著者はオルガ・シャルマ、テレサ・ハント、および技術サポートのためのDZNE顕微鏡および動物施設のスタッフに感謝します。

動物を含めたすべての手順は、ノルトライン=ヴェストファーレン州国家環境庁（LANUV）の動物保護委員会の承認を得て行きました。動物を飼育し、12時間の明/暗サイクル、食物および水に自由にアクセスできる標準条件に従って世話しました。 1.動物モデル<ol><li>マウス15、16：ヘミ接合バイジェニックTgは（GFAP -Luc +/- M83 +/-）を生成するためにヘミ接合のTg（M83 +/-）マウスとの交雑ヘミ接合性Tgは（GFAP -Luc +/-）マウス。 </li><li>トランスジーンエンコードヒトα-シヌクレインの存在およびホタルルシフェラーゼ12のための標準的なPCR、17でリアルタイムPCRを用いて子孫を遺伝子型。 </li><li> 6〜8週齢で動物を接種します。 </li></ol> 2.接種材料の調製<ol><li> MOを準備20mMのTris-HCl中150mMのNaClを含むトリス緩衝生理食塩水（TBS）緩衝液（pH 7.2）中にA53T変異を有するnomeric組換えヒトα-シヌクレイン以前14に記載されているように。 </li><li> 5日間800rpmで37℃でオービタルシェーカー中のモノマーの組換えヒトα-シヌクレインの3μg​​の/μLを攪拌することにより接種するための繊維状α-シヌクレインを調製します。 </li><li>腹腔内またはintraglossal接種のための2μg/μLのために1μg/μLの最終濃度に達するようにリン酸緩衝生理食塩水（PBS）中にフィブリルアセンブリを希釈します。 </li><li>氷上で1分（0.5秒毎のパルス間1秒のポーズと持続時間と50％に設定された振幅の40個のパルス）用ロッドソニケーターでα-シヌクレイン線維を断片。クロスシーディングによる汚染を避けるために。別の接種物を準備するためにクリーンな超音波処理プローブを使用してください。 </li></ol> 3. Intraglossal注射<ol><li> intrapeで動物を麻酔100mg / kgのケタミンおよび10mg / kgのキシラジンのritoneal注射。動物のつま先をつまんで、それが適切な麻酔を確保するために、その後肢を撤回しないことを確認してください。麻酔下ながら乾燥を防ぐために、動物の目に獣医軟膏を使用してください。 </li><li>慎重に調査員の方を向いて頭と背臥位での加熱プレート上に粘着テープで麻酔し、動物を修正。 </li><li>超音波処理α-シヌクレイン原線維またはPBSの5μLと27 G使い捨て皮下注射器を充填します。 </li><li>動物の口が開いて保持するための鋸歯状の先端を有する1鈍い鼻の親指の鉗子を使用し、鉗子の第二の小さいペアは慎重に注入するための舌の下側にアクセスできるように舌を引き出します。 </li><li>舌下神経に近接した舌の右または左の下側に注射器の針を注入します。ゆっくりと5秒後に接種材料を注入。ゆっくり接種物ことを保証するために、5秒後に針を引っ込めます組織を貫通した針を後退しながら、失われることはありません。 </li><li>その執着から動物を放し、完全に回復するまでに一定の監視の下で加熱プレート上でそれを残します。 </li></ol> 4.腹腔内注射<ol><li>腹腔内注射のために、2リットル/分の流量及び2％に設定気化器を使用して、イソフルラン/酸素で吸入により麻酔チャンバー内ですぐに動物をnarcotize。動物のつま先をつまんで、それが適切な麻酔を確保するために、その後肢を撤回しないことを確認してください。 </li><li>超音波処理α-シヌクレイン原線維またはPBS50μlの27 G使い捨て皮下注射器を充填します。 </li><li>直接マウスの腹膜内に注入し、研究者から離れて面するヘッドと背面位置に動物を保持し、下方に約45°腹壁の背後に位置小腸もしくは盲腸を貫通避けます。彼らが回復するまで動物を監視麻酔から。 </li></ol> 5.生物発光イメージング<ol><li>画像のTg（M83 +/-：GFAP -luc +/-）マウス生物発光イメージングシステムを用いて、2〜4週間ごと。 </li><li>まもなく撮像2L / minの流量及び2％に設定気化器とイソフルラン/酸素チャンバ内でマウスを麻酔前。動物のつま先をつまんで、それが適切な麻酔を確保するために、その後肢を撤回しないことを確認してください。 </li><li>脱毛クリームでマウスの頭を剃るとdepilate。非特異的生物発光をブロックする非刺激性マーカーと黒に耳を着色。 </li><li> 30mg / mLのストック溶液をPBS中のD-ルシフェリンカリウム塩、ルシフェラーゼの基質を希釈します。 -20°Cで1 mLのアリコートでこれを保管してください。溶解したD-ルシフェリンは、光への暴露から保護します。 </li><li>注射前に、動物を計量。 150 mg / kg体重の注入のためのD-ルシフェリンの正確な量を計算します。腹腔Dを注入-luciferinと麻酔室に動物を返します。 </li><li>イメージングソフトウェアを起動します。撮像システムは、その動作温度に達した後、コントロールパネルの「初期化」ボタンをクリックすることによって、システムを初期化します。 </li><li>ボタンの発光」と「写真」を選択します。 、60秒に露光時間を設定して「中」にビニング、F /ストップ「1」に、そして「オフ」にEMゲイン。 </li><li>励起フィルタを「開く」に「ブロック」と発光フィルタに設定されていることを確認し、1.50 cmの被写体の高さを設定します。 </li><li> D-ルシフェリンの注射後10分で、撮像チャンバ内の加熱プレート上に動物を配置し、それらのグリグリが正しく麻酔出口に配置されていることを確認。吸入室からイソフルラン/酸素の流れを閉じ、0.25リットル/分の流量及び2％の蒸発と撮像チャンバのためのフローを開きます。撮像チャンバPRのドアを閉めますoperly。 </li><li> 60秒を要する生物発光を測定するために「獲得」ボタンをクリックしてください。イソフルランの流れを停止し、彼らは完全に自分のケージに戻ってそれらを戻した後回復するまで動物を監視します。 </li><li>生物発光を定量化するためにイメージングソフトウェアを使用してください。 「ツールパレット」の中で「ROIツール」を選択します。 「ROIのツール」内の「タイプ」の下に「測定ROI」を選択します。 </li><li> 「ROIのツール」内の「円」ツールをクリックし、プルダウンメニューから描画するROIの数を選択する - 理想的に「3」を3匹のマウスのために。 </li><li>画像では、各ROIを右クリックして「プロパティ」は1.25センチメートルに幅と高さを調整する下。定量化された脳の領域の上に、各ROIを置きます。 </li><li>画像の左上に、「放射（光子）」に単位パネルを設定します。 </li><li> 「ツールパレット」の中で、選択し「画像調整」と最小値を調整し、0.20e6から1.00e6に例えば「カラースケール」の最大値。 </li><li> 「ファイル」の下に「保存」を使用してデータを保存します。 </li></ol> 6.生化学分析<ol><li>確立されたプロトコル18、19による脳と脊髄を分離します。 </li><li>組織ホモジナイザーで6,000rpmで有する2回の30秒サイクルで（PBS中で1倍に希釈）、プロテアーゼおよびホスファターゼ阻害剤の存在下でPBSに別々に全脳および全脊髄サンプルを均質化します。脳および脊髄サンプルを20％（重量/体積）の最終濃度に均質化されていることを確認します。 </li><li> 10秒の一定のパルスと50％に設定された振幅と氷上でそれらを維持しながら二回サンプルを超音波処理。 </li><li> PBS中の1.5 M NaClで1：それらの1に希釈することによりPBSおよび750mMのNaCl中10％ホモジネートを調整します。 </li><li>細胞破片を分離するために、5分で1000×gでホモジネートを遠心4°C。次のステップのために上清を保持し、ペレットを捨てます。 </li><li>製造業者の指示に従ってビシンコニン酸（BCA）アッセイを用いてホモジネート中のタンパク質濃度を測定します。氷上で15分間、10％（重量/体積）の最終濃度でN-laurylsarcosyl脊髄ホモジネートからの脳ホモジネートからの総タンパク質の1.0ミリグラムまたは0.8ミリグラムをインキュベートします。 </li><li>蒸留水中の10％（wt / vol）の3mLのスクロースクッション上にホモジネートをオーバーレイし、4℃で1時間、465,000×gでそれらを超遠心。 </li><li> 4％ドデシル硫酸ナトリウム（SDS）、2％βメルカプトエタノール、192 mMグリシン、25 mMトリス、5％（重量/容量）のスクロースを含有する緩衝液中にペレットを再懸濁します。 </li><li> 5分間、100℃でサンプルを沸騰およびモルホリンエタンスルホン酸（MES）緩衝液系中での電気泳動のためにSDSポリアクリルアミドゲル上にロードします。 </li><li> SEMにおけるポリビニリデンジフルオリド（PVDF）膜に分離されたタンパク質を転送しますidryブロッティングシステム。 </li><li>膜と架橋タンパク質に35回転ロータに室温で30分間PBS中の0.4％（vol / vol）のホルマリン溶液中で膜をインキュベートします。 </li><li>室温で1時間、0.05％（体積/体積）のTween 20および5％（wt / vol）のミルクを含むTBS緩衝液中で膜をブロックします。 </li><li> 4℃で一晩ブロッキング緩衝液中で一次抗体と共にブロットをインキュベートします。 </li><li> TBS緩衝液中で膜を3回洗浄し、室温で1時間、TBS中のペルオキシダーゼまたは蛍光団コンジュゲート二次抗体でそれらをインキュベートします。 </li><li>確立されたプロトコール20に従って化学発光又は蛍光によって結合した二次抗体を可視化します。 </li></ol> 7.免疫蛍光分析<ol><li>組織処理ステーションでの解剖脳と脊髄を脱水し、パラフィンステーションを使用してパラフィンにそれらを埋め込みます。 </li><li>パラフィン包埋TISをカット厚さ8μmの冠状切片でミクロトームで提訴し、スライドガラス上のセクションをマウントします。 </li><li> 5〜10分間、二つの別々のキシロール浴でそれらをインキュベートすることにより組織切片を脱パラフィン化し、段階的エタノール浴のシリーズを介してそれらを再水和（100％、90％、70％、50％）、そして最後にH 2 Oで</li><li>室温で5分間、0.01 Mクエン酸緩衝液（pH6.0）で切片をインキュベートし、さらに電子レンジで10分間、それらを沸騰。 </li><li>セクションでは、室温まで冷却し、内因性ペルオキシダーゼを阻害する30分間3％過酸化水素溶液でそれらをインキュベートしてみましょう。 </li><li>室温で1時間PBS中の20％（vol / vol）の正常ヤギ血清、1％（vol / vol）のウシ血清アルブミン（BSA）とのインキュベーションによって、組織切片をブロックし、そして0.5％のTriton X-100。 </li><li> 4℃で一晩、1％に希釈した一次抗体（vol / vol）の正常ヤギ血清でセクションをインキュベートし、1％（体積/体積）BSA、PBS中の0.25％トリトンX-100。 </li> &lt;LI&gt; PBSで一度0.25％（vol / vol）のPBS中のTriton X-100で二回セクションを洗浄し。 </li><li>室温で1時間、対応するフルオロフォア結合二次抗体と1％に希釈した核色素DAPI（vol / vol）の正常ヤギ血清、1％（体積/体積）BSA、PBSで切片を染色します。 </li><li> 0.25％（容量/容量）のTriton X-100 PBSで一度PBSで2回洗浄した後、包埋媒体とスライドをカバースリップおよび共焦点レーザー走査顕微鏡で染色を可視化します。 </li></ol>

<ol>
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F., Gravel, M., Kriz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+amyotrophic+lateral+sclerosis+pathogenesis:+disease+onset+is+characterized+by+marked+induction+of+GFAP+in+Schwann+cells.">Live imaging of amyotrophic lateral sclerosis pathogenesis: disease onset is characterized by marked induction of GFAP in Schwann cells.</a> Glia. 57 (10), 1130-1142 (2009).</li><li>St&#246;hr, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+synthetic+Abeta+prion+strains+producing+different+amyloid+deposits+in+bigenic+mice.">Distinct synthetic Abeta prion strains producing different amyloid deposits in bigenic mice.</a> Proc Natl Acad Sci U S A. , (2014).</li><li>Luk, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+inoculation+of+pathological+alpha-synuclein+initiates+a+rapidly+progressive+neurodegenerative+alpha-synucleinopathy+in+mice.">Intracerebral inoculation of pathological alpha-synuclein initiates a rapidly progressive neurodegenerative alpha-synucleinopathy in mice.</a> J Exp Med. 209 (5), 975-986 (2012).</li><li>Recasens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lewy+body+extracts+from+Parkinson+disease+brains+trigger+alpha-synuclein+pathology+and+neurodegeneration+in+mice+and+monkeys.">Lewy body extracts from Parkinson disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys.</a> Ann Neurol. 75 (3), 351-362 (2014).</li><li>Sacino, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+CNS+alpha-synuclein+pathology+by+fibrillar+and+non-amyloidogenic+recombinant+alpha-synuclein.">Induction of CNS alpha-synuclein pathology by fibrillar and non-amyloidogenic recombinant alpha-synuclein.</a> Acta Neuropathol Commun. 1 (1), 38 (2013).</li><li>Peelaerts, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Synuclein+strains+cause+distinct+synucleinopathies+after+local+and+systemic+administration.">alpha-Synuclein strains cause distinct synucleinopathies after local and systemic administration.</a> Nature. 522 (7556), 340-344 (2015).</li></ol>

著者は、開示することは何もありません。

Tgは（M83 +/-：GFAP -Luc +/-）の腹膜内へのα-シヌクレイン線維の末梢注射したマウスは、シヌクレイノパチーの重要な特性を再現するために、神経炎症を伴う神経疾患を誘発するための容易な方法を表します。同様に、舌の注射は、トランスジェニックマウスにおけるα-シヌクレインprionoidsによって神経侵入のための別のルートを表すが、あまり効率的です。私たちは、?...

α-シヌクレインは、特に自己シードする能力で、プリオンタンパク質のものと類似しており、細胞間および神経経路に沿ったミスフォールディングを伝搬特性を有することが成長の証拠があります。 α-シヌクレインのこの特性はまた、「プリオン様」または「prionoid」と呼ばれ、罹患ニューロンから新たに移植され、健康なニューロン1にミスフォールドα-シヌクレインの透過性を示唆している移植実験において観察によって支持され、2 、3、4。また、脳または周囲、 例えば後肢筋肉または腸壁の遠位部分にα-シヌクレイン病理の広がりにおける結果に誤って折り畳まれたα-シヌクレインの直接注入CNS 5、6、7、&lt;SUPクラス= &quot;外部参照&quot;&gt; 8、9、10。我々は、より詳細には、末梢経路を介してα-シヌクレインprionoidsの伝送を分析し、ミスフォールドα-シヌクレインは、単一intraglossalまたは腹腔内注射後にCNSをneuroinvadeできるかどうかを疑問に対処し、以前にプリオンのためではなく、ミスフォールドアルファのために示された機能シヌクレイン。舌へのプリオンの注入後の、CNSの神経侵入は、脳幹11に位置する舌下神経の核につながる舌の舌下神経に沿って伝播を介して達成されます。プリオンプロモーターからのヒトα-シヌクレインのA53T変異体を過剰発現マウス、及びホタルによってアストロサイトの活性化をモニターするために、GFAPプロモーターの制御下でルシフェラーゼ：マウスモデルとして、我々はTgが（GFAP -Luc +/- M83 +/-）を選択しました生物発光、以前の脳に示すように、プリオン感染マウス12。我々の手ではバイジェニックTgは（M83 +/-：GFAP -luc +/-）他13で示されているようマウスは生後23ヶ月まで、疾患を発症しませんでした。 α-シヌクレインprionoidsという仮説を支持するマウス：脳及びTgは（GFAP -Luc +/- M83 +/-）の脊髄での病理と神経疾患を誘発しintraglossalまたは腹腔内経路を介してヒトα-シヌクレイン線維の単回注射プリオン14との重要な特徴を共有します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-actin antibody</td>
			<td>Merck Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [pSyn#64]</td>
			<td>Wako</td>
			<td>015-25191</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha-synuclein, phospho S129 antibody [EP1536Y]</td>
			<td>Abcam</td>
			<td>ab51253</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFAP antibody</td>
			<td>Dako</td>
			<td>Z0334 01</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-IBA-1 antibody</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sequestosome-1 (p62) antibody</td>
			<td>Proteintech</td>
			<td>18420-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ubiquitin antibody [Ubi-1]</td>
			<td>Merck Millipore</td>
			<td>MAB1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>100 mg/kg</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Ratiopharm</td>
			<td></td>
			<td>10 mg/kg</td>
		</tr>
		<tr>
			<td>27-gauge syringe</td>
			<td>VWR</td>
			<td>613-4900</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal Healthcare</td>
			<td>PZN&#160; 4831850</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secureline lab marker&#160;</td>
			<td>Neolab</td>
			<td>25040</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin potassium salt</td>
			<td>Acris</td>
			<td>LK10000</td>
			<td>30 mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5776671</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonopuls Mini20 sonicator</td>
			<td>Bandelin</td>
			<td>3648</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II imaging system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image 3.0 Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(M83+/-) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>004479</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>N-laurylasarcosyl</td>
			<td>Sigma</td>
			<td>L5125-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>TLA-110 rotor&#160;</td>
		</tr>
		<tr>
			<td>Thickwall polycarbonate tubes</td>
			<td>Beckman Coulter</td>
			<td>362305</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAG Novex 4-12% Bis-Tris Midi Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>WG1401BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP conjugated antibody</td>
			<td>Cayman</td>
			<td>Cay10004301-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR Dye 680 conjugated antibody&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Dura Extended Duration Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34075</td>
			<td></td>
		</tr>
		<tr>
			<td>Stella 3200 imaging system</td>
			<td>Raytest</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey infrared imaging system&#160;</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td>MP Biomedicals</td>
			<td>TWEEN201</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SA/T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF membrane</td>
			<td>Merck Millipore</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol</td>
			<td>Sigma</td>
			<td>Roth</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma</td>
			<td>SA/00216763/000500</td>
			<td>working solution 3%</td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A3294-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td>working dilution 1:50,000</td>
		</tr>
		<tr>
			<td>Fluoromount media&#160;</td>
			<td>Omnilab</td>
			<td>SA/F4680/000025</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HALT protease and phosphatase inhibitors</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>10516495</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys 24-Dual homogenizer&#160;</td>
			<td>Peqlab</td>
			<td>91-PCS24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31619</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 conjugated antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#160;BCA&#160;Protein&#160;Assay&#160;Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>10741395</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome RM2255</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

bioluminescence imaging of neuroinflammation in transgenic mice after peripheral inoculation of alpha-synuclein fibrils

ミスフォールドα-シヌクレインのプリオンのような挙動を研究するために、マウスモデルは、中枢神経系（CNS）内の神経病理を引き起こすα-シヌクレインprionoids、迅速かつ簡単な伝送を可能にする必要があります。プリオンタンパク質プロモーターからA53T変異を有するヒトα-シヌクレインを過剰発現し、ホタル由来のルシフェラーゼマウス、：ここでは、バイジェニックのTg（GFAP -Luc +/- M83 +/-）にα-シヌクレイン原線維のintraglossalまたは腹腔内注射を記述しますグリア線維性酸性タンパク質（GFAP）のプロモーターは、神経病理学的疾患を誘導するのに十分です。 8ヶ月の年齢で始まる深刻な神経症状を発症ホモ接合体のTg（M83 + / +）マウスと比較して、ヘテロ接合のTg（M83 +/-：GFAP -luc +/-）、彼らが到達するまでの動物は、自発的な病気の自由なまま22ヶ月の年齢。興味深いことに、intraperitone介しα-シヌクレイン線維の注射229±17日の中央値はインキュベーション時間をマウス：アルルートは4 5のTg（GFAP -luc +/- M83 +/-）で麻痺と神経疾患を誘発しました。病気にかかった動物は自分の脳や脊髄にリン酸化αシヌクレインの深刻な沈着を示しました。 α-シヌクレインの蓄積は、サルコシル不溶性であり、ユビキチンおよびP62と共局在し、そして星状細胞グリオーシスおよび小膠細胞を生じる炎症応答を伴いました。驚くべきことに、舌へのα-シヌクレイン線維の接種285日後にα-シヌクレイン病理を示す5注射した動物の一方のみで疾患を引き起こすにはあまり効果的でした。マウス。：私達の調査結果は、intraglossal経路を介して接種し、より多くのように腹腔内経路では、Tgのシヌクレイノパチーの関連する顕著な特徴（GFAP -luc +/- M83 +/-）で神経学的な病気を誘発するのに適していることを示していますこれは、誘導プリオンなどの病因を研究するための新しいモデルを提供しますより詳細にα-シヌクレインprionoidsによってDは

マウス（ 表1及び図1）：舌又はバイジェニックTgはCNS（GFAP -Luc +/- M83 +/-）で腹膜誘導される神経病理を介してα-シヌクレインprionoidsの周辺注射。 α-シヌクレイン線維を持つ単一の腹腔内注射した後、5匹のマウスの4つが229±17日の中央値はインキュベーション時間と神経疾患を発症しました。驚くべきことに、唯一の5匹?...

Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

Tgは（M83 +/-：GFAP -Luc +/-）の腹膜または舌にα-シヌクレイン原線維の末梢注入家族A53T変異ホタルルシフェラーゼでヒトα-シヌクレインを発現するマウスは、神経炎症を含む、神経病理を誘導することができます、その中枢神経系インチ

α-シヌクレイン線維の末梢接種後のトランスジェニックマウスにおける神経炎症の生物発光イメージング

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

bioluminescence-imaging-neuroinflammation-transgenic-mice-after

Research

JoVE Journal

Neuroscience

8.5K ビュー.  German Center for Neurodegenerative Diseases (DZNE).  Tgは（M83 +/-：GFAP -Luc +/-）の腹膜または舌にα-シヌクレイン原線維の末梢注入家族A53T変異ホタルルシフェラーゼでヒトα-シヌクレインを発現するマウスは、神経炎症を含む、神経病理を誘導することができます、その中枢神経系インチ

ビデオ: Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

慢性二光子用薄型頭蓋骨のウィンドウテクニック in vivoで</em神経炎症のモデルのマウスミクログリアの>イメージング

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

神経科学

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

腸神経系ではα-シヌクレインの介在物の強制経口および画像解析を用いたロテノンの経口投与

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

根切断術後の後根の軸索のライブイメージング

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

局所トランスジェニックラットの発生により<em&gt;子宮内</em&gt;エレクトロポレーションおよび<em&gt;インビボ</em&gt;生物発光スクリーニング

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

末梢神経損傷後の機能回復を評価する嗅神経鞘細胞の移植

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

細胞内のCaの蛍光と生物発光イメージング<sup&gt; 2+</sup&gt;高齢者における海馬ニューロン

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

パーキンソン脳からの可溶性および不溶性のα-シヌクレインの逐次抽出

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニック マウス

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

線維形成の強力な細胞モデルとして α-シヌクレインのミクロソーム関連付けられた集計一次ニューロンの外因性投与

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

自由浮遊免疫蛍光アッセイによる皮下末梢神経線維におけるアルファシヌクレイン凝集体の標的化

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...