Inc.
  • K SHINODA; M NAGANO; Y OSAWA
    JOURNAL OF COMPARATIVE NEUROLOGY WILEY-LISS 343 (1) 113 - 129 0021-9967 1994/05 
    Brain aromatase has been considered to be an important clue in elucidating the actions of androgen on brain sexual differentiation. Using highly specific anti P450arom antiserum, the regional and subcellular distributions were immunohistochemically evaluated in the preoptic, strial, and amygdaloid regions of developing rat brains. Aromatase-immunoreactive (AROM-I) neurons were classified into three groups. The first, in which immunostaining; occurs only during certain pre- or neonatal days (E16-P2), included the anterior medial preoptic nucleus the periventricular preoptic nucleus, neurons associated with the strial part of the preoptic area, and the rostral portion of the medial preoptic nucleus. The second is a striking AROM-I cell group in the ''medial preopticoamygdaloid neuronal are,'' which extends from the medial preoptic nucleus to the principal nucleus of the bed nucleus of the stria terminalis and the posterodorsal part of the medial amygdaloid nucleus. The AROM-I neurons appeared by E16, reaching a peak in staining intensity between E18 and P2 and diminishing after the perinatal stage, After P14, a third group of AROM-I neurons emerged in the lateral septal nucleus,,the oval nucleus of the bed nucleus of the stria terminalis, and the central amygdaloid nucleus. The second group was thought to be the major aromatization center in developing rat brains, while the center might partly shift to the third group bf neurons after the late infantile stage. The distribution and developmental patterns were basically similar in males and females, suggesting that the neonatally prominent aromatase is not induced by male-specific androgen surges occurring around birth. On immunoelectron microscopy, subneuronal aromatase was predominantly localized on the nuclear membrane and endoplasmic reticulum, which appeared to be appropriate for the efficient conversion of androgen into estrogen just prior to binding to the nuclear receptors. (C) 1994 Wiley-Liss, Inc.
  • M NAGANO; K SHINODA
    BRAIN RESEARCH ELSEVIER SCIENCE BV 634 (2) 296 - 304 0006-8993 1994/01 
    Recent immunohistochemical studies have suggested that the forebrain distribution of stigmoid bodies, marked by an antibody against placental aromatase-associated antigen X-P-2 (PAX), overlaps with that of the common binding sites of androgen and estrogen. In the present light- and electron-microscopy study the coexistence of stigmoid bodies and estrogen receptors (EsR) is immunohistochemically examined and quantitatively analyzed in the medial preoptic region, part of the bed nucleus of the stria terminalis and part of the medial amygdaloid nucleus of young female rats. Light microscopy with double immunostaining for PAX and EsR in all three regions indicates that 75-84% of the total of PAX-immunoreactive stigmoid structures are present in neurons which also contain EsR-immunoreactive nuclei, and that 75-78% of EsR-immunoreactive neurons contain PAX-immunoreactive inclusions. Electron microscopic analysis confirms that 70-80% of stigmoid body-containing neurons have EsR-immunoreactive nuclei. These results indicate that the majority of the stigmoid bodies and EsRs intimately coexist, strongly suggesting a functional interrelationship in brain regions which are involved in rat reproductive functions. Stigmoid bodies may play a role in subneuronal EsR mechanisms associated with aromatization in these sex steroid targets in rat brain.
  • K SHINODA; T OHTSUKI; M NAGANO; T OKUMURA
    BRAIN RESEARCH ELSEVIER SCIENCE BV 618 (1) 160 - 166 0006-8993 1993/07 
    The relationship between placental antigen X-P2 (PAX)-immunoreactive glomeruli and intensely acetylcholinesterase-reactive (IAE) patchy regions was evaluated by comparison of neighboring cryostat sections of the rat olfactory bulb. Both groups of distribution show similar necklace patterns. Each IAE region consists of heterologous glomerulus-like structures with variable acetylcholinesterase reactivity: strongly and less-reactive (IAE-S and IAE-L) structures. The PAX-immunoreactive glomeruli were detected as parts of the IAE-L portions. Three heterologous PAX/IAE glomeruli or glomerulus-like structures (IAE-S, IAE-L/PAX and IAE-L/non-PAX structures) locally form a distinct glomerular complex, the 'PAX/IAE glomerular complex'. At the caudal end of the main olfactory bulb, nine to sixteen such complexes occur at intervals and form a circumferential 'necklace'. Since one of them corresponds to the 'modified glomerular complex' involved in rat suckling behavior, the entire 'necklace' may be associated with processing olfactory stimuli eliciting or suppressing the suckling response.
  • K SHINODA; M NAGANO; Y OSAWA
    JOURNAL OF COMPARATIVE NEUROLOGY WILEY-LISS 329 (1) 1 - 19 0021-9967 1993/03 
    The ultrastructure of aromatase-associated ''stigmoid (dot-like) structures,'' which were detected in a previous study using light-microscopic immunohistochemistry (Shinoda et al.: J. Comp. Neurol. 322:360-376, '92), were examined in the rat medial preoptic region, bed nucleus of the stria terminalis, medial amygdaloid nucleus, and arcuate nucleus by pre- and post-embedding marking with a polyclonal antibody against human placental antigen X-P2 (hPAX-P2) for immuno-electron microscopic analysis. The immunoreactive stigmoid structure was identified as a distinct, non-membrane-bounded cytoplasmic inclusion (approximately 1-3 mum in diameter), which has a granulo-fuzzy texture with moderate-to-low electron density in non-immunostained preparations. It consists of at least four distinct granular and three distinct fibrillo-tubular elements forming a granulo-fibrillar conglomerate. This type of inclusions was formally termed the ''stigmoid body'' under the electron microscope. The stigmoid body is composed of the outer granulo-fibrillar and inner hyaloplasmic compartments. The immunoreactivity for hPAX-P2 is mainly localized to the former, especially to the low density granulo-fuzzy materials associated with the fibrillo-tubular elements. Identification of the ultrastructure of stigmoid body clarified their prevalence not only in the limbic and hypothalamic regions, but also in sex-steroid-sensitive peripheral tissues (e.g., peripheral sensory ganglia, ovary, testis) by consulting earlier electron-microscopic studies. Reviewing the history and nomenclature of this inclusion body, we reorganized the terminology of related neuronal cytoplasmic inclusions, the terms of which have often been confused, and discussed its functional significance on the basis of the present and previously accumulated data. In conclusion, we emphasized the importance of the stigmoid bodies in the sex-steroid-sensitive neural system because of their large size, high frequency, specific distribution in brains and peripheral tissues, effects of sex-steroids, and immunological and histochemical characteristics of the antibody marking the inclusion. The stigmoid bodies may provide a subcellular site for sex-steroid metabolism in their target tissues and play a critical role in cytosolic modulation of their actions (e.g., by aromatization) prior to their receptor binding.
  • M NAGANO; A FUJIOKA; S MORI
    ACTA HISTOCHEMICA ET CYTOCHEMICA JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY 24 (1) 77 - 83 0044-5991 1991 
    Ca2+-ATPase activity in the choroid plexus of the third ventricle of the rat was cytochemically detected. Reaction products were detected in the apical and basolateral plasma membranes of the choroid plexus epithelial cells. The activity was calcium-dependent in both membranes. High substrate specificity for ATP was observed only in the apical plasma membrane. It is speculated that Ca2+-ATPase in the apical plasma membrane acts as a Ca2+-pump and regulates cerebrospinal fluid Ca2+ concentration.
  • Midori Ohtsuki; Atsuko Fujioka; Mamoru Nagano; Shiro Mori
    Microscopy 35 (3) 292 - 297 2050-5701 1986 [Refereed]
     
    Membrane specialization was found on the luminal surfaces of the apical invaginations, vesicles, vacuoles, and tubules of the proximal tubule cells prepared by a rapid-freezing and freeze-substitution method. The appearance of the specialization depended on sectional planes of the membrane. Some appeared to be triangular protrusions from the outer leaflet of the membrane and some appeared to be particles regularly arranged in linear rows. The findings suggest that the specialization consists of conical bodies regularly arranged on the luminal surface of the membrane. © 1986 Oxford University Press. All rights reserved.

Books etc

  • 顕微鏡, 時間差を捉える哺乳類体内時計中枢
    重吉 康史; 長野 護; 升本 宏平; 鯉沼 聡 (Joint work)社団法人日本顕微鏡学会 2012 
    視交叉上核(SCN)は,視交叉の直上に存在する一組の神経核であり,哺乳類概日リズムの中枢である.SCN は末梢時計を制御することだけでなく,外界の光環境の変化を認識する機構を備えている.概日リズムはリミットサイクルとよばれる物理現象と考えられており,視交叉上核における概日リズム発振細胞の振る舞いに理論的裏付けを与えている.視交叉上核の時計の位相を大きく変動させる入力は光である.光反応部と非光反応部をそなえる視交叉上核の解剖学的構築が時差ぼけの原因となっている.非光反応部では概日リズムが最大で1 日2 時間程度しかシフトしないため環境の明暗周期に同期するのに日数を要する.これが時差ぼけ(時差症候群)である.また視交叉上核内部には位相波とよばれる現象が現れ,同期を維持しながら日長を振動子間の位相差として視交叉上核内に蓄えるシステムであることが明らかになって来た.
  • Circadian clock as multi-oscillation system, Circadian and Desynchrony in the Mammalian Circadian Center
    重吉 康史; 長野 護; 中浜健一 (Joint work)北海道出版 2003/05 
    2001年に行われた時間生物学札幌国際シンポジウムのproceedingである。体内時計の中枢である視交叉上核が、光条件に迅速に追随する部分となかなか環境に同調できない2つの領域によってなっており、それが時差ボケを引き起こしていることを述べた。

Conference Activities & Talks

  • Which region of the suprachiasmatic nucleus determines the phase of the dead zone, shell or core?  [Not invited]
    長野護; 池上啓介; 南陽一; 金澤佑治; 鯉沼聡; 筋野貢; 重吉康史
    第26回日本時間生物学会学術大会  2019/10
  • SCN regulates circadian sensitivity to light by adrenocorticoid in mouse retina  [Not invited]
    IKEGAMI Keisuke; NAGANO Mamoru; SHIGEYOSHI Yasufumi
    第24回日本時間生物学会学術大会  2017/10
  • Gating mechanism of responses to light in murine inner retina  [Not invited]
    池上啓介; 長野護; 杉本典明; 西郷和真; 重吉康史
    第23回日本時間生物学会学術大会  2016/11
  • An abrupt shift in the LD cycle causes desynchrony of core and shell in the mouse suprachiasmatic nucleus  [Not invited]
    長野護; 池上啓介; 重吉康史
    第22回日本時間生物学会学術大会  2015/11
  • Dorsomedial region of the SCN determines the phase of dead zone after an abrupt shift of a light dark cycle  [Not invited]
    長野護; 升本宏平; 重吉康史
    第21回日本時間生物学会学術大会  2014/11
  • 哺乳類体内時計中枢における領域的周期解析  [Not invited]
    鯉沼 聡; 長野 護; 古河恵一; 重吉 康史
    第87回日本解剖学会近畿支部学術集会  2012/12  兵庫県西宮市  第87回日本解剖学会近畿支部学術集会
     
    哺乳類概日リズムの中枢である視交叉上核(SCN)は、構成するそれぞれの神経細胞が固有の周期を持って概日リズムを発振している。細胞間の同期シグナルによって等周期の概日リズムを刻んでいる。一方、位相についてはサブ領域間で異なり、背側部の内側から外側に向かって位相波を生じていることを時計遺伝子Per1, Per2の発現解析により明らかになっている。 SCNの細胞間の同期にはcAMPによる位相伝達が必要であることが報告されていることから、今回、このシグナル伝達系を撹乱した際にSCN内の周期や位相がどのような影響を受けるかについて検索した。 (方法)新生児Per2::Lucラットの冠状断SCNスライスを作製し、その発光を冷却CCDカメラで経時的に記録した。cAMPシグナルを受容体非依存的に活性化するForskolinをSCNに継続的に作用させることで同期シグナルを撹乱した。周期と位相の解析には、SCNが外接するような格子状の長方形を設定し、各領域における発光の振動をsineの減衰
  • 視交叉上核における周期分布と位相波形成  [Not invited]
    鯉沼 聡; 長野 護; 古河恵一; 筋野 貢; 升本 宏平; 重吉 康史; 浅川 剛; 八木田和弘
    第19回日本時間生物学会学術大会  2012/09  札幌市  第19回日本時間生物学会学術大会
     
    哺乳類体内時計の中枢である視交叉上核(SCN)は構成する神経細胞が同期し、概日のリズムを発振する。これらの細胞は個々に解離した状態では様々な周期を示すことが知られているが、それらがSCNでどのように分布しているかは不明であった。我々はラットPer2::lucのSCNスライスにcAMP合成酵素であるアデニル酸シクラーゼを活性化するホルスコリンを継続的に添加した際の周期への影響を測定した。観察したSCNの画像を格子状に分割し、それぞれの区画においてlucの発光が示す振動を解析すると、SCNの同期状態が失われていることを発見した。この時、24h未満の周期を持つ短周期の領域(SPR)はSCNの内側の小領域に限局し、24時間よりも長周期の領域(LPR)は外側に存在するという領域特異性を持っていた。興味深いことに、SPRはSCNでPerの発現が内側から外側へと位相差を持って伝播する位相波の起点領域と一致していた。また、Per1, Per2の発現解析からこの位相差は短日条件から長日条件
  • ラットの体内時計中枢である視交叉上核における光入力制御機構の局在  [Not invited]
    長野 護; 升本 宏平; 重吉 康史
    第19回日本時間生物学会大会  2012/09  札幌  第19回日本時間生物学会大会
     
    哺乳類において、主観的夜の光照射は概日リズムのシフトを生じ、その際には視交叉上核にてPer遺伝子の誘導に伴いシフトが生じると考えられている。一方、主観的昼の光照射に対しては概日リズム位相は変化せず、主観的夜の光照射で誘導されるcfos、Per1遺伝子誘導も生じない。このように光入力があっても視交叉上核がシフトしない機構(ゲート)についてはその機序がほとんど明らかになっていない。今回、このゲート位相を規定しているのが視交叉上核の背内側部(光非反応領域)か腹外側部(光反応領域)かを検討した。そのために明暗サイクルを10時間後退させて腹外側部と背内側部を脱同期させ、その後恒暗条件下で1、3,7日目(シフト当日を1日目)に、あるいは明暗条件を保ったまま1日目から4日目までの暗期に2時間おきに30分間光照射を行った後サンプルを採取し、視交叉上核におけるcfos、Per1遺伝子の誘導を in situ hybridization法を用いて観察した。その結果は背内側部がゲート
  • マウス視交叉上核における光応答プログラム  [Not invited]
    升本 宏平; 長野 護; 重吉 康史; 辻野 薫; 上田泰己
    第19回日本時間生物学会学術大会  2012/09  北海道札幌市  第19回日本時間生物学会学術大会
     
    哺乳類体内時計における最大の同調因子は光である。しかしながら視交叉上核(SCN)において光の変化、強さ、長さ等の情報がどのように表現されているのか、その詳細は明らかではない。そこでこの問題を明らかにするために、恒暗条件下のマウスに主観的夜の前半及び後半で光照射を与えた後、SCNに発現するRNAを抽出し、GeneChipを用いて光応答遺伝子の包括的解析を行った。その結果、光照射後に発現が上昇する240遺伝子及び発現が抑制される45遺伝子を同定した。さらに光照射後に発現が上昇する遺伝子群に対して主成分分析を行った結果、光照射30分後に発現がピークに達し急速に減少する遺伝子(early type)と、120分後に発現がピークに達しゆっくりと減少する遺伝子(late type)の2つの反応パターンがあることを見出した。その反応パターンよりearly typeは光の変化、late typeは光の強さ、長さを感知しているのではないかと考え、マウスに光の長時間照射を与えたところ、SCNにおいてearly type
  • The underlying mechanism that generate phase waves in the mammalian circadian center  [Not invited]
    重吉 康史; 長野 護; 升本 宏平; 八木田和弘; 浅川 剛
    The 3rd International conference on cognitive neurodynamics(ICCN2011)  2011/06  北海道(Hilton Niseko Village)  The 3rd International conference on cognitive neurodynamics(ICCN2011)
     
    視交叉上核における周期の異なる2振動体とそれによって生じる視交叉上核内の位相波について数理モデルを構築し発表した。
  • REGIONAL PERIOD DIFFERENCE THAT GENERATES A PHASE GRADIENT IN THE MAMMALIAN CIRCADIAN CENTER  [Not invited]
    重吉 康史; 鯉沼 聡; 升本 宏平; 長野 護; 古河恵一; 浅川剛; 八木田和弘
    第三回世界時間生物学会(World congress of chronobiology)  2011/05  メキシコ プエブラ  第三回世界時間生物学会(World congress of chronobiology)
     
    視交叉上核における時差ぼけと視交叉上核背側部の周期の異なる領域について述べた。
  • グルココルチコイド刺激による肝腎末梢時計の位相変化の違い  [Not invited]
    筋野 貢; 古河恵一; 鯉沼 聡; 藤岡 厚子; 長野 護; 重吉 康史; 飯郷雅之
    日本時間生物学会  2010/11  東京  日本時間生物学会
     
    視交叉上核からの位相情報を末梢時計に伝える主要な因子の1つに、グルココ ルチコイドがある。我々は末梢時計の位相制御におけるグルココルチコイドの関 与を調べるために、以下の実験を行った。 内因性のグルココルチコイドの影響を避けるため、実験にはADXラットを用い た。7日間にわたり、本来の内因性リズムとは逆位相であるZT0.5にコルチコステ ロン3mg/kgまたは27mg/kgを投与し、7日目にサンプリングを行った。肝臓、腎臓 のrPer1, rPer2, rCry1, rBmal1mRNA量を測定したところ、肝臓ではいずれの遺 伝子の発現リズムの位相も対照群と変化が見られなかった。しかし、腎臓では rPer2, rCry1, rBmal1の遺伝子発現リズムが対照群とは逆位相に変化していた。 肝臓の末梢時計は位相の変化が見られなかったことから、他の同調因子によって 位相が固定されていることが示された。我々はこの同調因子を摂食性のものと考 え、次にZT4-7の制限給餌とZT11.5のコルチコステロ
  • Abnormal development of olfactory dopaminergic interneurons in Prokineticin receptor type 2 knock out mice.  [Not invited]
    重吉 康史; 久保厚子; 長野 護; 筋野 貢; 山崎千尋; 松本俊一郎
    日本解剖学会第86回近畿支部学術集会  2010/11  大阪  日本解剖学会第86回近畿支部学術集会
     
    プロキネティシン受容体2を欠損したKOマウスでは嗅球での発生異常が生じる。とくに、糸球体層が欠損することが特徴的である。今回、このKOマウスにおける嗅球ドーパミン細胞は、成体では、野生型と比較して、細胞数が減少している。このドーパミン細胞の発生時の異常について検討を行った。ドーパミンニューロンを検出するためには、ドーパミンの合成酵素であるチロシン水酸化酵素の抗体を用いた。KOマウスの嗅球において、胎生13.5日, 16.5日では陽性細胞の数、サイズにおいて、野生型マウス(WT)との差は無かった。しかし、出生後1日では、野生型で明らかな数およびサイズの増大があったのに対して、WTでは、胎生期との変化はほとんど無かった。これは、嗅球ドーパミン細胞の主な障害は遊走時の障害ではなく、嗅球における成熟の異常であることを示唆する。
  • A mechanism coupling two major subregions of the mammalian central circadian pacemaker  [Not invited]
    重吉 康史; 鯉沼 聡; 長野 護; 八木田 和弘
    米国神経科学学会  2010/11  サンディエゴ  米国神経科学学会
     
    視交叉上核の腹外側部と背内側部の同期を保つメカニズムを明らかにするために、micropunchを用いてDMSCNとVLSCNのRNAを採取し、広範囲の遺伝子発現分析を行った。その結果、VIPとVpac2レセプターが抽出された。in situ hybridizationによってVIPとVpac2レセプターmRNAがVLSCNとDMSCNに限定されることが証明された。培養SCNにVIPとVPAC2作用薬を作動させた。すると、DMSCNとVLSCNを分離することによって、DMSCNのみ10時間もの大きなシフトを示した。VIPとVpac2システムがおそらくLDサイクル・シフトの後の視交叉上核再同期のメディエーターであることを示唆する。
  • Mathematical model of suprachiasmatic nucleus to describe the asymmetric resynchronization after an abrupt shift of the light: dark cycle  [Not invited]
    浅川 剛; 重吉 康史; 鯉沼 聡; 升本 宏平; 長野 護
    米国神経学会  2010/11  サンディエゴ  米国神経学会
     
    時差ぼけの際に体内時計中枢である視交叉上核の腹外側部と背内側部の脱同期が生じる。それが同期状態になるまでに要する期間が前進して同期する場合と、後退して同期する場合で異なることが知られている。この現象について、シミュレーションによって、解析を行った。
  • 視交叉上核組織切片培養における細胞間同期機構の解析  [Not invited]
    鯉沼 聡; 長野 護; 古河 惠一; 重吉 康史; 八木田 和弘; 大橋 陽子; 橋本 誠一
    第17回日本時間生物学会学術大会  2010/11  東京  第17回日本時間生物学会学術大会
     
    視交叉上核(SCN)は網膜から直接投射する腹外側部(VLSCN)と投射のない背内側部(DMSCN)に分けられ、これらが強固に同期していることが知られている。しかし、SCN組織切片を作製し、長期間培養すると安定した振動の後に、やがて振動の減衰が観察される。このことは薬物添加による再同調では振動が回復することから、細胞間の脱同期の結果と考えられる。 今回、SCN切片作製後に各領域の周期変化を追跡することで、細胞間がどのように脱同期するかを観察した。実験はmPer2::lucラットのSCN組織培養を作製し、細胞レベルの発光をCCDカメラによって経時的に捉えることによって周期を測定した。 この結果、切片作製僅か数日後において周期が大きく異なる細胞が現れた。このときDMSCNの最背側部では24時間よりも短い周期の細胞が、またVLSCNでは24時間よりも長周期の細胞が存在した。さらに新生児SCNにおいて同様の解析を行ったところ、成体とは異なりDM-VL間は切片作製後も長期間同期
  • C6細胞におけるsynaptotagmin17の概日振動  [Not invited]
    藤岡 厚子; 長野 護; 堀内 喜高; 重吉 康史; 橋本 誠一
    第17回日本時間生物学会学術集会  2010/11  東京  第17回日本時間生物学会学術集会
     
    すでにC6細胞、および視交叉上核におけるsynaptotagmin17(SYT17) mRNA の概日リズムについて報告した。本研究ではSYT17タンパクの局在について検討した。SYT17抗体による免疫電顕では、C6細胞細胞体の小胞膜および、細胞突起の細胞膜とその直下の小胞膜上にSYT17を検出した。視交叉上核では、視交叉上核全体に広く発現し、さらに外側に伸びる突起にも陽性反応を観察した。GFAP抗体との二重染色では、視交叉上核の多くの細胞でGFAP,SYT17の共発現を示した。アストロサイトでのSYT17の発現が示唆された。
  • 視交叉上核における領域間同期機構の探索―ペプチド産生細胞の働きー  [Not invited]
    長野 護; 重吉 康史; 鯉沼 聡; 升本 宏平
    第17回日本時間生物学会学術大会  2010/11  東京  第17回日本時間生物学会学術大会
     
    視交叉上核(SCN)の個々の神経細胞は細胞間同期が存在しなければ、多様な周期の概日リズムを発振することが明らかになっている。よって、SCNが単一周期を発振するためには細胞間の同期を必要とする。今回、ペプチド受容体の局在を検討することによってSCNにおける領域間同期機構を検討した。SCNは、網膜からの投射がある腹外側部(VLSCN)と投射のない背内側部(DMSCN)に区分され、VLSCNにはVIP、GRP産生細胞が、DMSCNにはAVP産生細胞が局在する。そこで、これらの受容体であるVIP受容体 (VPAC2)、AVP受容体(V1a)、GRP受容体(GRPR)の局在をin situ hybridization 法を用いてラットSCNにおいて検討した。その結果、V1a mRNA発現細胞は主にDMSCNに強く発現した。また、VPAC2 mRNA発現細胞はDMSCNおよびVLSCN内の背側領域、GRPR mRNA発現細胞はVLSCNに密に局在した。これらの結果は、VIP産生細胞がVLSCNからDMSCNへの情報伝達に関与していることを示す。さらにAVPとGRP産生細胞はそれぞれDMSCNおよび、VLSCNにおけるautocrineまたはpara
  • C6細胞および視交叉上核におけるsynaptotagmin17の概日振動  [Not invited]
    藤岡 厚子; 長野 護; 堀内 喜高; 重吉 康史; 橋本 誠一
    第51回日本組織細胞化学会学術集会  2010/09  東京  第51回日本組織細胞化学会学術集会
     
    C6細胞において、Dex刺激後、synaptotagmin17(SYT17) mRNA の概日リズムを検出した。刺激後16時間および40時間をピークとする振動であった。同様な振動は視交叉上核(SCN)でも認められ、夜に高く昼に低い発現を得た。。SYT17はCa2+依存的に小胞の膜融合に働くと考えられている。Astrogliaからは神経伝達物質であるglutamateが放出されることも知られており, 日内変動を持って発現するSyt17は、何らかの液性因子の日内変動を示す分泌に関与しているかもしれない。
  • 視交叉上核における領域間カップリング機構-哺乳類体内時計中枢固有の位相情報伝達様式―  [Not invited]
    長野 護; 重吉 康史; 鯉沼 聡
    第115回日本解剖学会全国学術集会  2010/03  岩手  第115回日本解剖学会全国学術集会
     
    体内時計の中枢である視交叉上核(SCN)は、網膜からの投射がある腹外側部(VLSCN)と投射のない背内側部(DMSCN)に区分される。急激な明暗サイクルのシフトに対してVLSCNの概日リズムは1日で新しい明暗条件に同調するが、DMSCNはより日数を費やして位相変位して再同調に至る。この再同調は、VLSCNの位相にDMSCNが引き込まれる様式になっており、VLSCNから DMSCNへの一方向性の位相情報の伝達様式を示唆する。そこで、VLSCNからDMSCNへ作用する伝達物質を同定するために各領域の包括的遺伝子解析を行い、DMSCNに有意に高く発現している遺伝子を検索した。その結果、VIPの受容体であるVPAC2がDMSCNに強く発現していた。さらに、in situ hybridization法によってVPAC2 mRNA発現細胞はDMSCNのみに密に存在することが確認できた。VLSCNにはVIPが強く発現していることから、VLSCNからDMSCNにVIPがVPAC2受容体を介して位相情報の伝達を行っていることが示唆された。
  • 哺乳類体内時計中枢における領域特異的入力応答  [Not invited]
    鯉沼 聡; 長野 護; 重吉 康史; 八木田 和弘; 橋本 誠一
    日本分子生物学会第32回年会  2009/12  横浜市  日本分子生物学会第32回年会
     
    【目的】光刺激に対する視交叉上核(SCN)は網膜からの直接の投射が存在する腹外側部(VLSCN)と投射のない背内側部(DMSCN)に分けられる。すでに我々は、この2つの領域の概日リズムが明暗サイクルのシフトによって脱同期すること、それが原因で時差症候群を引き起こしていることを明らかにしている(J. Neurosice. Nagano et al. 2003)。今回、VLSCNとDMSCNとの外部入力に対する応答の差異がどこに起因するのかを検討した。【方法】mPer2 promoterの制御下でluciferase遺伝子を発現するラットSCNの組織スライス培養を作製し、発光を経時的にモニターすることによって、概日リズムを捉えた。組織スライス培養にadenylate cyclaseを賦活化するforskolinを作用させ、概日リズムの変化を微量光測定装置およびCCDカメラを用いて検討した。一方でラット VLSCNとDMSCNの組織を個別にパンチアウトしたのちRNAを採取し、DNAマイクロアレイによって発現解析を行った。得られた結果から領域特異的に発現する受容体
  • 腎臓の末梢時計におけるグルココルチコイドの影響  [Not invited]
    筋野 貢; 古河恵一; 藤岡 厚子; 鯉沼 聡; 長野 護; 重吉 康史; 飯郷雅之
    第16回日本時間生物学会学術大会合同大会  2009/10  大阪  第16回日本時間生物学会学術大会合同大会
     
    視交叉上核からの位相情報を伝える制御因子には様々なものが報告され ているが、主要な因子の1つとして副腎から分泌されるグルココルチコイドがあ る。我々はグルココルチコイドが末梢臓器の時計機構に与える影響を調べるため に、以下の実験を行った。 副腎を除去(ADX)したラットを作成し、対照群と共に一日を通して4時間おきに6 点でサンプリングを行った。肝臓、腎臓からmRNAを抽出し、定量的PCR法で時計 遺伝子rPer1, rPer2, rCry1, rBmal1のmRNA量の日内変動を測定した。その結 果、いずれの遺伝子とも位相に変化は見られなかったが、ADXラットのrPer1は肝 臓、腎臓ともに日内変動の振幅が大幅に減衰していた。rPer2, rCry1, rBmal1 は、肝臓では大きな変化が見られなかった。しかし、興味深いことに腎臓では日 内変動の振幅が大きく減衰していた。 次に、末梢時計の位相制御におけるグルココルチコイドの関与を調べた。内因性 のグルココルチコイドの影響を
  • ラットにおける中枢時計の入力応答性―視交叉上核組織切片を用いた検討  [Not invited]
    鯉沼 聡; 長野 護; 重吉 康史; 八木田 和弘; 橋本 誠一
    第16回日本時間生物学会学術大会  2009/10  大阪市  第16回日本時間生物学会学術大会
     
    視交叉上核(SCN)は網膜から直接の投射が終止する腹外側部(VL-SCN)と直接の投射のない背内側部(DM-SCN)の領域に明瞭に区分される。生物の体内時間に対して明暗周期を大きく変動させると、VL-SCNにおける時計遺伝子の発現リズムは新たな明暗周期に短時間で同調するのに対し、DM-SCNにおける同調には長時間を要するため、このことが時差症候群あるいは時差ぼけの原因になることを我々はこれまでに示してきた。しかしながらSCN内における領域的な同調様式の違いがどのように生じるかについてはまだ明らかになっていない。今回我々はmPer2::lucを発現するラットを用い、SCNの組織スライス培養を作成することで刺激に対するSCNの反応性を検索した。まず、cAMPのシグナル経路を賦活化するforskolinをSCNに作用させ、luciferineの概日発光リズムを微量光測定装置によって測定することで位相応答を調べたところ、forskolinへの刺激応答には明確な位相依存性が存在することが明らかになった。次に、mPer
  • Modeling of the generalized photic Perturbation  [Not invited]
    浅川 剛; 重吉 康史; 鯉沼 聡; 長野 護
    IX.Congeress of the European Biological Rhythm for, Chronobiology  2009/08  Strassbourg, France  IX.Congeress of the European Biological Rhythm for, Chronobiology
     
    時差ぼけの際に視交叉上核の腹外側部と背内側部が脱同期し、背内側部の位相の変位が遅延して、時差ぼけ症候を生じることが知られている。これらの時日から2つの振動体を視交叉上核に想定して、数理モデルを作成した。時差ぼけの修復、すなわち視交叉上核の再同期にかかる日数は後退に比較して、前進に日数をより要することが知られている。視交叉上核の遺伝子発現を忠実に再現しようと、非線形性を強める課程で、前進と後退の非対称性が表れた。
  • Two distinct oscillators in the suprachiasmatic nucleus  [Not invited]
    重吉 康史; 鯉沼 聡; 長野 護; 浅川 剛
    IX. Congress of the European Biological Rhythm for Chronobiology  2009  Strassbourg, France  IX. Congress of the European Biological Rhythm for Chronobiology
     
    視交叉上核が日の長さを記憶し、それに伴って動物の行動に変化が生じることが知られている。視交叉上核にはMオシレーターとEオシレーターが存在し、その二つの振動子の位相差が調整されることによって、行動に変化が生じると考えられている。さて、視交叉上核は腹外側部と背内側部に別れる。我々は、背内側部が日長によって、その内側と外側部の位相差が拡大することを発見した。すなわちMオシレーターとEオシレーターがこの位相が乖離した2つの部分で構成されている可能性が高い。さらに、どうして、このような位相差が生まれるかについて、モデルを作成した。
  • 腎臓の時計遺伝子発現にたいするグルココルチコイドの影響  [Not invited]
    筋野 貢; 古河恵一; 藤岡 厚子; 鯉沼 聡; 長野 護; 重吉 康史; 飯郷雅之
    第15回日本時間生物学会学術大会  2008/11  岡山  第15回日本時間生物学会学術大会
     
    我々はグルココルチコイドの時刻情報伝達因子としての性質に注目し、 その血中濃度の日内リズムが臓器にリズム情報を伝えている可能性を検証した。 グルココルチコイドを産生する副腎を除去(ADX)したラットを作成し、肝臓、 腎臓での遺伝子発現を径時的に調べたところ、肝臓で時計遺伝子rPer1の発現の 減衰が認められた。一方、腎臓ではrPer1, rPer2, rCry1, rBmal1において、発 現リズムの振幅が大きく減衰していた。リズムの位相は肝臓、腎臓とも変化がな かった。ADXラット腹腔内へのコルチコステロン投与により、肝臓、腎臓での rPer1の発現が誘導された。しかし、rPer2の発現は誘導されなかった。この結果 から、rPer1発現リズムの減衰はグルココルチコイドの欠失によるものと考えら れたが、腎臓ではrPer2, rCry1, rBmal1の発現リズムも減衰していたことから、 rPer1の発現リズムがフィードバックループ全体の振幅の維持に関与している、 あるいはADXによって消
  • 日長が視交叉上核背内側部のPer1発現に及ぼす影響  [Not invited]
    長野 護; 重吉 康史; 堀川和政
    第15回日本時間生物学会学術大会  2008/11  岡山  第15回日本時間生物学会学術大会
     
    体内時計の中枢である視交叉上核に日長の変化がどのような影響を持つかを明らかにするために、時計遺伝子Per1遺伝子発現をin situ hybridizationを用いて検討した。その結果、背内側部の脳室近傍領域におけるPer1遺伝子の発現開始が早まり、それより外側の領域では、発現の終了が遅くなっていた。また長日の1日目では、腹外側部のPer1遺伝子の誘導時刻が早まったのに加え、背内側部の脳室近傍領域におけるPer1遺伝子の発現開始も早くなった。このことから、長日条件においては光条件の変化に伴い、背内側部のPer1発現が修飾され、発現ピーク時刻のずれおよび発現の延長をもたらしていることが示唆された。
  • 概日リズムの位相依存性にリン酸化されるタンパクの網羅的解析  [Not invited]
    藤岡 厚子; 本田映子; 長野 護; 重吉 康史
    第15回日本時間生物学会学術大会  2008/11  岡山  第15回日本時間生物学会学術大会
     
    生物時計の分子機構を明らかにするために、概日時計の位相に依存してリン酸化、脱リン酸化されるタンパクに注目しプロテオーム解析を行った。リン酸化の変動をうかがわせるいくつかのタンパクのうち、Elongation factor2 (EF2)について検討し、リン酸化の概日リズムを明らかにした。
  • Morphological dynamism of the mammalian central clock mapped onto the time axis  [Not invited]
    長野 護; 重吉 康史
    第113回日本解剖学会総会・全国学術集会  2008/03  大分  第113回日本解剖学会総会・全国学術集会
     
    我々は、体内時計の位相の指標として視交叉上核におけるPer1、Per2遺伝子の発現量を解析することで体内時計の示す位相を知ることができることを見出した。そこで、本シンポジウムでは、光が体内時計中枢の視交叉上核に及ぼす影響について、急激な明暗周期のシフトにより生じる時差症候群の分子機序、夜間の一回の光照射によって視交叉上核に一週間以上の影響を及ぼすこと、明期を延長することによって、視交叉上核の腹外側部と背内側部の非同期があらわれてくることなど、最近我々の教室で得た知見を空間軸および時間軸で解説した。
  • 「抹消時計の制御システム」  [Not invited]
    重吉 康史; 長野 護; 筋野 貢; 井上慎一
    第85回日本生理学会大会  2008/03  京王プラザホテル  第85回日本生理学会大会
     
    シンポジウム 「生理機能を支配する末梢の時計:生物リズム研究の新展開」にての発表。哺乳類の体内時計は中枢である視床下部視交叉上核がマスタークロックとして末梢の時計を制御する階層的システムをなしている。このような末梢時計のコントロールがどのような機序で生じているのかについて、検討を加えた。視交叉上核を破壊したマウスに対して、胎児の視交叉上核を移植し、末梢時計の運行が開腹するかと行った検討、および、副腎から分泌される液性因子(ホルモン)によって、肝、腎の時計が制御されているのかどうかについての検討について発表した。
  • Clock gene daily expression in the suprachiasmatic nucleus is differentially affected in the dorsomedial and ventrolateral regions by photoperiod  [Not invited]
    長野 護; 重吉 康史
    2nd World Coongress of Chronobiology  2007/11  東京  2nd World Coongress of Chronobiology
     
    体内時計の中枢である視交叉上核において、Per1, Per2遺伝子発現の日周変動における日長時間の影響をin situ hybridization法を用いて検討した。その結果、長日明暗サイクル(明期16時間、暗期8時間)で飼育したラット視交叉上核において、Per1, Per2遺伝子発現周期に網膜からの直接入力がある腹外側部領域と投射のない背内側部領域との脱同調が認められた。これらのことから、日長時間の変化は、網膜からの直接入力がない背内側部領域においても影響を及ぼすことが明らかとなった。(英文)
  • Dynamism of the suprachiasmatic nucleus induced by light  [Not invited]
    重吉 康史; 長野 護; 升本宏平
    第14回日本時間生物学会  2007/11  東京  第14回日本時間生物学会
     
    光環境の違いによって生じる体内時計中枢視交叉上核の多様性について述べた。(英文)
  • Prokineticin receptor 2変異マウスは嗅脳および生殖器の形成異常を生じる  [Not invited]
    長野 護; 重吉 康史; 松本俊一郎; 山崎千尋; 升本宏平
    第112回日本解剖学会総会・全国学術集会  2007/03  大阪  第112回日本解剖学会総会・全国学術集会
     
    Prokineticin(PK1/PK2)は分泌性のタンパク質で、Gタンパク質共役受容体(GPCR)であるProkineticin 受容体(PKR1/PKR2)を介して、様々な生理的機能に関与する。PKR1とPKR2の生体内での働きを検討するため、PKR1とPKR2遺伝子欠損マウスを作成し、形態学的解析を行った。その結果、PKR2遺伝子欠損マウスのみに、明らかな形態異常、すなわち、嗅球および、生殖器の形成不全が生じていた。さらに生殖器の形成不全の原因について、検討を加え、中枢性の性腺機能不全であることが明らかとなった。また、胎児のnasal regionを検索すると、鋤鼻神経が前脳にいたらず途絶していることが判明し、GnRHニューロンの前脳への遊走の障害が、生殖腺の形成不全の原因と考えられた。
  • REGULATORY MECHANISM OF PHYSIOLOGICAL EVENTS BY THE SUPRACHIASMATIC NUCLEUS  [Not invited]
    重吉 康史; 長野 護
    国際生物学賞記念シンポジウム  2006/12  東京  国際生物学賞記念シンポジウム
     
    体内時計中枢である視交叉上核に発現し、行動を抑制する作用があるとされるProkineticin2(PKR2)とその中枢神経系での受容体であるPK受容体2(PKR2)の視交叉上核における分布について報告した。PK2が背内側部、腹外側に発現するのに対して、PKR2は背内側部に発現していた。背外側部は、発現が概日振動を示す時計遺伝子Per1が遅れて発現することから、視交叉上核内部における位相差にPK2ーPKR2の相互作用が何らかの役割を持つことを示唆する。さらに、PKR2遺伝子KO mouseでは、活動のリズムは残るものの変動が小さいこと、総活動量が減ること、夜の終わりに活動が上昇することなどを報告した。
  • 視交叉上核腹外側部は夜間の光照射によって振動体となる  [Not invited]
    長野 護; 重吉 康史; 古河 惠一
    日本時間生物学会学術大会  2006/12  東京  日本時間生物学会学術大会
     
    主観的夜の一回の光照射の位相変位に対する影響を検討した。そして、恒常暗条件においては光照射によって、腹外側部が振動体として働くようになり、その位相情報が数日間にわたって背内側部に伝達され最終的に位相変位が達成されることが示唆される結果を得た。
  • α-2,8-sialyltransferase 8F遺伝子における視交叉上核の局在と発現周期について  [Not invited]
    西郷 和真; 渥美 正彦; 升本 宏平; 長野 護; 足立 明人; 早坂 直人; 重吉 康史; 楠 進
    第47回日本神経学会総会  2006/12  東京  第47回日本神経学会総会
  • Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNA in the rat suprachiasmatic nucleus  [Not invited]
    重吉 康史; 長野 護; 早坂 直人; 升本宏平; 高嶋直敬
    米国神経科学大会  2006/10  米国・アトランタ コンベンションホール  米国神経科学大会
     
    体内時計中枢である視交叉上核に発現し、行動を抑制する作用があるとされるProkineticin2(PKR2)とその中枢神経系での受容体であるPK受容体2(PKR2)の視交叉上核における分布を検討した。PK2が背内側部、腹外側に発現するのに対して、PKR2は背内側部に発現していた。背外側部は、発現が概日振動を示す時計遺伝子Per1が遅れて発現することから、視交叉上核内部における位相差にPK2ーPKR2の相互作用が何らかの役割を持つことを示唆する。
  • Regulation of implanted suprachiasmatic nucleus on behavior and peripheral clock gene expression  [Not invited]
    筋野 貢; 重吉 康史; 藤岡 厚子; 長野 護; 井上慎一
    第29回日本神経科学大会  2006/07  京都  第29回日本神経科学大会
     
    視交叉上核(SCN)は哺乳類の概日時計の中枢である。SCNの破壊によって概日リ ズムが失われたマウスに他のマウスのSCNを移植すると、ホストマウスと移植さ れたSCNとの神経連絡が不完全であるにもかかわらず概日活動リズムが回復す る。われわれは移植されたSCNがホストマウスの末梢組織にどのような影響を与 えるのかを調べた。その結果、移植SCNはホストの末梢組織に概日リズムを発生 させることが明らかとなった。ホストマウスの末梢組織の時計遺伝子発現はイン タクトマウスと同様に日周変動していた。しかし、Period1, Period2では振幅が 大きく減少していた。これらのことから、移植されたSCNからの時刻情報は末梢 組織の時計機構を制御するが、時計遺伝子によって異なる制御を受けていること が示唆された。(英文)
  • Neuron or Glia? Involvement of two different cell-types in circadian rhythm generation in the SCN.  [Not invited]
    早坂 直人; 長野 護; 重吉 康史; 升本宏平
    Tenth Meeting Society for Research on Biological Rhythms (SRBR)  2006/05  Destin, Florida  Tenth Meeting Society for Research on Biological Rhythms (SRBR)
     
    視交叉上核における神経とグリアの概日リズム形成への関与について、樹立したグリア細胞株や視交叉上核の培養スライスの共培養等で検討した結果、グリア細胞が概日リズムに積極的に影響を与えているという知見が得られ、神経のみで説明されていた概日時計機構に新たなモデルを提唱することとなった。
  • 明暗サイクルへの同調に対するPer1、Per2の役割  [Not invited]
    長野 護; 重吉 康史; 早坂 直人; 足立 明人; 升本宏平
    第12回日本時間生物学会  2005/11  つくば市  第12回日本時間生物学会
     
    T-cycleを用いて、前進、あるいは後退を必要とする明暗条件を作り、Per1, Per2遺伝子の光同調での使い分けについて検討した。そして、主観的夜の終わりにPer1にのみ依存した、主観的夜の始まりにPer2にのみに依存した位相変位が生じる概日時計の位相があり、その位相ではPer1が前進に、Per2が後退といった使い分けが生じていることを示唆する結果を得た。
  • 視交叉上核移植マウスにおける末梢組織の概日リズム  [Not invited]
    筋野貢; 重吉 康史; 長野 護; 藤岡 厚子
    第12回日本時間生物学会  2005/11  つくば市  第12回日本時間生物学会
     
    視交叉上核(SCN)移植動物における、末梢組織の概日振動の有無とその位相を調べるために、SCN移植マウスの肝臓、腎臓、骨格筋をサンプリングし、時計遺伝子のmRNA量をリアルタイムPCR法で測定した。時計遺伝子は各臓器とも、対照群と同位相の概日リズムを示したことから、移植されたSCNは行動リズムだけでなく、末梢組織の概日振動を制御することが明らかとなった。
  • ラットSCNにおけるPK2とPKR2の局在  [Not invited]
    升本宏平; 重吉 康史; 長野 護; 早坂 直人
    第12回日本時間生物学会  2005/11  つくば市  第12回日本時間生物学会
     
    PK2 の視交叉上核(SCN)における役割を明らかにするためPK2と、PK2の受容体であるprokineticin receptor 2 (PKR2)のラットSCNでの局在を調べた。rPK2 mRNAは主観的昼にピークを持つ日周変動を示した。一方、rPKR2 mRNAは日周変動を示さず、SCNの背外側の小領域にのみ、陽性細胞が密に存在していた。これらの所見はSCNでPK2による神経伝達があることを示唆する。
  • Circadian Oscillation and Light Induction of Immature Per2 mRNA in the Suprachiasmatic Nucleus  [Not invited]
    足立 明人; 長野 護; 重吉 康史
    第28回日本神経科学大会  2005/07  横浜  第28回日本神経科学大会
     
    体内時計を制御する重要な時計遺伝子であるmPer1とmPer2の視交叉上核における発現をそれらのイントロンの部分をコードしたプローブを用いて検討した。
  • Daily induction of Per1 and Per2 in the SCN in a steady light: dark condition  [Not invited]
    早坂 直人; 長野 護; 重吉 康史
    Ninth Meeting Society for Research on Biological Rhythms (SRBR)  2005/05  カナダ ウィスラー  Ninth Meeting Society for Research on Biological Rhythms (SRBR)
     
    マウスやラットの体内時計中枢である視交叉上核において、12時間明期: 12時間暗期の条件下では、Per1、Per2遺伝子の発現が明期と暗期の開始の前後に異なる様式で誘導されており、両遺伝子が内因性のリズムを24時間に同調させる機能を果たしている可能性を強く示唆した。
  • Per1 gene expression in Suprachiasmatic nucleus (ventrolateral) at DD condition  [Not invited]
    古河 惠一; 長野 護; 重吉 康史
    第11回日本時間生物学会  2005  滋賀  第11回日本時間生物学会
     
    視交叉上核(SCN)の腹外側部(VLSCN)において、背内側部(DMSCN)と同様に明暗(LD)、恒暗(DD)条件下においてもPer 1の日周変動が認められるが、DD条件下ではその振動はLD条件と比較してかなり減弱する。そこで、in situ hybridizationを用いて同様の領域が存在するかを検討した。特にVLSCNの最も腹側に、1日を通してほとんど陽性細胞のない小領域を認めた。このことから、VLSCNにおいて自律性リズムを持つ細胞群と自身では振動せず、光刺激によりPer 遺伝子が誘導される細胞群が存在すると考えられる。
  • localization of PK2 mRNA-positive neurons in the rat SCN  [Not invited]
    升本宏平; 重吉 康史; 長野 護; 井上慎一; 高嶋直敬
    第11回日本時間生物学会  2004/11  滋賀  第11回日本時間生物学会
     
    ラットSCNにおけるrPK2 mRNAの発現リズムとその局在について、Digoxigenin-labeledプローブを用いたin situ hybridization調べた結果、rPK2 mRNA発現ニューロンはSCNにおいて広く散在していることがわかった。またSCNにおけるPK2の光応答について調べた。その結果、PK2は主観的夜の前半よりも主観的夜の後半で強い光応答をすることが確認できた。
  • Daily photic resetting of the circadian clock are associated with Per genes expression  [Not invited]
    長野 護; 足立 明人; 早坂 直人; 重吉 康史; 升本 宏平
    第11回日本時間生物学会  2004/11  大津  第11回日本時間生物学会
     
    光同調メカニズムにおけるPer1、Per2の役割について検討するため、ラットにおいて、明暗周期を延長して、体内時計を後退させなければならない環境を作り、その際のPer1、Per2発現をin situ hybridization で観察した。その結果、25時間の明暗サイクルで明期の終わりに、視交叉上核の腹外側部にPer2の強い発現を観察した。そして、Per1が位相前進にPer2が位相後退に働くことを示唆した。
  • Differential localization of Prokineticin 2-containing neurons in the rat suprachiasmatic nucleus  [Not invited]
    升本宏平; 重吉 康史; 長野 護; 井上慎一
    Ninth Meeting Society for Research on Biological Rhythms (SRBR)  2004/06  カナダ ウィスラー  Ninth Meeting Society for Research on Biological Rhythms (SRBR)
     
    Prokineticin 2 (PK2)はSCNにおいて強い発現リズムを示し、哺乳類の行動活動を抑制することが知られている。そこで我々はDigoxigenin-labeledプローブを用いたin situ hybridizationにより、ラットSCNにおけるrPK2 mRNAの発現リズムとその局在について調べた。その結果、rPK2 mRNA発現ニューロンはSCNの背内側部、腹外側部のどちらの領域にも広く散在し、各領域において強い振動を示した。またrPK2 mRNA発現ニューロンは複数のペプチドのmRNAと共発現していることがわかった。
  • Circadian oscillation and light induction of immature Per2 mRNA in the suprachiasmatic nucleus  [Not invited]
    足立 明人; 長野 護; 重吉 康史
    Ninth Meeting Society for Research on Biological Rhythms (SRBR)  2004/06  カナダ ウィスラー  Ninth Meeting Society for Research on Biological Rhythms (SRBR)
     
    これまでの研究でRNAの安定性が概日リズムの制御に重要なことが明らかとなった。我々は遺伝子の転写をより正確にとらえるために、重要な時計遺伝子の一つであるmPer2のイントロンをコードしたプローブを用いたin situ hybridization法により、mRNA前駆物質の発現リズム及び光による誘導を検討した。その結果、イントロンの反応はmRNAプローブを用いた場合より先に発現のピークが見られることが明らかとなった。そのため、イントロンを用いたプローブは転写の制御を検討する上で非常に有効な方法であると考えられる
  • Transient Dissociation in the DorsomedialRegion o the SCN  [Not invited]
    重吉 康史; 足立 明人; 長野 護
    Ninth Meeting Society for Research on Biological Rhythms (SRBR)  2004/06  カナダ ウィスラー  Ninth Meeting Society for Research on Biological Rhythms (SRBR)
     
    体内時計の中枢は視交叉上核に存在する。そのうち背内側部は網膜からの投射が無く、急激な明暗変化にすぐには同期できないため時差ぼけの原因となっている。今回我々は、明暗周期の10時間の前進が、視交叉上核の背内側部の分裂を生じ、前進して同期するもの、後退して同期するものが生じることを明らかにした。
  • Oscillator formation in the ventrolateral region of the SCN after an abrupt shift of light:dark cycle  [Not invited]
    重吉 康史; 長野 護
    解剖学会近畿支部地方会  2003/11  大阪  解剖学会近畿支部地方会
     
    急激な明暗周期の変位に伴い、視交叉上核光反応部、腹外側部に振動体が形成される。それによって位相情報を背内側部に伝えると考えられた。
  • Rhythmic expression of Id2 mRNA in the mouse SCN  [Not invited]
    足立 明人; 長野 護; 藤岡 厚子; 重吉 康史
    1st World Congress of Chronobiology  2003/09  札幌  1st World Congress of Chronobiology
     
    概日リズムは約24時間周期をもつ生理的リズムで、その制御にはbHLHを持つ転写因子が重要な役割を示していることが知られている。Id2は HLHに結合し、転写を抑制することが知られ、概日リズムの制御への関与を調べるためにその発現の局在を検討した。
  • Daily photic resetting of the circadian clock are associated with Per gene expression.  [Not invited]
    長野 護; 重吉 康史
    1st World Congress of Chronobiology  2003/09  札幌  1st World Congress of Chronobiology
     
    哺乳類における体内時計の光同調時期を同定するため、視交叉上核におけるPer1 の発現を検討した。その結果、24時間より長い周期をもつラットは早朝の光刺激、24時間より短い周期をもつマウスは夕暮れの光刺激が同調機構に重要な関係があると考えられた。
  • Spontaneous locomotor and free running rhythms of stroke-prone spontaneously hypertensive rats  [Not invited]
    大島 佳奈; 東野 英明; 長野 護; 重吉 康史
    第54回近畿大学医学会学術講演会  2003/07  大阪狭山市  第54回近畿大学医学会学術講演会
     
    SHRSPの自由行動および自発運動についてWKYと比較検討した。その結果、運動量はWKYより少なく、加齢により低下した。
  • Comparison of spontaneous locomotor rhythms, activities and patterns between SHRSP and WKY  [Not invited]
    大島 佳奈; 東野 英明; 長野 護; 重吉 康史
    第39回高血圧自然発症ラット学会総会  2003/06  東京都  第39回高血圧自然発症ラット学会総会
     
    SHRSPの自由行動リズムとパターンをWKYと比較した。その結果、概日周期に差が無かったが、運動量はSHRSP1の方で少なかった。
  • Expression profiles of heat shock protein (HSP) mRNA in rat suprachiasmatic nucleus  [Not invited]
    長野 護; 重吉 康史; 足立 明人
    第78回日本解剖学会近畿支部学術集会  2002/12  京都  第78回日本解剖学会近畿支部学術集会
     
    ラット視交叉上核においてHSP遺伝子の時間依存的発現及び光照射による影響について検討した。その結果、視交叉上核においてHsp40 mRNAは、光刺激により発現が誘導され、網膜からの投射部位へのグルタミン酸の放出に対して細胞保護に働くことが示唆された。
  • Expression of Per 1 in the suprachiasmatic Nucleus (Ventlateral portion) of enucleation rat.  [Not invited]
    古河 惠一; 長野 護; 重吉 康史
    第9回日本時間生物学会  2002/11  名古屋  第9回日本時間生物学会
     
    長期にわたって光入力を断つことにより視交叉上核腹外側部のper1の日周変動にどのような変化を生じるかを検討した。結果から視交叉上核腹外側部のper1の振動の振幅は、光入力に依存したものであることが考えられた。
  • Circadian rhythm of heat shock protein ( HSP) mRNA expression in rodent suprachiasmatic nucleus  [Not invited]
    長野 護; 重吉 康史; 足立 明人; 上田 泰巳; 橋本 誠一
    日本時間生物学会  2002/11  名古屋  日本時間生物学会
     
    熱ショックタンパク質の遺伝子がマウス、ラット視交叉上核においてサーカディアンリズムを示しているかを検討した。その結果、マウスにおいて、HSP40 、HSP105、HSPa5の遺伝子発現は主観的夜に高く、主観的昼に低いリズミカルな発現パターンを示すことが明らかとなった。
  • Appearance of Oscillators in Ventrolateral Suprachiasmatic Nucleus after LD Cycle Shift  [Not invited]
    重吉 康史; 長野 護; 足立 明人
    日本時間生物学会  2002/11  名古屋  日本時間生物学会
     
    体内時計の中枢は視床下部の視交叉上核に存在する。 急激な明暗サイクルのシフト後に腹外側部に概日振動子が一時的に生じる事を報告した。また、腹外側部の時計が一時的に大きな振動を生じてその位相を背内側部に伝えるとのモデルを提出した。
  • A role of the C-terminus of aquaporin 4 in its membrane expression in cultured astrocytes.  [Not invited]
    藤岡 厚子; 長野 護; 古河 惠一; 重吉 康史; 中浜 健一; 佐藤 伸介
    日米組織化学学会(アメリカ)  2002/07  アメリカ  日米組織化学学会(アメリカ)
     
    aquaporin 4は脳にみられる代表的な水チャンネルタンパクである。このタンパクが培養アストログリア細胞膜に発現するためには、C末端での二つの重要な部分が関与している。一つは疎水性部分、もう一つはPDZ結合領域である
  • JRhythmic expression of Dexras1 mRNA in the mouse SCN  [Not invited]
    足立 明人; 重吉 康史; 長野 護
    5th Meeting of Society for Research on Biological Rhythms  2002/05  Florida  5th Meeting of Society for Research on Biological Rhythms
     
    マイクロアレイ法により視交差上核と肝臓において概日リズムを示す遺伝子をin situ法を用いて周期的発現及び発現部位の特徴を検討した。
  • Jet lag is cause by the dessociation of the cental clock in the SCN  [Not invited]
    重吉 康史; 長野 護; 中浜健一
    第107回日本解剖学会  2002  浜松  第107回日本解剖学会
     
    時差ボケは哺乳類の体内時計が存在する視交叉上核の背内側領域が環境の変化に取り残されることから生ずると考えられる。
  • Rat Per1 mRNA expression in the suprachiasmatic nucleus under light-dark cycle  [Not invited]
    長野 護; 重吉 康史
    第77回日本解剖学会近畿支部学術集会  2001/12  大阪狭山市  第77回日本解剖学会近畿支部学術集会
     
    今回我々は明暗条件(12:12)で飼育した場合のラットの同調機構をrPer1の発現を示標として分子レベルで検討した。その結果、ラットは早朝と夕暮れの光刺激によって体内時計を環境のリズムに同調すると考えられる。
  • The modulation of Cry1 expression in the SCN after a light pulse  [Not invited]
    重吉 康史; 藤岡 厚子; 長野 護; 中浜健一
    第77回日本解剖学会近畿支部学術集会  2001/12  大阪  第77回日本解剖学会近畿支部学術集会
     
    時計遺伝子の一つであるCry1が体内時計の位相変位の際どのような反応形式で位相変位を生じるのかについて検討した。
  • Abnormal structure in the suprachiasmatic nucleus of anophthalmic rats  [Not invited]
    古河 惠一; 重吉 康史; 長野 護
    日本時間生物学会  2001/11  山口  日本時間生物学会
     
    眼球欠損のラット(LOU/CN/Krm)を用い、視交叉上核における形態的変化について検討した。その結果、網膜からの投射が存在しないために各種のペプチド産生ニューロンの遊走に異常が生じた可能性があると考えられた。
  • Daily photic resetting of the circadian clock : Rat Per1 mRNA expression in the suprachiasmatic nucleus  [Not invited]
    長野 護; 重吉 康史
    第8回日本時間生物学会山口大会  2001/11  山口  第8回日本時間生物学会山口大会
     
    ラットにおける体内時計の光による同調時期を同定するため、明暗条件下と恒暗条件下での視交叉上核におけるrPer1 の発現を検討した。その結果、ラットは早朝の光刺激が同調機構に重要な関係があると考えられた。
  • The induction of rCry1 mRNA in Rat1 fibroblasts and rat SCN  [Not invited]
    藤岡 厚子; 長野 護; 重吉 康史; 中浜 健一
    日本時間生物学会  2001/11  山口  日本時間生物学会
     
    体内時計におけるCry1 の役割を明らかにすることを目的として、 馬血清処理後のrCry1発現の誘導について検討した。Cry1発現のピークは処理後4~8時間で、Per2のピークに約4時間遅れることが解った。
  • Unusual morphology in the suprachiasmatic nuclei of congenitally anophthalmic rat.  [Not invited]
    古河 惠一; 重吉 康史; 長野 護
    第50回近畿大学医学会学術講演会 第50回近畿大学医学会学術講演会  2001/06  大阪狭山  第50回近畿大学医学会学術講演会 第50回近畿大学医学会学術講演会
     
    元来、発生の途中に脳室から遊走する神経細胞は、視交叉上核を形成するが、眼球欠損の場合、網膜からの直接投射に関係しないためVIP. GRPの細胞局在に変化を生じたものと思われる。
  • A profile of rat Par1 mRNA expression in the suprachiasmatic nucleus(SCN) after a rapid change of the light -dark cycle  [Not invited]
    長野 護; 藤岡 厚子; 重吉 康史; 中浜 健一; 佐藤 伸介
    第50回近畿大学医学会学術講演会  2001/06  大阪狭山市  第50回近畿大学医学会学術講演会
     
    ラットの明暗サイクルを急激にずらすと、視交叉上核に2つの相異の異なる時計が生ずることが、明らかとなった。そしてこの2つの時計が再同調するのに5~7日かかることが、時差ボケの原因になると考える。
  • A profile of rat Per1 (rPer1) mRNA expression in the suprachiasmatic nucleus (SCN) after an abrupt shift of the light-dark cycle.  [Not invited]
    長野 護; 藤岡 厚子; 重吉 康史; 中浜建一; 佐藤伸介
    第41回日本組織細胞化学会総会・学術集会  2000/12  高知市  第41回日本組織細胞化学会総会・学術集会
     
    時差ボケの機序を検討するために急激に明暗サイクルをずらし、ラット視交叉上核におけるPer1mRNAの変化を観察した。その結果、明暗サイクルのシフト後、視交叉上核に位相の異なる2つの時計を生じ、それが再同調するのに5~7日かかることが判明した。
  • A study on cell membrane localization mechanisms of aquaporin 4 in cultured astrocytes  [Not invited]
    藤岡 厚子; 長野 護; 重吉 康史; 古河 惠一; 中浜 健一; 佐藤 伸一
    第41回日本組織細胞化学会総会・学術集会  2000/12  高知市  第41回日本組織細胞化学会総会・学術集会
     
    アクアポリン4(AQP4)の細胞膜局在機構を明らかにするため、種々の長さのAQP4変異体を作成し、蛍光顕微鏡下であるいは免疫細胞化学的に局在を検討した。AQP4の全アミノ酸301のうち、C末26アミノ酸を欠損させると、膜に局在しなかった
  • The internal dissociation and reassociation of the internal clocks after an abrupt light dark cycle shift  [Not invited]
    重吉 康史; 長野 護; 藤岡 厚子; 中浜健一; 佐藤伸介
    第7回日本時間生物学会  2000/11  東京  第7回日本時間生物学会
     
    体内時計の光同調、視交叉上核の存在する体内時計の内的脱同調によって時差ボケの生じる仕組みについて述べた。
  • A role of COOH-terminal amino acid sequence in membrane localization of aquaporin 4.  [Not invited]
    中浜健一; 重吉 康史; 長野 護; 藤岡 厚子; 古河 惠一; 佐々木 宏; 佐藤伸介
    第73回日本生化学大会  2000/10  横浜  第73回日本生化学大会
     
    様々なアクアポリン4のC末変異体とGFPの融合タンパクを作製し、培養アストロサイトに発現させた。その結果、PDZドメインに結合すると考えられていたC末の配列はアクアポリン4の細胞膜への移行には関与しないことが分かった。
  • The mechanism of membrane targeting of aquaporin 4-GFP fusion protein  [Not invited]
    中浜健一; 重吉 康史; 長野 護; 藤岡 厚子; 古河 惠一
    第47回近畿大学医学会学術講演会  2000/01  大阪狭山市  第47回近畿大学医学会学術講演会
     
    アクアポリン4(AQP4)の膜局在化機構についてAQP4-GFP融合タンパクを培養細胞に発現させ、その局在を共焦点レーザー顕微鏡で観察した。様々な欠損変異体を作製し調べたところAQP4のC末部分に膜局在に重要だと思われる配列を見つけた。

MISC

Research Grants & Projects

  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2020/04 -2023/03 
    Author : 長野 護
     
    我々は以前にマウス(C57BL/6J)で明暗サイクルを10時間後退させ、詳細に視交叉上核の水平断切片でPer1遺伝子発現パターンを観察し、視交叉上核のコア領域とシェル領域間の脱同期に加えて、シェル領域においてPer1遺伝子発現のピークが吻側部は尾側部より速く後退することを報告した。今回、Per2:: lucノックインマウス(Per2プロモーターの制御下でルシフェラーゼ遺伝子を発現するコンス トラクトを導入したマウス)を用いて、明暗サイクルの10時間後退1日目と2日目において視交叉上核のスライス培養を作成し発光リズムを観察した結果、コア領域とシェル領域間の脱同期を認められたが、シェル領域内において明確な脱同期を確認することはできなかった。そこで、明暗サイクルを後退させた時よりも再同期に期間を要する明暗サイクルを前進させてシェル領域内において脱同期が生じるかを検討した。まず、マウス(C57BL/6J)で明暗サイクルを8時間前進させ、視交叉上核の水平断切片でPer1遺伝子発現をin situ hybridization法を用いて経時的に観察した結果、明暗サイクルを後退させて時よりも明確なシェル領域内における脱同期が観察された。現在、Per2:: lucノックインマウスによる検証を進めている。 概日リズムの減退が時差ぼけの持続時間に及ぼす影響を調べるため、時計遺伝子BMAL1のdominant negative formを発現するトランスジェニックラットを作成して検討した結果、このトランスジェニックラットは、対照群よりも早くlight off直後に活動的になった。また、明暗周期を急激に変化させた時に、このトランスジェニックラットは対照群に比べ、新しい明暗周期への同調が早いことが明らかとなった。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : KOINUMA Satoshi
     
    Mammals have an intrinsic biological clock that shows a stable period of about 24 hours. Underlying this function is the suprachiasmatic nucleus (SCN) in the hypothalamus. We have elucidated that there are cell groups that oscillate rhythms shorter than this period in the dorso-medial region of the SCN (DM-SCN). And we have focused on a phase wave (the expression of the clock gene propagates from medial to lateral SCN) originating from DM-SCN. The presence of spreading of phase wave in the rostro-caudal axis direction in addition to the phase waves towards the lateral sides which was known so far was found by histochemical analysis using the mouse SCN. This result will deepen the basic understanding of the physiologically unknown function of the phase wave.
  • 体内時計の中枢である視交叉上核における光入力制御に係わるゲート機構の解明
    文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2013/04 -2016/03 
    Author : 長野 護
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : KOINUMA Satoshi; NAGANO Mamoru
     
    We had artificially disrupted the intercellular coupling among oscillating neurons in the suprachiasmatic nucleus (SCN) and observed regional differences in the periods of the SCN. The period analysis had divided the SCN into two regions - a region with periods shorter than 24 h (short-period region, SPR) and another with periods longer than 24 h (long period region). In this project, we focused on the SPR, since an endogenous phase wave (a propagation of the clock genes expression from the medial region to the lateral regions) seemed to start from the SPR, and importantly, the phase wave responds to a light-dark cycle change. We obtained several findings as follows, (1) a distribution of the SPR expands caudally, (2) cells are more densely packed in the SPR. Further, through a transcriptome and in situ hybridization analyses, we identified a ligand with an expression confined to an area around the SPR. Since the light-pulse type PRC was induced, it is likely an endogenous SCN signal.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011/04 -2013/03 
    Author : NAGANO Mamoru; KOINUMA Satoshi; MASUMOTO Kohei
     
    We investigated the mechanisms of light input and synchronization in the suprachiasmatic nucleus (SCN). Using rPer2 promoter::Luciferase rat, we have found that the SCN is divided into two regions in relevance to period length. One of which showed medial region of the SCN containing oscillators with a period shorter than 24 hours (SPR) and lateral region with a period longer than 24 hours (LPR). And we have found that synchrony between SPR and LPR is possibly attained by cAMP signaling. Then, We observed Per1 and cFos induction in the SCN by light when the environmental LD cycle (12:12-h) was abruptly changed to dissociate the dorsomedial SCN (DMSCN) and ventrolateral SCN (VLSCN). As a result, the phase shifting of the gate corresponded well with that of the DMSCN. The finding suggested that the DMSCN determines the phase of the gate.
  • 体内時計の中枢である視交叉上核における光シグナル伝達経路の形態学的解明
    文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2009/04 -2011/03 
    Author : 長野 護
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2008 -2010 
    Author : SHIGEYOSI Yasufumi; FUJIOKA Atsuko; NAGANO Mamoru; KOINUMA Satoshi; SUJINO Mitsugu
     
    To know the intercellular system that keeps the synchrony among oscillator neurons in the SCN, we developed two projects to delineate the intrinsic synchronizing systems. We got two bland new findings. (1) Synchrony between dorsomedial SCN (DMSCN) and ventrolateral SCN (VLSCN) is possibly attained by VIP and VPAC2 system. (2) We found that the SCN is divided into two parts in relevance to period of circadian rhythm. Medial small part containing oscillators with a period less than 24 hours and the rest of the SCN with a period longer than 24 hours.
  • 体内時計の中枢における二振動体の光入力制御機構の形態学的解明
    文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2007/04 -2009/03 
    Author : 長野 護
  • Gene Chipによって採取された体内時計中枢に発現する機能未知遺伝子の解析
    文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2004/04 -2006/03 
    Author : 長野 護
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : SHIGIYOSHI Yasufumi; FUJIOKA Atsuko; NAGANO Mamoru; ADACHI Akihito; HAYASAKA Naoto
     
    1. Cloning and functional analysis on a functionally unknown G-protein coupled receptor, GPRg1. We cloned GPRg1 by using genome database and localized the expression by using in situ hybridization. We found that the gene is expressed in the caudate-putamen, habenular nucleus and mammillary body. We also analyzed what G-protein is coupled with the receptor and found that Gq is coupled (BBRC 2005). 2. Resetting of the internal clock is initiated by Perl and/or Per2 gene induction in the suprachiasmatic nucleus by light. By using in situ hybridization technique, we found that Per1 is used for the advance of the clock and Per2 is used for the delay (submitted). 3. We revealed an intracellular signal transduction pathway utilized for Per2 induction that is resumed to cause phase shift of the circadian clock. The pathway that lead to Per2 induction consists of the activation of Gq-protein, phospholipase C, IP3, IP3 receptor, release of Ca2+ from the intracellular Ca2+ store and influx of Ca2+ from extracellular space (Genes to Cells 2006). 4. We found that C6 glioma cells makes circadian rhythm after the application by dexamethasone (BBRC 2006). 5. We localized Prokineticin 2 and its receptor PKR2 in the suprachiasmatic nucleus (SCN). We found that P1(2 is expression both in the ventrolateral SCN and dorsomedial SCN. PKR2 is expressed in the dorsolateral region (European J. Neurosci.; 2006). 6. We revealed that mutation of PKR2 gene causes undevelopment of olfactory bulb and hypogonadotropic hypogonadism. We also found that GnRH neurons are lost in PKR2 knockout mice. The finding suggests that the hypogonadism is caused by loss of migration of GnRH neurons from the olfactory pit to the hypothalamus (PNAS 2006).
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2005 
    Author : HAYASAKA Naoto; SHIGEYOSHI Yasufumi; NAGANO Mamoru
     
    We have previously identified circadianly regulated genes in the suprachiasmatic nucleus (SCN) and liver by Gene Chip analyses. To elucidate their functions in efficient and multidimensional ways, we have established methods using a lentiviral vector system to analyze gene functions both in vivo and in vitro. First, we constructed lentiviral vectors for overexpression or knockdown (siRNA) of candidate genes. Then we developed a technique to inject lentivirus into the mouse SCN using a stereotaxic instrument with a syringe pump. The method enabled us to analyze circadian phenotype (e.g., locomotor activity) in vivo right after injection of the virus in the SCN and made it possible to analyze functions of several candidate genes in a short term. Next, we further applied the virus infection to brain slices. We established a method to efficiently introduce a transgene into the SCN, which had been quite difficult like in other tissues. Using this new method, we studied roles of the candidate genes in circadian system by measuring different circadian outputs. By making SCN slices from transgenic rats/mice carrying Per2::luciferase, which demonstrate circadian rhythmicity of luciferase activity, we examine an effect of the candidate gene expression on the circadian rhythms of the Per2::luc. We also analyzed circadian activity of the SCN neurons in the same slices by electrophysiology. Lastly, we could also study roles of the candidate genes using dissociated neurons/glias infected with the lentivirus. Using these methods mentioned above, so far we have analyzed two candidate genes, Rgs16 and Hmg4 and are preparing to publish papers
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2002 -2003 
    Author : SHIGEYOSHI Yasufumi; ADACHI Akihito; NAGANO Mamoru; FUJIOKA Atsuko
     
    1.Dichotomy of the circadian center is associated with jet lag syndrome Dissociation of the suprachiasmatic nucleus, the circadian center, into two oscillators was observed after 10 hour delay and 6 hour advance of light-dark cycle. The ventrolateral region of the SCN(VLSCN) shifted rapidly whereas the dorsomedial regions of the SCN(DMSCN) shifted slowly, spent more than a week to regain synchronization of the VLSCN and DMSCN. The sluggish shift of the DMSCN corresponded to the suppressed locomotors activity during the subjective night, which suggested that jet lag was caused by the slow shift of the DMSCN after LD cycle shift. 2.A Transcription Factor Response Element for Gene Expression During Circadian Night We profiled suprachiasmatic nuclei(SCN) and liver genome-wide expression patterns under light/dark(LD) cycles and constant darkness(DD). We determined transcription start sites(TSS) of human orthologues for newly identified cycling genes and then performed bioinformatical searches for relationships between time-of-day specific expression and transcription factor response elements around TSS. We demonstrate the role of the Rev-ErbA/ROR response element in gene expression of nocturnally expressed genes in SCN and liver. 3.Phosphodiesterase type 4(PDE4) specifically degrade cAMP To elongate Per1 expression by the inhibition of cAMP degradation, we used Rolipram, a specific inhibitor of phosphodiesterase type 4, which degrades the cAMP. With Rolipram application, the amount of Perl transcripts increased transiently but the period length of Perl induction was not extended.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2001 -2002 
    Author : SHIGEYOSHI Yasufumi; NAKAHAMA Ken-ichi; NAGANO Mamoru; FUJIOKA Atsuko; SATO Shinsuke
     
    A. Dichotomy of the circadian center is associated with jet lag syndrome In both 10 hour delay and 6 hour advance of light-dark cycle, we found that there appeared the dissociation of the suprachiasmatic into two oscillators. The ventrolateral region of the SCN (VLSCN) shifted rapidly whereas the dorsomedial regions of the SCN (DMSCN) shifted very slowly, spent more than a week to regain synchronization of the VLSCN and VLSCN. The sluggish shift of the DMSCN corresponds to the suppressed locomotors activity, which suggests that jet lag is caused by the slow shift of the DMSCN after LD cycle shift. B. Transgene of mammalian Per2 genes to Per2-null Drosophila mutants recovered the circadian rhythmicity. We introduced transgene to Per2-null Drosophila of which activity is arrhythmic. In the Drosophila expressing mouse Per21 or Per22 gene, we found the recovery of locomotor rhythms. The finding suggests that Pa-21 and Per22 have similar roles of clock genes even in the mammalian species. C. A Transcription Factor Response Element for Gene Eypressinn During Circadian Night We profiled suprachiasmatic nuclei (SCN) and liver genome-wide expression patterns under light/dark (LD) cycles and constant darkness (DD). We extensively determined transcription start sites (TSS) of human orthologues for newly identified cycling genes and then Per2 formed bioinformatical searches for relationships between time-of-day specific expression and transcription factor response elements around TSS. We demonstrate the role of the Rev-ErbA/ROR response element in gene expression of nocturnally expressed genes in SCN and liver. D. Phosphodiesterase type,4 (PDE4) specifically degradg cAMP To enhance Per21 transcription by the inhibition of cAMP degradation, we used Rolipram, a specific inhibitor of phosphodiesterase type 4 which degrades the cAMP. We found that the Per21 transcript increased transiently but the Per2iod length of Per21 increase was not extended.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1999 -2000 
    Author : SHIGEYOSI Yasufumi; NAKAHAMA Ken-ichi; NAGANO Mamoru; FUJIOKA Atsuko; SATO Shinsuke
     
    Two-clock system in the SCN and cause of jet lag We found the restriction of Per1 gene induction to the ventrolateral region of the SCN.After brief light exposure in the night, rats showed high level of Per1 mRNA induction in the ventrolateral region of the suprachiasmatic nucleus (SCN). The results suggested that there are two types of clocks in the SCN ; light responsive and light unresponsive. To show the two-oscillator system in the SCN clearly, we utilized a large rapid light-dark cycle shift. After the 10 hours delay of LD cycle, we found the dissociation of the internal timing system in the SCN ; the Per1 expression in the ventrolateral region showed a rapid large shift while the dorsomedial regions were far less affected. The following resynchronization process of the two regions was rather slow, requiring several days. These findings suggest that there are potential two clocks in the SCN, which could be dissociated by a certain perturbation. These findings also suggest that jet lag is caused by delayed shift of the clocks in the dorsomedial region of the SCN. Investigation on the transgenic flies carrying mouse Per genes We investigated the functional conservation of per by introducing the mouse mPer1 and mPer2 genes, driven by the Drosophila timeless (tim) promoter, into Drosophila melanogaster. Behavioral assays showed that both mPer constructs restored rhythms in per^<01>flies that are otherwise arrhythmic due to a lack of endogenous per protein (PER). This study demonstrates that both mPer1 and mPer2 can function as clock components, and has implications for functional analysis of the different per genes. Analysis on the transgenic mouse carrying mPer1 genes We obtained the cDNAs containing coding region and promoter regions of mPer1, Per2, and Per3. These cDNAs corresponding regions of Per1, Per2 and Per3 tagged with FLAG are subcloned into expression vectors. We transfected these expression vectors into cell lines and obtained enough expression of these proteins. Now we are trying to establish the transgenic mice carrying these Per genes.
  • Mammalian circadian clock
    Date (from‐to) : 2000
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1996 -1998 
    Author : SHINODA Koh; NAKAHAMA Ken-ichi; NAGANO Mamoru; OTSUKI Tsukasa; KAWATA Keisuke
     
    The presence of neuronal aromatase has been immunohistochemically determined in the aldehyde-fixed rat and monkey brains. The most striking aromatase-immunoreactive (AROM-I) neurons are present in the medial preoptico-amygdaloid neuronal arcs (mPOAM arcs) in both animals. The regions have been considered as centers of reproductive functions and larger in size in males than in females, confirming that intracranial estrogen-biosynthesis is essential to development of reproductive neural substrates and functions in mammals. They appear by E15 in the rat, reaching a peak in staining intensity between El 8-P2 and diminishing after the perinatal stage. Expression in the mPOAM arc are clearly more prominent in males in number and immunoreactivity than in females, especially after young-to-adult stages. The AROM-I neurons have also been clarified to show sexually dimorphic expression for both alpha-estrogen and androgen receptors (EsR and AnR). In neonates, expression of EsR in the mPOAM arc is already more prominent in females than in males, whereas that of AnR is more prominent in males than in female. In young-to-adult stages, the sexually dimorphic expressions are further enhanced in accordance with increased expressions of EsR in females or AnR in males during adolescence. Neonatal castration in male rats was found to change the male's pattern to the female's one with respect to AROM, EsR and AnR in the mPOAM arc. Administration of 5alpha-dihydrotestosterone strongly increases the expressions of AROM and AnR and slightly increase EsR, while 17beta-estradiol slightly increases the expressions of AROM and AnR and strongly decreases the expression of EsR.Testosterone strongly increases the expressions of AROM and AnR, and strongly decreases the expression of EsR in the mPOAM arc, indicating that only testosterone can recovered expression of the male pattern in the neonatally castrated male rats. Thus, some testosterones are considered to be actually aromatized into estradiol and to down-regulate EsR, while others sparing aromatization can act directly on AnR in the mPOAM arc. A complete male probably requires not only masculinization by enhancement of an AnR-stimulation via the up-regulation of AnR but also defeminization by reduction of an EsR-stimulation via the down-regulation of EsR in the Iimbic arc.

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