Name: sensitive detection of proteopathic seeding activity with fret flow cytometry
Uploaded: 2015-12-08T13:00:00
Description: Watch this Scientific Journal Video about Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry at JoVE.com

This work was supported by the Tau Consortium (M.I.D); National Institutes of Health Grant 1R01NS071835 (M.I.D.), a Department of Defense Grant PT110816 (to M.I.D.), 1F32NS087805 (to J.L.F.), and 1F31NS079039 (to B.B.H.).

注：このプロトコルは、マウスの生体サンプルから播種活性を検出するためのFRETフローサイトメトリーの使用を強調しています。それはまた、組換え原線維と人間の生物学的サンプルと互換性があります。マウスを安楽死させ、脳を収穫はIACUC承認手順に従って実施しました。 1.脳抽出<ol><li>イソフルラン（2％）との深い麻酔の後、0.03％のヘパリンを含む氷冷PBSでマウスを灌流し、?...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeding+of+normal+tau+by+pathological+tau+conformers+drives+pathogenesis+of+Alzheimer-like+tangles.">Seeding of normal tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles.</a> Journal of Biological Chemistry. , (2011).</li><li>Holmes, B. 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FRETは、ここで説明するフローサイトメトリーシステムを迅速かつ定量的にタウ播種活性を評価するための強力なツールです。これは、中程度の細胞培養の経験とFRETの実用的な知識を必要とし、フローサイトメトリー。このようチオフラビンTのような他の播種アッセイ、 - βシート構造に結合したときに増強された蛍光を示す - 面倒であり、純粋な、組換えタンパク質基質を必要とします。?...

細胞内のタウアミロイドの蓄積がアルツハイマー病などのタウオパチーを定義します。疾患の早期段階で、病理学は、一般的に脳の別々の領域に局在しているが、疾患の進行と、病理学は、常に明確なニューラルネットワーク1-5に沿って広がります。証拠の蓄積は毒性タンパク質凝集体の細胞間伝播が（6-10のレビュー）この病理学の根底を示唆しています。このモデルでは、proteopathic種子（ 例えば 、タウ）は、テンプレートコンホメーション変化を経て11月15日ミスフォールド形式にネイティブタウタンパク質を変換する、ドナー細胞から放出され、隣接するセルを入力されています。ここに記載のアッセイは敏感にそのようなシーディング活性を検出するために開発されました。これは、組換えタンパク質および生物学的サンプルと互換性があり、proteopathic播種アクティビティ16の微小レベルの定量を可能にします。 安定タウ繰り返しdを表現するHEK 293T細胞CFPまたはYFPのいずれかに融合された疾患関連P301Sの突然変異を含むomain（RD）は、播種活性の安定したバイオセンサーとして機能する（以下、タウ-RD-CFP / YFP細胞と呼ばれます）。 proteopathic種子の非存在下では、細胞は、可溶性単量体としてタウを維持し、感知できるバックグラウンドFRETがありません。細胞への自発的な取り込みまたはタウ種のリポソーム媒介形質導入、しかし、フローサイトメトリーを介して、単一の細胞内で測定されるFRETシグナルを生成RD-CFPとRD-YFPの集約、で結果。 このアッセイの多数の構成要素は、感度を高め、ばらつきを低減するように設計されました。 1を有するモノクローナル細胞株：それは最適な信号を提供するよう1 RD-CFP / YFP発現率は、選択された：ノイズを。感度を高めるために、リン脂質は、直接細胞内に種子を導入するために使用された（取り込みの生物学的機構を研究するが、これを省略することができます）。最後に、フローサイトメトリーモニタは集団レベル、単一細胞にFRET<html>レベル、他のタンパク質凝集アッセイとは異なり。最終アウトカム指標、統合されたFRET密度は、高度に定量的であり、両方の集合を有する細胞の数、及び凝集は、各セル内で発生した程度を占めます。これらの最適化されたパラメータのすべては、感度を向上させ、再現性を確保します。 このシステムは、最近、他の一般的に使用されるタウ病理学的マーカー （例えば、MC1、AT8、PG5、およびチオフラビンS）にタウ播種活動の相対的な時間的な発症と進行を評価するトランスジェニックマウスP301Sタウオパチー17に包括的な研究で用いました。シーディング活性は、少なくとも6週間の組織学的検出の前に、これまでで評価しタウ病理の初期の、最も堅牢なマーカーです。種まき活動が発症および/ ​​または神経変性16の進行におけるproteopathic種子の因果的役割を示唆し、年齢とともに徐々に1.5ヶ月と増加に表示されます。 e_content &quot;&gt;生物学的サンプルからのシード材料の微細なレベルの正確な定量化は、初期の疾患の進行をモニターする研究を容易にすることができる試験の期間を短縮し、より若い動物の使用を可能にすることで、これは前臨床動物試験の効率及び精度を高めることができる。例えば、でP301Sマウス先に述べた、鉛化合物は、初期の4〜6週間ほどお届けすることができ、2〜4週間後に有効性について監視。アッセイが正確に播種内の任意の削減を定量化する必要があります（または播種活動の開始時の直前に）アクティビティ。フローサイトメトリーFRETは、同様にインビトロスクリーニングの用途を有する。例えば、抗タウ試薬（ 例えば、抗体 、小分子など ）、組換えタウを使用して、培養物中に直接播種誘導をブロックする能力について迅速に試験することができますシード源（ 図5）。このセットアップでは、シード材料を準備したら、元のような凝集体または脳由来の溶解物perimentは、データ分析など、完了するまでにわずか3日かかります。 proteopathic播種活動の急速な定量化は、このように神経変性の多くの研究を容易にすることができます。

<table>
	<tbody>
		<tr>
			<td>TBS</td>
			<td>Sigma</td>
			<td>T5912</td>
		</tr>
		<tr>
			<td>cOmplete Protease Inhibitors (EDTA-free)</td>
			<td>Roche</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>Cryo-vials</td>
			<td>Sarstedt</td>
			<td>72.694.006</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>XSE 105DU</td>
		</tr>
		<tr>
			<td>Weighing Boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>USA Scientific</td>
			<td>1475-0501</td>
		</tr>
		<tr>
			<td>Omni Sonic Ruptor Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>18-000-115</td>
		</tr>
		<tr>
			<td>Micro-Tip for Ultrasonic Homogenizer</td>
			<td>Omni International</td>
			<td>OR-T-156</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>A451</td>
		</tr>
		<tr>
			<td>Noise Cancelling Ear Muffs</td>
			<td>Fisher Scientific</td>
			<td>19-145-412</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>S47299</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>USA Scientific</td>
			<td><a href="http://www.usascientific.com/Seal-Rite-1.5-ml-tube-sterile.aspx">1615-5510</a></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424 000.215</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
		</tr>
		<tr>
			<td>50 mL Conical Tubes</td>
			<td>Phenix Research</td>
			<td>SS-PH15</td>
		</tr>
		<tr>
			<td>25 mL reagent resevoirs</td>
			<td>VWR</td>
			<td>41428-954</td>
		</tr>
		<tr>
			<td>Multi channel pipet</td>
			<td>Fisher Scientific</td>
			<td>TI13-690-049</td>
		</tr>
		<tr>
			<td>96 well flat bottom plates</td>
			<td>Corning</td>
			<td>3603</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td>Corning</td>
			<td>3788</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>ED2SS</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
		</tr>
		<tr>
			<td>Sorvall ST 40 Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004509</td>
		</tr>
		<tr>
			<td>BIOLiner Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003796</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>VWR</td>
			<td>15170-172</td>
		</tr>
		<tr>
			<td>MACSQuant VYB Flow Cytomter</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-116</td>
		</tr>
		<tr>
			<td>Chill 96 Rack</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-459</td>
		</tr>
		<tr>
			<td>Flow Jo analysis software</td>
			<td>Flow Jo</td>
			<td></td>
		</tr>
		<tr>
			<td>20 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-L10</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-250</td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>Rainin</td>
			<td>GPS-1000</td>
		</tr>
		<tr>
			<td>200 uL pipet tips</td>
			<td>USA Scientific</td>
			<td>1111-1800</td>
		</tr>
		<tr>
			<td>5 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-606180</td>
		</tr>
		<tr>
			<td>10 mL serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-607180</td>
		</tr>
		<tr>
			<td>25 mL Serological pipett</td>
			<td>Phenix Research</td>
			<td>SPG-760180</td>
		</tr>
	</tbody>
</table>

sensitive detection of proteopathic seeding activity with fret flow cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

FRETフローサイトメトリー敏感、定量、および組換えまたは生物学的サンプルからの播種活動の迅速な検出を可能にします。アッセイセットアップが容易である：タウ- RD-CFP / YFPを発現しているモノクローナル由来の安定な細胞株は、24〜48時間インキュベートし、シード材料を用いて形質導入し、分析（ 図1A）、フローサイトメトリーに供しています。種子の不在下では、バイオ...

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Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

FRETフローサイトメトリーでProteopathic播種活動の高感度検出

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

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