Axonal RNA study based on Dying back hypothesis for novel factors involved in motor neuron selective degeneration of Amyotrophic Lateral Sclerosis.

• Project/Area Number: 20K16593
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 52020:Neurology-related
• Research Institution: Tohoku University
• Principal Investigator: Mitsuzawa Shio 東北大学, 医学系研究科, 医員 (60869618)
• Project Period (FY): 2020-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2023: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2022: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2021: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2020: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
• Keywords: 筋萎縮性側索硬化症 / TARDBP / TDP-43 / iPS細胞 / 運動ニューロン選択的変性 / PHOX2B / 人工多能性幹細胞（iPS細胞） / 運動ニューロン脆弱性 / TAR-DNA binding protein / 人工多能性幹細胞 / 運動ニューロン / 軸索

Outline of Research at the Start

本研究では、なぜ筋萎縮性側索硬化症(ALS)では運動ニューロンが選択的に変性するのかを、その解剖学的な特徴である長大な軸索に着目して解析する。申請者はALS患者の人工多能性幹細胞（iPS細胞）を樹立し、分化誘導した変異運動ニューロンの突起伸長が抑制されることを確認した。さらに、RNAシークエンスより変異運動ニューロン軸索で発現が低下している新規病態関連因子を見出した。今回は、その因子がゼブラフィッシュなどの動物モデルにおいても神経突起や運動機能に影響するかを確認し、さらにマイクロアレイによる下流経路の解析により、どのように神経突起伸長抑制や運動ニューロン変性を来すかを解析する。

Outline of Final Research Achievements

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive and fatal neurodegenerative disease that causes generalized muscle weakness and atrophy, breathing and swallowing dysfunction. To elucidate the cause of selective degeneration of motor neurons in ALS, induced pluripotent stem cells (iPS cells) from TARDBP mutant ALS patients were differentiated to motor neurons (iMNs). Axon fractionated RNA sequencing (RNA-Seq) of iMNs revealed PHOX2B, which is down-regulated in the mutant axons and involved in neurite outgrowth.
Furthermore, we compared the RNA expression of iMNs with iPS cells derived autonomic neurons, which were rich in PHOX2B and maintained in ALS late-stage. Some downstream target candidate genes of PHOX2B have narrowed down.

Academic Significance and Societal Importance of the Research Achievements

筋萎縮性側索硬化症（ALS）は成人発症難治性進行性の致死的な神経変性疾患である。病態は未解明で根本的治療法はない。
PHOX2BはALSで発症後長期まで保たれる自律神経等に多く含まれるが、これまでALSとの関連が指摘されていなかった。PHOX2Bの下流に位置する治療標的を見出すことは、ALS病態解明と新規治療法開発につながると考える。さらにPHOX2B mRNAはTARDBP mRNAやそのタンパクTDP-43との相互作用や結合が確認され、PHOX2B下流の治療標的研究は、患者病理でみられるような細胞内TDP-43異常凝集をきたす神経筋疾患群の病態研究にも貢献する可能性がある。

Research Products

• [Journal Article] Reduced PHOX2B stability causes axonal growth impairment in motor neurons with TARDBP mutations (2021)
  • Author(s): Mitsuzawa S, Suzuki N, Akiyama T, Ishikawa M, Sone T, Kawada J, Funayama R, Shirota M, Mitsuhashi H, Morimoto S, Ikeda K, Shijo T, Ohno A, Nakamura N, Ono H, Ono R, Osana S, Nakagawa T, Nishiyama A, Izumi R, Kaneda S, Ikeuchi Y, Nakayama K, Fujii T, Warita H, Okano H, Aoki M.
  • Journal Title: Stem Cell Reports Volume: 16 Issue: 6 Pages: 1527-1541
  • DOI: 10.1016/j.stemcr.2021.04.021
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] iPS細胞由来運動ニューロン軸索を用いた筋萎縮性側索硬化症の病態研究 (2023)
  • Author(s): 光澤志緒、鈴木直輝、青木正志
  • Organizer: 第128回日本解剖学会総会・全国学術集会
• [Presentation] TARDBP変異 iPS細胞を用いた軸索に焦点をあてた筋萎縮性側索硬化症の運動ニューロン脆弱性の病態研究 (2023)
  • Author(s): 光澤 志緒、鈴木 直輝、割田 仁、岡野 栄之、青木 正志
  • Organizer: 第46回日本神経科学大会
• [Presentation] Axonal growth impairment in iPS-derived motor neurons with TARDBP mutations. (2021)
  • Author(s): Shio Mitsuzawa, Suzuki, Akiyama, Ishikawa, Sone, Kawada, Funayama, Mitsuhashi, Nishiyama, Ikeda, Shijo, Nakamura, H. Ono, R. Ono, Izumi, Ohno, Nakagawa, Nakayama, Warita, Okano, Aok.
  • Organizer: 62st Annual Meeting of the Japanese Society of Neurology
• [Presentation] Reduced PHOX2B stability causes axonal growth impairment in motor neurons with TARDBP mutations (2021)
  • Author(s): Shio Mitsuzawa, Naoki Suzuki, Tetsuya Akiyama, Mitsuru Ishikawa, Takefumi Sone, Jiro Kawada, Hiroaki Mitsuhashi, Satoru Morimoto, Kensuke Ikeda, Tomomi Shijo, Akiyuki Ohno, Naoko Nakamura, Hiroya Ono, Risako Ono, Ayumi Nishiyama, Rumiko Izumi, Masaaki Kato, Hitoshi Warita, Hideyuki Okano, Masashi Aoki
  • Organizer: PACTALS 2021
  • Int'l Joint Research
• [Presentation] REDUCED PHOX2B STABILITY CAUSES AXONAL GROWTH IMPAIRMENT IN MOTOR NEURONS WITH ALS-LINKED TARDBP MUTATIONS (2021)
  • Author(s): Shio Mitsuzawa, Suzuki, Akiyama, Ishikawa, Sone, Kawada, Funayama, Mitsuhashi, Morimoto, Ikeda, Shijo, Ohno, Nakamura, H. Ono, R. Ono, Nishiyama, Izumi, Kaneda, Ikeuchi, Nakayama, Fujii, Warita, Okano, Aoki.
  • Organizer: ISSCR Tokyo 2021
  • Int'l Joint Research
• [Presentation] Axonal growth impairment in motor neurons with TARDBP mutations were mediated by PHOX2B downregulation (2021)
  • Author(s): Shio Mitsuzawa, Suzuki, Akiyama, Ishikawa, Sone, Kawada, Funayama, Shirota, Mitsuhashi, Morimoto, Ikeda, Shijo, Ohno, Nakamura, H. Ono, R. Ono, Osana, Nakagawa, Nishiyama, Izumi, Kaneda, Ikeuchi, Nakayama, Fujii, Kato, Warita, Okano, Aoki.
  • Organizer: 32th international symposium on ALS/MND
  • Int'l Joint Research
• [Presentation] Axonal pathology in amyotrophic lateral sclerosis with TARDBP mutations. (2020)
  • Author(s): Shio Mitsuzawa, Naoki Suzuki, Tetsuya Akiyama, Mitsuru Ishikawa, Takefumi Sone, Jiro Kawada, Ryo Funayama, Hiroaki Mitsuhashi, Ayumi Nishiyama, Kensuke Ikeda, Tomomi Shijo, Naoko Nakamura, Hiroya Ono, Risako Ono, Rumiko Izumi, Tadashi Nakagawa, Keiko Nakayama, Hitoshi Warita, Hideyuki Okano, and Masashi Aoki
  • Organizer: 第61回日本神経学会学術大会（61st Annual Meeting of the Japanese Society of Neurology）
• [Remarks] 東北大学プレスリリース：筋萎縮性側索硬化症（ALS）の新しい病態関連候補因子を発見
  • URL: https://www.tohoku.ac.jp/japanese/2021/05/press20210528-01-als.html
• [Remarks] 筋萎縮性側索硬化症（ALS）の新しい病態関連候補因子を発見
  • URL: https://www.tohoku.ac.jp/japanese/2021/05/press20210528-01-als.html