Updated on 2022/05/24

写真a

 
OHKAWARA, Bisei
 
Organization
Graduate School of Medicine Designated associate professor
Title
Designated associate professor
External link

Degree 1

  1. 理学博士 ( 2002.3   総合研究大学院大学 ) 

Research Interests 5

  1. neuromuscular junctions

  2. Developmental Biology

  3. Synapse Formation

  4. Signaling pathways

  5. Wnt

Research Areas 1

  1. Life Science / Developmental biology

Research History 2

  1. Nagoya University Graduate School of Medicine   Division of Neurogenetics   Associate professor

    2017.4

  2. Nagoya University Graduate School of Medicine   Division of Neurogenetics   Lecturer

    2013.2 - 2017.4

Education 1

  1. The Graduate University for Advanced Studies

    - 2002.3

Professional Memberships 2

  1. Japan Muscle Society

  2. THE MOLECULAR BIOLOGY SOCIETY OF JAPAN

 

Papers 28

  1. Functional analysis of mutations in a glycosylation enzyme gene, GFPT1, underlying limb-girdle congenital myasthenic syndromes (CMS)

    Farshadyeganeh Paniz, Ohkawara Bisei, Kamon Masayoshi, Araki Toshiyuki, Komaki Hirofumi, Ohno Kinji

    EUROPEAN JOURNAL OF HUMAN GENETICS   Vol. 30 ( SUPPL 1 ) page: 271 - 271   2022.4

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    Language:Japanese  

    Web of Science

  2. Promethazine Downregulates Wnt/beta-Catenin Signaling and Increases the Biomechanical Forces of the Injured Achilles Tendon in the Early Stage of Healing

    Sakaguchi Takefumi, Ohkawara Bisei, Kishimoto Yasuzumi, Miyamoto Kentaro, Ishizuka Shinya, Hiraiwa Hideki, Ishiguro Naoki, Imagama Shiro, Ohno Kinji

    AMERICAN JOURNAL OF SPORTS MEDICINE   Vol. 50 ( 5 ) page: 1317 - 1327   2022.4

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  3. Possible Repositioning of an Oral Anti-Osteoporotic Drug, Ipriflavone, for Treatment of Inflammatory Arthritis via Inhibitory Activity of KIAA1199, a Novel Potent Hyaluronidase

    Koike Hiroshi, Nishida Yoshihiro, Shinomura Tamayuki, Ohkawara Bisei, Ohno Kinji, Zhuo Lisheng, Kimata Koji, Ushida Takahiro, Imagama Shiro

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   Vol. 23 ( 8 )   2022.4

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  4. Meclozine ameliorates skeletal muscle pathology and increases muscle forces in mdx mice

    Kawamura Yusuke, Hida Tetsuro, Ohkawara Bisei, Matsushita Masaki, Kobayashi Takeshi, Ishizuka Shinya, Hiraiwa Hideki, Tanaka Satoshi, Tsushima Mikito, Nakashima Hiroaki, Ito Kenyu, Imagama Shiro, Ito Mikako, Masuda Akio, Ishiguro Naoki, Ohno Kinji

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   Vol. 592   page: 87 - 92   2022.2

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  5. Regulated splicing of large exons is linked to phase-separation of vertebrate transcription factors

    Kawachi Toshihiko, Masuda Akio, Yamashita Yoshihiro, Takeda Jun-ichi, Ohkawara Bisei, Ito Mikako, Ohno Kinji

    EMBO JOURNAL   Vol. 40 ( 22 ) page: e107485   2021.11

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  6. Zonisamide upregulates neuregulin-1 expression and enhances acetylcholine receptor clustering at the in vitro neuromuscular junction. International journal

    Taro Inoue, Bisei Ohkawara, Samira Bushra, Shunsuke Kanbara, Hiroaki Nakashima, Hiroyuki Koshimizu, Hiroyuki Tomita, Mikako Ito, Akio Masuda, Naoki Ishiguro, Shiro Imagama, Kinji Ohno

    Neuropharmacology   Vol. 195   page: 108637 - 108637   2021.9

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    Language:Japanese   Publishing type:Research paper (scientific journal)  

    Decreased acetylcholine receptor (AChR) clustering compromises signal transmission at the neuromuscular junction (NMJ) in myasthenia gravis, congenital myasthenic syndromes, and motor neuron diseases. Although the enhancement of AChR clustering at the NMJ is a promising therapeutic strategy for these maladies, no drug is currently available for this enhancement. We previously reported that zonisamide (ZNS), an anti-epileptic and anti-Parkinson's disease drug, enhances neurite elongation of the primary spinal motor neurons (SMNs). As nerve sprouting occurs to compensate for the loss of AChR clusters in human diseases, we examined the effects of ZNS on AChR clustering at the NMJ. To this end, we established a simple and quick co-culture system to reproducibly make in vitro NMJs using C2C12 myotubes and NSC34 motor neurons. ZNS at 1-20 μM enhanced the formation of AChR clusters dose-dependently in co-cultured C2C12 myotubes but not in agrin-treated single cultured C2C12 myotubes. We observed that molecules that conferred responsiveness to ZNS were not secreted into the co-culture medium. We found that 10 μM ZNS upregulated the expression of neuregulin-1 (Nrg1) in co-cultured cells but not in single cultured C2C12 myotubes or single cultured NSC34 motor neurons. In accordance with this observation, inhibition of the Nrg1/ErbB signaling pathways nullified the effect of 10 μM ZNS on the enhancement of AChR clustering in in vitro NMJs. Although agrin was not induced by 10 μM ZNS in co-cultured cells, anti-agrin antibody attenuated ZNS-mediated enhancement of AChR clustering. We conclude that ZNS enhances agrin-dependent AChR-clustering by upregulating the Nrg1/ErbB signaling pathways in the presence of NMJs.

    DOI: 10.1016/j.neuropharm.2021.108637

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  7. Secreted Signaling Molecules at the Neuromuscular Junction in Physiology and Pathology. International journal

    Bisei Ohkawara, Mikako Ito, Kinji Ohno

    International journal of molecular sciences   Vol. 22 ( 5 )   2021.3

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    : Signal transduction at the neuromuscular junction (NMJ) is affected in many human diseases, including congenital myasthenic syndromes (CMS), myasthenia gravis, Lambert-Eaton myasthenic syndrome, Isaacs' syndrome, Schwartz-Jampel syndrome, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. The NMJ is a prototypic cholinergic synapse between the motor neuron and the skeletal muscle. Synaptogenesis of the NMJ has been extensively studied, which has also been extrapolated to further understand synapse formation in the central nervous system. Studies of genetically engineered mice have disclosed crucial roles of secreted molecules in the development and maintenance of the NMJ. In this review, we focus on the secreted signaling molecules which regulate the clustering of acetylcholine receptors (AChRs) at the NMJ. We first discuss the signaling pathway comprised of neural agrin and its receptors, low-density lipoprotein receptor-related protein 4 (Lrp4) and muscle-specific receptor tyrosine kinase (MuSK). This pathway drives the clustering of acetylcholine receptors (AChRs) to ensure efficient signal transduction at the NMJ. We also discuss three secreted molecules (Rspo2, Fgf18, and connective tissue growth factor (Ctgf)) that we recently identified in the Wnt/β-catenin and fibroblast growth factors (FGF) signaling pathways. The three secreted molecules facilitate the clustering of AChRs by enhancing the agrin-Lrp4-MuSK signaling pathway.

    DOI: 10.3390/ijms22052455

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  8. Zonisamide ameliorates neuropathic pain partly by suppressing microglial activation in the spinal cord in a mouse model. International journal

    Hiroyuki Koshimizu, Bisei Ohkawara, Hiroaki Nakashima, Kyotaro Ota, Shunsuke Kanbara, Taro Inoue, Hiroyuki Tomita, Akira Sayo, Sumiko Kiryu-Seo, Hiroyuki Konishi, Mikako Ito, Akio Masuda, Naoki Ishiguro, Shiro Imagama, Hiroshi Kiyama, Kinji Ohno

    Life sciences   Vol. 263   page: 118577 - 118577   2020.12

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    Neuropathic pain is caused by a lesion or a functional impairment of the sensory nervous system and allodynia is one of the frequently observed symptoms in neuropathic pain. Allodynia represents abnormal pain due to a non-noxious stimulus that does not normally provoke pain. Cellular mechanisms underlying neuropathic pain remain mostly elusive, and partial pain relief can be achieved in a limited number of patients by antidepressants, anticonvulsants topical anesthetics, and others. Zonisamide (ZNS) is widely used as an anti-epileptic and anti-Parkinson's disease drug. A recent report shows that ZNS suppresses neuropathic pain associated with diabetes mellitus in a mouse model. We made a mouse model of neuropathic pain in the hindlimb by cutting the nerve at the intervertebral canal at lumbar level 4 (L4). At 28 days after nerve injury, ZNS ameliorated allodynic pain, and reduced the expression of inflammatory cytokines and the nerve injury-induced increase of Iba1-positive microglia in the spinal dorsal horn at L4. In BV2 microglial cells, ZNS reduced the number of lipopolysaccharide-induced amoeboid-shaped cells, representing activated microglia. These results suggest that ZNS is a potential therapeutic agent for neuropathic pain partly by suppressing microglia-mediated neuroinflammation.

    DOI: 10.1016/j.lfs.2020.118577

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  9. Gene Expression Profile at the Motor Endplate of the Neuromuscular Junction of Fast-Twitch Muscle. International journal

    Kun Huang, Jin Li, Mikako Ito, Jun-Ichi Takeda, Bisei Ohkawara, Tomoo Ogi, Akio Masuda, Kinji Ohno

    Frontiers in molecular neuroscience   Vol. 13   page: 154 - 154   2020.9

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    The neuromuscular junction (NMJ) is a prototypic chemical synapse between the spinal motor neuron and the motor endplate. Gene expression profiles of the motor endplate are not fully elucidated. Collagen Q (ColQ) is a collagenic tail subunit of asymmetric forms of acetylcholinesterase and is driven by two distinct promoters. pColQ1 is active throughout the slow-twitch muscle, whereas pColQ1a is active at the motor endplate of fast-twitch muscle. We made a transgenic mouse line that expresses nuclear localization signal (NLS)-attached Cre recombinase under the control of pColQ1a (pColQ1a-Cre mouse). RiboTag mouse expresses an HA-tagged ribosomal subunit, RPL22, in cells expressing Cre recombinase. We generated pColQ1a-Cre:RiboTag mouse, and confirmed that HA-tagged RPL22 was enriched at the NMJ of tibialis anterior (TA) muscle. Next, we confirmed that Chrne and Musk that are specifically expressed at the NMJ were indeed enriched in HA-immunoprecipitated (IP) RNA, whereas Sox10 and S100b, markers for Schwann cells, and Icam1, a marker for vascular endothelial cells, and Pax3, a marker for muscle satellite cells, were scarcely detected. Gene set enrichment analysis (GSEA) of RNA-seq data showed that "phosphatidylinositol signaling system" and "extracellular matrix receptor interaction" were enriched at the motor endplate. Subsequent analysis revealed that genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins were enriched at the motor endplate. We first characterized the gene expression profile under translation at the motor endplate of TA muscle using the RiboTag technique. We expect that our gene expression profiling will help elucidate molecular mechanisms of the development, maintenance, and pathology of the NMJ.

    DOI: 10.3389/fnmol.2020.00154

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  10. CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction

    Ohkawara Bisei, Kobayakawa Akinori, Kanbara Shunsuke, Hattori Takako, Kubota Satoshi, Ito Mikako, Masuda Akio, Takigawa Masaharu, Lyons Karen M., Ishiguro Naoki, Ohno Kinji

    EMBO REPORTS   Vol. 21 ( 8 )   2020.8

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  11. CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction. Reviewed International journal

    Bisei Ohkawara, Akinori Kobayakawa, Shunsuke Kanbara, Takako Hattori, Satoshi Kubota, Mikako Ito, Akio Masuda, Masaharu Takigawa, Karen M Lyons, Naoki Ishiguro, Kinji Ohno

    EMBO reports   Vol. 21 ( 8 ) page: e48462   2020.8

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    At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf-/- ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf-/- embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.

    DOI: 10.15252/embr.201948462

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  12. Zonisamide ameliorates progression of cervical spondylotic myelopathy in a rat model. International journal

    Shunsuke Kanbara, Bisei Ohkawara, Hiroaki Nakashima, Kyotaro Ohta, Hiroyuki Koshimizu, Taro Inoue, Hiroyuki Tomita, Mikako Ito, Akio Masuda, Naoki Ishiguro, Shiro Imagama, Kinji Ohno

    Scientific reports   Vol. 10 ( 1 ) page: 13138 - 13138   2020.8

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    Cervical spondylotic myelopathy (CSM) is caused by chronic compression of the spinal cord and is the most common cause of myelopathy in adults. No drug is currently available to mitigate CSM. Herein, we made a rat model of CSM by epidurally implanting an expanding water-absorbent polymer underneath the laminae compress the spinal cord. The CSM rats exhibited progressive motor impairments recapitulating human CSM. CSM rats had loss of spinal motor neurons, and increased lipid peroxidation in the spinal cord. Zonisamide (ZNS) is clinically used for epilepsy and Parkinson's disease. We previously reported that ZNS protected primary spinal motor neurons against oxidative stress. We thus examined the effects of ZNS on our rat CSM model. CSM rats with daily intragastric administration of 0.5% methylcellulose (n = 11) and ZNS (30 mg/kg/day) in 0.5% methylcellulose (n = 11). Oral administration of ZNS ameliorated the progression of motor impairments, spared the number of spinal motor neurons, and preserved myelination of the pyramidal tracts. In addition, ZNS increased gene expressions of cystine/glutamate exchange transporter (xCT) and metallothionein 2A in the spinal cord in CSM rats, and also in the primary astrocytes. ZNS increased the glutathione (GSH) level in the spinal motor neurons of CSM rats. ZNS potentially ameliorates loss of the spinal motor neurons and demyelination of the pyramidal tracts in patients with CSM.

    DOI: 10.1038/s41598-020-70068-0

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  13. tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly

    Masuda Akio, Kawachi Toshihiko, Takeda Jun-ichi, Ohkawara Bisei, Ito Mikako, Ohno Kinji

    EMBO REPORTS   Vol. 21 ( 5 )   2020.5

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  14. tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly. Reviewed International journal

    Akio Masuda, Toshihiko Kawachi, Jun-Ichi Takeda, Bisei Ohkawara, Mikako Ito, Kinji Ohno

    EMBO reports   Vol. 21 ( 5 ) page: e49890   2020.5

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    RNA processing occurs co-transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome-wide identification of protein-RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP-seq) method that detects protein-RNA interaction sites in thousands of cells. The high sensitivity of tRIP-seq enables identification of protein-RNA interactions at functional subcellular levels. Application of tRIP-seq to the FUS-RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP-seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS-U1 snRNP interaction aberrantly activate the APA sites. tRIP-seq provides new insights into the regulatory mechanism of co-transcriptional RNA processing by RNA processing factors.

    DOI: 10.15252/embr.201949890

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  15. Congenital myasthenic syndrome-associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner. Reviewed International journal

    Bisei Ohkawara, XinMing Shen, Duygu Selcen, Mohammad Nazim, Vera Bril, Mark A Tarnopolsky, Lauren Brady, Sae Fukami, Anthony A Amato, Uluc Yis, Kinji Ohno, Andrew G Engel

    JCI insight   Vol. 5 ( 7 )   2020.4

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    Congenital myasthenic syndromes (CMS) are caused by mutations in molecules expressed at the neuromuscular junction. We report clinical, structural, ultrastructural, and electrophysiologic features of 4 CMS patients with 6 heteroallelic variants in AGRN, encoding agrin. One was a 7.9-kb deletion involving the N-terminal laminin-binding domain. Another, c.4744G>A - at the last nucleotide of exon 26 - caused skipping of exon 26. Four missense mutations (p.S1180L, p.R1509W, p.G1675S, and p.Y1877D) expressed in conditioned media decreased AChR clusters in C2C12 myotubes. The agrin-enhanced phosphorylation of MuSK was markedly attenuated by p.Y1877D in the LG3 domain and moderately attenuated by p.R1509W in the LG1 domain but not by the other 2 mutations. The p.S1180L mutation in the SEA domain facilitated degradation of secreted agrin. The p.G1675S mutation in the LG2 domain attenuated anchoring of agrin to the sarcolemma by compromising its binding to heparin. Anchoring of agrin with p.R1509W in the LG1 domain was similarly attenuated. Mutations of agrin affect AChR clustering by enhancing agrin degradation or by suppressing MuSK phosphorylation and/or by compromising anchoring of agrin to the sarcolemma of the neuromuscular junction.

    DOI: 10.1172/jci.insight.132023

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  16. Author Correction: Mianserin suppresses R-spondin 2-induced activation of Wnt/β-catenin signaling in chondrocytes and prevents cartilage degradation in a rat model of osteoarthritis. Reviewed International journal

    Toshiaki Okura, Bisei Ohkawara, Yasuhiko Takegami, Mikako Ito, Akio Masuda, Taisuke Seki, Naoki Ishiguro, Kinji Ohno

    Scientific reports   Vol. 10 ( 1 ) page: 2995 - 2995   2020.2

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    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    DOI: 10.1038/s41598-020-60112-4

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  17. Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells. Reviewed International journal

    Kun Huang, Akio Masuda, Guiying Chen, Samira Bushra, Masayoshi Kamon, Toshiyuki Araki, Masanobu Kinoshita, Bisei Ohkawara, Mikako Ito, Kinji Ohno

    Scientific reports   Vol. 10 ( 1 ) page: 2558 - 2558   2020.2

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    Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA-binding protein, which is highly expressed in skeletal muscle. Abnormally expanded CUG-repeats in the DMPK gene cause myotonic dystrophy type 1 (DM1) by sequestration of MBNL1 to nuclear RNA foci and by upregulation of another RNA-binding protein, CUG-binding protein 1 (CUGBP1). We previously reported that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone, upregulates MBNL1 expression in DM1 mouse model by demethylation of MeR2, an enhancer element in Mbnl1 intron 1. NSAIDs inhibit cyclooxygenase (COX), which is comprised of COX-1 and COX-2 isoforms. In this study, we screened 29 NSAIDs in C2C12 myoblasts, and found that 13 NSAIDs enhanced Mbnl1 expression, where COX-1-selective NSAIDs upregulated Mbnl1 more than COX-2-selective NSAIDs. Consistently, knockdown of COX-1, but not of COX-2, upregulated MBNL1 expression in C2C12 myoblasts and myotubes, as well as in myotubes differentiated from DM1 patient-derived induced pluripotent stem cells (iPSCs). Luciferase assay showed that COX-1-knockdown augmented the MeR2 enhancer activity. Furthermore, bisulfite sequencing analysis demonstrated that COX-1-knockdown suppressed methylation of MeR2. These results suggest that COX-1 inhibition upregulates Mbnl1 transcription through demethylation of the MeR2 enhancer. Taken together, our study provides new insights into the transcriptional regulation of Mbnl1 by the COX-1-mediated pathway.

    DOI: 10.1038/s41598-020-59517-y

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  18. Mianserin suppresses R-spondin 2-induced activation of Wnt/β-catenin signaling in chondrocytes and prevents cartilage degradation in a rat model of osteoarthritis. Reviewed International journal

    Toshiaki Okura, Bisei Ohkawara, Yasuhiko Takegami, Mikako Ito, Akio Masuda, Taisuke Seki, Naoki Ishiguro, Kinji Ohno

    Scientific reports   Vol. 9 ( 1 ) page: 2808 - 2808   2019.2

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    Aberrant activation of the Wnt/β-catenin signaling pathway promotes the progression of osteoarthritis (OA). We previously reported that R-spondin 2 (Rspo2), an activator of the Wnt/β-catenin signaling, facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification. However, the role of Rspo2 in OA remains elusive. Here, we showed that the amounts of Rspo2 protein in synovial fluid were increased in OA patients. We searched for a preapproved drug that suppresses Rspo2-induced Wnt/β-catenin signaling in chondrogenic cells and reduces joint pathology in a rat model of OA. In Rspo2-treated ATDC5 cells, mianserin, a tetracyclic antidepressant, inhibited Wnt/β-catenin signaling, increased proteoglycan production, and upregulated chondrogenic marker genes. Mianserin suppressed Rspo2-induced accumulation of β-catenin and phosphorylation of Lrp6. We identified that mianserin blocked binding of Rspo2 to its receptor Lgr5. We also observed that intraarticular administration of mianserin suppressed β-catenin accumulation and prevented OA progression in a rat model of OA. We conclude that mianserin suppresses abnormally activated Wnt/β-catenin signaling in OA by inhibiting binding of Rspo2 to Lgr5.

    DOI: 10.1038/s41598-019-39393-x

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  19. Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction. Reviewed International journal

    Jin Li, Mikako Ito, Bisei Ohkawara, Akio Masuda, Kinji Ohno

    Scientific reports   Vol. 8 ( 1 ) page: 13577 - 13577   2018.9

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    We recently reported that R-spondin 2 (Rspo2), a secreted activator of Wnt/β-catenin signaling, promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor, Lgr5. Rspo2 is expressed highly in spinal motor neurons (SMNs) and marginally in the skeletal muscle, but the origin of Rspo2 at the NMJ remains elusive. We rescued Rspo2-deficient (Rspo2-/-) mice by specifically expressing Rspo2 in the skeletal muscle and SMNs. SMN-specific Rspo2 mitigated or over-corrected abnormal features of the NMJs and AChR clusters observed in Rspo2-/- mice including (i) abnormal broadening of enlarged AChR clusters, (ii) three of six abnormal ultrastructural features, and (iii) abnormal expression of nine genes in SMNs and the diaphragm. In contrast, muscle-specific Rspo2 normalized all six abnormal ultrastructural features, but it had no effect on AChR clustering and NMJ formation at the light microscopy level or on abnormal gene expression in SMNs and the diaphragm. These results suggest that SMN-derived Rspo2 plays a major role in AChR clustering and NMJ formation in the postsynaptic region, and muscle-derived Rspo2 also plays a substantial role in juxtaposition of the active zones and synaptic folds.

    DOI: 10.1038/s41598-018-31949-7

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  20. Lack of Fgf18 causes abnormal clustering of motor nerve terminals at the neuromuscular junction with reduced acetylcholine receptor clusters. Reviewed

      ( 8 ) page: 434   2018.1

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    DOI: 10.1038/s41598-017-18753-5.

  21. Lack of Fgf18 causes abnormal clustering of motor nerve terminals at the neuromuscular junction with reduced acetylcholine receptor clusters. Reviewed International journal

    Kenyu Ito, Bisei Ohkawara, Hideki Yagi, Hiroaki Nakashima, Mikito Tsushima, Kyotaro Ota, Hiroyuki Konishi, Akio Masuda, Shiro Imagama, Hiroshi Kiyama, Naoki Ishiguro, Kinji Ohno

    Scientific reports   Vol. 8 ( 1 ) page: 434 - 434   2018.1

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:Nature Publishing Group  

    FGF receptor 2 is involved in the formation of the neuromuscular junction (NMJ), but its in vivo ligand remains to be determined. Laser capture microdissection of the mouse spinal motor neurons (SMNs) revealed that Fgf18 mRNA is highly expressed in SMNs in adults. Expression of Fgf18 mRNA was the highest in the spinal cord at embryonic day (E) 15.5, which gradually decreased to postnatal day 7. FGF18 protein was localized at the NMJs of the tibialis anterior muscle at E18.5 and in adults. Fgf18-/- mice at E18.5 showed decreased expressions of the NMJ-specific Chrne and Colq genes in the diaphragm. In Fgf18-/- diaphragms, the synaptophysin-positive areas at the nerve terminals and the acetylcholine receptor (AChR)-positive areas at the motor endplates were both approximately one-third of those in wild-type embryos. Fgf18-/- diaphragms ultrastructurally showed abnormal aggregation of multiple nerve terminals making a gigantic presynapse with sparse synaptic vesicles, and simplified motor endplates. In Fgf18-/- diaphragms, miniature endplate potentials were low in amplitude with markedly reduced frequency. In C2C12 myotubes, FGF18 enhanced AChR clustering, which was blocked by inhibiting FGFRs or MEK1. We propose that FGF18 plays a pivotal role in AChR clustering and NMJ formation in mouse embryogenesis.

    DOI: 10.1038/s41598-017-18753-5

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  22. Activated FGFR3 promotes bone formation via accelerating endochondral ossification in mouse model of distraction osteogenesis. Reviewed International journal

    Yusuke Osawa, Masaki Matsushita, Sachi Hasegawa, Ryusaku Esaki, Masahito Fujio, Bisei Ohkawara, Naoki Ishiguro, Kinji Ohno, Hiroshi Kitoh

    Bone   Vol. 105   page: 42 - 49   2017.12

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE INC  

    Achondroplasia (ACH) is one of the most common short-limbed skeletal dysplasias caused by gain-of-function mutations in the fibroblast growth factor receptors 3 (FGFR3) gene. Distraction osteogenesis (DO) is a treatment option for short stature in ACH in some countries. Although the patients with ACH usually show faster healing in DO, details of the newly formed bone have not been examined. We have developed a mouse model of DO and analyzed new bone regenerates of the transgenic mice with ACH (Fgfr3ach mice) histologically and morphologically. We established two kinds of DO protocols, the short-DO consisted of 5days of latency period followed by 5days of distraction with a rate of 0.4mm per 24h, and the long-DO consisted of the same latency period followed by 7days of distraction with a rate of 0.3mm per 12h. The callus formation was evaluated radiologically by bone fill score and quantified by micro-CT scan in both protocols. The histomorphometric analysis was performed in the short-DO protocol by various stainings, including Villanueva Goldner, Safranin-O/Fast green, tartrate-resistant acid phosphatase, and type X collagen. Bone fill scores were significantly higher in Fgfr3ach mice than in wild-type mice in both protocols. The individual bone parameters, including bone volume and bone volume/tissue volume, were also significantly higher in Fgfr3ach mice than in wild-type mice in both protocols. The numbers of osteoblasts, as well as osteoclasts, around the trabecular bone were increased in Fgfr3ach mice. Cartilaginous tissues of the distraction region rapidly disappeared in Fgfr3ach mice compared to wild-type mice during the consolidation phase. Similarly, type X collagen-positive cells were markedly decreased in Fgfr3ach mice during the same period. Fgfr3ach mice exhibited accelerated bone regeneration after DO. Accelerated endochondral ossification could contribute to faster healing in Fgfr3ach mice.

    DOI: 10.1016/j.bone.2017.05.016

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  23. Fluoxetine ameliorates cartilage degradation in osteoarthritis by inhibiting Wnt/β-catenin signaling. Reviewed International journal

    Kentaro Miyamoto, Bisei Ohkawara, Mikako Ito, Akio Masuda, Akihiro Hirakawa, Tadahiro Sakai, Hideki Hiraiwa, Takashi Hamada, Naoki Ishiguro, Kinji Ohno

    PloS one   Vol. 12 ( 9 ) page: e0184388   2017.9

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

    Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.

    DOI: 10.1371/journal.pone.0184388

    Web of Science

    PubMed

  24. SRSF1 suppresses selection of intron-distal 5' splice site of DOK7 intron 4 to generate functional full-length Dok-7 protein. Reviewed International journal

    Khalid Bin Ahsan, Akio Masuda, Mohammad Alinoor Rahman, Jun-Ichi Takeda, Mohammad Nazim, Bisei Ohkawara, Mikako Ito, Kinji Ohno

    Scientific reports   Vol. 7 ( 1 ) page: 10446 - 10446   2017.9

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Dok-7 is a non-catalytic adaptor protein that facilitates agrin-induced clustering of acetylcholine receptors (AChR) at the neuromuscular junction. Alternative selection of 5' splice sites (SSs) of DOK7 intron 4 generates canonical and frame-shifted transcripts. We found that the canonical full-length Dok-7 enhanced AChR clustering, whereas the truncated Dok-7 did not. We identified a splicing cis-element close to the 3' end of exon 4 by block-scanning mutagenesis. RNA affinity purification and mass spectrometry revealed that SRSF1 binds to the cis-element. Knocking down of SRSF1 enhanced selection of the intron-distal 5' SS of DOK7 intron 4, whereas MS2-mediated artificial tethering of SRSF1 to the identified cis-element suppressed it. Isolation of an early spliceosomal complex revealed that SRSF1 inhibited association of U1 snRNP to the intron-distal 5' SS, and rather enhanced association of U1 snRNP to the intron-proximal 5' SS, which led to upregulation of the canonical DOK7 transcript. Integrated global analysis of CLIP-seq and RNA-seq also indicated that binding of SRSF1 immediately upstream to two competing 5' SSs suppresses selection of the intron-distal 5' SS in hundreds of human genes. We demonstrate that SRSF1 critically regulates alternative selection of adjacently placed 5' SSs by modulating binding of U1 snRNP.

    DOI: 10.1038/s41598-017-11036-z

    Web of Science

    PubMed

  25. Wnt/β-catenin signaling suppresses expressions of Scx, Mkx, and Tnmd in tendon-derived cells. Reviewed International journal

    Yasuzumi Kishimoto, Bisei Ohkawara, Tadahiro Sakai, Mikako Ito, Akio Masuda, Naoki Ishiguro, Chisa Shukunami, Denitsa Docheva, Kinji Ohno

    PloS one   Vol. 12 ( 7 ) page: e0182051   2017.7

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

    After tendon injuries, biomechanical properties of the injured tendon are not fully recovered in most cases. Modulation of signaling pathways, which are involved in tendon development and tendon repair, is one of attractive modalities to facilitate proper regeneration of the injured tendon. The roles of TGF-β signaling in tendon homeostasis and tendon development have been elucidated. In contrast, the roles of Wnt/β-catenin signaling in tendon remain mostly elusive. We found that the number of β-catenin-positive cells was increased at the injured site, suggesting involvement of Wnt/β-catenin signaling in tendon healing. Activation of Wnt/β-catenin signaling suppressed expressions of tenogenic genes of Scx, Mkx, and Tnmd in rat tendon-derived cells (TDCs) isolated from the Achilles tendons of 6-week old rats. Additionally, activation of Wnt/β-catenin reduced the amounts of Smad2 and Smad3, which are intracellular mediators for TGF-β signaling, and antagonized upregulation of Scx induced by TGF-β signaling in TDCs. We found that Wnt/β-catenin decreased Mkx and Tnmd expressions without suppressing Scx expression in Scx-programmed tendon progenitors. Our studies suggest that Wnt/β-catenin signaling is a repressor for tenogenic gene expressions.

    DOI: 10.1371/journal.pone.0182051

    Web of Science

    PubMed

  26. Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms. Reviewed International journal

    Mohammad Nazim, Akio Masuda, Mohammad Alinoor Rahman, Farhana Nasrin, Jun-Ichi Takeda, Kenji Ohe, Bisei Ohkawara, Mikako Ito, Kinji Ohno

    Nucleic acids research   Vol. 45 ( 3 ) page: 1455 - 1468   2017.2

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

    DOI: 10.1093/nar/gkw823

    Web of Science

    PubMed

  27. Agrin-LRP4-MuSK signaling as a therapeutic target for myasthenia gravis and other neuromuscular disorders. Reviewed International journal

    Kinji Ohno, Bisei Ohkawara, Mikako Ito

    Expert opinion on therapeutic targets   Vol. 21 ( 10 ) page: 949 - 958   2017

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:TAYLOR & FRANCIS LTD  

    INTRODUCTION: Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including myasthenia gravis, Lambert-Eaton myasthenic syndrome, Isaacs' syndrome, congenital myasthenic syndromes, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. Except for sarcopenia, all are orphan diseases. In addition, the NMJ signal transduction is impaired by tetanus, botulinum, curare, α-bungarotoxin, conotoxins, organophosphate, sarin, VX, and soman to name a few. Areas covered: This review covers the agrin-LRP4-MuSK signaling pathway, which drives clustering of acetylcholine receptors (AChRs) and ensures efficient signal transduction at the NMJ. We also address diseases caused by autoantibodies against the NMJ molecules and by germline mutations in genes encoding the NMJ molecules. Expert opinion: Representative small compounds to treat the defective NMJ signal transduction are cholinesterase inhibitors, which exert their effects by increasing the amount of acetylcholine at the synaptic space. Another possible therapeutic strategy to enhance the NMJ signal transduction is to increase the number of AChRs, but no currently available drug has this functionality.

    DOI: 10.1080/14728222.2017.1369960

    Web of Science

    PubMed

  28. LRP4 third β-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner. Reviewed

    Ohkawara B1, Cabrera-Serrano M, Nakata T, Milone M, Asai N, Ito K, Ito M, Masuda A, Ito Y, Engel AG, Ohno K.

    Hum Mol Genet.   Vol. 23 ( 7 ) page: 1856-68   2014.4

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    Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)  

▼display all

Presentations 4

  1. 神経筋接合部形成における細胞外分泌因子の役割 Invited

    大河原 美静

    第6回日本筋学会学術集会  2020.12.19 

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    Event date: 2020.12

    Presentation type:Symposium, workshop panel (nominated)  

  2. Wnt/beta-catenin signaling in chondrocytes for cartilage degradation of osteoarthritis Invited International conference

    Bisei Ohkawara

    The Molecular Biology Society of Japan  2018.11 

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    Language:English   Presentation type:Symposium, workshop panel (nominated)  

  3. Mianserin suppresses R-spondin 2-induced activation of Wnt/β-catenin signaling in chondrocytes and prevents cartilage degradation in a rat model of osteoarthritis International conference

    Bisei Ohkawara

    European Wnt Meeting 2018  2018.9 

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    Language:English   Presentation type:Oral presentation (general)  

  4. CTGF binds to LRP4 to ameliorate NMJ formation International conference

    Bisei Ohkawara

    Gurdon Conference, Wnt meeting  2017.8 

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    Language:English   Presentation type:Poster presentation  

KAKENHI (Grants-in-Aid for Scientific Research) 11

  1. Elucidation of molecular mechanisms of neuromuscular junction formations in physiology and pathology

    Grant number:20H03561  2020.4 - 2023.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

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    Authorship:Coinvestigator(s) 

  2. Pathological analysis of limb girdle congenital myasthenia and development of new therapy

    Grant number:20K06925  2020.4 - 2023.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

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    Authorship:Coinvestigator(s) 

  3. Wnt/βカテニン・ADAM10阻害による新規作用機序の変性関節症治療薬の開発

    Grant number:19H03779  2019.4 - 2022.3

    科学研究費助成事業  基盤研究(B)

    石黒 直樹, 高橋 伸典, 水野 正明, 平岩 秀樹, 大野 欽司, 大河原 美静

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    Wntシグナルの抑制は関節炎の抑制効果など軟骨保護に加えて複合的な効果が期待でき、関節治療薬の開発が期待できる。一方、ヒアルロン酸の主たる受容体であるCD44のADAM10による断片化がOA軟骨において観察され,CD44断片化は細胞外マトリックス形成を阻害し、断片化の結果生成されたICD(intra cellular domain)が生理活性を持ち、MMPなど分解系酵素や炎症性サイトカイン産生に働き関節破壊を招来する。ADAM10阻害はCD44断片化抑制を介して、OA治療に応用可能である。ADAM10の阻害活性を持つ化合物をDrug repositioning により見出すことを目標とする。
    ①Wnt/βカテニン阻害によるOA治療薬の開発
    昨年に引き続き、1186種のFDA既認可薬を用い、Wntシグナル抑制効果のある薬剤のスクリーニングを行った。既に見出しているRaloxifeneを含めて一貫性のある結果が得られず、再現性に問題のある化合物ばかりであった。これについての考察を行い細胞種による違い、特に軟骨細胞ではWntとDDK-1の活性変化が刺激物質や培養条件(passage等)により異なる挙動を示す可能性を考えた。そこで、従来から着目していたRaloxifeneの軟骨保護作用がWnt/βカテニン阻害作用のみで説明出来ない可能性が示唆された。検討継続の可否について研究者間での議論している。
    ②ADAM10阻害によるOA治療薬開発
    ADAM10 knock out mouseの入手がCovid-19感染症の関係で想定以上に遅れたので実験開始がずれ込んだ。ADAM10阻害効果の陽性対照モデルとして本knock out mouseを使用して、動物実験モデルの最適化を行っている。通常の靭帯断裂モデルは関節軟骨の急性破壊モデルとなるために、十分にCD44断片化モデルには適さない。加齢による慢性的な軟骨破壊を近似するモデルの構築を目的に運動負荷状態での老齢化mouseモデルを検討した。対象に比較してADAM10knock out mouseは軟骨変性の抑制が確認された。ADAM10酵素活性抑制とmRNA発現抑制の両面からスクリーニングを行っている。動物実験モデルは完成したので、今後は速やかに進むと考えている。
    理由の一つにCOVID19感染症により大学からの要請により研究活動を縮小したことがある。それに加えて以下の個別の問題があり、予定通り伸展しなかった。
    ①Wnt/βカテニン阻害によるOA治療薬の開発
    新規の化合物発見を期待したが、出来ていない。過去にWnt/βカテニン経路の阻害活性を確認した物質を含めて構造展開の可能性を検討している。スクリーニングを繰り返しても候補化合物の発見に至らず、既存化合物の転用を計るdrug repositioning戦略の限界があり、戦術の見直しが必要である。同様の作用を持つ化合物で臨床試験を始めている製薬企業の進捗状況を注目している。方向性の見直しが必要となる可能性を含め、共同研究者と協議中である。
    ②ADAM10阻害によるOA治療薬開発
    ADAM10阻害による関節軟骨変性予防効果を判定するための動物モデルが完成した。今後はCD44断片化抑制を指標とした酵素活性評価とADMA10mRNA発現抑制の両面から化合物を探索する予定である。既にsimvastatin を確認しているが、次期の標的化合物の候補は複数確認されており、これを動物実験に展開する予定である。しかしながら動物実験には運動負荷、老齢化モデルを用いるので効率が悪く、結果を得るのに時間がかかった。
    ①Wnt/βカテニン阻害によるOA治療薬の開発
    米国で先行しているWnt/βカテニン阻害薬は全身的影響を考慮して、関節内注射薬として開発されているが、2相試験の結果は微妙なものであった。これから分かることは、強めのWnt/βカテニン阻害効果を持つと思われる物質も関節内で有意な効果が発揮出来ない可能性である。この結果は我々の知見とも一致する。研究期間内に終了する目途が立たないので全ての研究資源を②ADAM10阻害によるOA治療薬開発に振り分けることとする。
    ②ADAM10阻害によるOA治療薬開発
    候補化合物の動物実験による前臨床POC確立をめざす。既に候補化合物の目星はついているので、速やかに動物実験を開始出来るものと思っている。これによりCD44断片化抑制という全く新しい治療戦略の可能性が示せるものと期待している。年度内での前臨床POC確立に至るように全ての研究資源を本研究課題に振り分ける。

  4. 神経筋接合部における細胞外分泌因子の同定

    Grant number:18K06483  2018.4 - 2022.3

    科学研究費助成事業  基盤研究(C)

    大河原 美静

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    Authorship:Principal investigator 

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    神経筋接合部(NMJ)は運動神経と筋肉細胞を結ぶシナプスで、シナプスの信号伝達にはアセチルコリンとその受容体であるアセチルコリン受容体(AChR)を利用している。よって、シナプスの信号を効率よく伝達するために、筋肉側のシナプス領域(終板)でAChRが集積することが必要である。現在までにこのAChR集積に必要な細胞外分泌因子が20個弱知られているものの、未知の重要なたんぱく質はそれ以上あることが想像されている。昨年度までに、研究代表者は培養条件下で分化した筋肉細胞を用い、AChRが集積する領域に存在する細胞外のたんぱく質を特異的に特定する手法を確立し、AChRのサブユニットとMuSKやLRP4などのいくつかのタンパク質が共局在している事を明らかにした。今年度は、この方法を用いて、細胞外分泌因子中で筋肉から分泌されAChRが集積する領域に存在しAChR集積に必須の因子を多く同定することを目指している。 今年度の実施計画では、発生過程におけるマウス筋肉組織においてAChRのサブユニットと同時かつ同じ核で発現しているmRNAをデータセット化し、同定することで、AChRと共局在する可能性のあるたんぱく質の候補リストを作成した。現在は、この候補リストの中から、実際にAChRとたんぱく質レベルで共局在するたんぱく質をウェスタンブロッティングと免疫染色により同定することを行っている。また、筋肉細胞と神経細胞を共培養することで作製したin vitro NMJの作製も行い、一部の細胞外分泌因子の結果については、論文において報告を行った。
    作製したデータセットでは、細胞外分泌因子のmRNA量が少ないことが理由で、発現量の詳細があいまいであった。そこで、現在は発生段階を追って、NMJ領域に必要な既知のmRNAを発現している核を同定し、その核での細胞外分泌因子の発現量を細かく検討している。再検討自体はスムーズに行えたため「やや遅れている」とした。
    昨年度行う予定だった、①データセットよりわかる既知の因子の局在を検討する、に引き続き、②Rspo2、FGF18などの分泌因子と共に関わる細胞外分泌因子を同定という計画を今年度中に実施する予定である。培養細胞を用いたウェスタンブロットや細胞免疫染色を取り急ぎ行うことでAChR集積に 関わる複数の因子の役割を明らかにしていきたい。

  5. Pathological analysis of GFPT1 congenital myasthenia and development of new therapy using iPS cells.

    Grant number:17K07094  2017.4 - 2021.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    Ito Mikako

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    Authorship:Coinvestigator(s) 

    One of the causative molecules of congenital myasthesis syndrome (CMS) is a mutation in GFPT1, a glycosylation enzyme of proteins and lipids, which is characterized by limb-type muscle weakness. GFPT1-L isoform is formed in skeletal muscle and enzyme activity is reduced. In this study, in order to clarify the role of GFPT1-L in skeletal muscle, model mouse Gfpt1-L-/-was prepared and phenotypic analysis was performed.
    In addition, we identified the GFPT1 c.722 ^ 723insG mutation in CMS patients and prepared a model mouse of the mutation. The mice were decreased motor function, decreased amount of glycosylated protein, fragmentation of AChR cluster, muscle atrophy, and increased interstitial space. of muscle fiber. It showed a patient-like phenotype.

  6. 成体の神経筋接合部シナプスの維持におけるCcnファミリー因子の役割

    Grant number:21K06392  2021.4 - 2024.3

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    大河原 美静

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    Authorship:Principal investigator 

    Grant amount:\4160000 ( Direct Cost: \3200000 、 Indirect Cost:\960000 )

    神経筋接合部(NMJ)は運動神経と筋肉を結ぶシナプスである。本研究ではこのシナプスにおけるCcnファミリー因子、6つのシナプスにおける役割や運動能力における役割を検討する。Ccnファミリー因子の機能がNMJに必要であるならば、今後Ccnファミリー因子を標的としたGene Therapyも視野に入れたい。Ccnファミリー因子が結合するであろうLrp4が中枢神経系の神経同士のシナプスでも機能していることから、Ccnの神経同士のシナプスへの役割をも想像したい。

  7. 神経筋接合部における細胞外分泌因子の同定

    2018.4 - 2021.3

    学術研究助成基金助成金  基盤研究C 

    大河原 美静

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    Authorship:Principal investigator  Grant type:Competitive

  8. 神経筋接合部の機能改善を標的としたサルコペニア治療薬の探索

    Grant number:18K09026  2018.4 - 2020.3

    石井 久雄

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    先行研究にてAgrin-LRP4-MuSKシグナリングの促進効果を認めた40種類の1次候補薬を用いて、マウス筋芽細胞株C2C12の誘導筋管におけるアセチルコリンレセプター凝集試験を行い、促進作用を示した上位6剤を2次候補薬として2次スクリーニングを行った。C2C12細胞を培養し筋管を形成した後、neural agrinとともに2次候補薬を添加し、抗MuSK抗体を用いた免疫沈降後Western blottingを行い、抗リン酸化tyrosine抗体を用いてリン酸化MuSKの発現量を、投与後30分、1時間、3時間、6時間、24時間の5段階で定量評価し、MuSKのリン酸化効果を最も認めた最終候補薬剤1種(Drug A)を選定した。また同時に得られたWhole cell lysateを用いてWestern blotting法でDok7のリン酸化作用を評価した。Drug AはMuSKのリン酸化作用を有する一方でDok7のリン酸化においては抑制的に働くことが判明した。
    次いで名古屋大学神経遺伝情報学教室で作成したchrne(AChRεサブニユット)欠損マウスに対し、体重1gあたり200μgのDrug Aを0.5%メチルセルロースに溶解して出生後4週から28日間連続投与を行い、対照群(0.5%メチルセルロース投与群)と体重の推移、回転ホイールを用いて測定した1日運動量、生存日数を比較した。出生後AChRのγサブユニットがεサブユニットに置換されるためchrne欠損マウスは出生後8週~12週程度で死亡するが、薬剤の投与により運動機能の維持や生命予後の改善が期待されたものの、本試験では薬剤の有効性を認めなかった。
    最終年度に予定していた老齢マウス(24月齢C57BL/6マウス)での試験の準備のため、chrne欠損マウス試験と並行して、老齢モデルマウス作成のためのマウスの飼育を行った。

  9. ドラッグリポジショニング戦略によるR-spondin2(Respo2)を標的とした変形性関節症の治療薬の探索

    2016.4 - 2017.3

    日本医学会総会記念医学振興基金  研究助成 

    大河原 美静

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    Authorship:Principal investigator  Grant type:Competitive

  10. The roles of growth factors in neuromuscular junction

    Grant number:15K06707  2015.4 - 2019.3

    Ohkawara Bisei

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    Authorship:Principal investigator 

    Grant amount:\5070000 ( Direct Cost: \3900000 、 Indirect Cost:\1170000 )

    The neuromuscular junction (NMJ) is a synapse that connects the spinal motor and skeletal muscle fiber. Factors involved in NMJ morphogenesis and maturation has not been yet elucidated, and the mechanism of the known factors in NMJ has not been fully elucidated.
    In this study, we investigated to identify growth factors involved in NMJ formation and signal transduction pathways downstream of them. We showed that the secretion factors such as Rspo2 and FGF18 are required for NMJ function to activate MuSK activation and/or AChR. We also showed that these factors are involved in the maturation and maintenance of NMJ. In addition, we suggested that IGF, TGFbeta and hedgehog may be involved in NMJ formation.

  11. 神経筋接合部における増殖因子の役割

    2015.4 - 2018.3

    学術研究助成基金助成金  基盤研究C 

    大河原 美静

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    Authorship:Principal investigator  Grant type:Competitive

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Social Contribution 1

  1. 伊勢志摩サミット 通訳ボランティア

    Role(s):Organizing member