筋衛星細胞における機能維持メカニズムのグローバル解析

• Project/Area Number: 21K06961
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 49030:Experimental pathology-related
• Research Institution: National Center of Neurology and Psychiatry
• Principal Investigator: 林 晋一郎 国立研究開発法人国立精神・神経医療研究センター, 神経研究所 疾病研究第一部, 室長 (10732381)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Project Status: Granted (Fiscal Year 2022)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2023: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2022: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2021: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: 骨格筋 / 筋幹細胞 / Pax7 / 骨格筋幹細胞 / 筋分化 / 筋衛星細胞

Outline of Research at the Start

骨格筋は優れた再生能力を有しており、骨格筋中の組織幹細胞である筋衛星細胞がその中心的な役割を担う。筋衛星細胞はPax7と呼ばれる転写因子を発現し、Pax7は筋衛星細胞の機能維持に必須であることが知られているが、その標的因子やPax7と相互作用して転写活性を調節するタンパク質などは殆ど明らかではない。本研究では新たに作成したタグノックインマウスを活用し、これまで不明であったPax7の標的遺伝子や転写共役因子等を明らかにすることで筋衛星細胞の機能維持機構を解明を目指す。

Outline of Annual Research Achievements

筋ジストロフィーの根本的治療法の一つとして、骨格筋幹細胞(筋衛星細胞)を用いた再生医学的治療法が挙げられる。移植された筋幹細胞は増殖・分化・融合 して筋再生に寄与するだけでなく、その一部は移植先の筋組織内で再び休止状態となり、組織幹細胞として定着することができる。しかしながら生体外で幹細胞 機能を維持したまま増幅させることが困難なことや移植後の定着率の低さなど乗り越えるべき課題も多く、実現に至っていない。Pax7は筋幹細胞で発現する転写因子であり、筋幹細胞の機能維持に必須であるが、その共役因子、標的因子、および発現制御機構については殆ど明らかにされていない。そこで、本研究では、 申請者が作製したPax7にHAタグを付加したノックインマウス(Pax7-HA KIマウス)を活用し、筋幹細胞におけるPax7の転写調節共役因子および標的因子を明らかにすることで骨格筋幹細胞の機能調節・機能維持メカニズムを明らかにすることを目的とした。今年度はPax7-HA KIホモ胚（E12.5）の体節を用いて、 抗HA抗体を用いた免疫沈降-質量分析法によりPax7結合タンパク質を網羅的に解析した。その結果、クロマチン制御因子を含むこれまで報告のない324種のタンパク 質を同定した。さらに、昨年度行ったPax7-HA KIホモマウス由来の筋幹細胞を用いた免疫沈降-質量分析法の結果を合わせて解析し、両者で共通するタンパク質を34種同定した。本研究の進展に伴い、骨格筋幹細胞の増殖・分化制御、筋形成の全容が解明されるとともに、その成果が再生医療へと応用されることが強く期待される。

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

抗HA抗体を用いた免疫沈降-質量分析法により特定したタンパク質の確認のため、免疫沈降-ウェスタンブロット法による確認を行ったが、現在までにPax7との結合が見られたものは得られていない。この理由として、①Pax7-HAの発現量が少なく、高感度の免疫沈降-質量分析法ではバックグラウンドを拾っていた可能性があること、②購入したPax7共役因子候補タンパク質の抗体がうまく反応していないことが考えられる。また、単離したPax7-HA筋幹細胞で抗HA抗体を用いたChIP-seqを行っているが、免疫沈降得られるDNA量が充分でなくシークエンスを行える状況にない。

Strategy for Future Research Activity

抗HA抗体を用いた免疫沈降-ウェスタンブロット法による解析については、新たな抗体を購入し再度解析を行う。Pax7との結合が確認でき次第、過剰発現およびsiRNAによる発現抑制をした時の筋幹細胞の増殖・分化を解析する。また、E10.5-E12.5胚および筋幹細胞において、免疫蛍光染色により当該因子とPax7との共局在を解析する。ChIP-seqに関しては微量のDNAからライブラリ作製可能なキットを用いて行うことを検討している。

Research Products

• [Journal Article] Distinctive chaperonopathy in skeletal muscle associated with the dominant variant in DNAJB4 (2022)
  • Author(s): Inoue Michio、Noguchi Satoru、Inoue Yukiko U.、Iida Aritoshi、Ogawa Megumu、Bengoechea Rocio、Pittman Sara K.、Hayashi Shinichiro、Watanabe Kazuki、Hosoi Yasushi、Sano Terunori、Takao Masaki、Oya Yasushi、Takahashi Yuji、Miyajima Hiroaki、Weihl Conrad C.、Inoue Takayoshi、Nishino Ichizo
  • Journal Title: Acta Neuropathologica Volume: 145 Issue: 2 Pages: 235-255
  • DOI: 10.1007/s00401-022-02530-4
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy (2022)
  • Author(s): Okubo M, Noguchi S, Awaya T, Hosokawa M, Tsukui N, Ogawa M, Hayashi S, Komaki H, Mori-Yoshimura M, Oya Y, Takahashi Y, Fukuyama T, Funato M, Hosokawa Y, Kinoshita S, Matsumura T, Nakamura S, Oshiro A, Terashima H, Nagasawa T, Sato T, Shimada Y, Tokita Y, Hagiwara M, Ogata K, Nishino I
  • Journal Title: Human Genetics Volume: 142 Issue: 1 Pages: 59-71
  • DOI: 10.1007/s00439-022-02485-2
  • Peer Reviewed
• [Journal Article] Multidimensional analyses of the pathomechanism caused by the non-catalytic GNE variant, c.620A>T, in patients with GNE myopathy (2022)
  • Author(s): Yoshioka Wakako、Iida Aritoshi、Sonehara Kyuto、Yamamoto Kazuki、Oya Yasushi、Mori-Yoshimura Madoka、Kurashige Takashi、Okubo Mariko、Ogawa Megumu、Matsuda Fumihiko、Higasa Koichiro、Hayashi Shinichiro、Nakamura Harumasa、Sekijima Masakazu、Okada Yukinori、Noguchi Satoru、Nishino Ichizo
  • Journal Title: Scientific Reports Volume: 12 Issue: 1 Pages: 21806-21806
  • DOI: 10.1038/s41598-022-26419-0
  • Peer Reviewed / Open Access
• [Journal Article] Intranuclear inclusions in muscle biopsy can differentiate oculopharyngodistal myopathy and oculopharyngeal muscular dystrophy (2022)
  • Author(s): Ogasawara Masashi、Eura Nobuyuki、Iida Aritoshi、Kumutpongpanich Theerawat、Minami Narihiro、Nonaka Ikuya、Hayashi Shinichiro、Noguchi Satoru、Nishino Ichizo
  • Journal Title: Acta Neuropathologica Communications Volume: 10 Issue: 1 Pages: 176-176
  • DOI: 10.1186/s40478-022-01482-w
  • Peer Reviewed / Open Access
• [Journal Article] Simultaneous measurement of the size and methylation of chromosome 4qA-D4Z4 repeats in facioscapulohumeral muscular dystrophy by long-read sequencing (2022)
  • Author(s): Hiramuki Yosuke、Kure Yuriko、Saito Yoshihiko、Ogawa Megumu、Ishikawa Keiko、Mori-Yoshimura Madoka、Oya Yasushi、Takahashi Yuji、Kim Dae-Seong、Arai Noriko、Mori Chiaki、Matsumura Tsuyoshi、Hamano Tadanori、Nakamura Kenichiro、Ikezoe Koji、Hayashi Shinichiro、Goto Yuichi、Noguchi Satoru、Nishino Ichizo
  • Journal Title: Journal of Translational Medicine Volume: 20 Issue: 1 Pages: 517-517
  • DOI: 10.1186/s12967-022-03743-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] RILPL1-related OPDM is absent in a Japanese cohort (2022)
  • Author(s): Eura Nobuyuki、Iida Aritoshi、Ogasawara Masashi、Hayashi Shinichiro、Noguchi Satoru、Nishino Ichizo
  • Journal Title: The American Journal of Human Genetics Volume: 109 Issue: 11 Pages: 2088-2089
  • DOI: 10.1016/j.ajhg.2022.10.005
  • Peer Reviewed / Open Access
• [Journal Article] Intranuclear inclusions in skin biopsies are not limited to neuronal intranuclear inclusion disease but can also be seen in oculopharyngodistal myopathy (2022)
  • Author(s): Ogasawara Masashi,Eura Nobuyuki,Nagaoka Utako,Sato Tatsuro,Arahata Hajime,Hayashi Tomohiro,Okamoto Tomoko,Takahashi Yuji,Mori‐Yoshimura Madoka,Oya Yasushi,Nakamura Akinori,Shimazaki Rui,Sano Terunori,Kumutpongpanich Theerawat,Minami Narihiro,Hayashi Shinichiro,Noguchi Satoru,Iida Aritoshi,Takao Masaki,Nishino Ichizo
  • Journal Title: Neuropathology and Applied Neurobiology Volume: 48 Issue: 3
  • DOI: 10.1111/nan.12787
  • Peer Reviewed
• [Journal Article] Deep convolutional neural network-based algorithm for muscle biopsy diagnosis (2021)
  • Author(s): Kabeya Yoshinori,Okubo Mariko,Yonezawa Sho,Nakano Hiroki,Inoue Michio,Ogasawara Masashi,Saito Yoshihiko,Tanboon Jantima,Indrawati Luh Ari,Kumutpongpanich Theerawat,Chen Yen-Lin,Yoshioka Wakako,Hayashi Shinichiro,Iwamori Toshiya,Takeuchi Yusuke,Tokumasu Reitaro,Takano Atsushi,Matsuda Fumihiko,Nishino Ichizo
  • Journal Title: Laboratory Investigation Volume: 102 Issue: 3 Pages: 220-226
  • DOI: 10.1038/s41374-021-00647-w
  • Peer Reviewed
• [Journal Article] Dermatomyositis (2021)
  • Author(s): Tanboon Jantima、Inoue Michio、Saito Yoshihiko、Tachimori Hisateru、Hayashi Shinichiro、Noguchi Satoru、Okiyama Naoko、Fujimoto Manabu、Nishino Ichizo
  • Journal Title: Neurology Volume: 98 Issue: 7
  • DOI: 10.1212/wnl.0000000000013176
  • Peer Reviewed / Open Access
• [Journal Article] A recurrent homozygous ACTN2 variant associated with core myopathy (2021)
  • Author(s): Inoue Michio、Noguchi Satoru、Sonehara Kyuto、Nakamura-Shindo Keiko、Taniguchi Akira、Kajikawa Hiroyuki、Nakamura Hisayoshi、Ishikawa Keiko、Ogawa Megumu、Hayashi Shinichiro、Okada Yukinori、Kuru Satoshi、Iida Aritoshi、Nishino Ichizo
  • Journal Title: Acta Neuropathologica Volume: 142 Issue: 4 Pages: 785-788
  • DOI: 10.1007/s00401-021-02363-7
  • Peer Reviewed / Open Access
• [Journal Article] Identification of a KLF5-dependent program and drug development for skeletal muscle atrophy (2021)
  • Author(s): Liu L, Koike H, Ono T, Hayashi S, Kudo F, Kaneda A, Kagechika H, Manabe I, Nakashima T, Oishi, Y.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 118 Issue: 35
  • DOI: 10.1073/pnas.2102895118
  • Peer Reviewed / Open Access
• [Journal Article] Clinicopathologic Features of Oculopharyngodistal Myopathy With LRP12 CGG Repeat Expansions Compared With Other Oculopharyngodistal Myopathy Subtypes. (2021)
  • Author(s): Kumutpongpanich T, Ogasawara M, Ozaki A, et al., Hamano T, Sugie K, et al.,
  • Journal Title: JAMA Neurol. Volume: 78(7) Issue: 7 Pages: 853-853
  • DOI: 10.1001/jamaneurol.2021.1509
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] CRISPR/Cas9-targeted single molecule long-read sequencing reveals allelic microheterogeneity of triplet repeat expansion in oculopharyngodistal myopathy. (2022)
  • Author(s): Eura N, Noguchi S, Ogasawara M, Iida A, Hayashi S, Nishino I
  • Organizer: 27th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Screening of small molecules for activation of GNE protein carrying non-catalytic site mutation based on molecular docking simulation. (2022)
  • Author(s): Yoshioka W, Yamamoto K, Hayashi S, Sekijima M, Nishino I, Noguchi S
  • Organizer: 27th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Dermatomyositis-specific autoantibodies and muscle MRI findings. (2022)
  • Author(s): Une H, Hayashi S, Noguchi S, Nishino I
  • Organizer: 27th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Innervation defect: new pathomechanism of centronuclear myopathy? (2022)
  • Author(s): Saito Y, Hayashi S, Noguchi S, Nishino I
  • Organizer: 27th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Multi-dimensional analyses of the pathomechanism of GNE myopathy with non-catalytic site variant, GNE: c.620A>T. (2022)
  • Author(s): Yoshioka W, Iida A, Sonehara K, Yamamoto K, Oya Y, Mori-Yoshimura M, Kurashige T, Okubo M, Ogawa M, Matsuda F, Higasa K, Hayashi S, Nakamura H, Sekijima M, Okada Y, Nishino I, Noguchi S
  • Organizer: MYOLOGY 2022
  • Int'l Joint Research
• [Presentation] Intra-myonuclear inclusions differentiate oculopharyngeal muscular dystrophy from oculopharyngodistal myopathy. (2022)
  • Author(s): Eura N, Ogasawara M, Hayashi S, Noguchi S, Nishino I
  • Organizer: MYOLOGY 2022
  • Int'l Joint Research
• [Presentation] Innervation defect: new pathomechanism of centronuclear myopathy? (2022)
  • Author(s): Saito Y, Hayashi S, Noguchi S, Nishino I
  • Organizer: MYOLOGY 2022
  • Int'l Joint Research
• [Presentation] Genotype-phenotype correlations in NEB-related myopathies. (2022)
  • Author(s): Ogasawara M, Nishimori Y, Eura N, Hashizume L, Miyazaki N, Sugie K, Hayashi S, Noguchi S, Iida A, Nishino I
  • Organizer: MYOLOGY 2022
  • Int'l Joint Research
• [Presentation] Dermatomyositis-specific autoantibodies and muscle MRI findings. (2022)
  • Author(s): Une H, Hayashi S, Noguchi S, Nishino I
  • Organizer: MYOLOGY 2022
  • Int'l Joint Research
• [Presentation] 本邦における骨格筋切片を用いたPompe病スクリーニング (2022)
  • Author(s): 斎藤良彦，中村公俊，福田冬季子，杉江秀夫，林晋一郎，野口 悟，西野一三
  • Organizer: 第40回日本神経治療学会学術集会
• [Presentation] Intra-nuclear inclusions can differentiate between oculopharyngeal muscular dystrophy and oculopharyngodistal myopathy. (2022)
  • Author(s): Eura N, Ogasawara M, Hayashi S, Noguchi S, Nishino I
  • Organizer: 第63回日本神経病理学会総会学術研究会
• [Presentation] Inflammatory features on muscle pathology in laminopathy. (2022)
  • Author(s): Saito Y, Hayashi S, Noguchi S, Nishino I
  • Organizer: 第63回日本神経病理学会総会学術研究会
• [Presentation] Neuromuscular junction abnormalities in patients with centronuclear myopathy. (2022)
  • Author(s): Saito Y, Hayashi S, Noguchi S, Nishino I
  • Organizer: 第64回日本小児神経学会学術集会
• [Presentation] Clinicopathological findings of anti-mitochondrial antibody associated myositis. (2022)
  • Author(s): Nishimori Y, Oyama M, Motegi H, Hayashi S, Noguchi S, Suzuki S, Nishino I
  • Organizer: 第63回日本神経学会学術大会
• [Presentation] Frequent intra-myonuclear inclusions is a distinctive finding of oculopharyngeal muscular dystrophy. (2022)
  • Author(s): Eura N, Ogasawara M, Hayashi S, Noguchi S, Nishino I
  • Organizer: 第63回日本神経学会学術大会
• [Presentation] Generation and characterization of a Pax7-HA knock-in mouse line. (2021)
  • Author(s): Hayashi S, Inoue Y, Kosako H, Inoue T, Noguchi S, Nishino I
  • Organizer: 26th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Mutation profiling and immunohistochemical characterization of Japanese patients with sarcoglycanopathy. (2021)
  • Author(s): Saito Y, Hayashi S, Noguchi S, Nishino I
  • Organizer: 26th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] RNA-seq analysis for Dystrophinopathy. (2021)
  • Author(s): Okubo M, Noguchi S, Hayashi S, Komaki H, Nishino I
  • Organizer: 26th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] GNE pathogenic variant p.D207V rarely leads to myopathy in homozygotes; GNE might not be the only pathogenic determinant of GNE myopathy. (2021)
  • Author(s): Yoshioka W, Sonehara K, Iida A, Oya Y, Kurashige T, Okubo M, Ogawa M, Matsuda F, Higasa K, Mori-Yoshimura M, Nakamura H, Hayashi S, Okada Y, Noguchi S, Nishino I
  • Organizer: 26th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Dermatomyositis: muscle pathology according to antibody subtypes. (2021)
  • Author(s): Tanboon J, Inoue M, Saito Y, Hayashi S, Noguchi S, Okiyama N, Fujimoto M, Nishino I
  • Organizer: 26th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Clinicopathological findings of anti-mitochondrial antibody associated myositis. (2021)
  • Author(s): Nishimori Y, Hayashi S, Noguchi S, Nishino I
  • Organizer: 26th International Congress of the World Muscle Society
  • Int'l Joint Research
• [Presentation] Clinicopathologic features of oculopharyngodistal myopathy with LRP12 CGG repeat expansions. (2021)
  • Author(s): Ogasawara M, Kumutpongpanich T, Ishiura H, Tsuji S, Minami N, Hayashi S, Noguchi S, Iida A, Nishino I
  • Organizer: 日本人類遺伝学会第66回大会
• [Book] 医学のあゆみ. 283(10). RNA-seq技術を用いた遺伝性筋疾患診断． (2022)
  • Author(s): 大久保真理子，林晋一郎
  • Total Pages: 6
  • Publisher: 医歯薬出版