Elucidation of molecular mechanisms of defective neuromuscular signal transmission as a primary cause of sarcopenia

2020 Fiscal Year Research-status Report

• Project/Area Number: 19K22802
• Research Institution: Nagoya University
• Principal Investigator: 大野 欽司 名古屋大学, 医学系研究科, 教授 (80397455)
• Project Period (FY): 2019-06-28 – 2022-03-31
• Keywords: サルコペニア / 廃用性筋萎縮 / 神経筋接合部 / リボタグマウス / 遺伝子発現プロファイル

Outline of Annual Research Achievements

サルコペニアにおける神経筋接合部(NMJ)信号伝達障害が近年注目されており、廃用性筋萎縮を含めてNMJ信号伝達障害はこれら筋萎縮病態の一次的な原因である可能性が示されている。サルコペニアモデルマウスの脊髄運動神経細胞(SMN)とNMJ筋終板において、細胞特異的なリボゾームタンパクRPL22へのHAタグの付加を可能にするRiboTagマウスを用いた網羅的遺伝子発現プロファイリング解析を行い、サルコペニアにより発現が変動するSMN分子・筋終板分子を同定し、iPS細胞から構築をしたin vitro NMJならびにノックアウトマウスによる機能解析に繋げることを目的とした。
令和2年度に細胞特異的なリボゾームタンパクRPL22へのHAタグの付加を可能にするRiboTagマウスを用いたNMJ筋終板の網羅的な遺伝子発現プロファイリング解析を論文にまとめて報告した(Huang et al. Front Mol Neurosci 2020, 13: 154)。サルコペニアにおけるSMNとNMJ筋終板の網羅的な遺伝子発現解析を目的として、サルコペニアモデルマウスとRiboTagマウスのかけ合わせたマウスの脊髄と骨格筋のRNA-seq解析を現在行なっている。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

細胞特異的なリボゾームタンパクRPL22へのHAタグの付加を可能にするRiboTagマウスを用いたNMJ筋終板の網羅的な遺伝子発現プロファイリングを明らかにし、サルコペニアモデルマウスへの展開を開始できた。

Strategy for Future Research Activity

サルコペニアにより発現が変動するSNM分子・NMJ筋終板分子を明らかにする。その機能解析を行うために、CRISPR/Cas9によるiPS細胞のノックアウト実験系を確立を行なった。iPS細胞から作成するin vitro神経筋接合部の構築実験を開始しており、この系を使ってサルコペニアのNMJ病態解明を今後さらに進めていく。

Causes of Carryover

RNA-seq解析の低価格下などによりRiboTagマウスを用いた骨格筋特異的・神経筋接合部特異的・脊髄前角細胞特異的な遺伝子発現プロファイル解析が予定よりも安価に行うことができた。iPS細胞培養系・筋分化誘導系に当初の予想よりも高価な試薬が必要であることが判明し令和3年度への予算の繰越を行った。

Research Products

• [Journal Article] Screening of signaling pathways for agrin-induced AChR clustering in C2C12 myotubes (2022)
  • Author(s): Noto K, Ohkawara B, Ishii H, Ito M, Masuda A, Hirata H, Ohno K.
  • Journal Title: Genes Cells Volume: - Pages: -
  • Peer Reviewed
• [Journal Article] Secreted Signaling Molecules at the Neuromuscular Junction in Physiology and Pathology (2021)
  • Author(s): Ohkawara Bisei、Ito Mikako、Ohno Kinji
  • Journal Title: International Journal of Molecular Sciences Volume: 22 Pages: 2455～2455
  • DOI: 10.3390/ijms22052455
  • Peer Reviewed / Open Access
• [Journal Article] Efficacy of salbutamol monotherapy in slow‐channel congenital myasthenic syndrome caused by a novel mutation in CHRND (2021)
  • Author(s): Tawara Nozomu、Yamashita Satoshi、Takamatsu Koutaro、Yamasaki Yoshimune、Mukaino Akihiro、Nakane Shunya、Farshadyeganeh Paniz、Ohno Kinji、Ando Yukio
  • Journal Title: Muscle & Nerve Volume: 63 Pages: in press
  • DOI: 10.1002/mus.27166
  • Peer Reviewed
• [Journal Article] Hierarchical non-negative matrix factorization using clinical information for microbial communities (2021)
  • Author(s): Abe Ko、Hirayama Masaaki、Ohno Kinji、Shimamura Teppei
  • Journal Title: BMC Genomics Volume: 22 Pages: 104～104
  • DOI: 10.1186/s12864-021-07401-y
  • Peer Reviewed / Open Access
• [Journal Article] tRIP‐seq reveals repression of premature polyadenylation by co‐transcriptional FUS‐U1 snRNP assembly (2020)
  • Author(s): Masuda Akio、Kawachi Toshihiko、Takeda Jun‐ichi、Ohkawara Bisei、Ito Mikako、Ohno Kinji
  • Journal Title: EMBO reports Volume: 21 Pages: e49890
  • DOI: 10.15252/embr.201949890
  • Peer Reviewed / Open Access
• [Journal Article] Congenital myasthenic syndrome?associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner (2020)
  • Author(s): Ohkawara Bisei、Shen XinMing、Selcen Duygu、Nazim Mohammad、Bril Vera、Tarnopolsky Mark A.、Brady Lauren、Fukami Sae、Amato Anthony A.、Yis Uluc、Ohno Kinji、Engel Andrew G.
  • Journal Title: JCI Insight Volume: 5 Pages: e132023
  • DOI: 10.1172/jci.insight.132023
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Meta‐Analysis of Gut Dysbiosis in Parkinson's Disease (2020)
  • Author(s): Nishiwaki Hiroshi、Ito Mikako、Ishida Tomohiro、Hamaguchi Tomonari、Maeda Tetsuya、Kashihara Kenichi、Tsuboi Yoshio、Ueyama Jun、Shimamura Teppei、Mori Hiroshi、Kurokawa Ken、Katsuno Masahisa、Hirayama Masaaki、Ohno Kinji
  • Journal Title: Movement Disorders Volume: 35 Pages: 1626～1635
  • DOI: 10.1002/mds.28119
  • Peer Reviewed / Open Access
• [Journal Article] InMeRF: prediction of pathogenicity of missense variants by individual modeling for each amino acid substitution (2020)
  • Author(s): Takeda Jun-ichi、Nanatsue Kentaro、Yamagishi Ryosuke、Ito Mikako、Haga Nobuhiko、Hirata Hiromi、Ogi Tomoo、Ohno Kinji
  • Journal Title: NAR Genomics and Bioinformatics Volume: 2 Pages: lqaa038
  • DOI: 10.1093/nargab/lqaa038
  • Peer Reviewed
• [Journal Article] CTGF/CCN2 facilitates LRP4‐mediated formation of the embryonic neuromuscular junction (2020)
  • Author(s): Ohkawara Bisei、Kobayakawa Akinori、Kanbara Shunsuke、Hattori Takako、Kubota Satoshi、Ito Mikako、Masuda Akio、Takigawa Masaharu、Lyons Karen M、Ishiguro Naoki、Ohno Kinji
  • Journal Title: EMBO reports Volume: 21 Pages: e48462
  • DOI: 10.15252/embr.201948462
  • Peer Reviewed / Open Access
• [Journal Article] Zonisamide ameliorates progression of cervical spondylotic myelopathy in a rat model (2020)
  • Author(s): Kanbara Shunsuke、Ohkawara Bisei、Nakashima Hiroaki、Ohta Kyotaro、Koshimizu Hiroyuki、Inoue Taro、Tomita Hiroyuki、Ito Mikako、Masuda Akio、Ishiguro Naoki、Imagama Shiro、Ohno Kinji
  • Journal Title: Scientific Reports Volume: 10 Pages: 13138～13138
  • DOI: 10.1038/s41598-020-70068-0
  • Peer Reviewed / Open Access
• [Journal Article] Gene Expression Profile at the Motor Endplate of the Neuromuscular Junction of Fast-Twitch Muscle (2020)
  • Author(s): Huang Kun、Li Jin、Ito Mikako、Takeda Jun-Ichi、Ohkawara Bisei、Ogi Tomoo、Masuda Akio、Ohno Kinji
  • Journal Title: Frontiers in Molecular Neuroscience Volume: 13 Pages: 154～154
  • DOI: 10.3389/fnmol.2020.00154
  • Peer Reviewed / Open Access
• [Journal Article] Short-Chain Fatty Acid-Producing Gut Microbiota Is Decreased in Parkinson’s Disease but Not in Rapid-Eye-Movement Sleep Behavior Disorder (2020)
  • Author(s): Nishiwaki Hiroshi、Hamaguchi Tomonari、Ito Mikako、Ishida Tomohiro、Maeda Tetsuya、Kashihara Kenichi、Tsuboi Yoshio、Ueyama Jun、Shimamura Teppei、Mori Hiroshi、Kurokawa Ken、Katsuno Masahisa、Hirayama Masaaki、Ohno Kinji
  • Journal Title: mSystems Volume: 5 Pages: e00797-20
  • DOI: 10.1128/mSystems.00797-20
  • Peer Reviewed / Open Access
• [Journal Article] Identification of Qk as a Glial Precursor Cell Marker that Governs the Fate Specification of Neural Stem Cells to a Glial Cell Lineage (2020)
  • Author(s): Takeuchi Akihide、Takahashi Yuji、Iida Kei、Hosokawa Motoyasu、Irie Koichiro、Ito Mikako、Brown J.B.、Ohno Kinji、Nakashima Kinichi、Hagiwara Masatoshi
  • Journal Title: Stem Cell Reports Volume: 15 Pages: 883～897
  • DOI: 10.1016/j.stemcr.2020.08.010
  • Peer Reviewed / Open Access
• [Journal Article] Zonisamide ameliorates neuropathic pain partly by suppressing microglial activation in the spinal cord in a mouse model (2020)
  • Author(s): Koshimizu Hiroyuki、Ohkawara Bisei、Nakashima Hiroaki、Ota Kyotaro、Kanbara Shunsuke、Inoue Taro、Tomita Hiroyuki、Sayo Akira、Kiryu-Seo Sumiko、Konishi Hiroyuki、Ito Mikako、Masuda Akio、Ishiguro Naoki、Imagama Shiro、Kiyama Hiroshi、Ohno Kinji
  • Journal Title: Life Sciences Volume: 263 Pages: 118577～118577
  • DOI: 10.1016/j.lfs.2020.118577
  • Peer Reviewed
• [Presentation] InMeRF: A set of random forest models for each amino acid substitution to predict pathogenicity of missense variants in the human genome (2020)
  • Author(s): Takeda J, Nanatsue K, Yamagishi R, Ito M, Ohno K
  • Organizer: European Society of Human Genetics Virtual Conference
  • Int'l Joint Research