JP2005006542A - New gene alivin 1 participating in existence of neurocyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new gene alivin 1 which inhibits the apoptosis of neurocytes and plays an important role for the existence of the neurocytes, to provide a protein encoding the gene, and to provide a medicinal composition containing the same. <P>SOLUTION: There are provided a mutated alivin 1 gene and to a mutated Alivin 1 protein encoding the gene, the gene for inhibiting the apoptosis of neurocytes, and a medicinal composition containing the protein. Since it has been concluded that the above gene is a gene related to Alzheimer's disease and Parkinson's disease, the medicinal composition can be used for these diseases. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】図１は、小脳顆粒細胞の脱分極依存性およびＮＭＤＡ依存性生存を示すグラフである。小脳顆粒細胞を、５ｍＭ ＫＣｌおよび３０μＭ ＡＰ−５を含む培養液（■−■）、１５ｍＭ ＫＣｌおよび３０μＭ ＡＰ−５を含む培養液（□−□）、２５ｍＭ ＫＣｌおよび３０μＭ ＡＰ−５を含む培養液（○−○）、ならびに、１５０μＭ ＮＭＤＡおよび１５ｍＭ ＫＣｌを含む培養液（●−●）中で培養した。さらに、１５０μＭ ＮＭＤＡおよび１５ｍＭ ＫＣｌを含む培養液中で培養した細胞については、３．５日間培養を行った後、３００μＭ ＡＰ−５を加えてＮＭＤＡ受容体をブロックし、アポトーシスを誘導した（▲−▲）。細胞の生存率はＭＴＴ法で測定し、２５ｍＭ ＫＣｌおよび３０μＭ ＡＰ−５を含む培養液中で培養した細胞の細胞生存に対する相対的生存率をプロットした。
【図２】図２は、ディファレンシャルディスプレイの結果を示すゲル電気泳動の写真である。レーンＡは、５ｍＭ ＫＣｌの存在下で３．０ＤＩＶ培養した小脳顆粒細胞のトータルＲＮＡをｃＤＮＡに変換した後、ディファレンシャルディスプレイＰＣＲで増幅したＰＣＲ産物である。レーンＳは、１５０μＭ ＮＭＤＡ、１５ｍＭ ＫＣｌ存在下で３．０ＤＩＶ培養した小脳顆粒神経細胞のトータルＲＮＡをｃＤＮＡに変換した後、ディファレンシャルディスプレイＰＣＲで増幅したＰＣＲ産物である。６％シークエンシングゲルで分析した。
【図３】図３は、Ａｌｉｖｉｎ １ タンパク質のドメインを示す模式図である。ラットマウスおよびヒトａｌｉｖｉｎ １ ｃＤＮＡのオープンリーディングフレームはそれぞれ、５２０、５１９、５２２アミノ酸のタンパク質をコードする。ＬはＮＨ_２−末端のシグナルペプチド配列を、ＮＣおよびＣＣはシステイン−リッチドメインを、ＬＲＲはロイシンリッチリピートを、ＩＧは免疫グロブリンＣ２−様ドメインを、ＴＭは膜貫通ドメインを、ならびに、ＩＤはＣＯＯＨ−末端領域のおよそ１００アミノ酸の細胞内ドメインを表す。
【図４−１】図４−１Ａは、神経顆粒細胞におけるａｌｉｖｉｎ １ ｍＲＮＡ発現のノーザンブロッティングの結果を示す写真である。５ｍＭ ＫＣｌ、１５ｍＭＫＣｌ、２５ｍＭ ＫＣｌ、または、１５０μＭ ＮＭＤＡおよび１５ｍＭ ＫＣｌの両方、のいずれかの存在下で培養した顆粒神経細胞から抽出した全ＲＮＡサンプル（１０μｇ）をノーザンブロッティングに供した。ディファレンシャルディスプレイ解析で得られたｃＤＮＡ（ラットａｌｉｖｉｎ １ ｃＤＮＡの３’−非翻訳領域に対応する）をプローブとして用いた。下の枠はＧ６ＰＤＨ ｍＲＮＡの発現レベルを示す。
図４−１Ｂは、図４−Ａの結果を定量した値を示すグラフである。神経顆粒細胞は５ｍＭ ＫＣｌ（△−△）；１５ｍＭ ＫＣｌ（○−○）；２５ｍＭ ＫＣｌ（▲−▲）；または、１５ｍＭ ＫＣｌおよび１５０μＭ ＮＭＤＡの両方（●−●）；のいずれかの存在下で培養した。
【図４−２】図４−２Ａは、ＡＰ−５でのＮＭＤＡレセプターのブロッキングによるａｌｉｖｉｎ １ ｍＲＮＡ発現の早い下方制御を示すノーザンブロッティングの写真である。神経顆粒細胞は１５ｍＭ ＫＣｌおよび１５０μＭ ＮＭＤＡを含有する培地中で４．５日培養し、ＮＭＤＡレセプターは最終濃度３００μＭのＡＰ−５の添加によってブロックした。３．３ｋｂのバンドはａｌｉｖｉｎ １ ｍＲＮＡに対応するバンドである。下の枠はＧ６ＰＤＨ ｍＲＮＡの発現レベルを示す。
図４−２Ｂは、図４−２Ａの結果を定量化した値を示すグラフである。
【図４−３】図４−３Ａは、ａｌｉｖｉｎ １ ｍＲＮＡ発現についての様々な阻害剤の効果を示すゲル電気泳動の写真である。小脳の顆粒神経細胞は、２５ｍＭ ＫＣｌを含有する培地中で４日間培養した。培地は５ｍＭ ＫＣｌを含有するものに交換し、さらに２４時間培養を続け、その後その細胞は５ｍＭ ＫＣｌ（５）；１５ｍＭ ＫＣｌ（１５）；２５ｍＭ ＫＣｌ（２５）；または、１５ ｍＭ ＫＣｌおよび１５０μＭ ＮＭＤＡの両方（Ｎ）；で刺激した。阻害剤はＫＣｌおよび／またはＮＭＤＡによる刺激の３０分前に培養培地に加えた。刺激の後（２４時間）、全ＲＮＡを抽出し、競合ＰＣＲ解析に供した。使用した阻害剤は３５μＭ シクロヘキシイミド（ＣＨＸ）、４．０μＭ アクチノマイシンＤ（ＡｃＤ）または０．２μＭ ニフェジピン（ＮＦＤ）を含む。ニフェジピンのビヒクルとしてエタノールを用いた。
図４−３Ｂは、図４−３Ａに示したデータを定量化したグラフである。
【図５】図５は、テトロドトキシン存在下および非存在下でのａｌｉｖｉｎ １ ｍＲＮＡの発現量を評価したグラフである。ラットの大脳皮質神経細胞を１４日間培養した（ｃｏｎｔｒｏｌ）。テトロドトキシン（１μＭ）を培養液に加え、さらに３０時間培養を行った（＋ＴＴＸ）。その後、培養液を新しいものと取り替え、さらに３０時間培養した（−ＴＴＸ）。それぞれのサンプルからＲＮＡを抽出し、競合的ＰＣＲによりａｌｉｖｉｎ １ ｍＲＮＡの発現量を評価した。
【図６】図６Ａは、ａｌｉｖｉｎ １ ｍＲＮＡの組織分布をノーザンブロッティングにより評価した結果の写真である。全ＲＮＡ（１０μｇ）を成体ラットの様々な組織から抽出し、ノーザンブロッティングに供した。下の枠は２８Ｓ ｒＲＮＡのエチジウムブロマイド染色を示す。
図６Ｂは、ラット小脳におけるａｌｉｖｉｎ １ ｍＲＮＡの発生期の発現をノーザンブロッティングで評価した際の写真である。全ＲＮＡは生後の特定の日の小脳から抽出し、１０μｇ ＲＮＡをノーザンブロッティング解析に供した。下の枠は２８Ｓ ｒＲＮＡのエチジウムブロマイド染色を示す。
図６Ｃは、図６Ｂの結果を定量化したデータを示すグラフである。
【図７】図７Ａは、ラットの全脳抽出物を１０％ＳＤＳポリアクリルアミドゲル電気泳動に供し、抗ラットＡｌｉｖｉｎ １ 抗体を用いてウエスタンブロッティングを行った結果を示す写真である。
図７Ｂは、ラット小脳におけるＡｌｉｖｉｎ １ の免疫組織化学上の局在を示す写真である。ＭＬは分子層、ＰＬはプルキンエ細胞層、ＧＬは顆粒細胞層をそれぞれ表す。
図７Ｃは、ラット海馬におけるＡｌｉｖｉｎ １ の免疫組織化学上の局在を示す写真である。図中、ＣＡ、ＣＡ１およびＣＡ２における“ＣＡ”は、Ｃｏｒｎｕ Ａｍｍｏｎｉｓ（アンモン角）の略であり、“ＤＧ”はｄｅｎｔａｔｅ ｇｙｒｕｓ（歯状回）の略である。
【図８】図８は、脳におけるＡｌｉｖｉｎ １ の細胞下の分布を評価したウエスタンブロッティングの結果を示す写真である。１０週齢のラットの全脳を様々な細胞成分画分に分離した。タンパク質（１００μｇ）を１０％ＳＤＳポリアクリルアミドゲル電気泳動に供した。
【図９】図９Ａは、初代培養小脳顆粒細胞へＡｌｉｖｉｎ １ タンパク質発現ベクターを導入した細胞の写真を示す。発現ベクターを培養０日目に小脳顆粒細胞に導入し、５ｍＭ ＫＣｌおよび３０μＭ ＡＰ−５を含む培養液で培養した。培養３．０日に４％パラホルムアルデヒドで固定し、抗Ｆｌａｇ抗体およびヘキスト色素による染色を行った。トランスフェクションした細胞は抗Ｆｌａｇ抗体による免疫染色およびＥＧＦＰによる蛍光により同定した。アポトーシスを起こしている細胞はヘキスト色素による染色により、収縮した、あるいは断片化した核を持つ細胞として検出した。（ａ−ｄ）Ａｌｉｖｉｎ １−Ｆｌａｇ融合タンパク質発現ベクターをトランスフェクションした細胞、（ｅ−ｈ）対照ベクターをトランスフェクションした細胞、（ａおよびｅ）ＥＧＦＰの蛍光、（ｂおよびｆ）抗Ｆｌａｇ抗体およびアレキサ５９４フロアー結合二次抗体による免疫染色、（ｃおよびｇ）ヘキスト色素による染色、（ｄおよびｈ）ＥＧＦＰ蛍光像、抗Ｆｌａｇ抗体免疫染色像、ヘキスト染色色素のオーバーレイ。矢印は、遺伝子導入された細胞を示す。
図９Ｂは、Ａｌｉｖｉｎ １ 発現ベクター、または、対照ベクターを小脳顆粒細胞へトランスフェクションした後、５ｍＭ ＫＣｌ／３０μＭ ＡＰ−５の存在下での培養でアポトーシスする細胞の割合を示すグラフである。
図９Ｃは、小脳の顆粒神経細胞生存における抗Ａｌｉｖｉｎ １ 抗体の効果を示すグラフである。抗Ａｌｉｖｉｎ １ 抗体はＢＭＥ培地に対してよく透析し、０．２２μｍメンブラン（Ｍｉｌｌｉｐｏｒｅ）で濾過することにより滅菌した。顆粒神経細胞は２５ｍＭ ＫＣｌを含有する培地中で培養した。抗Ａｌｉｖｉｎ １ 抗体または非免疫抗体を培養液に１．０ＤＩＶで加え、５．０ＤＩＶにおいて生存を解析した。２回のの独立した実験の典型的な結果を示した。それぞれの値は二つの解析の平均±Ｓ．Ｄ．である。抗Ａｌｉｖｉｎ １ 抗体（●−●）、非免疫抗体（□−□）。図９Ｄは、ラットＡｌｉｖｉｎ １（５７−４０２）の小脳の顆粒神経細胞の生存における効果を示すグラフである。ラットＡｌｉｖｉｎ １（５２−４０２）をＢＭＥ培地に対してよく透析し、０．２２μｍメンブラン（Ｍｉｌｌｉｐｏｒｅ）を通じて濾過することにより滅菌した。小脳顆粒神経細胞は、２５ｍＭ ＫＣｌを含有する培地中で培養した。ラットＡｌｉｖｉｎ １（５７−４０２）を培養液に２．０ＤＩＶで加え、そして神経細胞の生存は６．０ＤＩＶで検定した。２回の独立した実験の典型的な結果を示した。それぞれの値は二つの実験の平均±Ｓ．Ｄ．である。
【図１０】図１０は、マウスａｌｉｖｉｎ １ 遺伝子の染色体上の局在のマッピングを示す模式図である。マーカーの間の隔たりをｃｅｎｔｉＲａｙｓ（ｃＲ）として示したマウス染色体１５の高分解能ＲＨマップ。マウスａｌｉｖｉｎ １ の遺伝子座は染色体１５のセントロメアからおよそ５５．３ｃＭに位置する。Ｍｉｔマーカーの相対的な位置はマウスゲノムデータベース（ＭＧＤ２００２）から取得した。
【図１１−１】図１１−１は、Ａｌｉファミリーの構成要素のそれぞれの間の相同性を示す概略図である。かっこの外側の数字は同一性％を示し、一方かっこの中の数字は類似性％を示す。
【図１１−２】図１１−２は、ヒトＡｌｉファミリーの構成要素のドメインの構成を示す模式図である。かっこの外側の数字は同一性％を示し、一方かっこの中の数字は類似性％を示す。Ｌ：シグナルペプチド配列を含むリーダー配列、ＮＣ：ＮＨ_２−末端隣接システイン−リッチドメイン、ＬＲＲ：ロイシン−リッチリピート、ＣＣ：ＣＯＯＨ−末端隣接システイン−リッチドメイン、ＩＧ：免疫グロブリンＣ２−様ループ、ＴＭ：膜貫通ドメイン、ＩＤ：細胞内ドメイン。
【図１１−３】図１１−３は、ヒトＡｌｉｖｉｎ １ のドメイン構成およびキイロショウジョウバエＫｅｋファミリーのドメインの構成を示す模式図である。かっこの外側の数字は同一性％を示し、一方かっこの中の数字は類似性％を示す。Ｌ：シグナルペプチド配列を含むリーダー配列、ＮＣ：ＮＨ_２−末端隣接システイン−リッチドメイン、ＬＲＲ：ロイシン−リッチリピート、ＣＣ：ＣＯＯＨ−末端隣接システイン−リッチドメイン、ＩＧ：免疫グロブリンＣ２−様ループ、ＴＭ：膜貫通ドメイン、ＩＤ：細胞内ドメイン。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel gene that plays an important role in the survival of nerve cells, and a protein encoded by the gene. The present inventors concluded from the function of this gene and its locus that this gene is a gene associated with Alzheimer's disease and Parkinson's disease.
[0002]
[Prior art]
Neural activity controls a wide range of functions, such as memory, nerve transmission, nerve growth, differentiation, synapse development, and nerve cell life and death. In particular, it is known that neural activity regulates the number and junction pattern of nerve cells by suppressing apoptosis and promoting the survival of neurons (see, for example, Non-Patent Document 1). It has been proved by experiments using many in vivo and in vitro systems that neural activity suppresses the death of nerve cells and promotes their survival (see, for example, Non-Patent Documents 2, 3, or 4). reference). In an in vitro experimental system, depolarization stimulation by high concentration KCl and stimulation to nerve cells by a glutamate receptor agonist are used. These stimuli are thought to mimic neural activity in vivo, and these stimuli have been extensively used in in vitro experimental systems. The mechanism by which these stimuli promote neuronal survival is considered as follows. When nerve cells are stimulated with high concentrations of KCl or glutamate receptor agonists, each stimulus is voltage-dependent Ca²⁺Ca into cells via channels and glutamate receptor channels²⁺Influx of Ca and thereby into and out of the cell²⁺Turnover of calcium (intracellular Ca²⁺Inflow and extracellular Ca²⁺(E.g., discharge) advances (see, for example, Non-Patent Document 5 or 6). This Ca²⁺The turnover signal is Ca²⁺Activating the / calmodulin-dependent protein kinase, protein kinase B (PKB / Akt) (see, eg, Non-Patent Document 7) and the RAS / MEK / MAPK cascade and their downstream transcription-dependent pathways and It suppresses apoptosis and promotes survival of neurons through both pathways that are transcription-independent (see, for example, Non-Patent Document 8). The transcription-dependent survival promotion pathway includes CREB (Ca²⁺/ CAMP response element binding protein (MEF2C) and a path mediated by MEF2C (myocyte-specific enhancer binding factor 2C) have been reported (for example, see Non-Patent Documents 8, 9, or 10). However, the downstream targets of these transcription factors that are thought to mediate neuronal activity-dependent survival have not yet been fully elucidated. BDNF (brain degenerated neurotrophic factor), PACAP (pituitary adenylate cyclase-activating polypeptide) and some parathyroid hormone related proteins (PTHrP: parathyroid hormone protein)²⁺It has been reported that the gene expression is dependent and has an effect of promoting the survival of nerve cells and a neuroprotective effect (see, for example, Non-Patent Documents 11, 12, or 13). These proteins are secreted from nerve cells in a neural activity-dependent manner and act in an autocrine and / or paracrine manner to promote nerve cell survival. For example, BDNF is Ca²⁺Expressed by transcription via CREB and CaRF (calcium-response transcription factor) existing downstream of the signal (see, for example, Non-Patent Documents 14, 15, or 16), and promote the survival of neuronal cells (for example, non-patented) (Ref. 11) has been shown by many studies. However, since BDNF alone is insufficient to sufficiently support the survival of neurons (see, for example, Non-Patent Document 17), it is considered that signal factors other than BDNF are involved in survival dependent on nerve activity. (See, for example, Non-Patent Document 18).
[0003]
Neuronal survival depends on neural activity or Ca²⁺In order to comprehensively identify the factors involved in the dependently promoted process, we specifically utilize mRNA differential displays in cerebellar granule cells cultured under conditions that promote survival (ie, depolarization). The expressed gene was screened. By this method, several candidate genes that have been thought to be involved in neural activity-dependent cell survival have been found. For example, the present inventors have already identified a known factor, PTHrP, by comparing the correlation between gene expression patterns and neural activity-dependent survival (see, for example, Non-Patent Document 12). ).
[0004]
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Ichikawa, D .; , Et al. Brain Res. Mol. Brain Res. , (1998), Vol. 56, p. 218-226.
[Non-Patent Document 18]
Franke, B.M. , Et al. Exp. Neurol. , (2000), Vol. 165, p. 125-135.
[0005]
[Problems to be solved by the invention]
The present invention provides a novel gene that plays an important role in neural activity-dependent survival of cerebellar granule cells designated as alivin 1 or ali1, a vector containing the gene, and a host cell transformed with the vector. Alivin 1 is “alive” and “activity-dependentLeucine-rich-repeat andimmunoglobulin superfamily survival-related proteinnIt was named after "".
[0006]
The present invention also provides a method for producing a novel protein Alivin 1 (Ali1) encoded by alivin 1 by culturing the host cell, and a novel protein produced by the method.
[0007]
The following inventions are encompassed by the present invention:
(1) DNA encoding Alivin 1 protein having the base sequence encoding the amino acid sequence shown in SEQ ID NO: 4, or one or more amino acids in the above amino acid sequence having deletion, substitution, insertion, addition, and Alivin 1 A DNA encoding a protein having substantially the same activity as the protein or an activity that inhibits the activity of Alivin 1.
(2) The DNA according to (1), which encodes the Alivin 1 protein or a protein having substantially the same activity as that derived from a rat.
(3) DNA encoding Alivin 1 protein having a base sequence encoding the amino acid sequence shown in SEQ ID NO: 5, or one or more amino acids in the above amino acid sequence having deletions, substitutions, insertions, additions, and Alivin 1 A DNA encoding a protein having substantially the same activity as the protein or an activity that inhibits the activity of Alivin 1.
(4) DNA encoding Alivin 1 protein having a base sequence encoding the amino acid sequence shown in SEQ ID NO: 6, or one or more amino acids in the amino acid sequence having deletion, substitution, insertion, addition, and Alivin 1 A DNA encoding a protein having substantially the same activity as the protein or an activity that inhibits the activity of Alivin 1.
(5) A vector having the DNA according to any one of (1) to (4).
(6) A transformed cell having the vector according to (5).
(7) A method for producing Alivin 1 protein, comprising culturing the transformed cell according to (6) under conditions for expressing Alivin 1 protein, and separating Alivin 1 protein from the cultured cell or a medium thereof.
(8) Alivin 1 protein consisting of the amino acid sequence shown in SEQ ID NO: 4 or a sequence having a deletion, substitution, insertion, or addition in one or more nucleotide sequences in the amino acid sequence.
(9) The Alivin 1 protein according to (8), which is a rat-derived protein.
(10) An Alivin 1 protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a sequence having a deletion, substitution, insertion, or addition in one or more nucleotide sequences in the amino acid sequence.
(11) An Alivin 1 protein comprising the amino acid sequence shown in SEQ ID NO: 6 or a sequence having a deletion, substitution, insertion, or addition in one or more nucleotide sequences in the amino acid sequence.
(12) An antibody specific for the protein according to any one of (8) to (11).
(13) A method for promoting the survival of nerve cells, comprising administering the DNA according to any one of (1) to (5).
(14) A method for suppressing neuronal cell death, comprising administering the DNA according to any one of (1) to (5).
(15) A neuronal cell death inhibitor comprising the DNA according to any one of (1) to (5).
(16) A method for treating Alzheimer's disease and Parkinson's disease, comprising administering the DNA according to any one of (1) to (5).
(17) A pharmaceutical composition for the treatment of Alzheimer's disease and Parkinson's disease, comprising the DNA according to any one of (1) to (5).
(18) A pharmaceutical composition comprising the protein according to any one of (8) to (11).
(19) The pharmaceutical composition according to (18), which is used for suppressing neuronal cell death.
(20) A non-human transgenic animal that inducibly or constitutively expresses the protein according to any one of (8) to (11).
(21) A non-human knockout animal in which the gene encoding the protein according to any one of (8) to (11) is deleted.
(22) DNA encoding an Alivin 1 protein having the base sequence shown in SEQ ID NO: 1, 2, or 3.
(23) Alivin 1 protein having the amino acid sequence shown in SEQ ID NO: 4, 5 or 6.
[0008]
[Means for Solving the Problems]
alivin 1 (ali1) Genes and proteins
The gene alivin 1 of the present invention is expressed in neural activity or Ca in cerebellar granule cells.²⁺It is expressed in a dependent manner and enhances the survival of neurons through the transcription process. In addition, the present inventor concluded that the gene has a relationship with Alzheimer's disease and Parkinson's disease from its function and its locus.
[0009]
The nucleotide sequences of the alivin 1 gene are shown in SEQ ID NOs: 1, 2 and 3, respectively, derived from rat, mouse and human. The amino acid sequences encoded by these nucleotide sequences are shown in SEQ ID NOs: 4, 5 and 6.
[0010]
The protein encoded by the alivin 1 gene (Alivin 1) has seven leucine-rich repeats, one immunoglobulin C2-like loop, and one transmembrane domain. Regions with leucine-rich repeats and immunoglobulin C2-like loops and transmembrane domains are homologous to Kek (kekkon) and Trk (tropomyosin receptor kinase).
[0011]
The expression of the alivin 1 gene of the present invention is frequently observed in the lung in vivo. In the primary culture system of cerebellar granule cells, the expression changes depending on the concentration of KCl and / or NMDA (N-methyl-D-aspartic acid) around the cells. For example, when cultured cerebellar granule cells are stimulated with 25 mM KCl, voltage-dependent L-type Ca²⁺Ca through the channel²⁺Flows into the cell and transcription of alivin 1 mRNA is promoted. This progression increases neuronal survival.
[0012]
From the various facts described above, it was revealed that the alivin 1 gene has an important role in the survival of nerve cells. Furthermore, the fact is that the addition of anti-Alivin 1 antibody or antiserum to the culture medium of cerebellar granule cells reduces the survival rate of neurons, and the addition of Alivin 1 extracellular domain instead of antibodies, etc. Even so, it was confirmed that the survival rate of cerebellar granule cells decreased.
[0013]
The human alivin 1 gene is present in a region corresponding to the locus of familial Alzheimer's disease type 5 and Parkinson's disease type 8 on chromosome 12. Therefore, we conclude that a mutation or decreased expression of alivin 1 is responsible for Alzheimer's disease type 5 in humans.
[0014]
From various animals or tissues alivin 1 Gene isolation and mutation alivin 1 Gene production
The mRNA of the gene of the present invention is distributed and expressed in organs or tissues including the brain and lungs of animals. Therefore, a genomic library or cDNA library from those organs or tissues is used as a material, and any nucleotide sequence of SEQ ID NO: 1 to 3 is used as a probe, or an appropriate part of the nucleotide sequence of SEQ ID NO: 1 to 3 is used. It can be used as a primer to isolate the alivin 1 gene from various animals.
[0015]
There may be a plurality of codons encoding each amino acid. Therefore, in addition to the nucleotide sequences of SEQ ID NOs: 1 to 3, there are nucleotide sequences encoding the same amino acid sequence. The present invention includes the alivin 1 gene consisting of such a nucleotide sequence and the protein encoded by the gene.
[0016]
Further, some of the natural proteins have gene mutations due to differences in the species that produce them, or one or more amino acid mutations, and have substantially the same activity as that of the protein before mutation or Alivin 1 It is also well known that there are proteins that have the activity of inhibiting the activity of. Therefore, proteins that are mutants of the native type of Alivin 1 protein and have the same activity as that of the natural type or an activity that inhibits the activity of Alivin 1 and the DNA encoding them are within the scope of the present invention. . Specifically, the present invention is encoded by a nucleotide sequence comprising a mutation by deletion, substitution, insertion or addition of one or more nucleotide residues in the nucleotide sequence of SEQ ID NOs: 1 to 3, and the Alivin 1 protein And a protein having substantially the same activity or that inhibits the activity of Alivin 1. Further, the present invention is the same as a biologically active Alivin 1 protein comprising a mutated amino acid sequence by deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequences of SEQ ID NOs: 4 to 6. Also included is a protein having an activity or an activity that inhibits the activity of Alivin 1. In addition, the present invention has a nucleotide sequence comprising a mutation by deletion, substitution, insertion or addition of one or more nucleotide residues in the nucleotide sequence of SEQ ID NOs: 1 to 3, and substantially A DNA encoding a protein having the same activity or an activity that inhibits the activity of Alivin 1. Further, the present invention is the same as a biologically active Alivin 1 protein comprising a mutated amino acid sequence by deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequences of SEQ ID NOs: 4 to 6. Also included is a protein having an activity or a DNA encoding an activity that inhibits the activity of Alivin 1.
[0017]
In the present specification, the term “Aliven 1 protein” includes not only a naturally-occurring Alivin 1 protein but also a protein comprising a mutated amino acid sequence and having the same activity as the Alivin 1 protein. In this case, mutation caused by deletion, substitution, insertion or addition of one or more nucleotide residues in a specific nucleotide sequence refers to a mutation that can be modified by a site-specific mutagenesis method, for example.
[0018]
Moreover, in this specification, the protein which has the activity which inhibits the activity of Alivin 1 is the Alivin which has the activity which exhibits the inhibitory activity by antagonizing the effect | action of Alivin 1 etc., and suppresses survival of a nerve cell. 1 mutant protein. In this case, the mutation includes deletion, substitution, insertion or addition of a plurality of amino acid residues in the amino acid sequence of Alivin 1 protein. The protein having the activity of inhibiting the activity of Alivin 1 includes, for example, an extracellular segment of Alivin 1 protein, preferably rat Alivin 1 (52-402).
[0019]
Furthermore, the Alivin 1 protein of the present invention includes all proteins as long as they have the properties described herein. For example, a protein whose similarity to the amino acid sequence of SEQ ID NOs: 1 to 3 is 40% or more (for example, Alivin 1 and Alivin 3 which are Alivin 1 analogues), preferably 80% or more, more preferably 90% or more, And a gene encoding the protein are included in the present invention as long as the protein has the same activity as the Alivin 1 protein or an activity that inhibits the activity of Alivin 1. The percent homology can be determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res., Vol. 25, p. 3389-3402, 1997). is there. Various conditions (parameters) for the homology search by the BLAST program are described in detail in the usage instructions of the program, and some settings can be appropriately changed. However, the search is usually performed using default values.
[0020]
The gene of the present invention can also be defined as nucleotides that hybridize under specific conditions to the nucleotide sequences of SEQ ID NOs: 1 to 3. For example, the present invention hybridizes with DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 3 under stringent conditions (eg, hybridization in 2 × SSC overnight at 50 ° C .; washed in 2 × SSC at 50 ° C.). A soybean gene that has seven leucine-rich repeats, one immunoglobulin C2-like loop, and one transmembrane domain, and cell adhesion and / or signal through protein-protein interactions at the cell surface It also includes a gene encoding a protein that plays a role in transmission.
[0021]
Furthermore, the present invention also includes a partial Alivin 1 protein having a part of the Alivin 1 protein (for example, a protein having an extracellular domain of the Alivin 1 protein or only an intracellular domain). The partial Alivin 1 protein has uses such as producing an anti-Aliven 1 antibody and evaluating the function of Alivin 1.
[0022]
Alivin 1 Protein production
In the present invention, an expression vector incorporating the gene of the present invention is used, and host cells transformed with the vector are cultured under conditions where the gene is expressed, and isolated and recovered from the culture to produce Alivin 1 protein. Also related to how to do.
[0023]
As the expression vector, a vector suitable for the host cell in which the Alivin 1 protein is to be expressed is appropriately selected and used. Vectors that can be used in various cells such as E. coli, yeast, animal cells, and insect cells are well known in the field of genetic engineering. In addition to the alivin 1 gene, the vector may contain a promoter, a terminator, and, if necessary, other expression control elements and markers for facilitating confirmation of gene expression.
[0024]
Those skilled in the art can appropriately set host cell transformation, medium, culture temperature, culture time, and the like. The full-length Alivin 1 protein has a transmembrane domain and is mainly present in the nuclear and cell membrane fractions in the host cell. Thus, for example, Alivin 1 protein can be isolated from cells after culture by purification by sucrose density gradient centrifugation or Ficoll density gradient centrifugation. A region encoding a transmembrane domain (parts 399 to 419 in the amino acid sequence of SEQ ID NOs: 4 and 6 (rat and human, respectively), and part 398 to 418 in the amino acid sequence of SEQ ID NO: 5 (mouse)) When producing the protein of the present invention not containing a transmembrane domain using the excluded alivin 1 gene, it can be expressed as a protein containing an appropriate leader sequence and secreted into the culture supernatant.
[0025]
Therefore, this invention manufactures the Alivin 1 protein of this invention using the Alivin 1 protein expression vector which has DNA which codes the Alivin 1 protein of this invention, the transformed cell which has the said expression vector, and the said transformed cell. To include a method.
[0026]
The present invention also provides an antibody against Alivin 1 protein. Production of antisera and / or polyclonal or monoclonal antibodies against the protein of the present invention can be carried out by known methods.
[0027]
Method of treatment and pharmaceutical composition
The present inventors have found that introduction of the gene of the present invention into nerve cells suppresses apoptosis of the nerve cells and promotes survival. Therefore, the protein of the present invention has a function of suppressing apoptosis of nerve cells and promoting survival.
[0028]
The protein of the present invention is administered to patients with various diseases caused by neuronal degeneration, such as Alzheimer's disease and Parkinson's disease, to promote the survival of nerve cells, and to be used for the prevention, improvement, and inhibition of progression of symptoms. Can be expected. The proteins and antibodies of the present invention are also useful as reagents for studying the mechanism of various diseases caused by neuronal degeneration.
[0029]
Therefore, the present invention relates to a method for suppressing death of nerve cells by administering the gene of the present invention to nerve cells and promoting survival, and a cell death inhibitor used in the method.
[0030]
In this aspect of the invention, the gene of the invention is administered to animals including humans to activate the neurons of the animals, suppress apoptosis and / or treat diseases such as Alzheimer's disease, Parkinson's disease, etc. Also encompassed are methods and pharmaceutical compositions for use in the methods of treatment.
[0031]
The inhibitor or pharmaceutical composition containing the gene of the present invention comprises, as an active ingredient, the alivin 1 gene incorporated into a vector that can be used for gene therapy. For example, an adenovirus, a retrovirus, or the like can be used as a viral vector from which toxicity has been removed while remaining infectious to an animal to be treated. The vector contains a promoter capable of expressing the gene of the present invention under specific conditions, for example, physical stimulation such as light or temperature, or chemical stimulation such as tetracycline, and after administration to animals including humans. It is possible to enhance the therapeutic effect by placing it in expression conditions at an appropriate time. Furthermore, the alivin 1 gene can be incorporated downstream of the tissue-specific promoter and expressed specifically in the target tissue. The composition may comprise a single chain antibody or peptide ligand or receptor for selectively delivering the alivin 1 gene to a site requiring treatment of the subject animal, preferably the cranial nerve, and the stability of the composition in the animal body. In order to enhance, other additives used as drugs may be included.
[0032]
In addition, the present invention provides the Alivin 1 protein of the present invention to animals including humans to suppress cell death of nerve cells of the animals and promote survival, and / or to treat diseases such as Alzheimer's disease and Parkinson's disease. It also relates to a method of treatment and a pharmaceutical composition for use in the method of treatment.
[0033]
The pharmaceutical composition containing the Alivin 1 protein of the present invention contains the protein of the present invention as an active ingredient, and includes various pharmaceutically acceptable carriers, diluents, excipients and the like that can be used pharmaceutically. May be. Pharmaceutically acceptable carriers or diluents and the like are essentially chemically inert and harmless compositions that do not affect the biological activity of the pharmaceutical composition of the present invention at all. The pharmaceutical composition may be in any form, and may be, for example, a tablet, a capsule, or the like, or may be a solution or a dry preparation used by dissolving at the time of use. The liquid preparation or the dry preparation used after dissolution is adjusted so that the osmotic pressure and pH in the solution state are compatible with those of the patient to be administered.
[0034]
The pharmaceutical composition of the present invention can be administered by intravascular administration, oral administration, topical administration, subcutaneous administration or the like. In addition, although the dosage of the pharmaceutical composition of the present invention varies depending on the symptoms, weight, age, etc. of each individual patient, a doctor can determine an amount suitable for each individual patient, Included in the invention.
[0035]
Animal models of Alzheimer's disease and Parkinson's disease
In accordance with the present invention, the inventors have concluded that mutation or abnormal expression of the alivin 1 gene is one of the causes of Alzheimer's disease and Parkinson's disease. Therefore, experimental animals such as rats and mice can be used as experimental models for Alzheimer's disease and Parkinson's disease by creating transgenic animals and / or knockout animals for the function of the alivin 1 gene. .
[0036]
A transgenic / knockout animal can be prepared by a method known to those skilled in the art (for example, Mouse Lab Manual Second Edition-Experimental Method in the Post-Genome Era-, Tokyo Metropolitan Institute of Medical Science, Experimental Animal) (See Research Division, Springer Fairlark Tokyo, (2003)).
[0037]
Such a model animal is useful for elucidating more detailed physiological functions of the alivin 1 gene and gene product. Since the mutation or abnormal expression of the alivin 1 gene is the cause of Alzheimer's disease and Parkinson's disease, elucidation of the mechanism of disease development as a model animal of these diseases, development of prevention methods, development of diagnostic methods, treatment methods Useful for early detection of diseases, development of treatment methods, and prevention of progression, such as screening for drugs for development.
[0038]
Drug screening
The present invention also relates to a method for screening a substance related to the protein of the present invention. Substances related to the protein of the present invention have the same action as the protein of the present invention, act on the protein of the present invention to promote or inhibit its activity, and control the activity of the protein of the present invention There are things that you can do. Such substances can also be expressed as agonists or antagonists. Here, antagonist means both competitive and non-competitive antagonists. These drugs can be screened using a method for measuring the survival promoting activity of Alivin 1 on nerve cells.
[0039]
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by an Example.
[0040]
【Example】
Example 1 Rat alivin 1 Gene identification
Measurement of primary culture and cell viability of rat cerebellar granule cells
Rat cerebellar granule cells are described in Levi et al. (Levi et al., Preparation of 98% pure cerebellar granule cell cultures, “A detection and tissue culture of the V. A. V. Eds), p. 211-214, New York: Alan R. Lis Inc., (1989)) (Ono et al., J. Biol. Chem., Vol. 272, p. 272). 14404-14411, (1997)). Cell viability was determined by the method described in the above-mentioned Ono et al. According to the description of Mosman (see Mosmann, T., J. Immunol. Methods., Vol. 65, p. 55-63, (1983)). It was measured.
[0041]
RNA isolation and Northern blot
Isolation of total cellular RNA was slightly modified by the method of Chomezyski and Sacchi (see Chomczynski, P. and Sacchi, N., Anal. Biochem., Vol. 162, p. 156-159, (1987)). Went in the way. Cells were lysed with a 1: 1 mixture of solution A and water saturated phenol. The obtained total RNA was separated on a 1% formaldehyde agarose gel and transferred to a Biodyne B membrane (Pall). A 0.24-9.5 kb RNA ladder (Invitrogen) was used as a size marker. cDNA probes were randomly generated from 9-mer (Takara) and [α-³²Labeled with P] dCTP. Hybridization was performed in a Rapid Hybridization buffer (Amersham) at 65 ° C. according to the manufacturer's instructions. After hybridization, the membrane was washed with 2 × SSC containing 0.1% SDS at room temperature, followed by 0.1 × SSC containing 0.1% SDS at 55 ° C. The membrane was reprobed with mouse glucose-6-phosphate dehydrogenase cDNA (G6PDH) to confirm that an equal amount of RNA sample was loaded (Inokuchi, K., et al., FEBS Lett., Vol. 382, p. 48-52, (1996)). Hybridization signals were analyzed using a FUJIX BAS2000 Bioimage analyzer (Fuji Photo film). mRNA levels were normalized to G6PDH mRNA levels.
[0042]
Differential display and cDNA cloning
The differential display is a method described in the literature (Ono et al., J. Biol. Chem., Vol. 272, p. 14404-14411, (1997); (See, Tokyo Institute of Clinical Medicine, Laboratory Animal Research, Springer Fairlark Tokyo, (2003)). The primer pairs used were 5'-ACA CGC TCA C-3 '(SEQ ID NO: 7) and 5'-TTTTTTTTTTTVA-3' (anchor primer, SEQ ID NO: 8; V is A, G, or C). The resulting PCR product was cloned into the pCR-Script Amp SK (+) vector (Stratagene). Positive clones were selected by Northern plotting analysis, and the DNA sequence of the obtained clones was determined with Thermo Sequenase fluoresced primer cycle kit (Amersham).
[0043]
Neural activity dependent gene alivin 1 Identification
Cerebellar granule cells survive very well when cultured in medium containing 25 mM KCl or 150 μM NMDA (N-methyl-D-aspartic acid) / 15 mM KCl (FIG. 1, ◯-○ and ●-●, respectively). Conversely, when these neurons are cultured in a medium containing 5 mM or 15 mM KCl, the cells gradually die from apoptosis (Gallo, V., et al., J. Neurosci., Vol. 7, p. 7). 2203-2213, (1987); Balazs, R., et al., Neuroscience, Vol. 27, p.437-451 (1988); or Ono et al., J. Biol. Chem., Vol.272. , P. 14404-14411, (1997)). Viability is higher in cultures containing medium containing 15 mM KCl than in cultures containing 5 mM KCl (FIG. 1, □-□ and ■-■, respectively). Moreover, apoptosis is induced by adding 300 μM AP-5 ((±) -2-amino-5-phosphonopentanoic acid) to the culture solution after 3.5 DIV (days in vitro) culture in 150 μM NMDA / 15 mM KCl medium. By the end of 36 hours, about half of the cells were killed (FIG. 1, ▲-▲). In order to identify a gene group induced by depolarization conditions or NMDA presence conditions as genes related to nerve activity-dependent survival of nerve cells, the commitment time of apoptosis was first determined. The commitment time was 3.0 DIV after the start of culture (see Ono et al., J. Biol. Chem., Vol. 272, p. 14404-14411, (1997)). Therefore, the present inventors transferred RNA to 3.0 DIV from a group of cells cultured under the conditions for promoting survival (medium containing 150 μM NMDA and 15 mM KCl) and apoptosis-inducing conditions (medium containing 5 mM KCl). Extracted. These RNA samples were converted to cDNA and then subjected to differential display analysis. As shown in FIG. 2, although a band was detected in the lane on which the PCR product derived from the cells cultured under the survival promoting condition was placed (FIG. 2, arrow on the lane S), it was cultured under the apoptosis-inducing condition. In the lane on which the cell-derived PCR product was placed, no band was detected at this position (FIG. 2, lane A). The arrowed band on lane S was removed from the gel and reconstituted as previously reported (see Ono et al., J. Biol. Chem., Vol. 272, p. 14404-14411, (1997)). Amplified. The PCR product was cloned into pCR-Script Amp SK (+) vector (Strategene) and sequence analysis was performed. The obtained cDNA was identified as a nucleotide fragment (sequence corresponding to 2957-3095 bp of SEQ ID NO: 1) which is the most downstream region of the 3'-untranslated sequence of rat alivin 1 cDNA.
[0044]
Rat alivin 1 Identification of full-length cDNA
A λZap II library constructed from RNA extracted from rat cerebellar granule cells cultured for 5.0 days in medium containing 15 mM KCl and 150 μM NMDA was screened with the cloned rat alivin 1 cDNA fragment. As a result, clones with several overlapping sequences were obtained. The same library was re-screened using the most upstream region of the resulting sequence, resulting in several clones with sequences approximately 500 bp upstream. As a result, full-length rat alivin 1 cDNA (SEQ ID NO: 1) was identified.
[0045]
Example 2 Mouse and human alivin 1 cDNA cloning
Database search
The full-length rat alivin 1 cDNA obtained in Example 1 was subjected to database search (DDBJ / EMBL / GenBank databases). As a result, it was revealed that there are a plurality of mouse and human EST clones having significant homology with respect to the 3 'region of rat alivin 1 cDNA. Furthermore, the entire rat alivin 1 cDNA sequence shows considerable identity (72% identity without a gap of 16 consecutive bases or more) with a part of the BAC clone of human chromosomal DNA (GenBank accession number: AC004010), This indicates that the full-length mRNA sequence of the human homolog of rat alivin 1 (human alivin 1) is encoded by a single exon.
[0046]
mouse alivin 1 cDNA cloning
Mouse alivin 1 cDNA was cloned by PCR using LA-Tag DNA polymerase (TaKaRa) using cDNA prepared from whole mouse brain as a template. The present inventors designed the following two primers so that mouse and human alivin 1 cDNA can be cloned with the same primer pair. The 5′-primer (5′-GCT AAG TAT TTC CGT TCA ATT GTT T-3 ′; sense: SEQ ID NO: 9) compares the genomic sequences of rat alivin 1 cDNA sequence and human alivin 1 Designed based on the consensus sequence of the region upstream of the sequence. In addition, the 3′-primer (5′-GGT ATT CAG GAC AGC GAG GAC GGA G-3 G; antisense: SEQ ID NO: 10) is derived from the consensus sequence of 3 ′ untranslated sequences of mouse and human EST clones from these alibin 1 It was designed as a primer characteristic of cDNA.
PCR conditions were 94 ° C. for 1 minute, followed by 35 cycles of 94 ° C. for 30 seconds; 66 ° C. for 30 seconds; 68 ° C. for 30 seconds, followed by heating at 68 ° C. for 3 minutes.
A full-length mouse alivin 1 cDNA (SEQ ID NO: 2) was obtained by PCR using the above 5'-primer and 3'-primer.
[0047]
Human alivin 1 cDNA cloning
First, an attempt was made to clone human alibin 1 cDNA using the same primer pair (SEQ ID NOs: 9 and 10) used in cloning mouse alivin 1 cDNA, but this primer could not be cloned. Therefore, based on the genome sequence of human alivin 1, 5′-GCT GGT CTT TTG GTA TCG TAG GC-3 ′ (sense 1-23) (SEQ ID NO: 11), 5′-TCC CCC TCG TGG ACT TTA GG 3 ′ (antisense 791-772) (SEQ ID NO: 12), 5′-GAG GGC ATC CTA AAG TCC ACG-3 ′ (sense 746-784) (SEQ ID NO: 13), and 5′-GTG TAG TCT ATT CAT TCT GGT CTA AAA CGG-3 ′ (antisense 1384-1355) (SEQ ID NO: 14) was designed and used.
PCR was carried out using the above two primer pairs, using human cDNA as a template, and under the same conditions as those for cloning mouse alivin 1 cDNA. By overlapping the overlapping portions of the base sequences of the two PCR products obtained using the above two primer pairs, a part of human alivin 1 cDNA was obtained (the 781 to 2163rd bases of SEQ ID NO: 3). ). Since it was confirmed that the human EST clone (GenBank accession number: BG724413) overlaps with this partial human alivin 1 cDNA, these two sequences were ligated to form a full-length human alivin 1 cDNA (SEQ ID NO: 3 ) Was built. The EST clone encodes a region containing the amino acid sequence 1-239 of SEQ ID NO: 6, while the partial human alibin 1 cDNA obtained by PCR encodes the amino acid sequence 240-522 of SEQ ID NO: 6. Yes.
[0048]
Example 3 Structural characteristics of rat, mouse and human Ali proteins
As shown in FIG. 3, the open reading frames of rat mouse and human alivin 1 cDNA encode proteins of 520, 519, and 522 amino acids, respectively. Each protein is NH₂-Signal peptide sequence (L) at the end, seven leucine rich repeats (LRR) surrounded by two cysteine-rich domains (NC and CC), one immunoglobulin C2-like domain (IG) and one transmembrane Has a domain (TM). The signal peptide cleavage site was determined using the method of Neilsen et al. (See Nielsen, H., et al., Protein Eng. Vol. 10, p. 1-6, (1997)). Expected to be between 38 and 39 residues for Alivin 1. In particular, the second half of the 5th LRR (the amino acid sequence of positions 169-190 of SEQ ID NO: 4 and 5 for rat and mouse, and the amino acid sequence of positions 170-191 of SEQ ID NO: 6 for human) is a consensus of leucine zipper sequences. (LxxxxxxxxLxxxxxxxxLxxxxXXXL; L represents leucine, x represents an α-amino acid of an arbitrary L form: SEQ ID NO: 15) and 100% coincidence. Each protein contained an approximately 100 amino acid intracellular domain (ID) in the COOH-terminal region, which was not homologous to other known proteins.
From the structural features, other members of the alivin family (alivin 2 and alivin 3) were also clarified by database search (see Example 11).
[0049]
Example 4 Neuronal activity-dependent survival of cerebellar granule cells alivin 1 Association of mRNA expression
Rat alivin 1 Competitive RT-PCR for expression analysis
Evaluation of the expression level of rat alivin 1 mRNA was performed by competitive RT-PCR. Total RNA (1 μg) extracted from cells was treated with DNase I (Gibco-BRL Life Technologies) and converted to cDNA with Superscript II (Gibco-BRL Life technologies) according to the manufacturer's manual. Competitive RT-PCR analysis was performed using a portion of the sample (1/20). Competitors were constructed using the 'competitive DNA construction kit' (Takara). The following sequences were used for competitive PCR analysis of rat alivin 1: sense primer (1992-2014): 5'-AAA CGC ACT GAA CCA AAG CAA CG-3 '(SEQ ID NO: 16) and antisense primer (2266- 2291): 5′-AAT CAC GAC ACC AGA CGA ACA CAG G-3 ′ (SEQ ID NO: 17), and complete sense primer: 5′-AAA CGC ACT GAA CCA AAG CAA CGT ACG CTC GTC C (SEQ ID NO: 18) and competent antisense primer: 5'-AAT CAC GAC ACC AGA CGA A A CAG GCG GTG AGT ATC TGC ATA TGA T-3 '(SEQ ID NO: 19). This sense and antisense primer pair yielded a 300 bp product, while PCR using the competitive primer pair amplified the 199 bp product. Competitive PCR is 10⁴This was done using 50 ng cDNA prepared from the copy competitor and sample.
[0050]
Rat alivin 1 Association between mRNA expression and neuronal activity-dependent survival of cerebellar granule cells
In order to elucidate the relationship between survival of cerebellar granule cells and expression of alivin 1 mRNA, temporal changes in the expression pattern were examined by Northern blotting. Alivin 1 mRNA has a size of 3.3 kb, and a minor transcript was also detected in the vicinity of 8.5 kb (FIG. 4A). Alivin 1 mRNA expression was limited to cells cultured under pro-survival conditions (ie, medium containing 25 mM KCl or 150 μM NMDA and 15 mM KCl), and its expression increased daily. In contrast, almost no alivin 1 mRNA was detected in cells cultured under apoptosis-inducing conditions (ie, culture medium containing 5 mM KCl or 15 mM KCl) (FIGS. 4-1A and B). When AP-5, a specific antagonist of the NMDA receptor, was added to cells cultured in the presence of NMDA at a final concentration of 300 μM, the expression of alivin 1 mRNA was down-regulated significantly faster (t_1/2= 1.4h, FIGS. 4-2A and B). This down-regulation occurs before the death of the majority of cerebellar granule cell cells, revealing that alivin 1 plays a critical role in neuronal cell survival. After being down-regulated, neurons were re-stimulated with either 25 mM KCl, or 150 μM NMDA and 15 mM KCl (FIGS. 4-3A, B). Alivin 1 mRNA was up-regulated, but it took 5-24 hours to return to the original level. In order to investigate the mechanism of up-regulation of alivin 1 mRNA, various inhibitors were added to the culture medium, and then stimulation with KCl and / or NMDA was performed, and the expression of transcripts was assayed 24 hours after stimulation.
[0051]
Alivin 1 mRNA expression was completely inhibited by actinomycin D and partially inhibited by cycloheximide (43% of the control level at 25 mM KCl stimulation and 49% of the control level at NMDA stimulation, respectively). Furthermore, up-regulation of alivin 1 mRNA is caused by voltage-dependent L-type Ca.²⁺It was inhibited by nifedipine, a specific inhibitor of the channel. These results show that alivin 1 is a voltage-dependent L-type Ca.²⁺Ca through the channel²⁺Inflow indicates that it is an early response gene whose expression is controlled during the transcription process.
[0052]
Example 5 alivin 1 Neural activity-dependent expression of mRNA
Primary culture of rat cerebral cortical neurons
Rat cerebral cortical neurons were cultured by the method of Ogura et al. (See Ogura, A., et al., Neurosci. Lett., Vol. 78, p. 69-74 (1987)). The cerebrum was removed from embryonic day 20 Wistar rats, and nerve cells were prepared using a nerve cell dispersion (Sumitomo Bakelite B-X9901). Dispersed neurons are 5x10 per 3.5cm dish⁶The cells were seeded at a density of 2 ml in DMEM medium containing fetal bovine serum and 5% horse serum and cultured.
[0053]
Rat alivin 1 Neural activity-dependent expression of mRNA
In many papers reported so far, long-term depolarization stimulation using a high concentration of KCl has been used to study neural activity-dependent genes. For example, Brosenitsch and Katz (see Brosenitsch, TA and Katz, DM, J. Neurosci., Vol. 21, p. 2571-2579 (2001)) are long-term depolarization stimuli: It has been reported that it induces a gene response different from the physiological nerve activity that occurs in vivo. See also Ogura et al. (Ogura, A., et al., Neurosci. Lett., Vol. 78, p. 69-74, (1987)) and Kuroda et al. (Kuroda, Y., et al., Neurosci). Lett., Vol.135, p.255-258, (1992)), respectively, primary cultured hippocampal neurons and cerebral cortical neurons form synapses that actually function between neurons, and It was reported that nerve cells were spontaneously and synchronously excited without any stimulation. They reported that the addition of tetrodotoxin to the culture system can suppress the spontaneous excitement of neurons, and that when the tetrodotoxin is removed from the culture medium, the spontaneous excitement of neurons is recovered.
[0054]
Therefore, the present inventors have used a primary culture system of cerebral cortical neurons to determine whether or not the expression of alivin 1 is controlled by physiological neural activity as described above, that is, the electrical properties of neurons. Whether it was controlled by various activities. Rat fetal cerebral cortex neurons prepared as described above were cultured for 2 weeks, which is the period during which neurons mature and synapse. As shown in FIG. 5, the cells cultured for 2 weeks expressed alivin 1 mRNA. Na into this cell²⁺Addition of tetrodotoxin, which is a channel inhibitor, significantly suppressed the expression of alivin 1 mRNA. When tetrodotoxin was removed from the culture solution, the expression of alivin 1 mRNA was restored to the original level. From these results, it was concluded that the expression of alivin 1 gene depends on the electrical activity of nerve cells, and thus alivin 1 is a gene whose expression is controlled depending on nerve activity.
[0055]
Example 6 alivin 1 Tissue distribution of mRNA and regulation of expression during development
Alivin 1 mRNA was most abundantly expressed in the lung (FIG. 6A). Except for the lung, low levels of alivin 1 mRNA expression were observed in all of the tissues tested.
The expression of alivin 1 mRNA in the cerebellum was subject to expression control with development. The rat cerebellum develops after birth and developmental morphogenesis is completed at 21 days of age. 7 to 14 days after birth is a period in which cerebellar granule neurons migrate from the outside of the granule layer to the inside, but during this period, cerebellar alibin 1 mRNA was down-regulated to approximately half of day 0 after birth (FIG. 6B, C). This level was maintained until 90 days after birth. These data suggest that alivin 1 has a role in cerebellar morphogenesis.
[0056]
Example 7 Immunohistochemical analysis
Rat Alivin 1 Bacterial expression and anti Alivin 1 Antibody production
Rat alivin 1 cDNA was amplified by PCR using Pfu polymerase. Appropriate restriction sites were incorporated into both primers and the His tag sequence was included in the antisense primer. Rat Alivin 1 (57-520) (in this specification, the expression “rat Alivin 1 (xy)” is a partial protein of rat Alivin 1 consisting of the amino acid sequence from the x-th to the y-th amino acid sequence of SEQ ID NO: 4. Primers for the production of specific antibodies for 5'-GGA ATT CCA TAT GAA CCT GTC TAA GGT GCC TGG G-3 '(SEQ ID NO: 20) and 5'-CCG CTC GAG AGT GGA TGC CAC GAA GGG G-3 ′ (SEQ ID NO: 21) was used. Primers for amplification of the extracellular domain (rat Alivin 1 (57-402)) were 5′-GGA ATT CCA TAT GAA CCT GTC TAA GGT GCC TGG G-3 ′ (SEQ ID NO: 22) and 5′-CCG CTC GAG GGT GGT GAA AGC-3 ′ (SEQ ID NO: 23) was used. The resulting PCR product was cloned into the pET22b vector with matching reading frames. This was transfected into Escherichia coli and induced with 1 mM IPTG for 3 hours for expression of Alivin 1 protein.²⁺-Purified by NTA column (QIAGEN) chromatography. Purified rat Alivin 1 (57-520) was further purified by SDS gel electrophoresis and immunized to rabbits to obtain anti-Alivin 1 antiserum. The obtained antiserum was used as an antibody as it was or after purification. It was confirmed by Western blot that this antibody cross-reacts with 66K and 63K Alivin 1 proteins of rat brain (FIG. 7A).
[0057]
Rat Alivin 1 Protein immunohistochemical analysis
A frozen section (15 μm thickness) of a mature rat brain was fixed with 100% methanol at −20 ° C. for 10 minutes. Sections were blocked with Dulbecco's phosphate buffered saline containing 5% bovine serum albumin and 5% goat serum. The sections were then reacted with the rabbit anti-Alivin 1 antibody described above, followed by anti-rabbit IgG conjugated to fluorescein isothiocyanate. Non-immune rabbit antibodies were used for negative control staining.
[0058]
In the rat brain Alivin 1 Immunohistochemical localization of proteins
In order to evaluate the distribution of Alivin 1 protein in the rat brain, rat brain immunohistochemistry was performed using the above-mentioned rabbit anti-Aliven 1 antibody (FIGS. 7B and 7C). In the rat cerebellum, the majority of Alivin 1 immunoreactivity was localized in the cell body of cerebellar granule neurons and Purkinje cells, whereas in the rat hippocampus, Alivin 1 immunoreactivity was measured in the CA1 and CA3 regions (CA is Cornu Ammonis). (Abbreviation of (Ammonian angle)) was localized in the body of cone cells and the cell body of granule cells in the dentate gyrus. There was no staining in the negative control specimen (data not shown).
[0059]
Example 8 Alivin 1 Intracellular localization of proteins
Cell fraction of rat brain
Brains were removed from 10-week-old male rats, and intracellular granules were collected from Gordon-Weeks (Gordon-Weeks, PR, (1987) Isolation of synaptosomes, growth conpens and cerp entral subcellular. (See Turner, AJ, Bachelard, HS, eds), pp 1-26. Oxford: IRL Press). The cell membrane fraction, synaptosome fraction and mitochondrial fraction were obtained by Ficoll concentration gradient centrifugation in the same manner as Gordon-Weeks. 5′-nucleotidase, a marker enzyme of cell membranes, is obtained from Graham et al. (See Graham, JM, et al., Biochim. Biophys. Acta., Vol. 150, p. 303-305, (1968)). The method was assayed.
[0060]
Western blot analysis
Cell extracts were electrophoresed on a 10% SDS gel. After electrophoresis, the protein was transferred to a PVDF membrane, and Alivi 1 immunoreactivity was detected with ECL or ECL-plus (Amersham) using a 3000-fold diluted anti-Aliven 1 antibody.
[0061]
In the rat brain Alivin 1 Intracellular localization of proteins
The entire rat brain was fractionated into various intracellular granule fractions by density gradient centrifugation as described above. Individual fractions were subjected to Western blot analysis with anti-Aliven 1 antibody. Most of the immunoreactivity of Alivin 1 was localized in the nuclear fraction (FIG. 8, lane 2) and the cell membrane fraction (FIG. 8, lane 6). The latter fraction showed the highest activity of 5'-nucleotidase, a marker enzyme in the cell membrane (data not shown). From these experimental results, it was found that Alivin 1 is localized in the nucleus and cell membrane.
[0062]
Examples of signaling receptors that were thought to be localized in the cell membrane were actually present in the cell nucleus are EGF receptor, FGF receptor, growth factor receptor, c-erb-4 / HER-4 growth factor receptor (Lobie) , P.E., et al., J. Biol.Chem., Vol.269, p.31735-31746, (1994); Xie, Y. and Hung, MC, Biochem.Biophys.Res. , Vol. 203, p. 1589-1598, (1994); Maher, PA, J. Cell Biol., Vol. 134, p. 529-536, (1996), Srinivasan, R., et al. , Cancer Res., Vol. 60, p.1483-1487, (2000); Lin, S.Y., et al., Nat.Cell Biol., Vol.3, p.802-808, (2001); et al., Thyroid, Vol. 11, p.137-145, (2001)). It has been reported that the EGF receptor itself exhibits transactivator activity and can act as a transcription factor (Lin, S. Y., et al., Nat. Cell Biol., Vol. 3, p. 802-808, (2001)). Due to the fact that Alivin 1 is localized in the nucleus, it is considered that Alivin 1 plays some role in the cell nucleus in addition to the function on the cell surface.
[0063]
Example 9 Alivin 1 Promotes depolarization-dependent survival of cerebellar granule cells
Alivin 1 Introduction of expression vector into primary cultured cerebellar granule cells
Using PCR, the nucleotide encoding the Flag epitope was fused to the 3'-side of the coding region of rat alivin 1 cDNA. The synthesized cDNA was cloned into pTriEx-1.1 vector (Novagen). The expression vector was introduced into cerebellar granule cells at the time of cell seeding (culture day 0) using Lipofectamine 2000 (Invitrogen). Alivin 1 expression vector or control vector (pTriEx-1.1 vector) was mixed 4: 1 with EGFP expression vector (pEGFP-N3; Clontech) and this was mixed with BME medium. Lipofectamine 2000 equivalent to the expression vector was diluted with BME medium and incubated at room temperature for 5 minutes. This diluted Lipofectamine 2000 solution was mixed with the DNA / BME solution and incubated at room temperature for 20 minutes to form a Liopfectamine / DNA complex. Cerebellar granule cells dispersed in BME medium containing 10% fetal bovine serum and Liopfectamine / DNA complex were mixed at a ratio of 4: 1. This mixture was seeded at a rate of 0.5 ml / well on a 12-well plate containing a cover glass coated with polyethyleneimine, and culture was started. 2.5x10 per well of culture⁶Cells and 1.25 μg of DNA. The culture medium was replaced with a new one 5 hours after the start of the culture. After 24 hours, 30 μM AP-5 and 100 μM AraC were added. Cells were fixed with 3% formaldehyde at 3.0 DIV and immunostained with anti-Flag antibody (M2; Sigma). Cell nuclei were stained with Hoechst 33342 by the method of See et al. (See See, V., et al., Faseb. J., Vol. 15, p. 134-144, (2001)). Identified dead cells.
[0064]
Alivin 1 Protein promotes depolarization-dependent survival of cerebellar granule cells
Alivin 1-Flag fusion protein expression vector and EGFP expression vector were simultaneously transfected into primary cultured cerebellar granule cells, and the effect of alivin 1 on cell viability was examined. The effect on the survival rate was examined by measuring the proportion of cells that caused apoptosis in the transfected cell group (Flag positive and EGFP positive) and died. A control vector and an EGFP expression vector were simultaneously transfected into a control experiment group, and cells into which the vector was introduced were identified by EGFP fluorescence. As shown in FIGS. 9A and B, when the Alivin 1-Flag expression vector was transfected and cultured in a culture medium containing 5 mM KCl / 30 μM AP-5, 47% of the transfected cells were apoptotic. Compared to 75% of the cells in the control group. These results confirmed that Alivin 1 protein suppresses cerebellar granule cell apoptosis and promotes survival.
[0065]
Since the result of FIG. 8 shows that some Alivin 1 proteins exist on the cell membrane, it was next examined whether the Alivin 1 proteins present on the cell membrane are related to cell survival. In order to inhibit the function of Alivin 1 protein on the surface of nerve cells, the inventors added anti-Aliven 1 antibody to the culture medium. As shown in FIG. 9C, when anti-Aliven 1 antibody was added to the culture medium of cells cultured in a culture medium containing 25 mM KCl, survival of cerebellar granule cells was inhibited in a dose-dependent manner. On the other hand, the survival of cerebellar granule cells cultured in a culture solution containing 5 mM KCl was not affected by the addition of antibody. From these results, it was revealed that Alivin 1 protein present on the cell surface is involved in the depolarization-dependent survival of cerebellar granule cells. In order to further confirm that Alivin 1 protein present on the cell membrane is involved in depolarization-dependent survival, examination was performed using the extracellular segment of Alivin 1 protein expressed in E. coli. The extracellular segment of Alivin 1 protein has domains (LRR and immunoglobulin domains) that act on protein-protein interactions, and these domains are thought to bind to the ligand of Alivin 1 protein, so that the extracellular segment is Alivin 1 protein. It is thought to act as a competitive inhibitor for. Similar experiments have been reported with TGFβ receptors and TrkA, and it has already been reported that the soluble extracellular domain of these proteins inhibits the function of these receptors. As shown in FIG. 9D, when an extracellular segment (rat Alivin 1 (52-402)) of Alivin 1 protein was added to a culture broth containing 25 mM KCl / 30 μMAP-5, culture was performed in a dose-dependent manner. Cerebellar granule cell survival was suppressed to 65%. From the above results, it was revealed that Alivin 1 protein present on the cell surface is involved in depolarization-dependent survival. These results do not deny the involvement of Alivin 1 protein present in the nucleus in depolarization-dependent survival. These results indicate that anti-Aliven 1 antibody inhibits cerebellar granule cell survival and interferes with the binding of Alivin 1 on the cell surface and the interaction between Alivin 1 and its specific ligand (s). It was shown that Moreover, since the soluble form of the extracellular domain of Alivin 1 inhibits the survival of primary cultured cerebellar granule neurons, the added extracellular domain of Alivin 1 competitively binds to Alivin 1 ligand (s), It has been found that the survival signal is inhibited by interfering with the interaction between Alivin 1 and Alivin 1 ligand (s).
[0066]
Example 10 Mouse alivin 1 Of the chromosomal locus
Radiation hybrid (RH) mapping and human alivin 1 Gene locus
The position of the mouse alivin 1 on the chromosome was determined by PCR analysis, and the T31 Radiation Hybrid (RH) panel (Research Genetics / Invitrogen) (McCarthy, LC, et al., Genome Res. Vol. 7, p. 7). The 94 DNA samples from 1153-1116 (1997) (excluding the reference cell lines 12, 25, 61, 62, 99 and 100) were type analyzed in duplicate. This PCR reaction was performed in a total volume of 15 μl, 20 ng genomic DNA, 0.55 μM each primer, 0.15 U AmpliTaq Gold (Applied Biosystems), 200 μM dNTP, 1.5 mM MgCl.₂And 1 × Gold buffer in a total volume of 15 μl. The cycle conditions were as follows: 95 ° C. 5 minutes (enzyme activation), 94 ° C. 30 seconds; 55 ° C. 40 seconds; 72 ° C. 12 minutes 40 cycles. PCR products were electrophoresed in 4% agarose gels (3% NuSierve agarose and 1% SeaKem ME agarose) in TAE buffer and visualized by staining with ethidium bromide. Data were recorded from the gel images by a special scoring system (0 = negative, 1 = positive, and 2 = no test line or no reproducibility). Using the obtained data, the Radiation Hybrid Framework Map was established. Following loci mapping, adjacent markers were retyped using the cell line of the RH panel. Analysis of RH mapping data in Map Manager QT (Manly, K. F. and Olson, J. M., Mamm. Genome Vol. 10, p. 327-334, (1999)) determines the distance in cR3000. did.
[0067]
mouse alivin 1 Chromosome mapping
In order to determine the chromosomal localization of alivin 1 in the mouse chromosome, we performed radiohybrid (RH) mapping (FIG. 10). As a result of comparing the obtained data with the mouse genome database (MGD2002 (http://www.informatics.jax.org/mgihome/)), the locus of mouse alivin 1 was about 55.3 cM from the centromere of chromosome 15. Mapped to the position. This region has synteny (gene homology) with human chromosome 12. According to the database, human alivin 1 is mapped to 12q13.11 (accession number AC004010). Consistent with this region, the causative genes of familial Alzheimer's disease type 5 (AD5: OMIM 602096, 12p11.23-q13.12) and Parkinson's disease type 8 (PARK8: OMIM 607060, 12p11.23-q13.11) Although present, the causative genes for AD5 and PARK8 have not been identified. AD5 is a late-onset (at least 60 years) Alzheimer's disease that develops at the age of 60 years or older, and is not caused by mutations of APP, presenelin 1/2, and apolipoprotein E reported so far. PARK8 has a different locus from α-synuclein, parkin, DJ1, and UCH-L1, which have been reported so far. Furthermore, no genes related to cell survival and death have been reported in the region where AD5 and PARK8 are mapped. Therefore, the inventor concluded that alivin 1 is the causative gene.
[0068]
Example 11 Alivin Family protein structure
Alivin 1 is a new member of the LRR and immunoglobulin superfamily. Searching the database also revealed other genes belonging to the alivin gene family: mouse alivin 2 (accession number BC010598), human alivin 2 (accession number LOC127001, AB032989), and human alivin 3 (accession number) AB058754). As shown in FIGS. 3 and 11-1, significant sequence homology was observed between the proteins encoded by the alivin gene family. Similarities in Alivin homologous species (orthologs) are between 92% and 95%, while similarities between Alivin paralogs are between 46% and 56%. The homology between the domain structures of human Alivin paralogs was highest in the transmembrane domain, followed by significant homology between the N-terminal adjacent cysteine-rich and LRR domains (FIG. 11-2). Furthermore, the domain structure of the entire Alivin protein was remarkably conserved among Alivin paralogs. Each Alivin paralog is NH₂-Signal peptide sequence at the end, NH₂-Consists of seven LRRs surrounded by terminal and COOH-terminal cysteine-rich sequences, one immunoglobulin C2-like domain, one transmembrane domain, and an intracellular domain of approximately 100 amino acids. The homologous sequence of the LRR domain of Alivin family proteins is LxxLxxLxLxxNxaxxxxa (SEQ ID NO: 24), which matches the sequence reported as a typical animal cell extracellular LRR sequence (Kobe, B. and Deisenhofer, J , Trends Biochem.Sci., Vol.19, p.415-421 (1994); or Kajava, A.V., J. Mol.Biol., Vol.277, p.519-527, (1998). ).
[0069]
To date, many LRR family components have been identified (see Kobe, B. and Deisenhofer, J., Trends Biochem. Sci., Vol. 19, p. 415-421, (1994)). ). However, only a few examples of proteins with both LRRs and immunoglobulin-like domains have been reported. These proteins are known as Kek 1/2 (Musacchio, M. and Perrimon, N., Dev. Biol., Vol. 178, p. 63-76, (1996); or Giglione, C., et al., Cell. Vol. 96, p. 847-856 (1999)), Trk A / B / C (Schneider, R. and Schweiger, M., Oncogene, Vol. 6, p. 1807-1811, (1991). )), ISLR (see Nagasawa, A., et al., Genomics, Vol. 44, p. 273-279, (1997)), NLRR 1/2/3 (Taguchi, A., et. Al.). al., Brain Res Mol. Brain Res., Vol.35, p.31-40, (1996); or Taniguchi, H., et al., Brain Res. Mol.Brain Res., Vol.36, p.45-52, (See 1996)), Pal (see Gomi, F., et al., J. Neurosci., Vol. 20, p. 3206-3213, (2000)), LIG-1 (Suzuki, Y. et al.). , Et al., J. Biol. Chem., Vol.271, p.22522-22527, (1996)) and peroxidasin (Nelson, RE, et al., Embo J., Vol. .13, p. 3438-3447, (1994). It is. In addition to LRRs and immunoglobulin-like domains, NLRR 1/2/3 and Pal have fibronectin type 3 domains. Peroxidasin also has a peroxidase and a thrombospondin / procollagen domain. In proteins with both LRRs and immunoglobulin-like domain (s), the Ali family extracellular domain shows significant similarity to those of Drosophila Kek1 and Kek2, the former of Drosophila melanogaster Inhibits the Grk / EFGR pathway, which regulates dorsoventral patterning by binding to the EGF receptor (EGFR) in the embryo and restricting ventralization signals (Ghiglione, C., et al., Cell, Vol. 96). , P. 847-856, (1999)). The structure of the primary structure is highly conserved between the Kek and Alivin families (Figure 11-3). The Kek family consists of one incomplete LRR adjacent to a cysteine-rich sequence, and six complete LRRs, one immunoglobulin C2-like loop, and one single-pass transmembrane domain (Musacchio, M. and Perrimon, N., Dev. Biol., Vol.178, p.63-76, (1996)). The highest homology was observed between the Alivin family and the Kek family in the transmembrane domain and the LRR domain, but no homology was observed in the intracellular domain. In addition, the extracellular domain of the Alivin family was homologous to that of the Trk family. Trk protein is a neurotrophic factor comprising a signal peptide sequence, three LRRs adjacent to a cysteine-rich sequence, two immunoglobulin C2-like loops, a single transmembrane transmembrane domain, and an intracellular tyrosine-kinase domain It is a receptor (see Schneider, R. and Schweiger, M., Oncogene, Vol. 6, p. 1807-1811, (1991)). The extracellular domain of human Trk C showed 21% amino acid identity and 37% similarity to that of human Alivin 1, showing the highest homology with human Alivin 1.
[0070]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph showing depolarization-dependent and NMDA-dependent survival of cerebellar granule cells. Cerebellar granule cells were cultured in medium containing 5 mM KCl and 30 μM AP-5 (■-■), medium containing 15 mM KCl and 30 μM AP-5 (□-□), medium containing 25 mM KCl and 30 μM AP-5. The cells were cultured in (○ − ○) and a culture solution (●-●) containing 150 μM NMDA and 15 mM KCl. Furthermore, for cells cultured in a culture medium containing 150 μM NMDA and 15 mM KCl, after culturing for 3.5 days, 300 μM AP-5 was added to block the NMDA receptor and induce apoptosis (▲- ▲). The cell viability was measured by the MTT method, and the relative viability against the cell viability of cells cultured in a culture medium containing 25 mM KCl and 30 μM AP-5 was plotted.
FIG. 2 is a gel electrophoresis photograph showing the results of a differential display. Lane A is a PCR product amplified by differential display PCR after converting total RNA of cerebellar granule cells cultured in 3.0 DIV in the presence of 5 mM KCl into cDNA. Lane S is a PCR product obtained by converting total RNA of cerebellar granule neurons cultured in 3.0 DIV in the presence of 150 μM NMDA and 15 mM KCl into cDNA and then amplifying by differential display PCR. Analyzed on a 6% sequencing gel.
FIG. 3 is a schematic diagram showing domains of Alivin 1 protein. The open reading frames of rat mouse and human alivin 1 cDNA encode proteins of 520, 519, and 522 amino acids, respectively. L is NH₂-Terminal signal peptide sequence, NC and CC for cysteine-rich domain, LRR for leucine rich repeat, IG for immunoglobulin C2-like domain, TM for transmembrane domain, and ID for COOH-terminal region Represents an approximately 100 amino acid intracellular domain.
FIG. 4-1 is a photograph showing the result of Northern blotting of alivin 1 mRNA expression in nerve granule cells. Total RNA samples (10 μg) extracted from granular neurons cultured in the presence of either 5 mM KCl, 15 mM KCl, 25 mM KCl, or both 150 μM NMDA and 15 mM KCl were subjected to Northern blotting. CDNA obtained by differential display analysis (corresponding to the 3'-untranslated region of rat alivin 1 cDNA) was used as a probe. The lower box shows the expression level of G6PDH mRNA.
FIG. 4-1B is a graph showing values obtained by quantifying the results of FIG. 4-A. Nerve granule cells in the presence of either 5 mM KCl (Δ-Δ); 15 mM KCl (O-O); 25 mM KCl (▲-▲); or both 15 mM KCl and 150 μM NMDA (●-●); Cultured.
FIG. 4-2 is a photograph of Northern blotting showing early down-regulation of alibin 1 mRNA expression by blocking NMDA receptor with AP-5. Neurogranular cells were cultured for 4.5 days in medium containing 15 mM KCl and 150 μM NMDA, and NMDA receptors were blocked by the addition of AP-5 at a final concentration of 300 μM. The 3.3 kb band is a band corresponding to alivin 1 mRNA. The lower box shows the expression level of G6PDH mRNA.
FIG. 4-2B is a graph showing values obtained by quantifying the results of FIG. 4-2A.
FIG. 4-3A is a gel electrophoresis photograph showing the effect of various inhibitors on alivin 1 mRNA expression. Cerebellar granule neurons were cultured in a medium containing 25 mM KCl for 4 days. The medium was changed to one containing 5 mM KCl and the culture continued for another 24 hours, after which the cells were 5 mM KCl (5); 15 mM KCl (15); 25 mM KCl (25); or 15 mM KCl and 150 μM NMDA. Stimulated with both (N); Inhibitors were added to the culture medium 30 minutes prior to stimulation with KCl and / or NMDA. After stimulation (24 hours), total RNA was extracted and subjected to competitive PCR analysis. Inhibitors used include 35 μM cycloheximide (CHX), 4.0 μM actinomycin D (AcD) or 0.2 μM nifedipine (NFD). Ethanol was used as the vehicle for nifedipine.
4-3B is a graph obtained by quantifying the data shown in FIG. 4-3A.
FIG. 5 is a graph evaluating the expression level of alivin 1 mRNA in the presence and absence of tetrodotoxin. Rat cerebral cortical neurons were cultured for 14 days (control). Tetrodotoxin (1 μM) was added to the culture and further cultured for 30 hours (+ TTX). Thereafter, the culture medium was replaced with a new one, and further cultured for 30 hours (-TTX). RNA was extracted from each sample, and the expression level of alivin 1 mRNA was evaluated by competitive PCR.
FIG. 6A is a photograph of the results of evaluation of the tissue distribution of alivin 1 mRNA by Northern blotting. Total RNA (10 μg) was extracted from various tissues of adult rats and subjected to Northern blotting. The lower box shows ethidium bromide staining of 28S rRNA.
FIG. 6B is a photograph when the developmental expression of alivin 1 mRNA in the rat cerebellum was evaluated by Northern blotting. Total RNA was extracted from the cerebellum on a specific day after birth and 10 μg RNA was subjected to Northern blotting analysis. The lower box shows ethidium bromide staining of 28S rRNA.
FIG. 6C is a graph showing data obtained by quantifying the result of FIG. 6B.
FIG. 7A is a photograph showing the results of subjecting a whole rat brain extract to 10% SDS polyacrylamide gel electrophoresis and performing Western blotting using an anti-rat Alivin 1 antibody.
FIG. 7B is a photograph showing the immunohistochemical localization of Alivin 1 in the rat cerebellum. ML represents a molecular layer, PL represents a Purkinje cell layer, and GL represents a granular cell layer.
FIG. 7C is a photograph showing the immunohistochemical localization of Alivin 1 in the rat hippocampus. In the figure, “CA” in CA, CA1 and CA2 is an abbreviation for Cornu Ammonis (Ammon angle), and “DG” is an abbreviation for dentate gyrus (dentate gyrus).
FIG. 8 is a photograph showing the results of Western blotting evaluating the subcellular distribution of Alivin 1 in the brain. The whole brain of 10 week old rats was separated into various cell component fractions. Protein (100 μg) was subjected to 10% SDS polyacrylamide gel electrophoresis.
FIG. 9A shows a photograph of cells obtained by introducing an Alivin 1 protein expression vector into primary cultured cerebellar granule cells. The expression vector was introduced into cerebellar granule cells on day 0 of culture and cultured in a culture medium containing 5 mM KCl and 30 μM AP-5. On day 3.0 of culture, the cells were fixed with 4% paraformaldehyde and stained with anti-Flag antibody and Hoechst dye. Transfected cells were identified by immunostaining with anti-Flag antibody and fluorescence with EGFP. Apoptotic cells were detected as cells with contracted or fragmented nuclei by staining with Hoechst dye. (Ad) cells transfected with Alivin 1-Flag fusion protein expression vector, (eh) cells transfected with control vector, (a and e) fluorescence of EGFP, (b and f) anti-Flag antibody and Immunostaining with Alexa 594 floor-bound secondary antibody, (c and g) staining with Hoechst dye, (d and h) EGFP fluorescence image, anti-Flag antibody immunostaining image, Hoechst staining dye overlay. Arrows indicate cells that have been transfected.
FIG. 9B is a graph showing the percentage of cells that apoptotic in culture in the presence of 5 mM KCl / 30 μM AP-5 after transfection of Alivin 1 expression vector or control vector into cerebellar granule cells.
FIG. 9C is a graph showing the effect of anti-Aliven 1 antibody on granule neuron survival in the cerebellum. Anti-Alivin 1 antibody was dialyzed well against BME medium and sterilized by filtration through a 0.22 μm membrane (Millipore). Granule neurons were cultured in a medium containing 25 mM KCl. Anti-Alivin 1 antibody or non-immune antibody was added to the culture medium at 1.0 DIV, and survival was analyzed at 5.0 DIV. Typical results from two independent experiments are shown. Each value is the mean ± SEM of two analyses. D. It is. Anti-Aliven 1 antibody (●-●), non-immune antibody (□-□). FIG. 9D is a graph showing the effect on survival of cerebellar granule neurons of rat Alivin 1 (57-402). Rat Alivin 1 (52-402) was dialyzed extensively against BME medium and sterilized by filtration through a 0.22 μm membrane (Millipore). Cerebellar granule neurons were cultured in a medium containing 25 mM KCl. Rat Alivin 1 (57-402) was added to the culture at 2.0 DIV and neuronal viability was assayed at 6.0 DIV. A typical result of two independent experiments was shown. Each value is the mean ± SEM of two experiments. D. It is.
FIG. 10 is a schematic diagram showing mapping of the localization of the mouse alivin 1 gene on the chromosome. High resolution RH map of mouse chromosome 15 showing the distance between markers as centrays (cR). The mouse alivin 1 locus is located approximately 55.3 cM from the centromere of chromosome 15. The relative position of the Mit marker was obtained from the mouse genome database (MGD2002).
FIG. 11-1 is a schematic showing the homology between each of the Ali family members. Numbers outside parentheses indicate% identity, while numbers inside parentheses indicate% similarity.
FIG. 11-2 is a schematic diagram showing the domain structure of the constituent elements of the human Ali family. Numbers outside parentheses indicate% identity, while numbers inside parentheses indicate% similarity. L: leader sequence including signal peptide sequence, NC: NH₂-Terminal adjacent cysteine-rich domain, LRR: leucine-rich repeat, CC: COOH-terminal adjacent cysteine-rich domain, IG: immunoglobulin C2-like loop, TM: transmembrane domain, ID: intracellular domain.
FIG. 11-3 is a schematic diagram showing the domain configuration of human Alivin 1 and the domain configuration of Drosophila melanogaster Kek family. Numbers outside parentheses indicate% identity, while numbers inside parentheses indicate% similarity. L: leader sequence including signal peptide sequence, NC: NH₂-Terminal adjacent cysteine-rich domain, LRR: leucine-rich repeat, CC: COOH-terminal adjacent cysteine-rich domain, IG: immunoglobulin C2-like loop, TM: transmembrane domain, ID: intracellular domain.

Claims (20)

DNA encoding Alivin 1 protein having a base sequence encoding the amino acid sequence shown in SEQ ID NO: 4, or one having a deletion, substitution, insertion, addition in one or more amino acids in the amino acid sequence, and Alivin 1 protein A DNA encoding a protein having substantially the same activity or an activity that inhibits the activity of Alivin 1. The DNA according to claim 1, which encodes the Alivin 1 protein or a protein having substantially the same activity as that derived from rat. DNA encoding Alivin 1 protein having a base sequence encoding the amino acid sequence shown in SEQ ID NO: 5, or one or more amino acids in the above amino acid sequence having deletion, substitution, insertion, addition, and Alivin 1 protein A DNA encoding a protein having substantially the same activity or an activity that inhibits the activity of Alivin 1. DNA encoding Alivin 1 protein having the base sequence encoding the amino acid sequence shown in SEQ ID NO: 6, or one or more amino acids having a deletion, substitution, insertion, or addition in the amino acid sequence, and Alivin 1 protein A DNA encoding a protein having substantially the same activity or an activity that inhibits the activity of Alivin 1. A vector comprising the DNA according to any one of claims 1 to 4. A transformed cell comprising the vector according to claim 5. A method for producing an Alivin 1 protein, comprising culturing the transformed cell according to claim 6 under conditions for expressing the Alivin 1 protein, and separating the Alivin 1 protein from the cultured cell or a medium thereof. An Alivin 1 protein comprising the amino acid sequence shown in SEQ ID NO: 4 or a sequence having a deletion, substitution, insertion, or addition in one or more nucleotide sequences in the amino acid sequence. The Alivin 1 protein according to claim 8, which is a rat-derived protein. An Alivin 1 protein comprising the amino acid sequence shown in SEQ ID NO: 5 or a sequence having a deletion, substitution, insertion, or addition in one or more nucleotide sequences in the amino acid sequence. An Alivin 1 protein comprising the amino acid sequence shown in SEQ ID NO: 6 or a sequence having a deletion, substitution, insertion, or addition in one or more nucleotide sequences in the amino acid sequence. An antibody specific for the protein according to any one of claims 8 to 11. A method for promoting the survival of nerve cells, comprising administering the DNA according to any one of claims 1 to 4. A method for suppressing neuronal cell death comprising administering the DNA according to any one of claims 1 to 4. An inhibitor of neuronal cell death, comprising the DNA according to any one of claims 1 to 4. A pharmaceutical composition for the treatment of Alzheimer's disease and Parkinson's disease comprising the DNA according to any one of claims 1 to 4. A pharmaceutical composition comprising the protein according to any one of claims 8 to 11. The pharmaceutical composition according to claim 17, which is used for suppressing neuronal cell death. A non-human transgenic animal that inducibly or constitutively expresses the protein according to any one of claims 8 to 11. A non-human knockout animal in which the gene encoding the protein according to any one of claims 8 to 11 is deleted.

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