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遺伝性痙性対麻痺28型のモデルとしてのDdhd1ノックアウトマウスの解析

森川, 拓弥 MORIKAWA, Takuya モリカワ, タクヤ 九州大学

2022.01.31

概要

両親がいとこ婚であり、その子が痙性対麻痺(下肢の痙縮と歩行障害を主な症状とする遺伝性神経疾患)を発症している家系から、エクソーム解析を用いて DDHD1 遺伝子内の新規 4 塩基欠失を責任変異として同定した (Miura et al 2016)。DDHD1 が責任遺伝子である痙性対麻痺を SPG28 (Spastic par aplegia type 28)という。この疾患は希少疾患であり、治療法もない。また、DDHD1はリン脂質の代謝酵素をコードしており、脂質の代謝異常がどのように神経疾患引き起こすのか、そのメカニズムが明らかになっていない。それらを明らかにする目的で、CRISPR/ Cas9 を用いて患者の持つ変異を模倣した Ddhd1 ノックアウトマウスを作成した。SPG28 は遅発性の疾患であるため、老齢マウスを用いてで実験を行う必要があった。

26 ヶ月の月齢のマウスで、FBA (Foot base angle)テスト(歩行時の後脚と地面の角度を測定する手法)を用いて歩行障害様症状の観察に成功した。同じく 26 ヶ月のマウスの大脳を用いてリピドーム解析(リン脂質の網羅的測定)と RNA sequencing を行ったところ、アラキドン酸を含む LPI (Lysophosphatidyl inositol) 20: 4 がノックアウトマウスで有意に低下しており、細胞間コミュニケーションやシナプス伝達に関わる遺伝子の発現が有意に変動していた。さらに、6 ヶ月の症状の出る前の段階においても、脊髄における軸索の脱落が起こっていることを免疫染色によって確認した(Morikawa et al 2021)。アラキドン酸を含む LPI が引き起こすシグナリングが神経回路の構成に関わっていることが知られており、アラキドン酸を含む LPI が減少することがこのシグナリングを脆弱化させ、神経細胞の衰退を引き起こしているという発症モデルを提案した。

さらに、SPG28 の血中バイオマーカーを同定する目的で、Ddhd1 ノックアウトマウスの血漿を用いてリピドーム解析を行った。その結果、予想に即して Ddh d1 の基質である PI (Phosphatidyl inositol)と LPA (Lysophosphatidic acid) 24: 0 が Ddhd1 ノックアウトマウス上昇していることを明らかにした(未発表)。これは、PI または LPA 24: 0 が SPG28 のバイオマーカーとして利用できる可能性を示唆している。実際にこれらを SPG28 のバイオマーカーとして使用するためには、ノックアウトマウスで見られた PI と LPA 24: 0 の上昇が、SPG28 患者の血漿においても再現される必要がある。2021 年 7 月に SPG28 患者の血液を採取することができ、現在リピドーム解析の準備中である。

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