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ヒトアポリポタンパク質Eが脳内Aβダイナミクスに与える影響の解析

粉川, 明日香 東京大学 DOI:10.15083/0002005002

2022.06.22

概要

【序⽂】
アルツハイマー病(Alzheimer’s disease; AD)発症のリスク遺伝⼦として、apolipoprotein E(APOE)が知られている。ヒトAPOE遺伝⼦には3種の遺伝⼦多型ε2、ε3、ε4が存在する。アレル頻度が80数%と最も⾼いε3アレルに対し、ε4アレルの頻度は約10%であるが、ADの発症率はε4アレルを1つ保有すると約3倍、2つ保有すると約12倍に上昇し、アレル数依存的に発症年齢が早まる。APOE遺伝⼦がコードするapoEタンパク質は、⾎液中で脂質輸送に関与するタンパク質として知られてきたが、apoEがAD発症に寄与するメカニズムは⼗分に明らかになっていない。本論⽂においては、apoEがAD発症の鍵分⼦であるAβと相互作⽤し、脳内におけるAβのダイナミクス、即ちその産⽣、凝集・蓄積、クリアランス(除去)のいずれかの過程に影響を与えることによりその蓄積を促進している可能性を検討した。

【結果】
1.apoEがAβ蓄積に与える影響の検討
ヒトAPOE3ノックインマウスをヒトアミロイド前駆体(APP)トランスジェニック(tg)マウス(APP/PS1マウス系統)と交配し、9か⽉齢時の梨状⽪質に占めるAβ免疫染⾊陽性領域を画像解析により定量化した。内因性マウスapoEのみ、apoE3をヘテロないしホモで発現するAPP/PS1マウスを⽐較したところ、APOE3アレル数依存的に脳Aβ蓄積が減少することを⾒出した。⼀⽅APOE2或いはAPOE4ノックインマウスとAPP/PS1マウスを交配したところ、脳Aβ蓄積の程度はAPOE3ノックインマウスと同程度であった。また、APOEノックアウトマウスとAPP/PS1マウスを交配するとAβ斑がびまん性の形態に変化したことから、apoEはAβ斑の蓄積形態を規定すること、特にコンパクトな斑状の形態の維持に関与すると考えられた。

2.apoEがAβクリアランスに与える影響の検討
3か⽉齢APP/PS1マウスの海⾺間質液中に放出されたAβ量を1,000kDaカットオフプローブのin vivo microdialysis法を⽤いて測定し、さらにγ-secretase阻害剤を還流することでAβの半減期を算出した。その結果、Aβ定常濃度及びAβ半減期は内因性マウスapoE及びapoE3を発現するAPP/PS1マウス間で有意な差は認められず、異なる遺伝⼦型のapoEがAβの脳内産⽣及びクリアランスに与える影響に差があることを⽰すことはできなかった。

3.apoEがAβ凝集に与える影響の検討
Aβがβシート構造をとって線維を形成する過程はAβ斑の出現に先⾏して重要なステップであるが、invivoでAβの線維形成を測定することは困難である。そこでβシート構造を特異的に認識する蛍光⾊素thioflavinT(ThT)を⽤いたinvitroAβ凝集実験により、apoEがAβ凝集に与える影響を検討した。リコンビナントAβを単独、或いはapoE3、apoE4、マウスapoEと混和してインキュベーションし、ThTを添加した時の蛍光強度を測定することによりAβ凝集を評価した。その結果Aβ単独に⽐べ、apoEの共存下ではAβ凝集開始が遅延し、そのAβ凝集抑制効果はapoE3、apoE4、マウスapoEの順に⾼いことが⽰された。

さらに、Aβ凝集核に富んだ⾼齢マウス脳TBS可溶画分を3か⽉齢APP/PS1マウス脳に接種するinvivoseeding実験を実施し、apoEがAβ線維伸⻑過程に与える影響をinvivoレベルで検討した。Aβ凝集核を海⾺に接種したAPP/PS1マウスでは⾮接種側に⽐べてAβ蓄積が増⼤する傾向を⽰した。⼀⽅APOE3ノックインAPP/PS1マウスにおいては、Aβ凝集核の接種はAβ蓄積を有意に増⼤させなかった。これらの結果から、apoE3はマウスapoEに⽐して、Aβの脳内蓄積を抑制する効果がより強いことが⽰唆された。

4.apoEとAβの相互作⽤の検討
ヒトapoEとマウスapoE間で、Aβとの相互作⽤に違いがあるかをinvitroで検討した。液相中でAβとapoEの相互作⽤を測定するため、split-luciferase complementation assayを利⽤して新規のapoE/Aβ複合体発光モニタリング系を樹⽴した。Gaussialuciferaseのアミノ末端断⽚(“luci”)をapoEのカルボキシ末端に融合させたcDNA(apoE-luci)、及びAβを効率的に分泌させるために、BRI前駆体タンパク質とluciferaseのカルボキシ末端断⽚(“ferase”)をAβのアミノ末端に融合させたcDNA(BRI-ferase-Aβ)を作出した。apoEとAβが相互作⽤するとluciferaseが再構築され、発光を呈することが期待された。

脳内でapoEは主にアストロサイト、Aβは神経細胞で産⽣・分泌され、両者は細胞外で相互作⽤すると考えられる。そこでapoE3-luci、apoE4-luci或いはマウスapoE-luciとBRI-ferase-Aβをそれぞれ別個に遺伝⼦導⼊したHEK293細胞の培養上清を混和して、両者の相互作⽤を検討した。その結果、apoE4-luciとBRI-ferase-Aβを混和した場合、apoE3-luciとBRI-ferase-Aβの混和時に⽐してluciferaseの発光が有意に低かったことから、apoE4はapoE3に⽐べてAβとの相互作⽤が弱いことが⽰唆された。⼀⽅、マウスapoE-luciとBRIferase-Aβの相互作⽤は、apoE3-luciとBRI-ferase-Aβのそれと同程度であった。

R61T変異をapoE4に導⼊すると、apoE4の⽴体構造がapoE3様に変わると推定されている。R61T変異型apoE4-luciはapoE3-luciと同程度にBRI-ferase-Aβと相互作⽤したことから、apoEとAβの相互作⽤は、apoEの⽴体構造に起因する可能性が⽰唆された。

5.細胞外apoEタンパク質量の検討
apoEタンパク質の安定性を⽐較するためapoE3、apoE4、apoE4(R61T)を発現するHEK293細胞のライセート及び培養上清中のapoE量をイムノブロット法により検討した。その結果、ライセート中のapoE3、apoE4及びapoE4(R61T)の量は同程度であるのに対して、培養上清中ではapoE4量がapoE3、apoE4(R61T)に⽐して低かった。

続いて、APOE3及びAPOE4ノックインマウスの海⾺間質液をin vivo microdialysis法により回収し、脳の細胞外腔に分泌されたapoE量を検討した。その結果APOE3ノックインマウス脳間質液中のapoE量はAPOE4ノックインマウスに⽐して、統計学的に有意ではないが⾼い傾向を⽰した(p=0.51)。これらの結果から細胞外へ放出されたapoE4はapoE3に⽐して不安定である可能性、あるいはapoE4がapoE3に⽐して放出の程度が低い可能性が考えられた。

【まとめ】
本論⽂においてAD発症の遺伝的危険因⼦であるapoEに関して、ヒトapoE3はマウスapoEに⽐して脳におけるAβ蓄積を減少させる作⽤を有することを⾒出した。Aβの脳内代謝において、apoE3とマウスapoEがAβのクリアランスに異なる影響を与えることは認められず、apoE3またはマウスapoEを発現するADモデルマウスでAβ蓄積が著しく異なった原因は、apoE3がマウスapoEと⽐べてAβ凝集核形成及び線維伸⻑過程を抑制することに起因する可能性が考えられた。これらの結果は、apoE3がAD病態の発症に対し防御的に働く因⼦である可能性を⽰唆し、今後apoE3のAβに対する作⽤を増強する、新たなAD予防・治療法の開発を試みたい。

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