JP7097613B2 - 疼痛治療のためのアルファ-チューブリンアセチル化の阻害剤 - Google Patents
疼痛治療のためのアルファ-チューブリンアセチル化の阻害剤 Download PDFInfo
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Description
(a)(i)α-チューブリンアセチルトランスフェラーゼ遺伝子を発現する生物学的細胞および/または(ii)α-チューブリンアセチルトランスフェラーゼタンパク質と、候補化合物を接触させる工程;
(b)α-チューブリンアセチルトランスフェラーゼ酵素活性または発現の少なくとも1つを決定する工程;および
(c)工程(b)で決定された活性および/または発現を、候補化合物の非存在下でのα-チューブリンアセチルトランスフェラーゼの活性または発現と比較する工程、
α-チューブリンアセチルトランスフェラーゼの測定された活性および/または発現の減少は、候補化合物がα-チューブリンアセチルトランスフェラーゼの阻害剤であり、かつ、該候補化合物は、機械的に誘発された神経学的感覚を阻害するために有用であることを示す、方法。
ATAT1遺伝子を欠失させる接触感受性に対する効果を研究目的で、我々は、Avil-Cre::ATAT1fl/+ (コントロール)およびAvil-Cre::Atat1fl/fl (cKO)動物を得るために、ATAT1fl/+ マウス17と末梢神経系特異的Cre系統Avilcre/+マウス18系統を交雑させた。マウスは、先の文献18、19に記載された遺伝子型であった、そしてイタリアの法律(9条、 1992年1月27日、116番)に従って、イタリア保健省のライセンスを受け、イタリアのモンテロトンドのEMBL Mouse Biology Unitで維持された。
皮膚神経製剤は、本質的に先の文献に記載27されているとおり使用した。簡単にいうと、マウスをCO2吸入を用いて犠牲にし、後肢の皮膚と共に伏在神経を自由に解剖し、浴槽(organ bath)に入れた。この容器をNaCl 123 mM; KCl 3.5 mM; MgSO4 0.7mM; NaH2PO4 1.7 mM; CaCl2 2.0 mM;グルコン酸ナトリウム 9.5 mM; グルコース 5.5 mM; スクロース 7.5 mM; およびHEPES(pH of7.4) 10mMからなる合成間質液(SIF緩衝液)で灌流した。皮膚を真皮側を上にし、線維を裂いたり(fiber teasing)単一ユニット記録するために隣接するチャンバーに神経を配置した。ガラスロッドを用いて機械的探索刺激を与えて単一ユニットを単離し、伝導速度、フォンフレイヘア(von Frey hair)閾値、および閾値上の刺激に対する適応特性によって分類した。コンピューター制御のナノモーター(Kleindiek Nanotechnik)を使用して、既知の振幅および速度の機械的なランプおよびホールド刺激を適用した。2秒または10秒間の標準化された変位刺激を一定の間隔(間隙時間、30秒)で受容野に適用した。プローブは、0.8mmの平坦な円形接触面積を有するステンレス鋼の金属棒であった。線形モーターの動きを駆動する信号と未処理の電気生理学的データを、Powerlab 4.0システムおよびLabchart 7.1ソフトウェア(AD instrument)で収集し、スパイクをソフトウェアのスパイクヒストグラム拡張でオフラインで識別した。
先の文献27に記載のとおり、DRG神経をマウスから採取し、解離させた。それらは、場合によっては、プレインストールされたプログラムSCN Basic Neuro program 6を使用して、室温でSCN nucleofector kit(Lonza AG)のマウス神経 Nucleofector溶液(20μl)と合計4~5μgのプラスミドDNAでNucleofectorシステム(Lonza AG)を用いてトランスフェクションされた。エレクトロポレーション後、37℃で10分間細胞懸濁液を500μlのRPMI 1640培地(Gibco)に移した。10%ウマ血清を補充したこの懸濁液を使用して、この細胞をガラスカバースリップ上にプレートして記録した。100ng/mlの神経成長因子(NGF)、50ng/mlのBDNFを補充したRPMI培地を、標準DRG培地で3~4時間後に置き換えた。電気生理学実験は、プレーティング後12時間に開始した。
DRG培養における微小管染色のために、細胞をPBSで1回洗浄し、次いで3%パラホルムアルデヒド、0.25%トリトンおよび0.2%グルタルアルデヒドを含む細胞骨格緩衝液(CB)pH6.3中で室温で15分間固定した。細胞をPBST(0.3%トリトン)で3回洗浄した。続いて、試料を室温で1時間、PBS中の5%正常ヤギ血清(NGS)でブロッキングした。それから、細胞を一次抗α-チューブリン(1:1000)(Sigma-Aldrich、T9026)または抗アセチル化-α-チューブリン(1:1000)(Sigma-Aldrich、T7451)を用いてPBS中で4℃で一晩静置した。それから、細胞をPBSで洗浄し、室温で1時間、蛍光標識二次抗体(1:1000)(Alexa Fluor 546 Lifetechnologies)と共に1時間インキュベートした。すべての画像は、ライカSP5共焦点顕微鏡上の40倍対物レンズを使用して取得した。画像の処理および表面プロットの生成はImageJを使用して実行された。画像はHuygensソフトウェアを用いてデコンボリューションした(deconvoluted)。
伏在神経を解剖し、4℃、0.1Mリン酸緩衝液中の新鮮な4%(w/v)PFA、2.5%(w/v)グルタルアルデヒド(TAAB)で24時間後固定した。後固定につづいて試料を1.5%(w/v)のフェロシアン化カリウムを補充した1%(w/v)OsO4と共に2時間インキュベートし、試料をエタノール中で脱水し、プロピレンオキシド/エポン(Epon、寒天)(1:1)で浸潤させた後、樹脂を包埋した。超薄切片を切断し(Ultracut S、Leica)、酢酸ウラニルおよびクエン酸鉛で対比染色し、MSC 791 CCDカメラ(Gatan)を備えた透過型電子顕微鏡(TEM)Jeol 1010で観察した。
DRG神経を10%FBS DMEM中の1:1 Matrigelで懸濁し、2つのチャンバーのマイクロ流体チップ(Xona Microfluidics, SD150)上に播種した。軸索は、2つのチャンバーを3~5日間接続しているマイクロチャンネルを横切って増殖することができた。実験の日に、細胞体および軸索チャンバーの両方の培地を、血清を含まない培地と3時間交換した。真核細胞から家で精製した1μMのモノビオチン化NGFを、1μMのストレプトアビジン結合量子ドット655(Life Technologies)と氷上で30分間結合させ、次いで画像化緩衝液(上記と同じ)で5nMに希釈し、 軸索チャンバー内の培地を置き換えるために使用した。軸索から細胞体チャンバーへの逆流を回避するために、細胞体と軸索チャンバーとの間に25%の体積差が維持された。5%CO2、37℃で1時間インキュベートした後、エンドソームを含むNGF-Qdot655の逆行輸送を、37℃、5%CO2加湿チャンバーを備えた共焦点Ultraview Vox(Perkin Elmer)を用いて画像化した。100秒の時間経過は300msの露光時間で記録した。 粒子追跡機能と自己回帰の動作追跡生成設定を使用してImarisソフトウェアで画像を解析した。
細胞を3mlの温かいPBSで1回洗浄した。続いて、細胞を固定し、0.3%グルタルアルデヒドおよび0.25%Triton X-100を含有する細胞骨格緩衝液中で2分間透過処理を行った。これに続いて、細胞を2%グルタルアルデヒドを含む細胞骨格緩衝液中で10分間固定し、PBS中の0.1%水素化ホウ素ナトリウム(NaBH 4)2mlで7分間処理した。それから、細胞をPBS中で10分間3回洗浄した。細胞をPBS + 2%BSA中で30分間一次抗体(マウス抗α-チューブリン、Neomarker、1:500)と共にインキュベートした。 PBSで10分間3回洗浄した後、細胞を室温で30分間、二次抗体(ヤギ抗マウスAlexa 647、1:500、Molecular Probes A21236)に移した。それから、細胞をPBSで10分間3回洗浄し、次にPALM画像化のためにマウントした。画像化の時、細胞をPALM点滅緩衝液(50mM Tris pH8.0、10mM NaCl、10%グルコース、100U/ml グルコースオキシダーゼ(Sigma-Aldrich)、40ug/ml カタラーゼ(Sigma-Aldrich))で覆った。
力分光法測定は、生細胞分析のための流体チャンバ(Biocell; JPK)および試料観察のための倒立光学顕微鏡(Axiovert 200; Zeiss)を備えたNanoWizard AFM(JPK Instruments、Berlin、Germany)を用いて行った。
培養したDRGを500nM C8 SIR-チューブリンを37℃、1時間でおよび/または37℃で30分間DRG 中の2μMカルセイン色素(Invitrogen C3100MP)で負荷した。それから、細胞を320mOsmで画像化緩衝液(10mM Hepes pH7.4、140mM NaCl、4mM KCl、2mM CaCl2、1mM MgCl2、5mM D-グルコース)に移した。5分間の順化期間の後、細胞を440mOsm(マンニトールで調整したモル浸透圧濃度)高浸透圧ショックに3分間供した。全ての画像処理は、ライカSP5共鳴スキャナを用いて行った。
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20 Garrison,S. R., Dietrich, A. & Stucky, C. L. TRPC1 contributes to light-touch sensationand mechanical responses in low-threshold cutaneous sensory neurons. JNeurophysiol 107, 913-922, doi:10.1152/jn.00658.2011 (2012).
21 Rugiero,F., Drew, L. J. & Wood, J. N. Kinetic properties of mechanically activatedcurrents in spinal sensory neurons. J Physiol 588, 301-314,doi:10.1113/jphysiol.2009.182360 (2010).
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23 Hu,J. & Lewin, G. R. Mechanosensitive currents in the neurites of culturedmouse sensory neurones. The Journal of physiology 577, 815-828, doi:10.1113/jphysiol.2006.117648(2006).
24 Kalebic,N. et al. The Tubulin Acetyltransferase alphaTAT1 Destabilizes MicrotubulesIndependently of its Acetylation Activity. Mol Cell Biol,doi:10.1128/MCB.01044-12 (2012).
25 Castro-Castro,A., Janke, C., Montagnac, G., Paul-Gilloteaux, P. & Chavrier, P.ATAT1/MEC-17 acetyltransferase and HDAC6 deacetylase control a balance ofacetylation of alpha-tubulin and cortactin and regulate MT1-MMP trafficking andbreast tumor cell invasion. Eur J Cell Biol 91, 950-960,doi:10.1016/j.ejcb.2012.07.001 (2012).
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Tape Test:テープ試験
Cotton Swab Test:綿棒試験
Number of responses:応答数
Von Frey test:フォンフレイ試験
Tail clip test:尾部クリップ試験
%response:応答%
Time (s) :時間(秒)
Hotplate assay:ホットプレートアッセイ
Rotorod:ロータロッド
Speed(RPM) :速度(RPM)
Ramp phase:ランプ段階
Spikes per s:1秒あたりのスパイク数
Displacement(μm) :変位(μm)
D-Hair:Dヘア
C fibres:C線維
% of total:全体の%
Percent size:パーセントサイズ
Before:前
After:後
Pixel intensity / area:ピクセル強度/面積
Claims (4)
- α-チューブリンアセチルトランスフェラーゼ1の発現を減じることが可能な核酸の有効量を含む、感覚神経によって媒介される機械的疼痛処置用組成物であって、ここで該核酸または該核酸の転写物がα-チューブリンアセチルトランスフェラーゼ1をコードするヌクレオチド配列に結合することができる、組成物。
- 核酸が、siRNAをコードする請求項1に記載の組成物。
- 機械的疼痛が、減少する請求項1に記載の組成物。
- 請求項1~3のいずれか1項に記載の組成物、および薬学的に許容される担体および/または賦形剤を含む、感覚神経によって媒介される機械的疼痛の予防または治療に使用するための医薬組成物。
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