研究者詳細 - 坂本　竜哉

MISC - 坂本　竜哉

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The mineralo-corticoid receptor knockout in medaka is further validated by glucocorticoid receptor compensation

Tatsuya Sakamoto, Madoka Yoshiki, Hirotaka Sakamoto

SCIENTIFIC DATA 4 924 - 928 2017年12月

記述言語：英語出版者・発行元：NATURE PUBLISHING GROUP

To study the critical role of mineralocorticoid signalling, we generated a constitutive mineralocorticoid receptor (MR)-knockout (KO) medaka as the first adult-viable MR-KO animal. This KO medaka displayed abnormal behaviours affected by visual stimuli. In contrast, the loss of MR did not result in overt phenotypic changes in osmoregulation, despite the well-known osmoregulatory functions of MR in mammals. Since glucocorticoid receptor (GR) has been suggested to compensate for loss of MR, we examined expression of duplicated GRs with markedly different ligand sensitivities, in various tissues. qRT-PCR results revealed that the absence of MR induced GR1 in the brain and eyes, but not in osmoregulatory organs. This reinforces the important functions of glucocorticoid signalling, but the minor role of mineralocorticoid signalling, in fish osmoregulation. Because both 11-deoxycorticosterone (DOC) and cortisol are ligands for MR, whereas GRs are specific to cortisol, GR1 signalling may compensate for the absence of cortisol-MR, rather than that of DOC-MR. Thus, this GR expression suggests that our MR-KO model can be used specifically to characterize DOC-MR signalling.

DOI： 10.1038/sdata.2017.189

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MORPHOLOGICAL AND MOLECULAR EVOLUTIONAL ANALYSES OF ITCH FOCUSED ON THE GASTRIN-RELEASING PEPTIDE SYSTEM IN MAMMALS

Keiko Takanami, Keita Satoh, Kazuyoshi Murata, Tatsuya Sakamoto, Hirotaka Sakamoto

ACTA DERMATO-VENEREOLOGICA 97 ( 8 ) 1049 - 1050 2017年9月

Identification of the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function in the mouse and Asian house musk shrew (Suncus murinus)

Kei Tamura, Yasuhisa Kobayashi, Asuka Hirooka, Keiko Takanami, Takumi Oti, Takamichi Jogahara, Sen-ichi Oda, Tatsuya Sakamoto, Hirotaka Sakamoto

JOURNAL OF COMPARATIVE NEUROLOGY 525 ( 7 ) 1586 - 1598 2017年5月

記述言語：英語出版者・発行元：WILEY

Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/cne.24138

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クルマエビの卵巣で発現するペプチドホルモン様因子

筒井直昭, 坂本竜哉

日本比較内分泌学会大会及びシンポジウムプログラム・講演要旨 42nd 43 2017年

コペプチンからバソプレシン遺伝子の転写-翻訳-プロセシングを解析する試み

坂本浩隆, 坂本浩隆, 佐藤慧太, 高浪景子, 大坪秋人, 坂本竜哉, MORRIS John F.

日本神経内分泌学会学術集会プログラム・抄録集 44th 2017年

蛍光タンパク質Venus導入ラットを用いた雄の性機能を司る脊髄神経回路系の解析

越智拓海, 高浪景子, 松田賢一, 河田光博, 坂本竜哉, 坂本浩隆

日本アンドロロジー学会総会記事 36th 2017年

Crystal structure of a crustacean hyperglycemic hormone (CHH) precursor suggests structural variety in the C-terminal regions of CHH superfamily members

Naoaki Tsutsui, Tatsuya Sakamoto, Fumio Arisaka, Masaru Tanokura, Hiromichi Nagasawa, Koji Nagata

FEBS JOURNAL 283 ( 23 ) 4325 - 4339 2016年12月

記述言語：英語出版者・発行元：WILEY

The crustacean hyperglycemic hormone (CHH) is one of the major hormones in crustaceans, and peptides belonging to the CHH superfamily have been found in diverse ecdysozoans. Although the basic function of CHH is to control energy metabolism, it also plays various roles in crustacean species, such as in molting and vitellogenesis. Here, we present the crystal structure of Pej-SGP-I-Gly, a partially active precursor of CHH from the kuruma prawn Marsupenaeus japonicus, which has an additional Gly residue in place of the C-terminal amide group of the mature Pej-SGP-I. The 1.6-angstrom crystal structure showed not only the common CHH superfamily scaffold comprising three a-helices, three disulfide bridges, and a hydrophobic core but also revealed that the C-terminal part has a variant backbone fold that is specific to Pej-SGP-I-Gly. The a-helix 4 of Pej-SGP-I-Gly was much longer than that of molt-inhibiting hormone (Pej-MIH) from the same species, and as a result, the following C-terminal helix, corresponding to a-helix 5 in MIH, was not formed. Unlike monomeric PejMIH, Pej-SGP-I-Gly forms a homodimer in the crystal structure via its unique a-helix 4. The unexpected dissimilar folds between Pej-SGP-I-Gly and Pej-MIH appear to be the result of their distinct C-terminal amino acid sequences. Variations in amino acid sequences and lengths and the resulting variety of backbone folds allow the C-terminal and sterically adjoining regions to confer different hormonal activities in diverse CHH superfamily members.

DOI： 10.1111/febs.13926

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Principal function of mineralocorticoid signaling suggested by constitutive knockout of the mineralocorticoid receptor in medaka fish

Tatsuya Sakamoto, Madoka Yoshiki, Hideya Takahashi, Masayuki Yoshida, Yukiko Ogino, Toshitaka Ikeuchi, Tomoya Nakamachi, Norifumi Konno, Kouhei Matsuda, Hirotaka Sakamoto

SCIENTIFIC REPORTS 6 2016年11月

記述言語：英語出版者・発行元：NATURE PUBLISHING GROUP

As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na+ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper-and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.

DOI： 10.1038/srep37991

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High levels of plasma cortisol and impaired hypoosmoregulation in a mutant medaka deficient in P450c17I

Hideya Takahashi, Tadashi Sato, Toshitaka Ikeuchi, Kazuhiro Saito, Mitsuru Sakaizumi, Tatsuya Sakamoto

MOLECULAR AND CELLULAR ENDOCRINOLOGY 430 ( C ) 25 - 32 2016年7月

記述言語：英語出版者・発行元：ELSEVIER IRELAND LTD

scl is a spontaneous medaka mutant deficient in P450c17I, which is required for production of sex steroids, but not of cortisol, the major role of which is osmoregulation in teleost fish. The scl mutant provides a new model to study the functions of these hormones. We first found that fish homozygous for this mutation have plasma cortisol constitutively at a high physiological level (1000 nM). Since we previously showed that this level reversed the seawater-type differentiation of the medaka gastrointestinal tract, hypoosmoregulation of the scl mutant was analyzed. Muscle water contents in freshwater were normal in scl homozygotes, but the contents were lower than those of the wild type (WT) after seawater transfer. There were no differences in gill mRNA levels of corticosteroid receptors or ion transporters between scl homozygotes and WT. In the intestine, expression of glucocorticoid receptors and Na+/K+/2Cl(-) cotransporter were induced in WT during seawater acclimation, but not in scl homozygotes. The high plasma cortisol may prevent hypoosmoregulation by inhibition of increased intestinal water absorption, essentially by the Na+/K+/2Cl(-) cotransporter, in seawater. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mce.2016.04.009

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Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors

Akiyoshi Takahashi, Perry Davis, Christina Reinick, Kanta Mizusawa, Tatsuya Sakamoto, Robert M. Dores

Data in Brief 7 1670 - 1677 2016年6月

記述言語：英語出版者・発行元：Elsevier Inc.

This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs) related to research published in "Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish" (Takahashi et al., 2016) [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.

DOI： 10.1016/j.dib.2016.04.050

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Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish

Akiyoshi Takahashi, Perry Davis, Christina Reinick, Kanta Mizusawa, Tatsuya Sakamoto, Robert M. Dores

GENERAL AND COMPARATIVE ENDOCRINOLOGY 232 115 - 124 2016年6月

記述言語：英語出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Melanocortin (MC) systems are composed of MC peptides such as adrenocorticotropic hormone (ACTH), several molecular forms of melanocyte-stimulating hormones (MSHs) and MC receptors (MCRs). Here we demonstrated that the cartilaginous fish, Dasyatis akajei (stingray) expresses five subtypes of MCR genes-mc1r to mc5r-as in the case of teleost and tetrapod species. This is the first evidence showing the presence of the full repertoire of melanocortin receptors in a single of cartilaginous fish. Expression of respective stingray mcr cDNAs in Chinese hamster ovary cells revealed that Des-acetyl-alpha-MSH exhibited cAMP-producing activity indistinguishable to ACTH(1-24) on MC1R and MC2R, while the activity of Des-acetyl-alpha-MSH on MC3R, MC4R, and MC5R were similar to or slightly greater than that of ACTH(1-24). Notably, in contrast to the other vertebrates, MC2R did not require coexpression with a melanocortin receptor-2 accessory protein 1 (mrap1) cDNA for functional expression. One of the roles of MC system resides in regulation of the pituitary-interrenal (PI) axis-a homologue of tetrapod pituitary adrenal axis. In stingray, interrenal tissues were shown to express mc2r and mc5r as major MCR genes. These results established the presence of functional PI axis in stingray at the level of receptor molecule. While MC2R participates in adrenal functions together with MRAP1 in tetrapod species, the fact that sensitivity of MC5R to Des-acetyl-alpha-MSH and ACTH(1-24) were two order of magnitude higher than MC2R without coexpression with MRAP1 suggested that MC5R could play a more important role than MC2R to transmit signals conveyed by ACTH and MSHs if MRAP1 is really absent in the stingray. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2016.03.030

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Postnatal development of the gastrin-releasing peptide system in the lumbosacral spinal cord controlling male reproductive function in rats

Nao Katayama, Takumi Oti, Keiko Takanami, Tatsuya Sakamoto, Hirotaka Sakamoto

PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES 92 ( 2 ) 69 - 75 2016年2月

記述言語：英語出版者・発行元：JAPAN ACAD

A sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord, which projects to the lower spinal centers, controls erection and ejaculation in rats. However, little is known about the postnatal development of this system. In this study, we therefore examined the postnatal development of the male-dominant spinal GRP system and its sexual differentiation in rats using immunohistochemistry. Our results show that male-dominant expression of GRP is prominent from the onset of puberty and that sexually dimorphism persists into adulthood. These results suggest that androgen surge during male puberty plays an important role in the development and maintenance of the male-specific GRP function in the rat spinal cord.

DOI： 10.2183/pjab.92.69

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Perinatal testosterone exposure is critical for the development of the male-specific sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that mediates erection and ejaculation

Takumi Oti, Keiko Takanami, Nao Katayama, Tomoca Edey, Keita Satoh, Tatsuya Sakamoto, Hirotaka Sakamoto

BIOLOGY OF SEX DIFFERENCES 7 ( 1 ) 2016年1月

記述言語：英語出版者・発行元：BIOMED CENTRAL LTD

Background: In rats, a sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord projects to spinal centers that control erection and ejaculation. This system controls the sexual function of adult males in an androgen-dependent manner. In the present study, we assessed the influence of androgen exposure on the spinal GRP system during a critical period of the development of sexual dimorphism.
Methods: Immunohistochemistry was used to determine if the development of the spinal GRP system is regulated by the perinatal androgen surge. We first analyzed the responses of neonates administered with anti-androgen flutamide. To remove endogenous androgens, rats were castrated at birth. Further, neonatal females were administered androgens during a critical period to evaluate the development of the male-specific spinal GRP system.
Results: Treatment of neonates with flutamide on postnatal days 0 and 1 attenuated the spinal GRP system during adulthood. Castrating male rats at birth resulted in a decrease in the number of GRP neurons and the intensity of neuronal GRP in the spinal cord during adulthood despite testosterone supplementation during puberty. This effect was prevented if the rats were treated with testosterone propionate immediately after castration. Moreover, treating female rats with androgens on the day of birth and the next day, masculinized the spinal GRP system during adulthood, which resembled the masculinized phenotype of adult males and induced a hypermasculine appearance.
Conclusions: The perinatal androgen surge plays a key role in masculinization of the spinal GRP system that controls male sexual behavior. Further, the present study provides potentially new approaches to treat sexual disorders of males.

DOI： 10.1186/s13293-016-0058-x

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Comparative Anatomy of Gastrin-releasing Peptide Pathways in the Trigeminal Sensory System of Mouse and the Asian House Musk Shrew Suncus murinus

Keiko Takanami, Kaihei Inoue, Hiroki Mukai, Kei Tamura, Takamichi Jogahara, Sen-ichi Oda, Mitsuhiro Kawata, Tatsuya Sakamoto, Hirotaka Sakamoto

ACTA HISTOCHEMICA ET CYTOCHEMICA 49 ( 6 ) 181 - 190 2016年

記述言語：英語出版者・発行元：JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY

Gastrin-releasing peptide (GRP) has recently been identified as an itch-signaling molecule in the primary afferents and spinal cord of rodents. However, little information exists on the expression and localization of GRP in the trigeminal somatosensory system other than in rats. We examined the generality of the trigeminal GRP system in mammals using two distinct species, suncus as a model of specialized placental mammals known to have a well-developed trigeminal sensory system and mice as a representative small laboratory animal. We first analyzed the gross morphology of the trigeminal somatosensory system in suncus to provide a brainstem atlas on which to map GRP distribution. Immunohistochemical analyses showed that 8% of trigeminal ganglion neurons in suncus and 6% in mice expressed GRP. Expression was restricted to cells with smaller somata. The GRP-containing fibers were densely distributed in the superficial layers of the caudal part of the trigeminal spinal nucleus (Vc) but rare in the rostral parts, both in suncus and mice. Expression of GRP receptor mRNA and protein was also detected in the Vc of suncus. Taken together, these results suggest that the trigeminal GRP system mediating itch sensation is conserved in mammals.

DOI： 10.1267/ahc.16030

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ニホンザル脊髄における痒み特異的伝達分子gastrin-releasing peptide受容体の発現

伊藤隆志, 高浪景子, 高浪景子, 越智拓海, 小林靖尚, 小林靖尚, 佐藤慧太, 高橋俊次, 上田康雅, 坂本竜哉, 坂本浩隆

日本組織細胞化学会総会・学術集会講演プログラム・予稿集 57th 2016年

雄の性機能を司る脊髄神経回路系の機能解析:Grp-promoter-Venusトランスジェニックラットの作出と利用

越智拓海, 高浪景子, 高浪景子, 松田賢一, 河田光博, 坂本竜哉, 坂本浩隆

日本神経内分泌学会学術集会プログラム・抄録集 43rd 2016年

Three-dimensional visualization of multiple synapses in thick sections using high-voltage electron microscopy in the rat spinal cord

Keita Satoh, Keiko Takanami, Kazuyoshi Murata, Mitsuhiro Kawata, Tatsuya Sakamoto, Hirotaka Sakamoto

Data in Brief 4 566 - 570 2015年9月

記述言語：英語出版者・発行元：Elsevier Inc.

This data article contains complementary figure and movies (Supplementary Movies 1-3) related to the research article entitled, "Effective synaptome analysis of itch-mediating neurons in the spinal cord: a novel immunohistochemical methodology using high-voltage electron microscopy" [7]. It is important to show the synaptic connections at the ultrastructural level to understand the neural circuit, which requires the three-dimensional (3-D) analyses in the electron microscopy. Here, we applied a new sample preparation method, a high-contrast en bloc staining according to the protocol of the National Center for Microscopy and Imaging Research (NCMIR), University of California, San Diego, CA, USA to high-voltage electron microscopy (HVEM) tomography in order to examine the 3-D chemical neuroanatomy of the rat spinal cord. Pre-embedding immunoelectron microscopy was used in this study. HVEM has an excellent potential to directly visualize the ultrastructures in semi-thin sections (~5. μm thick), and we have successfully visualized many itch-mediating synaptic connections and neural networks in the spinal cord using "HVEM tomography". Moreover, the methodology used in this study is simple and can be applied in multiple ways. This is an important contribution to ultrastructural investigations of the central nervous system in the present post-genomic age.

DOI： 10.1016/j.dib.2015.07.005

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Mudskipper to general behavioral neuroendocrinology

Tatsuya Sakamoto, Yukitoshi Katayama, Yasuhisa Kobayashi, Kazuhiro Saito, Yoshio Takei

NIPPON SUISAN GAKKAISHI 81 ( 5 ) 868 - 868 2015年9月

Osmotic/ionic status of body fluids in the euryhaline cephalopod suggest possible parallel evolution of osmoregulation

Tatsuya Sakamoto, Satoshi Ogawa, Yudai Nishiyama, Chiaki Akada, Hideya Takahashi, Taro Watanabe, Hiroyuki Minakata, Hirotaka Sakamoto

SCIENTIFIC REPORTS 5 2015年9月

記述言語：英語出版者・発行元：NATURE PUBLISHING GROUP

Acclimation from marine to dilute environments constitutes among the dramatic evolutionary transitions in the history of life. Such adaptations have evolved in multiple lineages, but studies of the blood/hemolymph homeostasis mechanisms are limited to those using evolutionarily advanced Deuterostome (chordates) and Ecdysozoa (crustaceans). Here, we examined hemolymph homeostasis in the advanced Lophotrochozoa/mollusc, the other unexplored taxa, and its possible regulation by the vasopressin/oxytocin superfamily peptides known to be implicated in fluid homeostasis in Chordata and Arthropoda. The hemolymph osmotic and ionic status in the euryhaline cephalopod (Octopus ocellatus) following transfer from 30-ppt normal seawater to 20 ppt salinity indicate hyperosmo- and hyperionoregulatory abilities for more than 1 week, as in crustaceans and teleost fish. While ventilation frequency decreased by 1 day, Na+/K+-ATPase activity, which has been generally implicated in ion transport, was induced in two of the eight posterior gills after 1 week. In addition, the octopuses were intravenously injected with 1 or 100 ng/g octopressin or cephalotocin, which are Octopus vasopressin/oxytocin orthologs. After 1 day, octopressin, but not cephalotocin, decreased the hemolymph osmolality and Ca concentrations, as well as urinary Na concentrations. These data provide evidence for possible parallel evolution in hyperionoregulatory mechanisms and coordination by conserved peptides.

DOI： 10.1038/srep14469

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In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS. 国際誌

Yuji Takihara, Masaru Inatani, Kei Eto, Toshihiro Inoue, Alexander Kreymerman, Seiji Miyake, Shinji Ueno, Masatoshi Nagaya, Ayami Nakanishi, Keiichiro Iwao, Yoshihiro Takamura, Hirotaka Sakamoto, Keita Satoh, Mineo Kondo, Tatsuya Sakamoto, Jeffrey L Goldberg, Junichi Nabekura, Hidenobu Tanihara

Proceedings of the National Academy of Sciences of the United States of America 112 ( 33 ) 10515 - 20 2015年8月

記述言語：英語

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

DOI： 10.1073/pnas.1509879112

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