JP2004107260A - Method for screening prophylactic and therapeutic agent for cerebral amyloidosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new substance which controls deposition of formed Aβ peptide and promotes decomposition and removal of the Aβ peptide, and to provide a method for screening the same. <P>SOLUTION: This method for screening the substance which inhibits HMG1 and exhibits promoting action on Aβ42 antiagglutination and/or Aβ42 opsonic action by microglia comprises using HMG1, its partial peptide or its salt. The method for screening a substance which controls the expression of HMG1 and exhibits promoting action on Aβ42 antiagglutination and/or Aβ42 opsonic action by microglia comprises using a nucleotide containing a base sequence encoding HMG1 or its partial peptide. The prophylactic and the therapeutic agent for cerebral amyloidosis contains a substance obtained by the method. <P>COPYRIGHT: (C)2004,JPO

Description

【０１１２】
Ｇｌｙ　　　　　　：グリシン
Ａｌａ　　　　　　　　　：アラニン
Ｖａｌ　　　　　　　　　：バリン
Ｌｅｕ　　　　　　　　　：ロイシン
Ｉｌｅ　　　　　　　　　：イソロイシン
Ｓｅｒ　　　　　　　　　：セリン
Ｔｈｒ　　　　　　　　　：スレオニン
Ｃｙｓ　　　　　　　　　　：システイン
Ｍｅｔ　　　　　　　　　：メチオニン
Ｇｌｕ　　　　　　　　　：グルタミン酸
Ａｓｐ　　　　　　　　　：アスパラギン酸
Ｌｙｓ　　　　　　　　　：リジン
Ａｒｇ　　　　　　　　　：アルギニン
Ｈｉｓ　　　　　　　　　：ヒスチジン
Ｐｈｅ　　　　　　　　　：フェニルアラニン
Ｔｙｒ　　　　　　　　　：チロシン
Ｔｒｐ　　　　　　　　　：トリプトファン
Ｐｒｏ　　　　　　　　　：プロリン
Ａｓｎ　　　　　　　　　：アスパラギン
Ｇｌｎ　　　　　　　　　：グルタミン
ｐＧｌｕ　　　　：ピログルタミン酸
＊　　　　　　　：終止コドンに対応する
Ｍｅ　　　　　　：メチル基
Ｅｔ　　　　　　：エチル基
Ｂｕ　　　　　　：ブチル基
Ｐｈ　　　　　　：フェニル基
ＴＣ　　　　　　：チアゾリジン−４（Ｒ）−カルボキサミド基
【０１１３】
また、本明細書中で繁用される置換基、保護基および試薬を下記の記号で表記する。

【０１１４】
本明細書の配列表の配列番号は、以下の配列を示す。
配列番号：１
ヒト由来ＨＭＧ１をコードするｃＤＮＡの塩基配列を示す。
配列番号：２
ヒト由来ＨＭＧ１のアミノ酸配列を示す。
【０１１５】
【実施例】
以下に実施例を示して、本発明をより詳細に説明するが、これらは本発明の範囲を限定するものではない。尚、イムノブロットのデンシトメトリー分析に関する結果は平均±標準誤差として示す。差の統計学的有意性は分散分析（ＡＮＯＶＡ）により決定した。事後比較のためのさらなる統計学的分析はＢｏｎｆｅｒｒｏｎｉ／Ｄｕｎｎテスト（ＳｔａｔＶｉｅｗ，　Ａｂａｃｕｓ　Ｃｏｎｃｅｐｔｓ，　Ｂｅｒｋｌｅｙ，　ＣＡ）を用いて行なった。また、共焦点顕微鏡観察は以下のように行った。ＡＤ脳切片は、ＨＭＧ１及びＨＬＡ−ＤＲに対する抗体と室温で一晩インキュベートし、Ａβ４２を注射したラット由来の海馬切片はＡβ及びＣＤ１１ｂに対する抗体とインキュベートした。一次抗体はローダミンもしくはＦＩＴＣ標識した抗ウサギＩｇＧ抗体及び抗マウスＩｇＧ抗体で検出し、蛍光をレーザー走査共焦点顕微鏡ＬＳＭ４１０（Ｃａｒｌ　Ｚｅｉｓｓ，　Ｇｅｒｍａｎｙ）を用いて観察した。
【０１１６】
実施例１　アルツハイマー病（ＡＤ）脳におけるＨＭＧ１のアップレギュレーション
臨床的且つ組織病理学的にＡＤと診断された６人の患者（年齢８１．２±３．５歳；検死の遅れ３．８±０．７時間）及び脳病変の臨床的且つ形態学的証拠のない６人のコントロール（年齢７１．０±４．２歳；検死の遅れ８．０±２．９時間）から検死により脳組織を得た。年齢及び検死の遅れはＡＤ及びコントロール群間で有意に相違していなかった。実験には側頭皮質を用いた。ＡＤの神経病理学的評価は、Ｃｏｎｓｏｒｔｉｕｍ　ｔｏ　Ｅｓｔａｂｌｉｓｈ　ａ　Ｒｅｇｉｓｔｒｙ　ｆｏｒ　Ａｌｚｈｅｉｍｅｒ’ｓ　Ｄｉｓｅａｓｅ　（ＣＥＲＡＤ）　の判断基準に従って行なった。細胞質画分（細胞質ゾル及び細胞外可溶性蛋白質を含む）及び粒子画分（核、膜及び不溶性蛋白質を含む）は、Ｂｒａｉｎ　Ｒｅｓ．　７８０：　２６０−２６９　（１９９８）に記載した方法に準じて調製した。両画分（１０μｇ蛋白質）をＳＤＳ−ＰＡＧＥにかけた。ＨＭＧ１又はＨＭＧ２に対する抗体（ＢＤ　Ｐｈａｒｍｉｎｇａｎ，　Ｓａｎ　Ｄｉｅｇｏ，　ＣＡ）でイムノブロットした後、ケミルミネッセンスアッセイ（ＥＣＬ　ｋｉｔ，　Ａｍｅｒｓｈａｍ　Ｐｈａｒｍａｃｉａ　Ｂｉｏｔｅｃｈ，　Ｂｕｃｋｉｎｇｈａｍｓｈｉｒｅ，　Ｕ．Ｋ．）を用いて蛋白質のバンドを検出した。蛋白質バンドの半定量的分析のため、Ｘ線フィルムをＣＣＤカラースキャナー（ＤｕｏＳｃａｎ，　ＡＧＦＡ）でスキャンし、濃さを測定した。
免疫組織化学的分析も、本質的にはＢｒａｉｎ　Ｒｅｓ．　７８０：　２６０−２６９　（１９９８）に記載した方法に準じて行なった。即ち、脳薄片を０．３％過酸化水素含有リン酸緩衝生理食塩水（ＰＢＳ）中で３０分間インキュベートし、０．３％トライトンＸ−１００含有ＰＢＳ（ＰＢＳ−Ｔ）で洗浄後、ＨＭＧ１に対する抗体、Ａβ（Ｃｈｅｍｉｃｏｎ，　Ｔｅｍｅｃｕｌａ，　ＣＡ）及びＨＬＡ−ＤＲ（Ｄａｋｏ，　Ｇｌｏｓｔｒｕｐ，　Ｄｅｎｍａｒｋ）を含有するＰＢＳ−Ｔとともに室温で一晩インキュベートした。ＰＢＳ−Ｔで洗浄し、Ｖｅｃｔａｓｔａｔｉｎ　ＡＢＣ　ｅｌｉｔｅ　ｋｉｔ（Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｂｕｒｌｉｎｇａｍｅ，　ＣＡ）とインキュベートした後、ニッケル増強及び過酸化水素とともに３，３’−ジアミノベンジジン（ＤＡＢ）を用いて該脳薄片を可視化した。
その結果、２９ｋＤａのＨＭＧ１はコントロール及びＡＤ脳の側頭皮質の細胞質画分と粒子画分の両方で検出された（図１）。粒子画分よりも細胞質画分に多く存在した。ＡＤ脳においては、細胞質画分及び粒子画分ともＨＭＧ１の発現レベルは、コントロール脳よりも有意に高く（図１ａ及びｂ，それぞれＰ＜０．０５及びＰ＜０．０１）、ＡＤ脳ではＨＭＧ１発現がアップレギュレートされていることを示している。２８ｋＤａのＨＭＧ２もまた細胞質画分と粒子画分の両方で検出されるが、その発現（ＨＭＧ１よりも低い）は、ＡＤ脳において顕著な相違はなかった（データは示さず）。
コントロール脳において、ＨＭＧ１免疫反応性はニューロンとグリア細胞の細胞質及び核でかすかに検出可能であった（図１ｃ）。ＡＤ脳では、ＨＭＧ１免疫反応性はこれらの位置で増大していた（図１ｄ）。さらに、ＨＭＧ１免疫反応性はプラーク様に拡散して観察されＡβ免疫反応性の領域で強く検出された（図１ｅを図１ｇと比較のこと）。活性化されたミクログリアのマーカーであるヒト白血球抗原（ＨＬＡ）−ＤＲもまた同様に観察された（図１ｆ）。二重免疫染色により、ＨＭＧ１は、ＨＬＡ−ＤＲを発現するミクログリア細胞内とその周辺の細胞外の両方に位置することが分かった（図１ｈ）。これらの免疫細胞化学的結果は、細胞外ＨＭＧ１が、老人斑においてミクログリアが蓄積する領域の周辺に蓄積することを示している。
【０１１７】
参考例１　トランスジェニックマウスにおけるＨＭＧ１の分布
最近、Ａβペプチドを過剰生産するいくつかのトランスジェニックマウスが作出された。Ｔｇ２５７６マウス及び変異プレセニリン−１（ＰＳ１）マウスは、それぞれＫ６７０Ｎ／Ｍ６７１Ｌ（スウェーデン型）変異ヒトアミロイド前駆蛋白質（ＡＰＰ）及びＭ１４６Ｌ変異ヒトＰＳ１を発現する。さらにＴｇ２５７６マウスとＰＳ１マウスを交配してダブルトランスジェニックＰＳ／ＡＰＰ系が作出されている。ＰＳ１マウスでは、マウスＡβ４２の産生は増加するが、Ａβ沈着は形成されないのに対し、ＰＳ／ＡＰＰマウスではヒト及びマウスのＡβ４０及びＡβ４２の著しい産生とＡβ沈着形成の加速が起こる。しかしながら、広範なニューロンの脱落はＰＳ１マウス及びＰＳ／ＡＰＰマウスのいずれでも検出されない。そこで、ニューロンの脱落なしにＡβ４２の甚大な産生を示すモデルとしてのトランスジェニックマウスにおけるＨＭＧ１及びＡβの免疫反応性を調べた。
実験には１２月齢のＰＳ１及びＰＳ／ＡＰＰマウスを用いた。これらのマウス由来の前頭脳切片を　Ａｍ．Ｊ．Ｐａｔｈｏｌ．，１５８：１３４５−１３５４（２００１）に記載したようにして調製した後、ＨＭＧ１、Ａβ及びＣＤ１１ｂに対する抗体（ＭＡＣ１，　Ｃａｌｔａｇ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｓａｎ　Ｆｒａｎｃｉｓｃｏ，　ＣＡ）で免疫染色した。二重標識した免疫組織化学的染色には、脳切片を、ＰＢＳ−Ｔ中で、マウスモノクローナルＡβ抗体とともにウサギ抗ＨＭＧ１抗体と４℃、４日間インキュベートした。洗浄後、切片を抗ウサギＩｇＧ抗体とインキュベートし、ＤＡＢ及びニッケルアンモニウムを用いてＨＭＧ１免疫反応性を検出した。ＨＭＧ１免疫染色完了後、切片を０．５％過酸化水素含有ＰＢＳ−Ｔで３０分間処理して第１のサイクルからの残余のペルオキシダーゼを破壊した。その後、切片と抗マウスＩｇＧ抗体との第２の免疫組織化学サイクルを実施した。ＤＡＢ反応はニッケル増強なしで行なった。
その結果、ＰＳ１マウスにおいては、ラット抗ＣＤ１１ｂ抗体（ＭＡＣ１）によって免疫染色される分枝型ミクログリアは観察されたが、Ａβ沈着及び反応性ミクログリアは１２月齢でさえ検出されなかった（図２ａ，ｃ及びｅ）。１２月齢のＰＳ／ＡＰＰマウスでは、ブッシュ及びアメーバ型ミクログリアに付随した過大なＡβ沈着が、ＡＤ脳におけるＡβ及びＨＬＡ−ＤＲの免疫反応性と類似したパターンで検出された（図２ｂ，ｄ及びｆを図１ｆ及びｇと比較のこと）。しかしながら、これらのトランスジェニックマウスにおけるＨＭＧ１免疫反応性は、ＡＤ脳におけるそれとはわずかに異なっていた。即ち、ＰＳ１及びＰＳ／ＡＰＰマウスの両方で、ＨＭＧ１免疫反応性はニューロン及びグリアの核で顕著に検出された（図２ｇ及びｈ）。ＰＳ／ＡＰＰマウスではＡβ沈着の周辺でも観察されたが、この免疫反応性は、おそらく細胞外ではなくグリア小核に見出されたものであろう（図２ｈ）。即ち、ＰＳ／ＡＰＰマウスはＡβ沈着を示すが、瀰漫性のＨＭＧ１免疫反応性は検出されなかった。これらの結果は、ＰＳ／ＡＰＰマウスにおけるＡβ沈着の形成は、主として細胞外ＨＭＧ１の関与しないＡβペプチドの過剰産生によって引き起こされ得ることを示唆している。
【０１１８】
参考例２　ラットモデルにおけるＨＭＧ１の分布
脳内の細胞外ＨＭＧ１とＡβとの関連を明らかにするために、我々は、さらに２つのラットモデル：１）カイニン酸（ＫＡ）の脳室内（ｉ．ｃ．ｖ．）注射の結果、Ａβ沈着なしに海馬ＣＡ３に甚大なニューロン脱落を生ずるラット及び２）Ａβ４２の海馬内注射の結果、中程度のニューロン脱落とともに大きなＡβ沈着を生ずるラットにおけるＨＭＧ１免疫反応性を調べた。ニューロン生存の指標として微小管関連蛋白質−２（ＭＡＰ２）を用いた。
体重約２８０ｇの雄性Ｗｉｓｔａｒラットを自由給水で一晩絶食させた。定位脳固定の顕微注入のためにラットを麻酔し（ペントバルビタールナトリウム５０ｍｇ／ｋｇを腹腔内注射）、Ｋｏｐｆ定位脳固定フレームに固定した。その後、１μｇ／２μｌ　ＫＡを右側脳室（即ち、ブレグマから３．８ｍｍ尾側、１．２ｍｍ右側、３．８ｍｍ下面）に注射した。あるいは、ラットの右海馬（即ち、ブレグマから０．２ｍｍ尾側、２．０ｍｍ右側、４．０ｍｍ下面）にモーター駆動の１０μｌハミルトンシリンジを介してＡβ４２（ＡｎａＳｐｅｃ，　Ｓａｎ　Ｊｏｓｅ，　ＣＡ）４．５μｇ／２μｌを注射した。３日後、処理したラットに動脈を通じて１０ｍＭ　リン酸緩衝生理食塩水（ＰＢＳ）１５０ｍｌを、続いて４％　パラホルムアルデヒド、０．３５％　グルタルアルデヒド及び０．２％　ピクリン酸（１００ｍＭ　リン酸緩衝液中）からなる冷固定液３００ｍｌを、ペントバルビタール（１００ｍｇ／ｋｇ腹腔内）による深麻酔下に灌流した。灌流後、速やかに脳を除去し、１００ｍＭ　ＰＢ中のパラホルムアルデヒドで２日間後固定した後、４℃で、１５％シュークロース溶液（０．１％　アジ化ナトリウム含有１００ｍＭ　ＰＢ中）に移した。脳片をクリオスタット中で２０μｍの切片に切り、ＰＢＳ−Ｔ中で集めた。その後、海馬切片を、ＭＡＰ２、ＨＭＧ１、Ａβ、ＣＤ１１ｂ（ＯＸ４２、Ｈａｒｌａｎ　Ｓｅｒａ−Ｌａｂ，　Ｌｏｕｇｈｂｒｏｕｇｈ，　Ｕ．Ｋ．）及びＧＦＡＰ（Ｃｈｅｍｉｃｏｎ　Ｉｎｔｅｒｎａｔｉｏｎａｌ，　Ｔｅｍｅｃｕｌａ，　ＣＡ）に対する抗体で免疫染色した。
その結果、ＫＡのｉ．ｃ．ｖ．注射は３日後に同側の海馬ＣＡ３におけるニューロン脱落を引き起こしたが（図３ｂ）、対側では引き起こさなかった（図３ａ）。Ａβ沈着は観察されなかったが（図３ｆ及びｈ）、活性化されたミクログリア及びアストロサイト（それらは、それぞれＣＤ１１ｂ（ＯＸ４２）に対する抗体及びグリア線維酸性蛋白質（ＧＦＡＰ）に対する抗体により免疫染色される）はＣＡ３に蓄積していた（図３ｉ−ｌ）。ＨＭＧ１免疫反応性はニューロン及びグリアの核で顕著に検出された（図３ｅ，ｇ及びｉ−ｌ）。ＨＭＧ１免疫反応性はまた、神経変性ＣＡ３のグリア突起でも検出されたが（図３ｇ，ｋ及びｌ）、瀰漫性の染色は検出されなかった。
Ａβ４２を海馬に顕微注入されたラットでは、マウス抗ＣＤ１１ｂ抗体により免疫染色されるアメーバ状ミクログリア（図４ｄ，ｆ及びｇ）が海馬内の大きなＡβ沈着（図４ａ，ｅ及びｇ）の周辺に観察された。さらに、ＭＡＰ２免疫反応性の中程度の喪失がＡβ沈着周辺の海馬歯状回（ＤＧ）で検出された（図４ｂ）。ＨＭＧ１免疫反応性はニューロン及びグリアの核、並びにグリア突起で観察された（図４ｃ，ｅ及びｆ）。さらに、ＨＭＧ１免疫反応性はＡβ沈着周辺の細胞外瀰漫性染色として検出された（図４ｃ，ｅ及びｆ）。これらの観察から、細胞外ＨＭＧ１は死滅したニューロンから主に漏出し、瀰漫性のＨＭＧ１沈着は甚大なニューロンの死滅とＡβ蓄積の両方によって引き起こされるのであろう。
【０１１９】
実施例２　ＨＭＧ１により誘導されるミクログリアによるＡβ４２食作用の阻害
ＨＭＧ１が存在することのメカニズム的意義を検討するため、ラットミクログリアの純粋培養を用いてミクログリアの機能に及ぼすＨＭＧ１の効果を調べた。実験では、Ａβ４０やＡβ４２は、トリフルオロ酢酸（ＴＦＡ）塩ではなく、容易に凝集する塩酸塩を用いた。本発明者らは、最近、Ａβ４０が１０μＭで腫瘍壊死因子α（ＴＮＦ−α）及びインターロイキン−６（ＩＬ−６）の産生を誘導することを見出した（ＦＡＳＥＢ　Ｊ．　１６：６０１−６０３　（２００２））。対照的に、細胞外Ａβ４２は、Ａβ４２曝露から６−２４時間後に、より低濃度（０．０３−０．３μＭ）でミクログリアによる貪食を顕著に引き起こした。インスリン又はペプスタチンＡは３日後のミクログリアにおけるＡβ４２レベルの減少を抑制したが、ホスホラミドン（ネプリライシンインヒビター）は抑制せず、貪食されたＡβ４２はミクログリア内でＩＤＥ様ペプチダーゼ及び／又はカテプシンＤ様アスパラギン酸プロテイナーゼにより分解されるであろうことを示唆している。比較的低濃度（１μＭ未満）のＡβ４０曝露はミクログリアによる取込みを引き起こさなかったが、より高濃度（３−１０μＭ）では、ミクログリアはＡβ４０を貪食するようになった。このように、ラットミクログリアはＡβ４０とＡβ４２に異なる応答を示す。
混合グリア細胞（アストロサイトとミクログリアの混合物）を、新生Ｗｉｓｔａｒラット（ＳＬＣ　Ｉｎｃ．，　Ｓｈｉｚｕｏｋａ，　Ｊａｐａｎ）の髄膜から注意深く除去した脳半球から調製した。該組織懸濁液を５０μｍ径のナイロンメッシュ（ｃｅｌｌ　ｓｔｒａｉｎｅｒｓ、　Ｆａｌｃｏｎ）を通して、５０ｍｌチューブ中に濾過し、２００×ｇで１０分間遠心して細胞を集めた。細胞を１０％ウシ胎仔血清添加ダルベッコ改変イーグル培地中に再懸濁し、１００ｍｍ径のディッシュにプレートして５％　ＣＯ_２／９５％大気中、３７℃でインキュベートした。エンドトキシンによるコンタミネーションを避けるため十分注意を払いながら、浮遊ミクログリアを混合グリア培養から回収して新しい培養皿にプレートした。
得られたラットミクログリア（純度９７％超）を、０．０１５−１．５μＭ精製仔ウシＨＭＧ１及び／又は０．４ｎＭ組換えヒトＴＧＦ−β１（Ｇｉｂｃｏ　ＢＲＬ，　Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，　Ｔｏｋｙｏ，　Ｊａｐａｎ）の存在下又は非存在下に、０．３μＭ　Ａβ４２又は３μＭ　Ａβ４０とインキュベートした。２４時間後、ミクログリアを集めて溶解した後、サンプルを抗Ａβ抗体（Ｃｈｅｍｉｃｏｎ，　Ｔｅｍｅｃｕｌａ，　ＣＡ）によるイムロブロットにかけた。モノマー及び凝集Ａβをケミルミネッセンスアッセイを用いて検出した後、Ａβの総量をデンシトメーターで測定した。
仔ウシ胸腺から精製したＨＭＧ１を低濃度（０．３μＭ）のＡβ４２を含むラットミクログリア培養に添加すると、ＨＭＧ１は６０％を超えるまで濃度依存的にＡβ４２の貪食を阻害した（図５ａ及びｂ）。アストロサイト由来ＴＧＦ−β１がＡβクリアランスに重要であることが報告されているので、さらにＴＧＦ−β１により誘導されるＡβクリアランスに及ぼすＨＭＧ１の影響を調べた。０．４ｎＭの組換えヒトＴＧＦ−β１はＡβ４２の貪食を有意に増大させたが、ＨＭＧ１は、ＴＧＦ−β１非存在下でのＨＭＧ１処理により生ずるレベルまで、ＴＧＦ−β１により誘導されるＡβ貪食を顕著に阻害した（図５ｃ及びｄ）。対照的に、Ａβ４０の貪食は、高濃度（３μＭ）で、ＴＧＦ−β１及びＨＭＧ１によっては変化しなかった（図６ａ及びｂ）。これらの結果は、もし老人斑に細胞外ＨＭＧ１が蓄積すれば、ＴＧＦ−β１のようなミクログリア賦活剤によるＡβ４２クリアランスは阻害されるが、Ａβ４０のクリアランスは阻害されないであろうことを示唆している。
【０１２０】
参考例３　Ａβ４０により誘導されるミクログリア活性化に及ぼすＨＭＧ１の影響
仔ウシ胸腺から精製した１．５μＭ　ＨＭＧ１（和光純薬）存在下又は非存在下に、ミクログリアを１０μＭ　Ａβ４０（ＡｎａＳｐｅｃ）で処理した。処理から２４時間後に各ウェルから培地を除去した。ＮＯ_２ ⁻量は、Ｇｒｉｅｓｓ試薬を用いて分光光学的に（ＤＵ−６４０分光光度計、Ｂｅｃｋｍａｎ）測定した。細胞から放出されたＴＮＦ−α及びＩＬ−６の量は、ラットＴＮＦ−α及びＩＬ−６用のＥＬＩＳＡキット（ＢｉｏＳｏｕｒｃｅ，　Ｃａｍａｒｉｌｌｏ，　ＣＡ）を製造業者の指示に従って用いて測定した。
その結果、１０μＭのＡβ４０は、亜硝酸塩（ＮＯ_２ ⁻）の蓄積とともに誘導性一酸化窒素シンターゼ（ｉＮＯＳ）の発現を誘導し、また、ＴＮＦ−α及びＩＬ−６の産生を誘導した（図６ｃ，ｄ及びｅ）。しかしながら、ＨＭＧ１は、Ａβ４０の存在下及び非存在下でこれらの化合物の産生に影響を与えなかった（図６ｃ，ｄ及びｅ）。したがって、ＨＭＧ１は、Ａβ４０の存在下及び非存在下でミクログリアによるＮＯ、ＴＮＦ−α及びＩＬ−６の産生に影響しない。
【０１２１】
実施例３　ＨＭＧ１のインビトロでのＡβペプチドとの結合及びそれらの凝集
精製仔ウシＨＭＧ１（１．５μｇ）を、３μｇのＡβ４０又はＡβ４２（ＰＢＳ（最終容量２０μｌ）中）と３７℃でインキュベートした。０、６及び２４時間後に、サンプルをＨＭＧ１又はＡβに対する抗体によるイムノブロットにかけた。また、１．５μｇのＨＭＧ１を３μｇのＡβ４０又はＡβ４２と３７℃で２４時間インキュベート後、ＨＭＧ１又はＡβに対する抗体（１０μｇ）を混合物に添加して、さらに２時間、４℃でインキュベートした。次いで、プロテインＡ−セファロース（５０％スラリー５０μｌ）を加えて、４℃で一晩インキュベートした。遠心後、免疫沈降物を冷緩衝液１ｍｌ中で３回洗浄し、Ｌａｅｍｍｌｉのサンプル緩衝液に再懸濁して、ＳＤＳ−ＰＡＧＥ及びＨＭＧ１又はＡβに対する抗体によるイムノブロットにかけた。
ＨＭＧ１をＡβ４２とインキュベートすると、これらの蛋白質の複合体（およそ３３ｋＤａ）が６時間後に検出された（図７ａ及びｂ）。Ａβ４２をＨＭＧ１とインキュベートすると、２４時間後、そのオリゴマーの形成は変化せず、より高分子の凝集体の形成が顕著に増加した（図７ｂ）。対照的に、Ａβ４２単独でインキュベートすると、６時間後にそのオリゴマーのレベルは減少し、凝集体は増加した。反対に、２４時間後、Ａβ４０のオリゴマー及び凝集体は検出されなかったが、ＨＭＧ１とＡβ４０の複合体は検出された（図７ａ及びｂ）。
ＨＭＧ１をＡβ４０又はＡβ４２と２４時間インビトロでインキュベートした後、混合物をＨＭＧ１に対する抗体又はＡβに対する抗体で免疫沈降させた。これらの抗体はＨＭＧ１とＡβペプチドの両方を沈降させた（図７ｃ）。さらに、ＨＭＧ１とＡβ４０又はＡβ４２のいずれかとの複合体もまた免疫沈降した。したがって、ＨＭＧ１はＡβペプチドと結合してＡβ４２の凝集体の形成を促進したが、Ａβ４０の凝集体形成は促進しなかった。
【０１２２】
結論として、ＨＭＧ１蛋白質のレベルはＡＤ脳の細胞質画分と粒子画分の両方でアップレギュレートされ、ＨＭＧ１は老人斑と共存する。瀰漫性のＨＭＧ１免疫反応性はＡβ４２を注射したラットの海馬で観察されたが、ＫＡを注射したラットの海馬及びＰＳ／ＡＰＰマウスでは観察されず、細胞外ＨＭＧ１とＡβ４２が相乗的に相互作用して老人斑を形成することを示唆している。細胞外ＨＭＧ１は、ＴＧＦ−β１の存在下及び非存在下にミクログリアによるＡβ４２の貪食を阻害した。さらに、ＨＭＧ１はＡβペプチドと結合してＡβ４２凝集体の形成を促進した（図８）。これらの結果は、ＨＭＧ１のアップレギュレーションがミクログリアによるＡβ４２クリアランスを阻害し、その結果Ａβ沈着が維持されることを示唆している。このように、細胞外ＨＭＧ１は、ミクログリアによるＡβクリアランスの阻害とＡβ凝集の促進を通じてＡＤにおける病原的役割を果たしているであろうことが示された。
【０１２３】
【発明の効果】
ＨＭＧ１もしくはその部分ペプチドまたはその塩は、アルツハイマー病等の脳アミロイドーシスの予防・治療用のワクチンとして、また、ＨＭＧ１等をコードするヌクレオチドに相補的なヌクレオチド及びＨＭＧ１等に対する抗体は脳アミロイドーシスの予防・治療剤として有用である。
ＨＭＧ１等をコードするヌクレオチド及びＨＭＧ１等に対する抗体は脳アミロイドーシスの診断薬として有用である。
ＨＭＧ１等を用いるＨＭＧ１阻害剤のスクリーニング法及びＨＭＧ１等をコードするヌクレオチドを用いるＨＭＧ１発現抑制剤のスクリーニング法は、脳アミロイドーシスの予防・治療活性を有する化合物の探索ツールとして有用である。
【０１２４】
【配列表】

【図面の簡単な説明】
【図１】コントロール及びＡＤ脳におけるＨＭＧ１の発現レベル及び分布を示す図及び写真である。ａ及びｂ：側頭皮質の細胞質（ａ）及び粒子（ｂ）画分のイムノブロット写真（上図）及び２９ｋＤａのバンド濃度を示すグラフ（下図；縦軸はコントロールに対する％割合；各数値は６検体の平均±標準誤差；個々の数値は丸印で示す）。ｃ−ｈ：コントロール及びＡＤ脳切片のＨＭＧ１（ｃ−ｅ，ｈ）、ＨＬＡ−ＤＲ（ｆ，ｈ）又はＡβ（ｇ）に対する抗体を用いた免疫染色写真。切片（ｅ−ｈ）は同一個体由来の一連のものであり、＊（ｅ−ｇ）は同一の脈管を示す。矢印はインセットに高倍率で示されたと同じ老人斑を示す。ｈはＨＭＧ１及びＨＬＡ−ＤＲに対する抗体を用いた老人斑の二重染色。スケールバー：２５０μｍ（ｅ−ｇ）及び５０μｍ（ｈ及びｅ−ｇのインセット）。
【図２】トランスジェニックマウス脳におけるＨＭＧ１の局在性を示す写真である。１２月齢のＰＳ１（ａ，ｃ，ｅ，ｇ）及びＰＳ／ＡＰＰマウス（ｂ，ｄ，ｆ，ｈ）の前頭脳切片のＣＤ１１ｂ（ＭＡＣ１，ａ−ｄ）、Ａβ（ｅ−ｈ）又はＨＭＧ１（ｇ，ｈ）に対する抗体を用いた免疫染色。ボックスの高倍率像をインセットで示す（ｃ−ｈ）。スケールバー：１ｍｍ（ａ，ｂ）、２００μｍ（ｃ−ｈ）及び５０μｍ（ｃ−ｈ中のインセット）。
【図３】ＫＡを注射したラット脳におけるＨＭＧ１の局在性を示す写真である。海馬切片のＭＡＰ２（ａ，ｂ）、ＨＭＧ１（ｃ−ｅ，ｇ，ｉ−ｌ）、Ａβ（ｆ，ｈ）、ＣＤ１１ｂ（ＯＸ４２，ｊ，ｌ）又はＧＦＡＰ（ｉ，ｋ）に対する抗体を用いた免疫染色。脳室内ＫＡ注射と対側（ａ，ｃ，ｅ，ｆ，ｉ，ｊ）及び同側（ｂ，ｄ，ｇ，ｈ，ｋ，ｌ）。海馬ＣＡ３中のボックスの高倍率像（ｅ−ｌ）。ｉ−ｌ：ＨＭＧ１及びＣＤ１１ｂ（ｊ，ｌ）又はＧＦＡＰ（ｉ，ｋ）に対する抗体を用いた二重染色。スケールバー：５００μｍ（ａ−ｄ）及び５０μｍ（ｅ−ｌ）。
【図４】Ａβ４２を注射したラット海馬におけるＨＭＧ１の局在性を示す写真である。海馬切片のＡβ（ａ，ｅ，ｇ）ＨＭＧ１（ｃ，ｅ，ｆ）、ＭＡＰ２（ｂ）又はＣＤ１１ｂ（ＯＸ４２，ｄ，ｆ，ｇ）に対する抗体を用いた免疫染色。ｅ，ｆ：ＨＭＧ１及びＡβ（ｅ）又はＣＤ１１ｂ（ｆ）に対する抗体を用いた二重染色。免疫染色された各切片（ａ−ｆ）中のボックスの高倍率像をインセットで示す。矢印は抗ＨＭＧ１抗体による瀰漫性染色を示す。ｇ：Ａβ及びＣＤ１１ｂに対する抗体を用いた海馬中のＡβ沈着周辺の二重染色の共焦点顕微鏡像。スケールバー：５００μｍ（ａ−ｆ）及び５０μｍ（ａ−ｆ中のインセット，ｇ）。
【図５】ミクログリアによるＡβ４２食作用に対するＨＭＧ１の阻害作用を示す写真及び図である。ａ及びｂ：ビヒクル（ａ中のレーンＣ及びｂ中のＣｏｎｔｒｏｌ）又は種々の濃度のＨＭＧ１の存在下でＡβ４２とインキュベートしたラットミクログリアライセートのイムノブロット写真（ａ）及び総Ａβ４２量を示すグラフ（縦軸はコントロールに対する％割合；各数値は３つの実験の平均±標準誤差；＊＊、＊＊＊はコントロールの数値に対してそれぞれｐ＜０．０１及びｐ＜０．００１を示す）。ｃ及びｄ：ビヒクル（ｃ中のレーンＴＧＦ−β１（−）及びｄ中のＶｅｈｉｃｌｅ）又はＴＧＦ−β１（ｃ中のレーンＴＧＦ−β１（＋）及びｄ中のＴＧＦ−β１）の存在下で、Ａβ４２（ｃ中のＨＭＧ１（−）及びｄ中のＣｏｎｔｒｏｌ）又はＡβ４２及びＨＭＧ１（ｃ中のＨＭＧ１（＋）及びｄ中のＨＭＧ１）とインキュベートしたラットミクログリアライセートのイムノブロット写真（ａ）及び総Ａβ４２量を示すグラフ（縦軸はコントロールに対する％割合；各数値は３つの実験の平均±標準誤差；＊＊＊、†††はそれぞれビヒクル及びＴＧＦ−β１単独の数値に対してｐ＜０．００１を示す）。
【図６】ミクログリアにおけるＡβ４０の取込み及びＡβ４０による活性化に及ぼすＨＭＧ１の影響を示す写真及び図である。ａ及びｂ：ビヒクル、ＴＧＦ−β１又はＨＭＧ１存在下でＡβ４０とインキュベートしたラットミクログリアのライセートのイムノブロット写真（ａ）及び総Ａβ４０量を示すグラフ（縦軸はビヒクルに対する％割合；各数値は３つの実験の平均±標準誤差を示す）。ｃ−ｅ：Ａβ４０存在下又は非存在下で、ビヒクル（ＨＭＧ１（−））又はＨＭＧ１（ＨＭＧ１（＋））とインキュベートしたラットミクログリアのライセートの抗ｉＮＯＳ抗体に対するイムノブロット写真（ｃ上図）、並びに培養上清中のＮＯ_２ ⁻量（ｃ下図）ＴＮＦ−α量（ｄ）及びＩＬ−６量（ｅ）を示すグラフ（各数値は３つの実験の平均±標準誤差；＊＊＊、†††は、それぞれビヒクル及びＡβ４０処理の数値に対してｐ＜０．００１であることを示す）。
【図７】インビトロでのＨＭＧ１とＡβペプチドとの結合及びＡβペプチドの凝集を示す写真である。ａ及びｂ：Ａβ４０又はＡβ４２の存在下又は非存在下でＨＭＧ１をインキュベートしたサンプルについてのＨＭＧ１（ａ）又はＡβに対する抗体を用いたイムノブロット（矢印はＨＭＧ１とＡβ４０又はＡβ４２との複合体を示す）。ｃ：Ａβ４０又はＡβ４２とＨＭＧ１とをインキュベートした後、反応液のＨＭＧ１（上図）又はＡβ（下図）に対する抗体によって免疫沈降させ、ＨＭＧ１又はＡβに対する抗体を用いてイムノブロットしたもの。
【図８】老人斑形成過程における細胞外ＨＭＧ１の関与を示す模式図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a means for screening for a substance having an inhibitory effect on senile plaque formation, characterized by using High \ Mobility \ Group \ Protein-1 (hereinafter abbreviated as HMG1). The present invention also relates to the pharmaceutical use of the substance obtained by the screening, particularly to the prevention and treatment of cerebral amyloidosis such as Alzheimer's disease and Down's syndrome.
[0002]
[Prior art]
Senile plaques are one of the major neuropathological changes in Alzheimer's disease and are detected from the very beginning of the onset process. A major component of the senile plaque is an amyloid β (hereinafter abbreviated as Aβ) peptide, which is produced by cleavage of amyloid precursor protein (APP) by two kinds of (β- and γ-) secretases. . As its main molecular species, there are Aβ40 consisting of 40 amino acid residues and Aβ42 whose C-terminal side is two residues longer.
[0003]
Recent studies suggest that Aβ peptide deposition is controlled by complex interactions between regulators of the acellular and cellular (glial) clearance pathways. Non-cellular pathways include 1) secretory peptidases that degrade extracellular Aβ peptides, such as neprilysin and insulin degrading enzyme (IDE) (eg, Non-Patent Document 1), and 2) from the brain parenchyma to the circulatory system. There is an outflow route of Aβ by transport / discharge (for example, Non-Patent Document 2). In Alzheimer's disease, extracellular deposition of Aβ peptide significantly accumulates and is associated with microglia. Such involvement of microglia suggests that they play an Aβ phagocytic function (for example, Non-Patent Document 3) and / or are involved in inflammatory activation (for example, Non-Patent Document 4). . The discovery that anti-Aβ antibodies produced by Aβ vaccine therapy induce microglial phagocytosis through Fc receptors (eg, Non-Patent Document 5) and / or remove plasma Aβ peptides (eg, Non-Patent Document 6). The importance of Aβ clearance is of great interest. In addition, cytokines such as transforming growth factor β1 (TGF-β1) promote Aβ clearance by microglia (for example, Non-Patent Document 7). Recently, we have found that extracellular stress proteins such as Hsp90, Hsp70, Grp78 promote Aβ clearance by activating phagocytosis by microglia and Aβ degradation by NFκB activation and / or by chaperone activity. (For example, Non-Patent Document 8).
[0004]
Many of the proteins associated with amyloidosis are known to share similar structures and sequences, and they are believed to contribute to amyloid fibril formation. Such amyloidogenic consensus sequences are also found in proteins with no known association with amyloidosis. For example, Kalijarvi et al. Have reported that HMG1, a non-histone chromosomal protein, has a sequence motif homologous to the Aβ peptide, and an HMG1 fragment containing this sequence motif forms amyloid-like fibers in vitro, and Aβ40 is polymerized. It is reported to promote it (Non-Patent Document 9).
[0005]
On the other hand, Toyama et al. Detected an abnormal presenilin 2 (known as one of the causative genes of familial Alzheimer's disease (FAD)) protein in sporadic Alzheimer's disease (SAD) brain. To prevent normal splicing by binding to transcripts, and that when a decoy substance that binds to HMG1 acts on a cell model of Alzheimer's disease, normal presenilin 2 mRNA is synthesized and no abnormal presenilin 2 protein is generated. It has been announced (eg, Non-Patent Document 10) and is expected to lead to the development of a second-generation therapeutic drug for Alzheimer's disease that exceeds current therapeutic drugs for symptomatic treatment.
[0006]
[Non-patent document 1]
Journal of Biological Chemistry (J. Biol. Chem.), Vol. 273, p32730-32738 (1998)
[Non-patent document 2]
Procedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 96, pp. 14088-14093 (1999)
[Non-Patent Document 3]
American Journal of Pathology (Am. @ J. @ Pathol.), 148, p399-403 (1996)
[Non-patent document 4]
Neurobiological Aging (Neurobiol. @Aging), Volume 21, p383-421 (2000)
[Non-Patent Document 5]
Nature, Volume 400, pp. 173-177 (1999)
[Non-Patent Document 6]
Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 98, pp. 8850-8855 (2001)
[Non-Patent Document 7]
Nature Medicine (Nat. @ Med.), Vol. 7, p612-618 (2001)
[Non-Patent Document 8]
FASEB Journal (FASEB @ J.), Volume 16, p601-603 (2002)
[Non-Patent Document 9]
Biochemistry, Vol. 40, p10032-10037 (2001)
[Non-Patent Document 10]
Tokyo Yomiuri Shimbun, January 9, 2002, evening edition
[0007]
[Problems to be solved by the invention]
However, in cerebral amyloidosis such as Alzheimer's disease, a useful substance that inhibits the aggregation and deposition of the already generated Aβ peptide and promotes its decomposition and removal has not been found yet, and cerebral amyloidosis from such a viewpoint has not been found. There is very little research and development of therapeutics.
Therefore, an object of the present invention is to (1) promote Aβ aggregation, and (2) inhibit Aβ removal mechanism by suppressing phagocytosis of Aβ by microglia, thereby promoting senile plaque formation and It is an object of the present invention to provide a method for screening a substance having a prophylactic / therapeutic effect on cerebral amyloidosis, in which a factor causing amyloidosis is identified, Aβ clearance is promoted by inhibiting the action or expression of the factor, and a kit for the same is provided. .
[0008]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to achieve the above object, and as a result, it has been found that HMG1 levels are increased in the brain of Alzheimer's disease patients, and that extracellular HMG1 coexists with Aβ42 in senile plaques, HMG1 was found to inhibit the phagocytosis of Aβ42 by microglia in an in vitro microglial culture system, and HMG1 formed a complex with Aβ42 in vitro to promote the formation of Aβ42 aggregates. The present inventors have further studied based on these findings, and have completed the present invention.
[0009]
That is, the present invention
[1] a vaccine for inhibiting amyloid β42 aggregation and / or promoting amyloid β42 phagocytosis by microglia, which comprises HMG1 or a partial peptide thereof or a salt thereof;
[2] a diagnostic agent for amyloid β42 aggregation and / or microglia-induced amyloid β42 phagocytic abnormality, comprising a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof;
[3] 剤 an agent for inhibiting amyloid β42 aggregation and / or promoting amyloid β42 phagocytosis by microglia, comprising an antibody against HMG1 or a partial peptide thereof or a salt thereof;
[4] a diagnostic agent for amyloid β42 aggregation and / or microglia-induced amyloid β42 phagocytosis abnormality, which comprises an antibody against HMG1 or a partial peptide thereof or a salt thereof;
[5] 剤 an amyloid β42 aggregation-suppressing and / or microglia-mediated amyloid β42 phagocytosis accelerator comprising a nucleotide sequence containing a nucleotide sequence complementary to the nucleotide sequence encoding HMG1 or a partial peptide thereof or a part thereof;
[6] a method for screening a substance which inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia by inhibiting HMG1 or a partial peptide thereof or a salt thereof,
[7] A method for screening for a substance which inhibits HMG1 and exhibits amyloid β42 aggregation inhibitory activity, comprising contacting amyloid β42 and a test substance with HMG1 or a partial peptide thereof or a salt thereof, and assaying amyloid β42 aggregation. ,
[8] The method of the above-mentioned [7], wherein the comparison is made with the aggregation of amyloid β42 when amyloid β42 is brought into contact with ΔHMG1 or a partial peptide thereof or a salt thereof.
[9] ΔHMG1 or a partial peptide thereof or a salt thereof, amyloid β42 and a test substance are brought into contact with microglia, and the amyloid β42 phagocytosis by microglia is assayed. HMG1 is inhibited to promote amyloid β42 phagocytosis by microglia A method for screening a substance having an action,
[10] The method of the above-mentioned [9], wherein HMG1 or a partial peptide thereof or a salt thereof and amyloid β42 are compared with amyloid β42 phagocytosis by microglia when the microglia are brought into contact with the microglia.
[11] a screening kit for a substance comprising HMG1 or a partial peptide thereof or a salt thereof, which inhibits HMG1 and inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia;
[12] The kit of the above-mentioned [11], further comprising amyloid β42.
[13] A substance obtained by using the method according to the above [6] or the kit according to the above [11], which inhibits amyloid β42 aggregation by inhibiting HMG1 and / or promotes amyloid β42 phagocytosis by microglia.
[14] a substance which inhibits amyloid β42 aggregation by inhibiting HMG1 and / or promotes amyloid β42 phagocytosis by microglia,
[15] A pharmaceutical comprising the substance of the above-mentioned [13] or [14],
[16] The medicament according to the above [15], which is an agent for preventing or treating Alzheimer's disease, Down's syndrome or amyloid angiopathy.
[17] A substance which suppresses amyloid β42 aggregation by inhibiting HMG1 expression and / or promotes amyloid β42 phagocytosis by microglia, which comprises using a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof. Screening method,
[18] A test substance is added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, and the expression of HMG1 or a partial peptide thereof is assayed. The method according to the above [17],
[19] A test substance and amyloid β42 are added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, and expression of HMG1 or a partial peptide thereof and aggregation of amyloid β42 Screening method for a substance showing suppression of amyloid β42 aggregation by suppressing the expression of HMG1;
[20] Test substance, amyloid β42 and microglia are added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, and expression of HMG1 or a partial peptide thereof and microglia A method for screening for a substance that suppresses HMG1 expression and exhibits amyloid β42 phagocytosis-promoting action, characterized by assaying amyloid β42 phagocytosis,
[21] ΔFor screening a substance comprising a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, which suppresses the expression of HMG1 and inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia. kit,
[22] The kit of the above-mentioned [21], further comprising amyloid β42.
[23] A substance obtained by using the method according to [17] or the kit according to [21], which suppresses amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia,
[24] Δ a substance which suppresses amyloid β42 aggregation by inhibiting expression of HMG1 and / or promotes amyloid β42 phagocytosis by microglia;
[25] A medicament comprising the substance according to the above [23] or [24],
[26] The present invention provides the medicament according to the above [25], which is an agent for preventing or treating Alzheimer's disease, Down's syndrome or amyloid angiopathy.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
HMG1 used in the present invention is a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. HMG1 can be, for example, any cell (eg, splenocytes, eg, spleen cells, monkeys, pigs, horses, sheep, goats, dogs, cats, guinea pigs, rats, mice, rabbits, hamsters, etc.) in humans and other mammals. Nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells , B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells , Hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells), blood cells, or any tissue in which these cells are present, such as the brain, Sites (eg, olfactory bulb, acrosomal nucleus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain staining , Substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessels, heart, thymus, It may be a protein derived from spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., or a chemical synthesis or cell-free translation system May be a protein synthesized by Alternatively, it may be a recombinant protein produced from a transformant into which a polynucleotide encoding HMG1 has been introduced.
[0011]
As the "amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2", for example, about 85% or more, preferably 90% or more, more preferably the amino acid sequence represented by SEQ ID NO: 2 Amino acid sequences having about 95% or more, most preferably about 98% or more identity, and the like.
Alternatively, “an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2” includes a) one or more (preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 2 About 1 amino acid sequence, more preferably about 1 to 10 amino acids, and still more preferably several (1 to 5) amino acids, and b) one or two amino acids in the amino acid sequence represented by SEQ ID NO: 2. An amino acid sequence to which the above (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids are added; c) represented by SEQ ID NO: 2 One or two or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids in the amino acid sequence have been substituted with other amino acids Amino acid sequence, or d) Such as proteins comprising the amino acid sequence which is a combination of these is also used.
The “protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2” of the present invention is “a protein substantially identical to the amino acid sequence represented by SEQ ID NO: 2”. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 2.
Examples of “substantially the same activity” include Aβ42 aggregation promoting activity and Aβ42 phagocytosis-suppressing activity by microglia. "Substantially the same" indicates that their activities are the same in nature. Therefore, it is preferable that the above activities are equivalent, but the extent of these activities (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times) Quantitative factors such as protein and protein molecular weight may be different.
The aggregation promoting activity of Aβ42 and the activity of inhibiting Aβ42 phagocytosis by microglia can be measured according to a method known per se. For example, it can be measured according to the method described in the screening method of the present invention described below.
[0012]
In the present specification, HMG1 is described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end, according to the convention of peptide labeling. HMG1 including human HMG1 containing the amino acid sequence represented by SEQ ID NO: 2 has a carboxyl group (—COOH) and a carboxylate (—COO) at the C-terminus.⁻), Amide (-CONH₂)) Or an ester (—COOR).
Here, R in the ester is, for example, C, such as methyl, ethyl, n-propyl, isopropyl or n-butyl._1-6Alkyl groups such as C, cyclopentyl, cyclohexyl, etc._3-8Cycloalkyl groups such as phenyl, α-naphthyl and the like;_6-12Aryl groups, for example phenyl-C such as benzyl, phenethyl, etc._1-2An alkyl group or α-naphthyl-C such as α-naphthylmethyl_1-2C such as an alkyl group_7-14In addition to aralkyl groups, pivaloyloxymethyl groups commonly used as oral esters are used.
When HMG1 has a carboxyl group (or carboxylate) other than the C-terminus, HMG1 includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Furthermore, in the above-mentioned protein, HMG1 has an amino group at the N-terminal methionine residue in which a protecting group (for example, formyl group, acetyl or the like) is used._2-6C such as alkanoyl group_1-6Acyl group), a glutamyl group formed by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, a substituent on the side chain of an amino acid in the molecule (for example, -OH, -SH, An amino group, an imidazole group, an indole group, a guanidino group, etc. are suitable protecting groups (for example, formyl group, C_2-6C such as alkanoyl group_1-6(Eg, an acyl group), or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
As a specific example of HMG1, for example, human-derived HMG1 containing the amino acid sequence represented by SEQ ID NO: 2 and the like are used. Human HMG1 is described in Nucleic Acids Research, Vol. 17, No. 3, p1197-1214 (1989), and has GenBank accession number CAA31110.1 (cDNA is X12597). It is a known protein registered and published.
[0013]
The partial peptide of HMG1 (hereinafter sometimes abbreviated simply as “the partial peptide of the present invention”) is a peptide having the above-described partial amino acid sequence of HMG1 as long as it has substantially the same activity as HMG1. For example, among HMG1 protein molecules, those having a partial amino acid sequence containing a sequence motif homologous to the amyloid-forming consensus sequence of Aβ peptide described in Non-Patent Document 9 described above, etc. Used.
The number of amino acids in the partial peptide of the present invention is preferably a peptide having an amino acid sequence of at least 20 or more, preferably 50 or more, more preferably 100 or more of the constituent amino acid sequences of HMG1.
Here, “substantially the same activity” has the same meaning as described above. The “substantially the same activity” can be measured in the same manner as described above.
[0014]
In the partial peptide of the present invention, the C-terminus has a carboxyl group (—COOH), a carboxylate (—COO)⁻), Amide (-CONH₂)) Or an ester (—COOR). When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Further, the partial peptide of the present invention includes, as in the case of HMG1, the amino group of the N-terminal methionine residue protected with a protecting group, and the generation of Gln formed by cleavage of the N-terminal side in a living body with pyroglutamic acid. Also included are those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those in which a sugar chain is bonded, such as a so-called glycopeptide.
Examples of the salt of HMG1 or a partial peptide thereof include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
[0015]
HMG1 or a salt thereof can be produced from the above-mentioned human or other mammalian cells or tissues by a protein purification method known per se, or by culturing a transformant containing a DNA encoding HMG1 described later. Can also be manufactured. Further, the protein can also be produced according to the protein synthesis method described later or according thereto.
When producing from human or mammalian tissues or cells, the homogenized human or mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.
[0016]
For the synthesis of HMG1 or a partial peptide thereof, a salt thereof or an amide thereof, a commercially available resin for protein synthesis can be usually used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or its amide.
Regarding the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid is directly added to the resin together with a racemization inhibitor additive (for example, HOBt, HOOBt), or the protected amino acid is previously activated as a symmetric acid anhydride or a HOBt ester or a HOOBt ester. After that, it can be added to the resin.
[0017]
The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When a sufficient condensation cannot be obtained by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.
[0018]
The protection of the functional group that should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
Examples of the protecting group for the amino group of the raw material include, for example, Z, Boc, tertiarypentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazide, trityl hydrazide and the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for this esterification, for example, a lower alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, and a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group are used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, tert-butyl and the like are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
[0019]
Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.
As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0020]
As another method for obtaining an amide form of a protein, for example, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is extended to a desired chain length on the amino group side, and A protein from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein from which only the protecting group for the carboxyl group at the C-terminal has been removed, and these proteins are mixed in the above-mentioned mixed solvent. To condense. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. The crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
In order to obtain an ester of a protein, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, an ester of the desired protein is obtained in the same manner as the amide of the protein be able to.
[0021]
A partial peptide of HMG1 or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving HMG1 with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting HMG1 with the remaining portion and, when the product has a protecting group, removing the protecting group. Examples of the known condensation method and elimination of the protecting group include the methods described in the following a) to e).
a) M. {Bodanszky} and {M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York, 1966 b) Schroeder and Luebke, The Peptide, They, Nyc.
c) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
d) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemical Experiments 1, Protein Chemistry IV, 205, (1977)
e) Supervised by Haruaki Yajima, Development of Continuing Drugs, Vol. 14, Peptide Synthesis, Hirokawa Shoten
After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is in a free form, it can be converted to an appropriate salt by a known method, and when it is obtained in a salt, it can be converted to a free form by a known method. Can be.
[0022]
The nucleotide containing the nucleotide sequence encoding HMG1 (hereinafter, sometimes abbreviated as “nucleotide of the present invention”) may be any nucleotide containing the nucleotide sequence encoding the HMG1 protein described above. And may be DNA, RNA or DNA / RNA chimera, preferably DNA. The nucleotide may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
[0023]
Examples of the DNA containing the nucleotide sequence encoding HMG1 (hereinafter, sometimes abbreviated as “DNA of the present invention”) include genomic DNA, genomic DNA library, human or other mammals (eg, bovine, monkey, Horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, hamster, etc., any cells (eg, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells) , Epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils , Eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, Progenitor cells of these cells, stem cells or cancer cells, etc.), blood cells, or any tissue in which these cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb, squamous nucleus, basal sphere, hippocampus) , Thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney , Liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testicle , Testis, ovary, placenta, uterus, bones, joints, skeletal muscle, etc. (particularly, brain and various parts of the brain), cDNA libraries derived from the above-described cells and tissues, and synthetic DNAs. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid, retrovirus vector, adenovirus vector, adeno-associated virus vector and the like. Alternatively, amplification can be directly performed by using Reverse RNA / Transscriptase / Polymerase / Chain / Reaction (hereinafter abbreviated as RT-PCR) using a total RNA or mRNA fraction prepared from the cells and tissues described above.
[0024]
Specifically, as the DNA encoding HMG1, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 or a DNA hybridizing with the nucleotide sequence represented by SEQ ID NO: 1 under high stringency conditions Any DNA may be used as long as it encodes a protein having a soybean base sequence and having substantially the same activity as HMG1 (eg, Aβ42 aggregation promoting activity, Aβ42 phagocytosis inhibitory activity by microglia, etc.). Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 1 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more with the base sequence represented by SEQ ID NO: 1. And most preferably a DNA containing a nucleotide sequence having about 95% or more homology.
[0025]
Hybridization is performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can be. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
[0026]
More specifically, as the DNA encoding human-derived HMG1 containing the amino acid sequence represented by SEQ ID NO: 2, a DNA containing the base sequence represented by SEQ ID NO: 1 or the like is used.
[0027]
Nucleotides containing a base sequence complementary to the target region of the target nucleic acid, that is, nucleotides capable of hybridizing with the target nucleic acid, can be said to be "antisense" to the target nucleic acid. On the other hand, a nucleotide containing a base sequence having homology to the target region of the target nucleic acid (that is, when the target nucleic acid encodes a protein, a nucleotide encoding a partial peptide of the protein) is “sense” to the target nucleic acid. It can be said that. The term “corresponding” as used herein means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. As used herein, "having homology" or "complementary" means that at least about 70%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95%, between base sequences. Refers to having homology or complementarity. The term “corresponding” between a nucleotide, a base sequence or a nucleic acid and a peptide (protein) means that the peptide (protein) has an amino acid sequence translated from a nucleotide (nucleic acid) or its complement. Usually pointing.
[0028]
Nucleotides comprising all or part of the nucleotide sequence complementary to the nucleotides of the present invention (hereinafter also referred to as “antisense nucleotides of the present invention”) are cloned or determined nucleotides of the present invention. It can be designed and synthesized based on nucleotide sequence information. Such nucleotides can inhibit the replication or expression of a gene containing the nucleotide sequence of the nucleotide of the present invention. That is, the antisense nucleotide of the present invention can hybridize to RNA transcribed from the HMG1 gene and inhibit mRNA synthesis (processing) or function (translation into protein), or HMG1-related. The expression of the HMG1 gene can be regulated and controlled through the interaction with RNA. Nucleotides complementary to selected sequences of HMG1-related RNA and capable of specifically hybridizing to HMG1-related RNA are useful for regulating and controlling HMG1 gene expression in vivo and in vitro. Yes, and is useful for treating or diagnosing diseases and the like.
[0029]
The target region of the antisense nucleotide of the present invention is not particularly limited in its length as long as the translation of the HMG1 protein is inhibited as a result of the hybridization of the antisense nucleotide. The sequence may be a short sequence or a partial sequence, and includes a short sequence of about 15 bases and a long sequence of the entire mRNA or early transcript. Considering the ease of synthesis and the problem of antigenicity, oligonucleotides consisting of about 15 to about 30 bases are preferred, but not limited thereto. Specifically, for example, the 5 'end hairpin loop of HMG1 mRNA, the 5' end 6-base pair repeat, the 5 'end untranslated region, the polypeptide translation initiation codon, the protein coding region, the ORF translation initiation codon, 3' The end untranslated region, the 3 'end palindrome region, and the 3' end hairpin loop may be selected as target regions, but any region within the HMG1 gene may be selected as a target. For example, it is also preferable to use the intron portion of the gene as the target region.
Furthermore, the antisense nucleotide of the present invention not only hybridizes with HMG1 mRNA or an early transcript to inhibit translation into a protein, but also binds to the HMG1 gene, which is a double-stranded DNA, to form a triplex. And may inhibit RNA transcription.
[0030]
Antisense nucleotides are deoxynucleotides containing 2-deoxy-D-ribose, deoxynucleotides containing D-ribose, other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides. Other polymers having a backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymer is a pair of bases as found in DNA or RNA (Including a nucleotide having a configuration permitting attachment of a ring or base). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known. Such as labeled, capped, methylated, one or more naturally-occurring nucleotides replaced by analogs, intramolecular nucleotides Modified, eg, those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, phosphorodithioate, etc.) ), Such as proteins (nucleases, nuclease inhibitors, toxic , Antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides) having side chain groups, interacting compounds (for example, acridine, psoralen, etc.), chelates Those containing compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.
[0031]
Antisense nucleotides are RNA, DNA, or modified nucleic acids (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids. Many such modifications are known in the art, for example, see {J. {Kawakami} et al. Pharm Tech Japan, Vol. $ 8, $ pp. 247, {1992; $ 8, $ pp. 395, $ 1992; T. {Crook et al. Ed. , {Antisense} Research \ and \ Applications, {CRC Press, {1993}, and the like.
[0032]
Antisense nucleotides may contain altered or modified sugars, bases, or linkages and may be provided in special forms such as liposomes, microspheres, applied or added by gene therapy. It could be given in form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, crude water-based substances such as phospholipid and cholesterol can be used. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Examples of other groups include cap groups specifically arranged at the 3 'end or 5' end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
[0033]
A ribozyme capable of specifically cleaving the HMG1 mRNA or the initial transcript of the HMG1 gene inside the coding region (including the intron portion in the case of the initial transcript) can also be included in the antisense nucleotide of the present invention. The term "ribozyme" refers to RNA having an enzymatic activity for cleaving a nucleic acid. Recently, it has been revealed that an oligo DNA having the nucleotide sequence of the enzyme active site also has a nucleic acid cleaving activity. In this document, the term is used as a concept including DNA as long as it has a sequence-specific nucleic acid cleavage activity. The most versatile ribozymes include self-splicing RNAs found in infectious RNAs such as viroids and viruses, and hammerhead and hairpin types are known. The hammerhead type exerts enzymatic activity at about 40 bases, and converts several bases at both ends (about 10 bases in total) adjacent to the hammerhead structure into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the further advantage that it does not attack genomic DNA since it uses only RNA as substrate. When the mRNA corresponding to the nucleotide of the present invention has a double-stranded structure by itself, the target sequence can be reduced by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid capable of specifically binding to RNA helicase. Main chain [Proc. {Natl. {Acad. {Sci. {USA, {98 (10): {5572-5577} (2001)]. Further, when the ribozyme is used in the form of an expression vector containing a DNA encoding the ribozyme, a hybrid ribozyme in which a sequence modified from tRNA is further linked in order to promote the transfer of a transcript to the cytoplasm. [Nucleic Acids Res. , {29 (13): {2780-2788} (2001)].
[0034]
Double-stranded oligo RNA complementary to HMG1 mRNA or a partial sequence in the coding region of the early transcript of the HMG1 gene (including an intron portion in the case of the early transcript) can also be included in the antisense nucleotide of the present invention. . When a short double-stranded RNA is introduced into a cell, mRNA complementary to the RNA is degraded, a phenomenon called so-called RNA interference (RNAi), which has long been known in nematodes, insects, plants, and the like. Recently, it has been confirmed that this phenomenon also occurs in mammalian cells [Nature, {411 (6836): {494-498} (2001)], and is attracting attention as an alternative technique to ribozymes.
The antisense oligonucleotide and ribozyme of the present invention determine the target region of mRNA or early transcript based on the cDNA sequence of HMG1 or genomic DNA sequence information, and use a commercially available automatic DNA / RNA synthesizer (Applied Biosystems, Inc.). Using Beckman, Inc.) to synthesize a sequence complementary thereto. A double-stranded oligo RNA having RNAi activity is prepared by synthesizing a sense strand and an antisense strand with a DNA / RNA automatic synthesizer, and in a suitable annealing buffer, for example, at about 90 to about 95 ° C. for about 1 minute. After denaturation, it can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, a longer double-stranded polynucleotide can be prepared by synthesizing complementary oligonucleotide chains so as to alternately overlap each other, annealing them, and ligating them with a ligase.
[0035]
The HMG1 gene expression inhibitory activity of the antisense nucleotide of the present invention can be determined by using a transformant containing the nucleotide of the present invention, an HMG1 gene expression system in vivo or in vitro, or an HMG1 protein in vivo or in vitro translation system. You can find out.
[0036]
The DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the above-described cells / tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by RT-PCR using an mRNA fraction prepared from the cells and tissues described above.
Specifically, the DNA encoding the partial peptide of the present invention includes, for example, (1) a DNA having a partial nucleotide sequence of a DNA having the nucleotide sequence represented by SEQ ID NO: 1, or (2) a DNA having a partial nucleotide sequence: Has a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by No. 1 and has substantially the same activity as HMG1 (eg, Aβ42 aggregation promoting activity, Aβ42 phagocytosis inhibitory activity by microglia, etc.) For example, a DNA having a partial base sequence of a DNA encoding a protein having the following structure is used.
Examples of the DNA capable of hybridizing the base sequence represented by SEQ ID NO: 1 include about 70% or more, preferably about 80% or more, more preferably about 90% or more with the base sequence represented by SEQ ID NO: 1. Most preferably, a DNA containing a base sequence having about 95% or more homology is used.
[0037]
As a means for cloning DNA that completely encodes HMG1 or a partial peptide thereof (hereinafter sometimes abbreviated as "HMG1" in some cases), PCR using a synthetic DNA primer having a partial base sequence of HMG1 is used. The DNA can be selected by amplifying or hybridizing with a DNA incorporated into an appropriate vector and labeled with a DNA fragment encoding a part or the entire region of HMG1 or a synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
[0038]
Conversion of the base sequence of DNA can be performed by PCR or a known kit, for example, Mutan.^TM-Super @ Express @ Km (Takara Shuzo Co., Ltd.), Mutan^TMUsing -K (Takara Shuzo Co., Ltd.) or the like, the method can be carried out according to a method known per se such as the ODA-LA @ PCR method, the Gapped @ duplex method, the Kunkel method, or a method analogous thereto.
The cloned DNA encoding HMG1 can be used as it is depending on the purpose, or may be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
An HMG1 expression vector can be produced, for example, by (a) cutting out a DNA fragment of interest from DNA encoding HMG1, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. it can.
[0039]
Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage, etc. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Among them, it is preferable to use the CMV promoter, SRα promoter and the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP_LWhen the host is a bacterium belonging to the genus Bacillus, the promoter, lpp promoter and the like are preferably SPO1 promoter, SPO2 promoter and penP promoter. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
[0040]
In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used. it can. Examples of the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance] and an ampicillin resistance gene (hereinafter Amp).^rNeomycin resistance gene (hereinafter Neo).^rG418 resistance). In particular, CHO (dhfr⁻) When the dhfr gene is used as a selection marker using cells, the target gene can be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
Using the vector containing the DNA encoding HMG1 thus constructed, a transformant can be produced.
[0041]
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
As a specific example of the genus Escherichia, Escherichia coli (Escherichia coli) K12.DH1 [Procedures of the National Academy of Sciences of the USA (Proc. @ Natl. @ Acad. @ Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology)]. 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genete Box (Genetics), 39 vol., 440 (1954)], etc..
Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)]. )] Is used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces @ pombe NCYC1913, NCYC2036, Pichia pastoris and the like.
[0042]
As the insect cells, for example, when the virus is AcNPV, a cell line derived from the larva of night rob moth (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, and Hig derived from Trichoplusia ni ni egg.^TMCells, cells derived from Mamestra @ brassicae or cells derived from Estigmena @ acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx @ mori @ N; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC @ CRL1711), Sf21 cells (above, Vaughn, @JL, et al., In Vivo, 13, 213-217, (1977)) and the like are used. .
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), and dhfr gene-deficient Chinese hamster cells CHO (hereinafter CHO (dhfr).⁻) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, human HEK293 cells, and the like.
[0043]
In order to transform Escherichia, for example, Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69, 2110 ( 1972) and Gene, Vol. 17, 107 (1982).
Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
In order to transform yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Processings of the National Academy of Sciences of Science -The USA (Proc. Natl. Acad. Sci. USA), Vol. 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, $ 6, $ 47-55 (1988).
To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, 456 (1973).
Thus, a transformant transformed with the expression vector containing the DNA encoding HMG1 is obtained.
When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are included. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.
[0044]
Examples of a medium for culturing Escherichia include, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journal of Experimentals in in Molecular Genetics)]. , 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolyl peracrylic acid can be added in order to make the promoter work efficiently.
When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, as a medium, for example, a Burkholder minimum medium [Bostian, ΔK. L. Procurings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and 0.5% casamino acid. Medium [Bitter, @G. A. Pla, Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.
[0045]
When culturing a transformant in which the host is an insect cell or an insect, the culture medium is immobilized as Grace's Insect Medium (Grace, TTC, Nature, 195, 788 (1962)). For example, those to which an additive such as 10% bovine serum or the like is appropriately added are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration or stirring is added.
When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association (The American Journal of Association) Volume 199, 519 (1967)], 199 [Proceding of the Society for the Biological Medicine (7), "Proceeding of the Society for the Biological Medicine", 7 Winding, such as 1 (1950)] is used. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.
As described above, HMG1 can be produced in the transformant, in the cell membrane, or outside the cell.
[0046]
HMG1 can be separated and purified from the culture by, for example, the following method.
When HMG1 is extracted from the cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and sonicated, lysozyme and / or freeze-thaw, etc. Alternatively, a method of obtaining a crude extract of HMG1 by centrifugation or filtration after disrupting the cells is used as appropriate. In a buffer, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-100 is used.^TMAnd the like. When HMG1 is secreted into the culture solution, after the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.
The HMG1 contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific novelty such as affinity chromatography, use of difference in hydrophobicity such as reversed-phase high-performance liquid chromatography And a method utilizing the difference in isoelectric points such as isoelectric focusing.
[0047]
When the HMG1 thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or a method analogous thereto Can be converted to a free form or another salt.
The HMG1 produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
The activity of HMG1 thus produced can be measured by an enzyme immunoassay using a specific antibody or the like.
[0048]
The antibody against HMG1 may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize HMG1.
An antibody against HMG1 can be produced by using HMG1 as an antigen according to a known method for producing an antibody or antiserum.
[0049]
[Preparation of monoclonal antibody]
(A) Preparation of monoclonal antibody-producing cells
HMG1 is administered to a mammal at a site capable of producing an antibody by administration, itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the mammal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, an individual having an antibody titer was selected from a mouse, and the spleen or lymph node was collected 2 to 5 days after the final immunization. By fusing the contained antibody-producing cells with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled HMG1 described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
Examples of myeloma cells include NS-1, P3U1, SP2 / 0 and the like, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. Incubation at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes allows efficient cell fusion.
[0050]
Various methods can be used for screening a monoclonal antibody-producing hybridoma. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which the HMG1 antigen is directly or adsorbed together with a carrier, and then the radioactive substance or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A; A method in which a hybridoma culture supernatant is added to a solid phase to which a globulin antibody or protein A is adsorbed, HMG1 labeled with a radioactive substance, an enzyme, or the like is added, and a monoclonal antibody bound to the solid phase is detected.
The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal bovine serum, or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
[0051]
(B) Purification of monoclonal antibody
Monoclonal antibodies can be separated and purified by immunoglobulin separation and purification methods [eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which an antibody alone is collected using an antigen-bound solid phase or an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody. Can be performed according to
[0052]
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (HMG1 antigen) and a carrier protein is formed, a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody, and an antibody-containing substance for HMG1 is collected from the immunized animal to obtain an antibody. Can be produced by separation and purification of
Regarding a complex of an immunizing antigen and a carrier protein used to immunize a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten should be such that the antibody can be efficiently produced against the hapten immunized by crosslinking the carrier. Any kind may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. may be used in a weight ratio of about 0.1 to 20 with respect to 1 hapten. Preferably, a method of coupling at a ratio of about 1 to 5 is used.
Various coupling agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of the mammal immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the measurement of the antibody titer in the serum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.
[0053]
HMG1 is up-regulated in both the particle fraction (including nucleus, membrane and insoluble proteins) and the cytoplasmic fraction (including cytosolic and extracellular soluble proteins) of Alzheimer's disease brain, and extracellular HMG1 Coexist with spots. Furthermore, HMG1 inhibits the phagocytosis of Aβ42 by microglia and binds to Aβ42 to promote Aβ42 aggregate formation. Accordingly, HMG1 or a partial peptide thereof or a salt thereof (hereinafter, also referred to as “HMG1 or the like”), DNA encoding HMG1 or the like, an antisense nucleotide of the present invention, an antibody against HMG1 or the like (hereinafter, abbreviated as “the antibody of the present invention”) Have the following uses.
[0054]
[1] Use of HMG1 or its partial peptide or salt thereof
(A) Vaccine for inhibiting Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia
When HMG1 or a partial peptide thereof or a salt thereof is administered as a vaccine, an anti-HMG1 antibody can be produced in the body. Since the anti-HMG1 antibody can neutralize the Aβ42 aggregation promoting action of HMG1 and the Aβ42 phagocytosis inhibitory action of microglia, the HMG1 vaccine has an Aβ42 aggregation inhibitory action and / or an effect of promoting Aβ42 phagocytosis by microglia. However, it is useful as a prophylactic / therapeutic agent for diseases associated with senile plaque formation due to Aβ deposition using Aβ42 as a seed, specifically, cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.). .
When HMG1 and the like are used for the prevention and treatment of the above-mentioned diseases, they can be vaccinated according to conventional means.
For example, a) HMG1 or the like can be sterilized with orally as a sugar-coated tablet, capsule, elixir, microcapsule or the like, or with water or another pharmaceutically acceptable liquid, if necessary. It can be used parenterally in the form of injections, such as solutions or suspensions. For example, b) incorporation of HMG1 or the like with known physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of the formulation. It can be manufactured by doing. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
[0055]
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
[0056]
In addition, the vaccine includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, (Eg, polyethylene glycol), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
The dose of HMG1 or the like varies depending on the administration subject, target organ, symptom, administration method and the like. In the case of oral administration, for example, in an Alzheimer's disease patient (as 60 kg), about 0.01 to 1000 mg, Preferably it is about 0.1-100 mg, more preferably about 1-50 mg. When administered parenterally, it varies depending on the target organ, symptoms, administration method, etc., but for example, in the form of an injection, for example, in an Alzheimer's disease patient (as 60 kg), about 0.01 to 1000 mg at a time. It is convenient to administer, preferably about 0.1 to 100 mg, more preferably about 1 to 50 mg. In the case of other animals, the amount can be administered in terms of 60 kg. Administration can be performed 1 to 10 times at intervals of 1 to 21 days.
[0057]
[2] Use of nucleotides encoding HMG1 or a partial peptide thereof
(A) Gene diagnostic agent
The nucleotides and antisense nucleotides of the present invention can be used as probes to convert HMG1 or a partial peptide thereof in humans or mammals (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). Since abnormalities (encoding abnormalities) of the encoding DNA or mRNA can be detected, they can be used, for example, as genetic diagnostic agents for damage, mutation or decreased expression of the DNA or mRNA, and increased or excessive expression of the DNA or mRNA. Useful.
The above-described genetic diagnosis using the nucleotide or the antisense nucleotide of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processing). Proceedings of the National Academy of Sciences of the United States of America, Volume 86, 2766-2770, etc. (Procedings of the National Academy of Sciences, United States of America, USA), Vol. 86, pp. 2766-2770. Can be.
If overexpression of HMG1 is detected by Northern hybridization, for example, overexpression of HMG1 may cause Aβ42 aggregation and / or microglial abnormalities in Aβ42 phagocytosis, and thus the formation and accumulation of senile plaques Therefore, it can be diagnosed that there is a high possibility of having a disease associated with senile plaque, for example, cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0058]
[3] Use of an antibody against HMG1 or a partial peptide thereof or a salt thereof
(A) Quantification of HMG1 and diagnosis of Aβ42 phagocytosis abnormality by Aβ42 aggregation and / or microglia
Since the antibody of the present invention can specifically recognize HMG1, it can be used for diagnosis of abnormal Aβ42 phagocytosis due to Aβ42 aggregation and / or microglia by detecting extracellular HMG1.
That is, the present invention
(I) reacting the antibody of the present invention with a test solution and labeled HMG1 competitively, and measuring the ratio of labeled HMG1 bound to the antibody in the test solution. A method for quantifying the receptor, and
(Ii) After reacting a test solution with the antibody of the present invention immobilized on a carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. And a method for quantifying HMG1 in a test solution.
[0059]
In the quantification method (ii), when one antibody is an antibody that recognizes the N-terminus of HMG1, it is preferable that the other antibody is an antibody that recognizes another portion of HMG1 such as the C-terminus. .
In addition, the protein can be quantified using a monoclonal antibody against HMG1, and detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, and the F (ab ')₂, Fab ′ or Fab fraction may be used.
[0060]
The method for quantifying HMG1 using the antibody of the present invention is not particularly limited, and the amount of an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of an antigen (for example, the amount of HMG1) in a liquid to be measured is determined. Any measurement method may be used as long as it is detected by physical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are suitably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.
[0061]
As a labeling agent used in a measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [¹²⁵I], [¹³¹I], [³H], [¹⁴C] is used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins and the like may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of HMG1 in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.
[0062]
In the method for measuring HMG1 by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different binding site to HMG1 is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of HMG1, the antibody used in the primary reaction is preferably the C-terminal. In addition, for example, an antibody that recognizes the N-terminal is used.
The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.
In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. A solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble sediment produced as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The HMG1 measurement system may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, "Radio immunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radio immunoassay continued" edited by Hiroshi Irie (published in 1974), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) 62)), "Methods in ENZY MOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), Id. 73 (ImmunochemicalｍTechniques (Part B)); $ 74 (Immunochemical Techniques (Part C)), Ibid. Vol. 84 (Immunochemical Techniques (Part Dａ: Selected ａImmunoassays)), Id. 92 (Immunochemical Techniques (Part E): Monoclonal Antibodies and General Immunoassay Methods), Ibid. Vol. $ 121 (Immunochemical Techniques (Part I): Hybridoma Technology and Monoclonal Antibodies) (above, published by Academic Press) can be referred to.
As described above, HMG1 can be quantified with high sensitivity by using the antibody of the present invention.
[0063]
In the above-described quantification method using the antibody of the present invention, the extracellular fluid in the brain parenchyma or cerebral blood vessel wall is used as a test solution, and the concentration of the extracellular HMG1 in the brain parenchyma or cerebral blood vessel wall is quantified, whereby the protein concentration is determined. If an increase in Aβ42 is detected, for example, it is highly likely that Aβ42 aggregation and / or microglia cause abnormal Aβ42 phagocytosis, and consequently the formation and accumulation of senile plaques due to, for example, overproduction and / or leakage of HMG1. It can be diagnosed that there is a high possibility of suffering from a disease involving senile plaques, for example, cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0064]
(B) Aβ42 Aggregation Inhibition and / or Aβ42 Phagocytosis Promoter by Microglia The antibody of the present invention can neutralize the Aβ42 aggregation promotion effect of HMG1 and the Aβ42 phagocytosis inhibitory effect of microglia. A / 42 has an effect of promoting Aβ42 phagocytosis by microglia, and is related to senile plaque formation due to Aβ deposition using Aβ42 as a seed, specifically, cerebral amyloidosis (eg, Alzheimer's disease, Down syndrome, amyloid) Useful as a prophylactic / therapeutic agent for angiopathy).
When the antibody of the present invention is used for the prevention or treatment of the above-mentioned diseases, it can be formulated according to a conventional method.
For example, a) the antibody of the present invention can be used as a tablet, capsule, elixir, microcapsule or the like, if necessary, coated with sugar, or with water or another pharmaceutically acceptable liquid. It can be used parenterally in the form of injections, such as sterile solutions or suspensions. For example, b) a unit dosage form required for carrying out a generally accepted formulation of the antibody of the present invention with known physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like. And can be produced by mixing. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
[0065]
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
[0066]
The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like may be added. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
The dose of the antibody of the present invention varies depending on the administration subject, target organ, condition, administration method, and the like. In the case of oral administration, for example, in an Alzheimer's disease patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, condition, administration method and the like. For example, in the case of injection, for example, in an Alzheimer's disease patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, the amount can be administered in terms of 60 kg.
[0067]
Further, since the antibody of the present invention specifically recognizes HMG1, it can be used as a targeting agent for delivering those agents to HMG1 in combination with an HMG1 inhibitor described below.
[0068]
[4] Uses of the antisense nucleotide of the present invention
(A) Aβ42 aggregation inhibitory and / or Aβ42 phagocytosis promoter by microglia
Since the antisense nucleotide of the present invention inhibits the synthesis of HMG1 mRNA and the translation into HMG1 protein, it has an effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia, and uses Aβ42 as a seed. It is useful as a prophylactic / therapeutic agent for diseases associated with senile plaque formation due to Aβ deposition, specifically, cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
When the antisense nucleotide of the present invention is used as the above-described prophylactic / therapeutic agent, the antisense nucleotide is inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by conventional means. It can be formulated. The antisense nucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter.
[0069]
For example, the nucleotide may be a sugar-coated tablet, capsule, elixir, microcapsule, or the like as needed, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions. For example, the nucleotide is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form required for generally accepted pharmaceutical practice. Can be manufactured by The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.
[0070]
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
[0071]
The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like may be added. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
[0072]
The dose of the antisense nucleotide varies depending on the administration subject, the target organ, the condition, the administration method, and the like. In the case of oral administration, in general, for example, in an Alzheimer's disease patient (as 60 kg), about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in Alzheimer's disease patients (as 60 kg). It is convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, the amount can be administered in terms of 60 kg.
[0073]
[5] HMG1 inhibitor cleaning method / screening kit
A compound that inhibits the activity of HMG1 or a partial peptide thereof or a salt thereof (eg, Aβ42 aggregation promoting activity, Aβ42 phagocytosis inhibitory activity by microglia) or a salt thereof is an effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia. It is useful as a prophylactic / therapeutic agent for diseases involving senile plaque formation due to Aβ deposition using Aβ42 as seed, specifically, cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.) It is.
Therefore, HMG1 and the like are useful as reagents for screening compounds or salts thereof that inhibit the activity of HMG1 and the like (eg, Aβ42 aggregation promoting activity, Aβ42 phagocytosis inhibitory activity by microglia).
[0074]
That is, the present invention
(1) Screening of a substance that promotes Aβ42 aggregation promoting activity of HMG1 and / or promotes Aβ42 phagocytosis inhibitory activity of microglia, which is characterized by using HMG1 or the like (hereinafter sometimes abbreviated simply as “HMG1 inhibitor”). Methods, and more specifically, for example,
(2a) screening for an HMG1 inhibitor, comprising comparing the aggregation of Aβ42 between (i) contacting Aβ42 with HMG1 etc. and (ii) contacting Aβ42 and a test substance with HMG1 etc. Method, and
(2b) Aβ42 phagocytosis by microglia is compared when (i) HMG1 etc. and Aβ42 are brought into contact with microglia and (ii) when HMG1 etc. and Aβ42 and a test substance are brought into contact with microglia. And a method for screening for an HMG1 inhibitor.
[0075]
The specific description of the screening method (2a) of the present invention is as follows.
HMG1, its partial peptide or a salt thereof may be any one as described above. Aβ42 is isolated and purified by a method known per se from the brain parenchyma or cerebral blood vessel wall of human or other mammals, is chemically synthesized based on the known Aβ42 amino acid sequence, and encodes Aβ42. Any of recombinant cells or those produced using a cell-free protein synthesis system may be used.
As test substances, for example, peptides, proteins, nucleic acids, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like are used. Or a known compound.
To carry out the above screening method, a sample such as HMG1 is prepared by suspending HMG1 or the like in a buffer suitable for screening. The buffer may be a buffer that does not inhibit the binding between HMG1 or the like and Aβ42 and / or the aggregation of Aβ42, such as a phosphate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) and a Tris-HCl buffer. Any may be used.
Aβ42 aggregation promoting activity such as HMG1 can be determined by, for example, incubating HMG1 and Aβ42 (and a test substance) at room temperature to about 40 ° C. for about 1 to about 48 hours, and then subjecting the reaction solution to immunoblotting with an anti-Aβ antibody. Can be measured.
For example, in the case of the above (ii), a substance inhibiting the Aβ42 aggregation promoting activity such as HMG1 is a test substance which inhibits the Aβ42 aggregation promoting activity by about 20% or more, preferably 50% or more as compared with the above (i). Can be selected as
The screening kit of the present invention contains HMG1 or a partial peptide thereof or a salt thereof. Examples of the screening kit of the present invention include the following.
[Screening reagent]
(1) Buffer for measurement
pH 7.0 phosphate buffered saline (PBS)
(2) Protein sample
Purified HMG1 (eg, from calf thymus)
(3) Aβ42
10 μM PBS solution
(4) Anti-Aβ polyclonal antibody (from rabbit)
[0076]
A specific description of the screening method (2b) of the present invention will be given below.
As for HMG1 and the like, Aβ42 and a test substance, the same ones as those in the above method (2a) are used. Microglia is prepared by, for example, suspending mixed glial cells (a mixture of astrocytes and microglia) prepared from a brain hemisphere excised from a mammal such as a rat in a medium such as DMEM supplemented with FBS, and plating the cells in a culture dish or the like. For example, 5% CO₂/ 95% in air, at about 30 to about 40 ° C, and obtained by collecting suspended microglia from mixed glial cultures.
To carry out the above screening method, a sample such as HMG1 is prepared by suspending HMG1 or the like in a buffer or medium suitable for screening. As the buffer, a phosphate buffer or a Tris-hydrochloride buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) is used, and a microglial culture medium such as MEM or DMEM is preferably used as a medium.
Aβ42 phagocytosis inhibitory activity by microglia such as HMG1 can be determined, for example, by adding HMG1 and Aβ42 (and a test substance) to a microglia suspension culture solution, for example, by adding 5% CO 2.₂After incubating at about 30 to about 40 ° C. for about 1 to about 48 hours in a / 95% atmosphere, microglia can be recovered and dissolved, and the lysate can be measured by immunoblotting with an anti-Aβ antibody.
For example, in the case of the above (ii), Aβ42 phagocytosis by a microglia such as HMG1 inhibits a test substance which inhibits the activity of inhibiting Aβ42 phagocytosis by microglia by about 20% or more, preferably 50% or more as compared with the case of the above (i). It can be selected as a substance that inhibits the inhibitory activity.
The screening kit of the present invention contains HMG1 or a partial peptide thereof or a salt thereof. Examples of the screening kit of the present invention include the following.
[Screening reagent]
(1) Buffer for measurement
pH 7.0 phosphate buffered saline (PBS)
(2) Protein sample
Purified HMG1 (eg, from calf thymus)
(3) Aβ42
10 μM PBS solution
(4) Microglial culture medium
DMEM with 10% FBS
(5) Anti-Aβ polyclonal antibody (from rabbit)
[0077]
The substance obtained by using the screening method or the screening kit of the present invention is a compound selected from the above-described test substances or a salt thereof, and has an Aβ42 aggregation promoting activity such as HMG1 and / or an Aβ42 phagocytosis inhibitory activity by microglia. It is a compound that inhibits. Representative examples of such compounds include HMG1 binding nucleic acids. HMG1 is a non-histone chromosomal protein and has a DNA binding domain in its amino acid sequence. Therefore, a nucleic acid having a base sequence capable of binding to HMG1 inhibits AMG aggregation promoting activity of HMG1 and / or Aβ42 phagocytosis inhibitory activity of microglia by capturing HMG1.
As the salt of the compound which is an HMG1 inhibitor, a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid).
[0078]
[6] Uses of HMG1 inhibitors
Compounds that inhibit Aβ42 aggregation promoting activity such as HMG1 and / or Aβ42 phagocytosis inhibitory activity by microglia have an effect of inhibiting Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia, and use Aβ42 as a seed. It is useful as a prophylactic / therapeutic agent for diseases involving senile plaque formation due to Aβ deposition, specifically, cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0079]
When the HMG1 inhibitor obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be carried out according to conventional means. For example, the compound can be a sugar-coated tablet, capsule, elixir, microcapsule, or the like, as needed, orally, or a sterile solution of water or other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions. For example, the compound may be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in a unit dosage form generally required for the practice of pharmaceutical preparations. Can be manufactured by The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.
[0080]
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
[0081]
The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like may be added. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
[0082]
The dose of the HMG1 inhibitor varies depending on the administration subject, target organ, symptoms, administration method, and the like. In the case of oral administration, for example, an HMG1 inhibitor is generally administered to an Alzheimer's disease patient (60 kg). About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. For example, in the case of an injection, it is usually used, for example, for Alzheimer's disease patients (as 60 kg). It is convenient to administer the HMG1 inhibitor in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, the amount can be administered in terms of 60 kg.
[0083]
[7] A screening method and a screening kit for an expression inhibitor such as HMG1 Further, the present invention further provides a substance that inhibits the expression of HMG1 or the like and inhibits Aβ42 aggregation and / or promotes Aβ42 phagocytosis by microglia (hereinafter, referred to as “ HMG1 expression inhibitor), and a kit therefor. In this method, a change in the amount of mRNA such as HMG1 is measured by using a nucleotide containing a nucleotide sequence encoding HMG1 or the like as a probe or primer. Preferably, a change in the Aβ42 aggregation promoting activity of HMG1 or the like or the Aβ42 phagocytosis inhibitory activity by microglia is measured by the same method as described above for the method of screening for an HMG1 inhibitor.
[0084]
The measurement of the amount of mRNA such as HMG1 is specifically performed as follows.
(I) Administering a test substance to a normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc., more specifically, dementia rat, etc.) After a certain period of time (for example, 30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), specific tissues such as brain parenchyma and cerebral blood vessels are excised. The mRNA such as HMG1 contained in the tissue can be quantified by, for example, extracting mRNA from cells by a usual method and using, for example, a technique such as TaqMan PCR, and Northern blotting by a method known per se. The analysis can also be performed by performing.
(Ii) A transformant expressing HMG1 or the like produced according to the above method is cultured in the presence of a test substance, and after a certain period of time (for example, 1 to 7 days, preferably 1 to 3 days, more preferably 1 day to 3 days later) mRNA such as HMG1 contained in the transformant can be quantified and analyzed in the same manner.
(Iii) A nucleotide containing a nucleotide sequence encoding HMG1 or the like prepared according to the above method is added to a per se known in vitro transcription system in the presence of a test substance, and incubated for a certain period of time (for example, after 30 minutes to 3 minutes). After 1 day, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), mRNA such as HMG1 contained in the reaction solution can be similarly quantified and analyzed.
[0085]
When the Aβ42 aggregation promoting activity of HMG1 or the like is further measured, Aβ42 may be administered (added) in addition to the test substance, and a part of the cell extract or the reaction solution may be subjected to immunoblotting with an anti-Aβ antibody. When the activity of inhibiting Aβ42 phagocytosis by microglia is further measured, (i) Aβ42 is administered in addition to a test substance, and after a certain period of time, microglia is separated and dissolved from tissues such as brain parenchyma, and the lysate is subjected to anti-Aβ Immunoblotting with antibodies or preparing brain slices to detect the presence of Aβ42 in microglia by immunohistochemical staining (for example, see Examples below), (ii) (iii) in addition to the test substance Aβ42 and microglia are added, and after a certain period of time, microglia is collected and dissolved, and the lysate may be subjected to immunoblotting with an anti-Aβ antibody.
[0086]
The screening kit used in the above screening method comprises a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof. Examples of the screening kit of the present invention include the following.
[Screening reagent]
(1) nucleotide sample
Isolated HMG1 cDNA (eg, from calf thymus mRNA)
(2) Aβ42
10 μM PBS solution
(3) Microglial culture medium
DMEM with 10% FBS
(4) Anti-Aβ polyclonal antibody (from rabbit)
[0087]
A compound or a salt thereof obtained by using the screening method / screening kit is a compound that suppresses the expression of HMG1 and the like and exhibits Aβ42 aggregation suppression and / or microglia-induced Aβ42 phagocytosis promoting action.
As a salt of the compound which is an HMG1 expression inhibitor, a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid).
[0088]
[8] Uses of HMG1 expression inhibitor
Compounds that suppress the expression of HMG1 and the like have an effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia, and diseases involving senile plaque formation due to Aβ deposition using Aβ42 as a seed. Is useful as a prophylactic / therapeutic agent for cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0089]
When an HMG1 expression inhibitor is used as the above-mentioned prophylactic / therapeutic agent, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be prepared in the same manner as in the preparation containing the above-mentioned HMG1 inhibitor.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
[0090]
The dose of the HMG1 expression inhibitor varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, HMG1 expression inhibition is generally performed, for example, on Alzheimer's disease patients (60 kg). The agent is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. For example, in the case of an injection, it is usually used, for example, for Alzheimer's disease patients (as 60 kg). It is convenient to administer the HMG1 expression inhibitor in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, the amount can be administered in terms of 60 kg.
[0091]
[9] A method for screening a compound that reduces the promoter activity of the HMG1 promoter
Further, the present invention provides a reporter gene assay method using the HMG1 promoter, wherein the promoter activity is measured when a test substance is administered and when the test substance is not administered. A screening method is provided.
The reporter gene assay can be performed by a method known per se, for example, The Journal of Biological Chemistry (JBC), 272, 22800-22808, 1997, Development, 124, 793. -804, 1997) can be used.
Examples of the test substance include peptides, proteins, non-peptidic compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds, microbial cultures, cell extracts, plant extracts, and animal tissue extracts. The compound may be a novel compound or a known compound.
Promoter activity can be measured by examining the expression level of a reporter gene. For example, the expression level of the reporter gene can be measured according to a method such as Northern blotting, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), TaqMan Polymerase Chain Reaction, or a method analogous thereto.
In this method, a compound that decreases the expression level of the reporter gene when the test substance is administered by about 20%, preferably 30% or more, and more preferably about 50% or more as compared with the case where the test substance is not administered is used. , HMG1 promoter.
[0092]
The compound or a salt thereof that reduces the promoter activity of the HMG1 promoter has an effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia, and is involved in senile plaque formation by Aβ deposition using Aβ42 as a seed. It is useful as a prophylactic / therapeutic agent for diseases, specifically, cerebral amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0093]
When a compound that reduces the promoter activity of the HMG1 promoter or a salt thereof is used as the above-mentioned prophylactic / therapeutic agent, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be prepared in the same manner as in the preparation containing the above-mentioned HMG1 inhibitor.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
[0094]
The dose of the compound or a salt thereof varies depending on the administration subject, the target organ, the condition, the administration method, and the like. However, in the case of oral administration, generally, for example, the HMG1 promoter is used for Alzheimer's disease patients (60 kg) Is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. For example, in the case of an injection, it is usually used, for example, for Alzheimer's disease patients (as 60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day of a compound that reduces the promoter activity of the HMG1 promoter. . In the case of other animals, the amount can be administered in terms of 60 kg.
[0095]
[10] Production of DNA-introduced animal of the present invention
The present invention relates to a non-human mammal having a foreign DNA of the present invention (hereinafter abbreviated as the foreign DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the foreign mutant DNA of the present invention). provide.
That is, the present invention
[1] a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
[2] the animal of [1], wherein the non-human mammal is a rodent;
[3] the animal of [2], wherein the rodent is a mouse or a rat, and
[4] It is to provide a recombinant vector containing the exogenous DNA of the present invention or its mutant DNA and capable of being expressed in mammals.
Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and can be used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) B6C3F as a system₁System, BDF₁System, B6D2F₁Strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD etc.) is preferred.
"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
[0096]
The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA.
The abnormal DNA means a DNA that expresses abnormal HMG1, and for example, a DNA that expresses a protein that suppresses the function of normal HMG1 is used.
The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, DNA derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto is expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created.
[0097]
As the HMG1 expression vector, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as λ phage, a retrovirus such as Moloney leukemia virus, an animal virus such as vaccinia virus or baculovirus are used. Can be Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, but are not limited to, (1) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, polio virus, etc.); ▼ Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, Tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chains, metallothionein I and IIA, Metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin, α And β-myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, Pre Russia enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, a human and chicken β-actin promoter, and the like are preferable.
The vector preferably has a sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal (generally called a terminator). For example, a sequence of each DNA derived from a virus and various mammals can be used. A simian virus SV40 terminator or the like is preferably used.
[0098]
In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.
Further, by adding a sequence encoding a secretion signal 5 ′ upstream of the translation region, the translation product of the exogenous DNA can be secreted out of the cell (by removing the nuclear translocation signal portion of the DNA encoding HMG1. It is preferable to keep it). A transgenic animal that secretes exogenous HMG1 extracellularly will be extremely useful in that it can serve as an Alzheimer's disease model that reflects pathological changes in Alzheimer's disease better than conventionally known PS1 and PS / APP mice.
[0099]
The normal HMG1 translation region is obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and various commercially available genomes From the DNA library, it is possible to obtain the whole or a part of the genomic DNA, or the complementary DNA prepared from the liver, kidney, thyroid cells and fibroblast-derived RNA by a known method as a raw material. In addition, a translation region in which a normal HMG1 translation region obtained from the above cells or tissues is mutated by a point mutagenesis method can be prepared from the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in a transposed animal by a conventional DNA engineering technique in which it is ligated downstream of the above-mentioned promoter and, if desired, upstream of the transcription termination site.
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal cells and somatic cells. means. The offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably retained by mating. .
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.
[0100]
The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and eventually develops hyperfunction of HMG1 by promoting the function of endogenous normal DNA. And can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of HMG1 hyperfunction and diseases associated with HMG1, and to examine methods for treating these diseases.
In addition, since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of free HMG1, it can be used for a screening test for a therapeutic drug for a disease associated with HMG1.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in an ordinary breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed the cells so that all offspring have the DNA.
[0101]
The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, and finally inhibits the function of endogenous normal DNA, resulting in HMG1 function inactive refractory disease. It can be used as a disease model animal. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of HMG1 function-inactive refractory disease and to examine a method for treating this disease.
Further, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention is a model for elucidating the function inhibition (dominant negative action) of normal HMG1 by the abnormal HMG1 of the present invention in HMG1 function-inactive refractory disease. It becomes.
In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of free HMG1, it can be used for a therapeutic drug screening test for HMG1 function inactive refractory disease.
In addition, other possible uses of the above two DNA-transferred animals of the present invention include, for example,
(1) Use as a cell source for tissue culture,
{Circle around (2)} By directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing HMG1 expressed by DNA, a protein specifically expressed or activated by HMG1 and HMG1 are analyzed. Analysis of relevance,
{Circle around (3)} The cells of a tissue having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture.
(4) screening for a drug that enhances the function of the cell by using the cell described in (3) above, and
(5) Isolation and purification of the mutant HMG1 of the present invention and production of its antibody can be considered.
Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of HMG1-related diseases, including HMG1-function-inactive refractory disease, etc. A more detailed pathological finding is obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.
In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain the released DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to examine the specification of HMG1-producing cells, the relationship with apoptosis, differentiation or proliferation, or the signal transduction mechanism thereof, and to investigate their abnormalities. It becomes.
Furthermore, using the DNA transgenic animal of the present invention to develop a therapeutic agent for a disease associated with HMG1, including a functionally inactive refractory type of HMG1, using the above-described test method and quantitative method, It is possible to provide an effective and rapid screening method for the therapeutic agent for the disease. Further, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for HMG1-related diseases.
[0102]
[11] Knockout animal
The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
That is, the present invention
[1] a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
[2] the embryonic stem cell according to [1], wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli);
[3] the embryonic stem cell according to [1], which is neomycin-resistant;
[4] the embryonic stem cell according to [1], wherein the non-human mammal is a rodent;
[5] the embryonic stem cell of [4], wherein the rodent is a mouse;
[6] a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA is inactivated;
[7] the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention [6]. The non-human mammal according to the item,
[8] the non-human mammal according to [6], wherein the non-human mammal is a rodent;
[9] the non-human mammal of [8], wherein the rodent is a mouse, and
[10] A method for screening a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention, which comprises administering a test compound to the animal according to [7] and detecting the expression of a reporter gene. I will provide a.
A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal, thereby suppressing the expression ability of the DNA, or By substantially losing the activity of HMG1 encoded by the DNA, the non-human mammal which does not substantially have the ability to express HMG1 (hereinafter sometimes referred to as the knockout DNA of the present invention) can be obtained. Refers to embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same one as described above is used.
The method of artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
[0103]
Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); Inability to synthesize complete messenger RNA Thus, a DNA strand having a DNA sequence constructed so as to eventually destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells PCR using the DNA sequence on or near the DNA of the present invention as a probe, and using the DNA sequence on the targeting vector and the DNA sequence in the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as primers And can be obtained by selecting the knockout ES cells of the present invention.
As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known method of Evans and Kaufma. It may be a newly established one. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. For example, for the purpose of obtaining ES cells in which BDF is clearly known, for example, BDF in which the number of eggs collected by C57BL / 6 mice or C57BL / 6 has been improved by hybridization with DBA / 2₁Mouse (F of C57BL / 6 and DBA / 2)₁) Can also be used favorably. BDF₁In addition to the advantage that the number of eggs collected and the eggs are robust, the mice have C57BL / 6 mice as a background, and thus, the ES cells obtained using the C57BL / 6 mice were used to produce C57BL / 6 mice. / 6 mice can be advantageously used in that the genetic background can be replaced by C57BL / 6 mice by backcrossing.
In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them until blastocysts are used. Early embryos can be obtained.
Either male or female ES cells may be used, but generally male ES cells are more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.
[0104]
As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. If this method is used, it is conventionally necessary to perform about 10 karyotype analyses.⁶Since the number of ES cells is required, the number of ES cells of about 1 colony (approximately 50) is sufficient, so that primary selection of ES cells in the early stage of culture can be performed by discriminating between male and female. In addition, by enabling selection of male cells at an early stage, labor in the initial stage of culture can be significantly reduced.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the normal cells (for example, chromosomes in mice) are knocked out. It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this way usually has very good growth properties, but it must be carefully subcultured because it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast, in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably, 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM @ EDTA Preferably, a method is used in which cells are made into single cells by treatment with about 0.1% trypsin / 1 mM @EDTA) and seeded on newly prepared feeder cells. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [M. {J. {Evans and M.A. H. Kaufman, Nature, 292, 154, 1981; R. {Martin} Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; C. Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], and the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is obtained by in vitro In HMG1 or cell biology of HMG1.
[0105]
The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
As the non-human mammal, those similar to the aforementioned can be used.
The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.
Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. It can be determined by analysis by PCR using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individuals obtained in this manner are usually individuals with a heterozygous expression of HMG1 heterozygous, and can be bred with individuals with a heterozygous expression of HMG1 to obtain a homozygous expression-deficient individual of HMG1 from their offspring.
[0106]
When an egg cell is used, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method, and these transgenic non-human mammals can be obtained. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
In this manner, the individual in which the DNA of the present invention is knocked out can be bred in an ordinary breeding environment after confirming that the DNA of the animal obtained by the breeding is also knocked out.
Further, the acquisition and maintenance of the germ line may be performed according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.
In addition, since the non-human mammal deficient in expressing DNA of the present invention lacks various biological activities that can be induced by HMG1, it can serve as a model for a disease caused by inactivation of the biological activity of HMG1. It is useful for investigating the causes of diseases and studying treatment methods.
[0107]
(11a) A method for screening a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention
The non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by deficiency or damage of the DNA of the present invention.
That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. These compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
[0108]
In the screening method, when a test compound is administered to a test animal, if the index value of the test animal changes by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is Can be selected as compounds having a therapeutic / preventive effect on the above diseases.
The compound obtained by using the screening method is a compound selected from the test compounds described above, and may be used as a drug such as a safe and low-toxic therapeutic or prophylactic agent for a disease caused by HMG1 deficiency or damage. Can be. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). ) Is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing an HMG1 inhibitor.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
[0109]
(11b) A method for screening a compound that reduces the activity of a promoter for DNA of the present invention
The present invention relates to a compound or a salt thereof, which comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention and detecting the expression of a reporter gene, thereby reducing the activity of the promoter for the DNA of the present invention. A screening method is provided.
In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound include the same compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding HMG1 is replaced by a β-galactosidase gene (lacZ) derived from Escherichia coli, β-galactosidase is originally expressed instead of HMG1 in a tissue where HMG1 is expressed. Therefore, for example, by staining with a reagent serving as a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), HMG1 can be easily prepared. The expression state in the animal body can be observed. Specifically, an HMG1-deficient mouse or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal-containing staining solution at room temperature or about 37 ° C. After reacting for 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM @ EDTA / PBS solution, and the coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that reduces the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an organic acid). And particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
[0110]
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful in screening for a compound or a salt thereof that reduces the activity of the promoter for the DNA of the present invention, and the excessive expression of the DNA of the present invention is involved. It can greatly contribute to investigating the causes of various diseases or developing preventive and therapeutic drugs.
[0111]
In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on the abbreviations of IUPAC-IUB {Commission} on {Biochemical} Nomenclature} or commonly used abbreviations in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.

[0112]
Gly: glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile II: Isoleucine
Ser: Serine
Thr: Threonine
Cys II: cysteine
Met: Methionine
Glu: glutamic acid
Asp: Aspartic acid
Lys: lysine
Arg: Arginine
His: histidine
Phe: phenylalanine
Tyr: Tyrosine
TrpII: tryptophan
Pro II: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: pyroglutamic acid
*: Corresponds to stop codon
Me: methyl group
Et: ethyl group
Bu: butyl group
Ph: phenyl group
TC: thiazolidine-4 (R) -carboxamide group
[0113]
Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

[0114]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
SEQ ID NO: 1
1 shows the nucleotide sequence of cDNA encoding human-derived HMG1.
SEQ ID NO: 2
1 shows the amino acid sequence of human-derived HMG1.
[0115]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but these do not limit the scope of the present invention. In addition, the result regarding the densitometry analysis of the immunoblot is shown as mean ± standard error. Statistical significance of the differences was determined by analysis of variance (ANOVA). Further statistical analysis for post-hoc comparisons was performed using the Bonferroni / Dunn test (StatView, Abacus Concepts, Berkeley, CA). In addition, confocal microscopic observation was performed as follows. AD brain sections were incubated with antibodies to HMG1 and HLA-DR overnight at room temperature, and hippocampal sections from rats injected with Aβ42 were incubated with antibodies to Aβ and CD11b. Primary antibodies were detected with rhodamine or FITC-labeled anti-rabbit IgG and anti-mouse IgG antibodies, and fluorescence was observed using a laser scanning confocal microscope LSM410 (Carl Zeiss, Germany).
[0116]
Example 1 Up-regulation of HMG1 in Alzheimer's disease (AD) brain
6 patients clinically and histopathologically diagnosed with AD (age 81.2 ± 3.5 years; delay of necropsy 3.8 ± 0.7 hours) and clinical and morphological findings of brain lesions Brain tissue was obtained by necropsy from six controls without evidence (age 71.0 ± 4.2 years; necropsy delay 8.0 ± 2.9 hours). Age and necropsy delay were not significantly different between the AD and control groups. The temporal cortex was used for the experiment. The neuropathological evaluation of AD was performed in accordance with the criteria of Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Cytoplasmic fractions (including cytosolic and extracellular soluble proteins) and particle fractions (including nuclear, membrane and insoluble proteins) were obtained from Brain @ Res. {780: {260-269} (1998). Both fractions (10 μg protein) were subjected to SDS-PAGE. After immunoblotting with an antibody against HMG1 or HMG2 (BD @ Pharmingan, @San Diego, CA), a chemiluminescence assay (ECL @ kit, Amersham @ Pharmacia Biotech, @Buckinghamshire, using a protein of the band using a protein from the Buckinghamshire, U.K.) was performed. For semi-quantitative analysis of protein bands, X-ray films were scanned with a CCD color scanner (DuoScan, AGFA) and the density was measured.
Immunohistochemical analysis was also performed essentially as described in Brain @ Res. {780: {260-269} (1998). That is, brain slices were incubated in phosphate buffered saline (PBS) containing 0.3% hydrogen peroxide for 30 minutes, washed with PBS containing 0.3% Triton X-100 (PBS-T), and then washed against HMG1. Incubated overnight at room temperature with PBS-T containing antibody, Aβ (Chemicon, @Temecula, CA) and HLA-DR (Dako, @Glostrup, @Denmark). After washing with PBS-T and incubating with Vectastatin ABC Elite kit (Vector Laboratories, Burlingame, CA), the brain slices were visualized using 3,3'-diaminobenzidine (DAB) with nickel enhancement and hydrogen peroxide. .
As a result, 29 kDa HMG1 was detected in both the cytoplasmic fraction and the particulate fraction of the temporal cortex of the control and AD brains (FIG. 1). It was more abundant in the cytoplasmic fraction than in the particle fraction. In the AD brain, the expression level of HMG1 in both the cytoplasmic fraction and the particle fraction was significantly higher than in the control brain (FIGS. 1a and b, P <0.05 and P <0.01, respectively). This indicates that expression is up-regulated. A 28 kDa HMG2 is also detected in both cytoplasmic and particulate fractions, but its expression (lower than HMG1) was not significantly different in AD brains (data not shown).
In control brains, HMG1 immunoreactivity was faintly detectable in the cytoplasm and nucleus of neurons and glial cells (FIG. 1c). In AD brains, HMG1 immunoreactivity was increased at these locations (FIG. 1d). In addition, HMG1 immunoreactivity was observed in a plaque-like diffused manner and was strongly detected in the Aβ immunoreactive region (compare FIG. 1e with FIG. 1g). Human leukocyte antigen (HLA) -DR, a marker of activated microglia, was also observed (FIG. 1f). Double immunostaining revealed that HMG1 was located both within and outside of microglial cells expressing HLA-DR (FIG. 1h). These immunocytochemical results indicate that extracellular HMG1 accumulates around the area where microglia accumulates in senile plaques.
[0117]
Reference Example 1 Distribution of HMG1 in transgenic mice
Recently, several transgenic mice that overproduce Aβ peptides have been created. Tg2576 mice and mutant presenilin-1 (PS1) mice express K670N / M671L (Swedish type) mutant human amyloid precursor protein (APP) and M146L mutant human PS1, respectively. Furthermore, a double transgenic PS / APP system has been created by crossing a Tg2576 mouse and a PS1 mouse. PS1 mice increase mouse Aβ42 production but do not form Aβ deposits, whereas PS / APP mice result in significant production of human and mouse Aβ40 and Aβ42 and accelerated Aβ deposit formation. However, extensive neuronal loss is not detected in either PS1 or PS / APP mice. Thus, the immunoreactivity of HMG1 and Aβ in transgenic mice as a model showing extensive production of Aβ42 without neuronal loss was examined.
12-month-old PS1 and PS / APP mice were used for the experiment. Frontal brain sections from these mice were labeled ΔAm. J. Pathol. , 158: 1345-1354 (2001), and then immunostained with antibodies to HMG1, Aβ and CD11b (MAC1, Caltag Laboratories, San Francisco, CA). For double-labeled immunohistochemical staining, brain sections were incubated with a rabbit anti-HMG1 antibody with a mouse monoclonal Aβ antibody in PBS-T at 4 ° C. for 4 days. After washing, sections were incubated with anti-rabbit IgG antibodies and HMG1 immunoreactivity was detected using DAB and nickel ammonium. After completion of HMG1 immunostaining, sections were treated with 0.5% hydrogen peroxide in PBS-T for 30 minutes to destroy residual peroxidase from the first cycle. Thereafter, a second immunohistochemical cycle of the sections and anti-mouse IgG antibodies was performed. The DAB reaction was performed without nickel enhancement.
As a result, in PS1 mice, branched microglia immunostained with rat anti-CD11b antibody (MAC1) were observed, but Aβ deposition and reactive microglia were not detected even at 12 months of age (FIGS. 2a, c). And e). In 12 month old PS / APP mice, excessive Aβ deposition associated with bush and amoeba-type microglia was detected in a pattern similar to Aβ and HLA-DR immunoreactivity in AD brains (FIGS. 2b, d and f). Compare FIGS. 1f and g). However, the HMG1 immunoreactivity in these transgenic mice was slightly different from that in AD brain. Thus, in both PS1 and PS / APP mice, HMG1 immunoreactivity was significantly detected in neurons and glial nuclei (FIGS. 2g and h). Although also observed around Aβ deposits in PS / APP mice, this immunoreactivity was probably found in glial micronuclei rather than extracellularly (FIG. 2h). That is, PS / APP mice showed Aβ deposition, but diffuse HMG1 immunoreactivity was not detected. These results suggest that the formation of Aβ deposits in PS / APP mice can be primarily caused by overproduction of Aβ peptides without extracellular HMG1 involvement.
[0118]
Reference Example 2 Distribution of HMG1 in rat model
To elucidate the relationship between extracellular HMG1 and Aβ in the brain, we further investigated two rat models: 1) Intraventricular (icv) injection of kainate (KA), resulting in Aβ HMG1 immunoreactivity was examined in rats that produced massive neuronal loss in hippocampal CA3 without deposition and 2) rats that produced large Aβ deposition with moderate neuronal loss as a result of intrahippocampal injection of Aβ42. Microtubule-associated protein-2 (MAP2) was used as an indicator of neuronal survival.
Male Wistar rats weighing approximately 280 g were fasted overnight with free access to water. Rats were anesthetized for microinjection of stereotaxic fixation (intraperitoneal injection of sodium pentobarbital 50 mg / kg) and fixed in a Kopf stereotaxic fixation frame. Thereafter, 1 μg / 2 μl @ KA was injected into the right ventricle (ie, 3.8 mm caudal from bregma, 1.2 mm right, 3.8 mm lower surface). Alternatively, Aβ42 (AnaSpec, San Jose, CA) 4.5 μg / via a 10 μl Hamilton syringe driven by a motor into the right hippocampus of the rat (ie, 0.2 mm caudal, 2.0 mm right, 4.0 mm underside from bregma). 2 μl were injected. Three days later, the treated rats received 150 ml of 10 mM phosphate buffered saline (PBS) through the artery, followed by 4% paraformaldehyde, 0.35% glutaraldehyde and 0.2% picric acid in 100 mM phosphate buffer. Was perfused under deep anesthesia with pentobarbital (100 mg / kg intraperitoneally). After perfusion, the brain was immediately removed, fixed with paraformaldehyde in 100 mM PB for 2 days, and then transferred to a 15% sucrose solution (in 100 mM PB containing 0.1% sodium azide) at 4 ° C. Brain sections were cut into 20 μm sections in a cryostat and collected in PBS-T. The hippocampal slices were then immunostained with antibodies to MAP2, HMG1, Aβ, CD11b (OX42, Harlan @ Sera-Lab, @Lowbrough, UK) and GFAP (Chemicon International, @Temecula, CA).
As a result, i. c. v. The injection caused neuronal loss in the ipsilateral hippocampal CA3 three days later (FIG. 3b) but not contralaterally (FIG. 3a). No Aβ deposition was observed (FIGS. 3f and h), but activated microglia and astrocytes, which were immunostained by antibodies against CD11b (OX42) and glial fibrillary acidic protein (GFAP), respectively. Was accumulated in CA3 (FIG. 3i-1). HMG1 immunoreactivity was prominently detected in neurons and glial nuclei (FIGS. 3e, g and i-1). HMG1 immunoreactivity was also detected in the glial processes of neurodegenerative CA3 (FIGS. 3g, k and l), but no diffuse staining was detected.
In rats in which Aβ42 was microinjected into the hippocampus, amoeboid microglia immunostained with mouse anti-CD11b antibody (FIGS. 4d, f and g) were observed around large Aβ deposits (FIGS. 4a, e and g) in the hippocampus. Was done. In addition, a moderate loss of MAP2 immunoreactivity was detected in the hippocampal dentate gyrus (DG) around Aβ deposition (FIG. 4b). HMG1 immunoreactivity was observed in neurons and glial nuclei, as well as in glial processes (FIGS. 4c, e and f). Furthermore, HMG1 immunoreactivity was detected as extracellular diffuse staining around Aβ deposits (FIGS. 4c, e and f). These observations suggest that extracellular HMG1 mainly leaks from dead neurons, and diffuse HMG1 deposition may be caused by both extensive neuronal death and Aβ accumulation.
[0119]
Example 2 Inhibition of Aβ42 Phagocytosis by Microglia Induced by HMG1
To examine the mechanistic significance of the presence of HMG1, the effects of HMG1 on microglial function were examined using pure cultures of rat microglia. In the experiments, Aβ40 and Aβ42 were not trifluoroacetic acid (TFA) salts, but hydrochlorides that readily aggregated. The present inventors have recently found that Aβ40 induces the production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) at 10 μM (FASEB {J. {16: 601-603} ( 2002)). In contrast, extracellular Aβ42 significantly induced microglial phagocytosis at lower concentrations (0.03-0.3 μM) 6-24 hours after Aβ42 exposure. Insulin or pepstatin A suppressed the reduction of Aβ42 levels in microglia after 3 days, but not phosphoramidon (neprilysin inhibitor), and phagocytosed Aβ42 was reduced by IDE-like peptidase and / or cathepsin D-like aspartate proteinase in microglia. Suggests that it will be broken down. Exposure to relatively low concentrations (less than 1 μM) of Aβ40 did not cause uptake by microglia, whereas at higher concentrations (3-10 μM), microglia became phagocytic to Aβ40. Thus, rat microglia shows different responses to Aβ40 and Aβ42.
Mixed glial cells (mixture of astrocytes and microglia) were prepared from hemispheres that were carefully removed from the meninges of neonatal Wistar rats (SLC Inc., Shizuoka, Japan). The tissue suspension was filtered through a 50 μm diameter nylon mesh (cell strainers, Falcon) into a 50 ml tube and centrifuged at 200 × g for 10 minutes to collect cells. Cells were resuspended in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, plated in 100 mm₂Incubation was carried out at 37 ° C./95% atmosphere. With great care to avoid endotoxin contamination, suspended microglia were recovered from mixed glial cultures and plated on new culture dishes.
The resulting rat microglia (purity greater than 97%) was purified in the presence of 0.015-1.5 μM purified calf HMG1 and / or 0.4 nM recombinant human TGF-β1 (Gibco BRL, Life Technologies, Tokyo, Japan). Alternatively, the cells were incubated with 0.3 μM ΔAβ42 or 3 μM ΔAβ40 in the absence. Twenty-four hours later, after collecting and lysing the microglia, the samples were subjected to immunoblot with an anti-Aβ antibody (Chemicon, Temecula, CA). After monomer and aggregated Aβ were detected using a chemiluminescence assay, the total amount of Aβ was measured with a densitometer.
When HMG1 purified from calf thymus was added to rat microglial cultures containing low concentrations (0.3 μM) of Aβ42, HMG1 inhibited Aβ42 phagocytosis in a concentration-dependent manner to over 60% (FIGS. 5a and b). Since it has been reported that astrocyte-derived TGF-β1 is important for Aβ clearance, the effect of HMG1 on TGF-β1-induced Aβ clearance was further investigated. While 0.4 nM recombinant human TGF-β1 significantly increased Aβ42 phagocytosis, HMG1 inhibited TGF-β1 induced Aβ phagocytosis to levels resulting from HMG1 treatment in the absence of TGF-β1. It was significantly inhibited (FIGS. 5c and d). In contrast, Aβ40 phagocytosis was not altered by TGF-β1 and HMG1 at high concentrations (3 μM) (FIGS. 6a and b). These results suggest that if extracellular HMG1 accumulates in senile plaques, Aβ42 clearance by microglial activators such as TGF-β1 will be inhibited, but Aβ40 clearance will not. .
[0120]
Reference Example 3 Influence of HMG1 on microglial activation induced by Aβ40
Microglia were treated with 10 μM ΔAβ40 (AnaSpec) in the presence or absence of 1.5 μM ΔHMG1 (Wako Pure Chemical Industries) purified from calf thymus. Medium was removed from each well 24 hours after treatment. NO₂ ⁻Quantities were measured spectrophotometrically (DU-640 spectrophotometer, Beckman) using the Griess reagent. The amount of TNF-α and IL-6 released from the cells was measured using an ELISA kit for rat TNF-α and IL-6 (BioSource, Camarillo, CA) according to the manufacturer's instructions.
As a result, 10 μM Aβ40 was converted to nitrite (NO₂ ⁻) Induced the expression of inducible nitric oxide synthase (iNOS) and the production of TNF-α and IL-6 (FIGS. 6c, d and e). However, HMG1 did not affect the production of these compounds in the presence and absence of Aβ40 (FIGS. 6c, d and e). Thus, HMG1 does not affect the production of NO, TNF-α and IL-6 by microglia in the presence and absence of Aβ40.
[0121]
Example 3 In Vitro Binding of HMG1 to Aβ Peptides and Their Aggregation
Purified calf HMG1 (1.5 μg) was incubated at 37 ° C. with 3 μg of Aβ40 or Aβ42 in PBS (final volume 20 μl). After 0, 6 and 24 hours, the samples were immunoblotted with antibodies to HMG1 or Aβ. After 1.5 μg of HMG1 was incubated with 3 μg of Aβ40 or Aβ42 at 37 ° C. for 24 hours, an antibody against HMG1 or Aβ (10 μg) was added to the mixture, and the mixture was further incubated at 4 ° C. for 2 hours. Then protein A-Sepharose (50 μl of 50% slurry) was added and incubated at 4 ° C. overnight. After centrifugation, the immunoprecipitates were washed three times in 1 ml of cold buffer, resuspended in Laemmli's sample buffer and subjected to SDS-PAGE and immunoblot with antibodies to HMG1 or Aβ.
When HMG1 was incubated with Aβ42, a complex of these proteins (approximately 33 kDa) was detected after 6 hours (FIGS. 7a and b). Incubation of Aβ42 with HMG1 did not change its oligomer formation and significantly increased the formation of higher molecular aggregates after 24 hours (FIG. 7b). In contrast, when incubated with Aβ42 alone, the levels of the oligomers decreased and aggregates increased after 6 hours. Conversely, after 24 hours, no oligomers and aggregates of Aβ40 were detected, but a complex of HMG1 and Aβ40 was detected (FIGS. 7a and b).
After in vitro incubation of HMG1 with Aβ40 or Aβ42 for 24 hours, the mixture was immunoprecipitated with antibodies against HMG1 or antibodies against Aβ. These antibodies precipitated both HMG1 and Aβ peptide (FIG. 7c). In addition, complexes of HMG1 with either Aβ40 or Aβ42 also immunoprecipitated. Thus, HMG1 bound to the Aβ peptide and promoted the formation of Aβ42 aggregates, but not Aβ40 aggregates.
[0122]
In conclusion, HMG1 protein levels are up-regulated in both cytoplasmic and particulate fractions of AD brain, and HMG1 coexists with senile plaques. Diffuse HMG1 immunoreactivity was observed in the hippocampus of rats injected with Aβ42, but not in the hippocampus of rats injected with KA and PS / APP mice, and extracellular HMG1 and Aβ42 interact synergistically. To form senile plaques. Extracellular HMG1 inhibited phagocytosis of Aβ42 by microglia in the presence and absence of TGF-β1. Furthermore, HMG1 bound to the Aβ peptide and promoted the formation of Aβ42 aggregates (FIG. 8). These results suggest that up-regulation of HMG1 inhibits Aβ42 clearance by microglia, thereby maintaining Aβ deposition. Thus, it was shown that extracellular HMG1 may play a pathogenic role in AD through inhibition of Aβ clearance by microglia and promotion of Aβ aggregation.
[0123]
【The invention's effect】
HMG1 or a partial peptide thereof or a salt thereof is used as a vaccine for prevention and treatment of cerebral amyloidosis such as Alzheimer's disease, and a nucleotide complementary to a nucleotide encoding HMG1 and the like and an antibody against HMG1 and the like are used for prevention and treatment of cerebral amyloidosis. Useful as an agent.
Nucleotides encoding HMG1 and the like and antibodies against HMG1 and the like are useful as diagnostic agents for cerebral amyloidosis.
The screening method for an HMG1 inhibitor using HMG1 or the like and the screening method for an HMG1 expression inhibitor using a nucleotide encoding HMG1 or the like are useful as a tool for searching for a compound having a prophylactic or therapeutic activity for cerebral amyloidosis.
[0124]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram and a photograph showing the expression level and distribution of HMG1 in control and AD brains. a and b: immunoblot photographs of cytoplasmic (a) and particle (b) fractions of temporal cortex (upper figure) and graphs showing 29 kDa band concentration (lower figure; vertical axis is% ratio to control; each value is 6) Mean of sample ± standard error; individual values are indicated by circles). ch: immunostaining photographs of the control and AD brain sections using antibodies against HMG1 (ce, h), HLA-DR (f, h) or Aβ (g). Sections (eh) are a series from the same individual, and * (eg) indicates the same vessel. The arrow indicates the same senile plaque as shown at high magnification in the inset. h, Double staining of senile plaques using antibodies against HMG1 and HLA-DR. Scale bar: 250 μm (eg) and 50 μm (inset of h and eg).
FIG. 2 is a photograph showing the localization of HMG1 in the transgenic mouse brain. 12 months old PS1 (a, c, e, g) and CD11b (MAC1, ad), Aβ (eh) or HMG1 (MB) of the frontal brain section of PS / APP mice (b, d, f, h) Immunostaining with antibodies to g, h). The high magnification image of the box is shown in inset (ch). Scale bars: 1 mm (a, b), 200 μm (ch) and 50 μm (inset in ch).
FIG. 3 is a photograph showing the localization of HMG1 in the rat brain injected with KA. Antibodies to MAP2 (a, b), HMG1 (ce, g, i-1), Aβ (f, h), CD11b (OX42, j, l) or GFAP (i, k) in hippocampal slices were used. Immunostaining. Intraventricular KA injection contralateral (a, c, e, f, i, j) and ipsilateral (b, d, g, h, k, l). High magnification image (el) of the box in hippocampus CA3. i-1: Double staining with antibodies to HMG1 and CD11b (j, 1) or GFAP (i, k). Scale bars: 500 μm (ad) and 50 μm (el).
FIG. 4 is a photograph showing the localization of HMG1 in the rat hippocampus injected with Aβ42. Immunostaining of hippocampal slices with antibodies against Aβ (a, e, g) HMG1 (c, e, f), MAP2 (b) or CD11b (OX42, d, f, g). e, f: Double staining with antibodies to HMG1 and Aβ (e) or CD11b (f). High magnification images of the boxes in each section (af) immunostained are shown in insets. Arrows indicate diffuse staining with anti-HMG1 antibody. g: Confocal micrograph of double staining around Aβ deposition in hippocampus using antibodies to Aβ and CD11b. Scale bar: 500 μm (af) and 50 μm (inset in af, g).
FIG. 5 is a photograph and a diagram showing the inhibitory effect of HMG1 on Aβ42 phagocytosis by microglia. a and b: Immunoblot photographs of rat microglia lysate incubated with Aβ42 in the presence of vehicle (Control in lanes C and b in a) or various concentrations of HMG1 (a) and graph showing total Aβ42 content (vertical). The axis is the percentage relative to the control; each value is the mean of three experiments ± standard error; **, *** indicate p <0.01 and p <0.001, respectively, for the control value. c and d: in the presence of vehicle (lane TGF-β1 (−) in c and vehicle in d) or TGF-β1 (lane TGF-β1 (+) in c and TGF-β1 in d) Immunoblot photograph of rat microglial lysate incubated with Aβ42 (HMG1 (−) in c and Control in d) or Aβ42 and HMG1 (HMG1 (+) in c and HMG1 in d) (a) and total Aβ42 amount (Vertical axis is% relative to control; each value is mean ± standard error of three experiments; ***, ††† indicates p <0.001 with respect to vehicle and TGF-β1 alone, respectively) Shown).
FIG. 6 is a photograph and a diagram showing the effect of HMG1 on Aβ40 uptake and activation by Aβ40 in microglia. a and b: Immunoblot photographs of rat microglia lysates incubated with Aβ40 in the presence of vehicle, TGF-β1 or HMG1 (a) and graphs showing total Aβ40 content (vertical axis is% ratio to vehicle; each value is three The mean of the experiment ± standard error is shown). ce: Immunoblot photographs of rat microglia lysates incubated with vehicle (HMG1 (-)) or HMG1 (HMG1 (+)) in the presence or absence of Aβ40 against anti-iNOS antibody (upper panel c), and NO in culture supernatant₂ ⁻A graph showing the amount of TNF-α (d) and the amount of IL-6 (e) (each numerical value is the average of three experiments ± standard error; ***, ††† indicates vehicle and Aβ40 treatment, respectively) P <0.001 for the value of
FIG. 7 is a photograph showing the binding between HMG1 and Aβ peptide and the aggregation of Aβ peptide in vitro. a and b: immunoblot using an antibody against HMG1 (a) or Aβ for samples incubated with HMG1 in the presence or absence of Aβ40 or Aβ42 (arrows indicate complexes of HMG1 with Aβ40 or Aβ42) . c: Aβ40 or Aβ42 was incubated with HMG1, then immunoprecipitated with an antibody against HMG1 (upper panel) or Aβ (lower panel), and immunoblotted with an antibody against HMG1 or Aβ.
FIG. 8 is a schematic diagram showing the involvement of extracellular HMG1 in the process of senile plaque formation.

Claims (26)

A vaccine for inhibiting amyloid β42 aggregation and / or promoting amyloid β42 phagocytosis by microglia, comprising HMG1 or a partial peptide thereof or a salt thereof. A diagnostic agent for amyloid β42 aggregation and / or microglia-induced amyloid β42 phagocytic abnormality, comprising a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof. An agent for inhibiting amyloid β42 aggregation and / or promoting amyloid β42 phagocytosis by microglia, comprising an antibody against HMG1 or a partial peptide thereof or a salt thereof. A diagnostic agent for amyloid β42 aggregation and / or microglia-induced amyloid β42 phagocytic abnormality, comprising an antibody against HMG1 or a partial peptide thereof or a salt thereof. An agent for inhibiting amyloid β42 aggregation and / or promoting amyloid β42 phagocytosis by microglia, comprising a nucleotide containing a nucleotide sequence complementary to the nucleotide sequence encoding HMG1 or a partial peptide thereof or a part thereof. A method for screening a substance that inhibits HMG1 and inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia, which comprises using HMG1 or a partial peptide thereof or a salt thereof. A method of screening for a substance that inhibits HMG1 and exhibits amyloid β42 aggregation inhibitory activity, comprising contacting amyloid β42 and a test substance with HMG1 or a partial peptide thereof or a salt thereof, and assaying amyloid β42 aggregation. The method according to claim 7, wherein a comparison is made with the aggregation of amyloid β42 when amyloid β42 is brought into contact with HMG1 or a partial peptide thereof or a salt thereof. HMG1 or a partial peptide thereof or a salt thereof, amyloid β42 and a test substance are brought into contact with microglia, and the amyloid β42 phagocytosis by microglia is assayed. A method for screening substances. The method according to claim 9, wherein the comparison is made with microglia phagocytosis of amyloid β42 when HMG1 or its partial peptide or a salt thereof and amyloid β42 are brought into contact with microglia. A kit for screening a substance comprising HMG1 or a partial peptide thereof or a salt thereof, which inhibits HMG1 and inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia. The kit according to claim 11, further comprising amyloid β42. A substance obtained by using the method according to claim 6 or the kit according to claim 11, which inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia. A substance that inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia by inhibiting HMG1. A medicament comprising the substance according to claim 13. The medicament according to claim 15, which is an agent for preventing or treating Alzheimer's disease, Down's syndrome or amyloid angiopathy. A method for screening a substance that suppresses amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia by suppressing the expression of HMG1 using a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof. . A test substance is added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, and the expression of HMG1 or a partial peptide thereof is assayed. 18. The method according to 17. A test substance and amyloid β42 are added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, and expression of HMG1 or a partial peptide thereof and aggregation of amyloid β42 are assayed. A method for screening a substance that suppresses amyloid β42 aggregation by suppressing HMG1 expression. A test substance, amyloid β42 and microglia are added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, and expression of HMG1 or a partial peptide thereof and amyloid β42 diet by the microglia are added. A method for screening for a substance that suppresses HMG1 expression and exhibits microglia-induced amyloid β42 phagocytosis. A screening kit for a substance comprising a nucleotide containing a nucleotide sequence encoding HMG1 or a partial peptide thereof, which suppresses the expression of HMG1 and inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia. 22. The kit according to claim 21, further comprising amyloid β42. A substance obtained by using the method according to claim 17 or the kit according to claim 21, which suppresses amyloid β42 aggregation by inhibiting HMG1 expression and / or promotes amyloid β42 phagocytosis by microglia. A substance that suppresses amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia by suppressing the expression of HMG1. A medicament comprising the substance according to claim 23 or 24. 26. The medicament according to claim 25, which is an agent for preventing or treating Alzheimer's disease, Down's syndrome or amyloid angiopathy.

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