JP7429404B2 - タウ関連疾患モデルの製造方法 - Google Patents
タウ関連疾患モデルの製造方法 Download PDFInfo
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Description
[1]MAPT遺伝子に変異を有する多能性幹細胞を3次元培養し、神経オルガノイドを形成する工程と、前記神経オルガノイドを単一細胞に解離させて2次元で接着培養し、神経細胞を得る工程と、を含み、前記神経細胞がタウ関連疾患モデルである、タウ関連疾患モデルの製造方法。
[2]前記変異が、エクソン9~13又はイントロン10に存在する1つ又は複数の変異である、[1]に記載の製造方法。
[3]エクソン9~13に存在する前記変異が、K257T、I260V、G272V、N279K、K280Δ、L284L、S285R、N296H、P301L、P301S、S305N、S303S、S305S、V337M、E342V、G389R及びR406Wからなる群より選択される変異型タウタンパク質をコードする変異であり、イントロン10に存在する前記変異が、イントロン10の5’側から第1番目~第20番目のヌクレオチドのいずれか1つ又は複数の塩基における変異である、[2]に記載の製造方法。
[4]野生型の神経細胞と比較して、タウタンパク質のリン酸化の程度、タウタンパク質の断片化の程度、軸索の変質の程度又は軸索輸送機能が有意に異なり、MAPT遺伝子に変異を有する神経細胞からなるタウ関連疾患モデル。
[5][1]~[3]のいずれかに記載の製造方法により製造された、[4]に記載のタウ関連疾患モデル。
[6]被験物質の存在下で[4]又は[5]に記載のタウ関連疾患モデルを培養する工程と、前記タウ関連疾患モデルの、タウタンパク質のリン酸化の程度、タウタンパク質の断片化の程度、軸索の変質の程度又は軸索輸送機能を測定する工程と、測定されたタウタンパク質のリン酸化の程度、タウタンパク質の断片化の程度、軸索の変質の程度又は軸索輸送機能が、前記被験物質の非存在下と比較して有意に異なることが、前記被験物質がタウ関連疾患の予防剤又は治療剤であることを示す、タウ関連疾患の予防剤又は治療剤のスクリーニング方法。
1実施形態において、本発明は、MAPT遺伝子に変異を有する多能性幹細胞を3次元培養し、神経オルガノイドを形成する工程と、前記神経オルガノイドを単一細胞に解離させて2次元で接着培養し、神経細胞を得る工程と、を含み、前記神経細胞がタウ関連疾患モデルである、タウ関連疾患モデルの製造方法を提供する。本明細書において、タウ関連疾患としては、前頭側頭型認知症、アルツハイマー病等が挙げられる。
1実施形態において、本発明は、野生型の神経細胞と比較して、タウタンパク質のリン酸化の程度、タウタンパク質の断片化の程度、軸索の変質の程度又は軸索輸送機能が有意に異なり、MAPT遺伝子に変異を有する神経細胞からなるタウ関連疾患モデルを提供する。本実施形態のタウ関連疾患モデルは、例えば、上述した製造方法により製造することができる。
1実施形態において、本発明は、被験物質の存在下で上述したタウ関連疾患モデルを培養する工程と、前記タウ関連疾患モデルの、タウタンパク質のリン酸化の程度、タウタンパク質の断片化の程度、軸索の変質の程度又は軸索輸送機能を測定する工程と、測定されたタウタンパク質のリン酸化の程度、タウタンパク質の断片化の程度、軸索の変質の程度又は軸索輸送機能が、前記被験物質の非存在下と比較して有意に異なることが、前記被験物質がタウ関連疾患の予防剤又は治療剤であることを示す、タウ関連疾患の予防剤又は治療剤のスクリーニング方法を提供する。
(MAPT R406W iPS細胞の作製)
同じ血統の2人の日本人の前頭側頭型認知症患者(以下、「患者#1」及び「患者#2」という場合がある。)からMAPT R406W iPS細胞を樹立した。iPS細胞はエピソーマルベクターを用いて作製した。これらの患者の初期の症状は記憶障害であった。DNAシーケンシングの結果、これらの患者のMAPT遺伝子における変異はヘテロ接合であることが確認された。MAPT遺伝子には、R406W以外の変異は存在しなかった。
(ゲノム編集によるiPS細胞株の同質遺伝子系統の作製)
CRISPR/Cas9システムを用いたゲノム編集により、実験例1で作製した各iPS細胞から、野生型株であるiPS細胞、及び、ホモ接合の変異型株であるiPS細胞の同質遺伝子系統をそれぞれ作製した。ターゲッティングベクターとして、3’アームがMAPT遺伝子の変異部位を含み、3’アームと5’アームの間に薬剤選択可能なセレクションカセットを含むベクターを設計して使用した。セレクションカセットは、両末端にPiggyBac ITRを配置することにより除去可能に設計した。図1(a)及び(b)は、ターゲッティングベクターの構造を示す模式図である。図1(a)は、MAPT遺伝子座を野生型に組換えるためのターゲッティングベクターの模式図であり、図1(b)は、MAPT遺伝子座を変異型に組換えるためのターゲッティングベクターの模式図である。
(タウ関連疾患モデルの作製)
実験例2で作製した各iPS細胞株を神経細胞に分化させた。図3は神経細胞への分化のスケジュールを示す模式図及び細胞の写真である。
(R406W変異型タウタンパク質は様々なキナーゼによるリン酸化の程度が低下していた)
続いて、皮質神経細胞におけるタウタンパク質のリン酸化を検討した。アルツハイマー病や前頭側頭型認知症等の神経変性疾患では、タウタンパク質は複数の位置で高度にリン酸化されていることが知られている。一方、興味深いことに、R406W変異型タウタンパク質は変異部位の近傍の特定の位置においてリン酸化の程度が低下することが報告されている。
(R406W変異型タウはC末端側が切断された)
実験例3と同様にして作製したタウ関連疾患モデルにおけるR406W変異型タウタンパク質をウエスタンブロッティングにより検出した結果、R406W変異型タウタンパク質は、35kDから45kDに断片化されることが明らかとなった。これは特に、タウタンパク質のN末端を認識するTau12抗体を用いてウエスタンブロッティングした場合に顕著であった。したがって、タウタンパク質のC末端側が切断され断片化されたと考えられた。
(変異型神経細胞の表現型の検討)
実験例3と同様にして作製したタウ関連疾患モデルを用いて、R406W変異型タウタンパク質により誘導される細胞表現型を検討した。
(微小管の不安定化の検討)
実験例3と同様にして作製したタウ関連疾患モデルを、微小管安定化剤であるエポチロンD(EpoD)で処理し、免疫染色によりβIIIチューブリンを染色した。具体的には、タウ関連疾患モデルを、終濃度20nMのエポチロンD(アブカム社)の存在下及び非存在下で24時間インキュベートした後、免疫染色を行った。
(変異型神経細胞におけるミトコンドリア輸送の検討)
変異型神経細胞におけるミトコンドリア輸送を検討した。まず、実験例3と同様にして作製したタウ関連疾患モデルに、Mito-eYFPの発現ベクター及びtdTomatoの発現ベクターを導入した。続いて、蛍光顕微鏡観察により、軸索上のミトコンドリアの数を測定した。Mito-eYFPは、ミトコンドリアに特異的に局在する蛍光タンパク質である。
Claims (3)
- Microtubule Associated Protein Tau(MAPT)遺伝子に変異を有する多能性幹細胞を3次元培養し、神経オルガノイドを形成する工程と、
前記神経オルガノイドを単一細胞に解離させて2次元で接着培養し、神経細胞を得る工程と、
を含み、前記神経細胞がタウ関連疾患モデルであり、
前記変異が、エクソン9~13又はイントロン10に存在する1つ又は複数の変異であり、
エクソン9~13に存在する前記変異が、K257T、I260V、G272V、N279K、K280Δ、L284L、S285R、N296H、P301L、P301S、S305N、S303S、S305S、V337M、E342V、G389R及びR406Wからなる群より選択される変異型タウタンパク質をコードする変異であり、
イントロン10に存在する前記変異が、Ex10+3、Ex10+12、Ex10+13、Ex10+14、Ex10+16、Ex10+19及びEx10+29からなる群より選択される塩基における変異である、タウ関連疾患モデルの製造方法。 - 請求項1に記載の製造方法により製造された、タウ関連疾患モデル。
- 被験物質の存在下で請求項2に記載のタウ関連疾患モデルを培養する工程と、
前記タウ関連疾患モデルの、タウタンパク質のリン酸化の程度、タウタンパク質の断片化の程度、軸索の変質の程度又は軸索輸送機能を測定する工程と、を備え、
前記被験物質の非存在下と比較して測定されたタウタンパク質のリン酸化が有意に減少又は増加し、タウタンパク質の断片化が増加し、軸索が変質し、又は軸索輸送機能の不全が生じていることが、前記被験物質がタウ関連疾患の予防剤又は治療剤であることを示す、タウ関連疾患の予防剤又は治療剤のスクリーニング方法。
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