本研究は、山東省卓越した科学者 (JQ201421) と NSFC (81371226) からの助成金の助成金からの助成金によって支えられました。

1. ミトコンドリアの分離
注: すべてのソリューションや機器 0 - 4 ° C に冷却、氷で保たれます。
<ol><li>10 cm 細胞培養ディッシュあたり種子約 3 x 10 の6 SH SY5Y 細胞。高グルコース ダルベッコの培養細胞の 10% 牛胎児血清ペニシリン (100 U/mL) とイーグル培地、変更された-ストレプトマイシン (100 μ g/mL)。一晩 5% CO2を含むインキュベーターで 37 ° C で維持します。 注: 少なくとも 1-2 107 SH SY5Y セル x は各分離アッセイに必要な。</li>
	<li>メディアを取り出して、3 回約 1 ml の 10 cm 細胞培養皿で氷冷 PBS のセルを洗浄します。</li>
	<li>10 cm 細胞培養皿に氷冷 PBS の新しい 1 mL を追加し、新しい 1.5 mL チューブに付着性のセルをこすり。800 x g で 4 ° C で 10 分間遠心</li>
	<li>、遠心分離中にプロテアーゼ阻害剤在庫の 50 μ L を添加した 5 mL ミトコンドリア分離バッファー (250 mM ショ糖、10 mM HEPES (4-(2-hydroxyethyl) 1 ピペラジン ethanesulfonic 酸), 10 mM KCl, 1.5 mM MgCl2、1 mM EDTA) の準備します。(100 倍の濃度; ddH2O の 500 μ L に溶解し 1 EDTA 無料タブレット) のソリューションです。</li>
	<li>上澄みを除去し、ミトコンドリア分離バッファーの 1 mL にペレットを再懸濁します。30 分間氷の上 1.5 mL チューブを孵化させなさい。</li>
	<li>上下に手動で氷の上ガラス ホモジナイザー (15-25 ストローク) と浮遊細胞を溶解します。 注: 細胞をホモジナイズ時に気泡がないことを確認します。視覚的に顕微鏡でそのままなセルとむく核をカウントし、ことを確認 > 90% 細胞破砕が発生しました。</li>
	<li>1.5 mL チューブと (核と残りのままなセル) の残骸を削除する 4 ° C で 5 分間 1,000 × g で遠心分離機に磨砕液を転送します。</li>
	<li>新しい 1.5 mL チューブに 1,000 x g で 5 分間遠心上清を転送 4 ° C</li>
	<li>上清を新しいチューブと 14,000 × g で 15 分間遠心するに転送 4 ° C</li>
	<li>上澄みを除去し、ミトコンドリア ミトコンドリア分離バッファーの 1 つの mL を含むペレットを再懸濁します。</li>
	<li>14,000 x g 4 ° C で 15 分のためのソリューションを遠心分離機、ミトコンドリアを沈殿させます。</li>
	<li>結果ミトコンドリア ペレットを氷で冷やして、50-100 お尻の前にペレットの量に応じて KCl メディアを μ L (125 mM KCl、2 ミリメートル K2HPO4、1 mM MgCl2、20 mM HEPES、5 mM グルタミン酸 5 mM マラテと 2 μ M ロテノン、pH 7.4) で再停止される必要があります。そう。</li>
	<li>標準 BCA アッセイ、OD562 を測定によってミトコンドリア蛋白質の集中を測定するミトコンドリア ソリューションの 5 μ L を使用してプレート リーダー13を使用して。</li>
</ol>2. ミトコンドリアの Ca2 +の保持容量の決定
<ol><li>KCl メディアを準備し、Ca2 +を追加-実験前に 0.5 μ M の最終的な集中に緑色の蛍光色素を結合します。</li>
	<li>0.5 μ M の Ca2 +を含む KCl メディア分離ミトコンドリア (0.4 mg/mL) の 1 mL を転送-よく各 6 ウェル プレートに緑色の蛍光色素を結合します。</li>
	<li>ミトコンドリアと Ca2 +を孵化させなさい-室温 20 mm CaCl2ソリューション (20 mM CaCl2, 127 mM KCl、1 mM MgCl2 1 分追加 4 μ 因数の周囲の光から保護 6 ウェル プレートで緑の蛍光色素を結合、20 mM HEPES、グルタミン酸 5 mM、5 mM のリンゴ酸、pH 7.4) 各 6 ウェル プレートに自動を使用してよく分配設定 200 nmol Ca2 +/mg ミトコンドリア蛋白質を紹介します。</li>
	<li>蛍光分析装置を使用するには、すべて 3 蛍光の変化を監視して s 励起波長 506 の 2 分を nm、発光波長 531 nm。プレートは 3 の 600 rpm で揺れ装備ソフトウェア (図 1) によって制御の測定値間の s。</li>
	<li>各ウェルに 20 mM CaCl2ソリューションの追加 4 μ 因数を追加し、すべての 3 の蛍光の変化を監視 2 分の s は、蛍光性は時間とともに増加するまでこの手順を繰り返します。 注: ミトコンドリアと追加された Ca2 +増加記録蛍光によって示される挑戦されています。MPTP が開いたら、蛍光性は時間とともに増加します。</li>
	<li>Ca2 +追加 Ca2 +保持能力として、mPTP が開くまでの総額を解釈します。</li>
</ol>3. Ca 2 +の定量-ミトコンドリアの膨潤を誘発
<ol><li>実験前に KCl メディアを準備します。<ol>
<li>KCl メディアでの分離ミトコンドリア (0.4 mg/mL) の 1 つの mL を各 6 ウェル プレートにも転送します。</li>
	</ol>
</li>
	<li>ミトコンドリアの膨潤をトリガーするミトコンドリアのタンパク質に 10 μ L の 20 mM CaCl2水溶液 (500 nmol/mg ミトコンドリア蛋白質) を追加します。</li>
	<li>540 で吸光度の減少を記録 nm 毎の 3 10 分 (図 2) のためのソフトウェアによって制御されるマイクロ プレート リーダーの s。蛍光変化は、ミトコンドリアの内膜に溶質の流入によって引き起こされます。 注: 540 で吸光度の低下腫れミトコンドリアに対応する nm。読者は、吸光度の減少を観察しない長い間隔を読書や読書の時間が適用できます。</li>
</ol>

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X. 太陽監督実験です。魏の李は、実験を行った。陳張及び X 太陽を書いた紙。

ここでは、我々 はミトコンドリアの Ca2 +の保持の能力分析と Ca2 +のシンプルで効果的なプロトコルを記述した-ミトコンドリアの膨潤アッセイをトリガーされます。
ミトコンドリアの分離セルを均質化する場合は特にすべての材料およびチューブは氷の上が存在することを確認します。0.25% トリプシン-EDTA は、37 ° C で 5 分間料理から細胞を切り離す場合にも使用できます。15 25 ストローク後観察顕微鏡の下でそのまま細胞膜とミトコンドリア膜の破損を避けるために必要です。しかし、我々 が得られる分離ミトコンドリア粗ミトコンドリアと精製ミトコンドリアは、1.0 M を得ることができる/1.5M 蔗糖不連続密度勾配。生きている細胞内のミトコンドリアの生物学的特性は、ミトコンドリアを囲む培地成分の濃度のためそれら体内より分離ミトコンドリアのような詳細です。ミトコンドリアの Ca2 +の保持容量または Ca2 +-誘発ミトコンドリア膨潤試験通電条件下で決定されました。機能しているミトコンドリアの定量、5 mM コハク酸はグルタミン酸とリンゴ酸の6の代わりに呼吸基質として追加できます。
Ca2 +-緑の蛍光色素をバインド (バインド Ca2 + 4.29 ± の親和性を持つ 0.67 mM)、Ca2 + fura 2 7などより高い親和性染料よりマイクロモル濃度の測定に適しています。Ca2 +の付加の後で-緑の蛍光色素を結合孵化は 1-3 分間隔で注入された Ca2 + (25-200 nmol から変化) 未満 240 s. 連続する追加する必要があります。検量線は、ミトコンドリアの Ca2 +パルス取り込みを示すミトコンドリアが、隔離することができる Ca2 +の濃度を示します。そうしないと、Ca2 +-蛍光染料もカルシウム シグナル伝達、細胞内 Ca2 +濃度測定、次の Ca2 +流入やリリースでは、多光子励起などの調査に使用された緑のバインドCa2 +生体組織のイメージング。
Ca2 +の定量-ミトコンドリアの膨潤による腫れを 200 mM ATR、CaCl2添加による誘導もことができます。コントロールとしてミトコンドリアは 1 mm CsA を開く mPTP を抑制測定前に、5 分間インキュベートでした。までの研究は、ミトコンドリアの膨潤14の割合の関数として吸光度の較正曲線を示した。それは 1 mM 0.1 mM ルテニウム赤、CsA を報告され、腫れが完了したときは、反応で新鮮なミトコンドリア (0.5 mg/mL) の等量を追加ことができます。CsA とルテニウム赤たて追加ミトコンドリアで開く mPTP を抑制することができます、検量線は腫れてミトコンドリアとミトコンドリアの膨潤以外15の両方を示します。Ca2 +過負荷時にミトコンドリアの膨潤のプロトコルはまた Ca2 +の効果的かつ直接測定-mPTP オープニングを誘発します。
以上 50 年前に Ca2 +哺乳類ミトコンドリアによっておよび他の高等脊椎動物の輸送の発見以来のメカニズムとミトコンドリアのカルシウム流出と流入の機能に多くの調査がずっとあります。ミトコンドリアの Ca2 +細胞、高速モードまたは RaM、およびリアノジン受容体 (mRyR)16ミトコンドリアの Ca2 +吸収メカニズムが含まれています。我々 は、することができます上記の試金は評価 Ca2 +-単にかつ効果的にミトコンドリアの機能を関連します。

ミトコンドリアは、酸化的リン酸化による哺乳類細胞で使用される ATP の約 95% を生産する主要な細胞器官です。合成 ATP1に ADP をリン酸化して使用できます、ADP と無機リン酸の存在、人里離れたマイクロモル濃度 Ca2 +ミトコンドリアによって報告されます。細胞内 Ca2 +濃度がしきい値を超えると、ミトコンドリアが取り込み Ca2 +急速および流出それゆっくり。したがって、機能しているミトコンドリアは、増加した細胞内 Ca2 +の流入をことができます。無関係酸化的リン酸化に関与する、ミトコンドリア Ca2 +もゾル性細胞質カルシウム信号の部分を取るし、mPTP のミトコンドリアの内側の開口部を誘導することによってミトコンドリアを介したアポトーシスのメカニズムをアクティブにします。膜2。細胞内 Ca2 +の異常上昇が mPTP3を開くことによってミトコンドリアの大規模な腫れを引き起こすことができる広く認識されています。したがって、このプロトコルがミトコンドリアの Ca2 +保持能力と Ca2 +を定量化することでミトコンドリアの機能を評価することを目的-ミトコンドリアの膨潤が起きるというものです。
Ca2 +保持能力ゾル性細胞質カルシウムを摂取するミトコンドリアの機能の測定であります。ミトコンドリアは細胞内遊離カルシウムをバッファーし、使用頻度の低い沈殿物の形でカルシウム摂取によってカルシウム依存性細胞プロセスを調整できます。障害者 Ca2 +保持能力にエネルギー制限とも神経変性疾患4,5に関連するストレス現象に発生します。ミトコンまたはジギトニン グリセリン細胞 Ca2 +保持能力を識別するために使用できます、追加グルタミン酸とリンゴ酸分離ミトコンドリアによる Ca2 +を蓄積する高い能力は受けない、ミトコンドリアの分離手順6。ジギトニンの滴定された量を使用して、異なる種類の細胞の細胞膜を permeabilize ください。Hexapotassium 塩 Ca2 +-緑色蛍光染料、Ca2 +の結合-敏感な細胞非透過性可視光興奮インジケーターは、広く使用されている7をされています。Ca2 +-(5 分) 刺激8延長バインド緑蛍光染料は低親和性インジケーターで、無料の細胞内 Ca2 +濃度が上昇し続けることを示すために使用します。このメソッドは、放射性フィルター技術よりも敏感だったが、大幅に簡略化します。追加 Ca2 +カルシウム緑色蛍光を高め、ミトコンドリアの Ca2 +吸収がベースラインに蛍光を返します。逐次追加の Ca2 +は、ミトコンドリア吸収 extramitochondrial Ca2 + 9に失敗するまで行われました。蛍光マイクロ プレート リーダーは、継続的にカルシウム緑色蛍光を報告する使用できます。
Ca2 +蓄積の後、ミトコンドリアが脱分極し、中に Ca2 +放出ははれだした。Ca2 +-腫れ、mPTP 開口部を示すためミトコンドリアをトリガーします。電子顕微鏡、Ca2 +の測定に使用に 540 nm の吸光度の減少-ミトコンドリアの膨潤10,11をトリガーします。前方角度光散乱で12、吸光度の減少がミトコンドリアのマトリックスの受動的な腫れを反映ミトコンドリア ボリュームを直接的に確認することができます。
ここで、我々 はミトコンドリアの Ca2 +保持能力と Ca2 +を検討する方法論を説明-SH SY5Y 細胞から分離ミトコンドリア ミトコンドリアの膨潤をトリガーします。

<table>
	<tbody>
		<tr>
			<td>SH-SY5Y cell line</td>
			<td>ATCC (The Global Bioresource Center)</td>
			<td>ATCC Number: CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium green-5N</td>
			<td>Life Technologies</td>
			<td>c-3737</td>
			<td>Ca2+-binding green fluorescent dye</td>
		</tr>
		<tr>
			<td>complete protease inhibitors</td>
			<td>Roche Molecular Biochemicals</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>glass homogenizer</td>
			<td>Kimble Chase</td>
			<td>9885303002</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>RES5063G-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>malate</td>
			<td>Sigma-Aldrich</td>
			<td>46940-U</td>
			<td></td>
		</tr>
		<tr>
			<td>rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>00457</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>V900050</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>V900266</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan flash instruments</td>
			<td>Thermo Scientific</td>
			<td>IC100E</td>
			<td></td>
		</tr>
		<tr>
			<td>Bongkrekate</td>
			<td>Biovision</td>
			<td>1820-100</td>
			<td></td>
		</tr>
		<tr>
			<td>atractyloside</td>
			<td>Sigma-Aldrich</td>
			<td>C4992</td>
			<td></td>
		</tr>
		<tr>
			<td>high-glucose Dulbecco&#8217;s modified Eagle medium</td>
			<td>Hyclone</td>
			<td>SH30022</td>
			<td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SV30087</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Invitrogen</td>
			<td>11860-038</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Thermo Scientific</td>
			<td>NCI3225CH</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay

酸化的リン酸化による ATP の生産は、ミトコンドリアの主要な機能です。高等真核生物のミトコンドリアは細胞内 Ca2 +のバッファリングにも参加し、ミトコンドリアで、ATP を生産することができます無料のミトコンドリア内 Ca2 +濃度によって媒介されます。Ca2 +保持能力は、ミトコンドリアのマトリックスでカルシウムを保持するミトコンドリアの機能としてみなすことができます。蓄積された細胞内 Ca2 +の内ミトコンドリア膜透過性につながるでは、ミトコンドリア膜透過性遷移孔 (mPTP)、分子と分子の漏れにつながる重量未満 1.5 kDa の開口部と呼ばれます。Ca2 +-腫れ、mPTP 開口部を示すためミトコンドリアをトリガーします。ここで、Ca2 +保持能力と Ca2 +を検討する 2 つの試金を記述-分離ミトコンドリア ミトコンドリアの膨潤をトリガーします。一定量に Ca2 +が追加されますすべての手順を実行することができます 1 日で完了なりマイクロ プレート リーダーによって記録されました。したがって、これら 2 つのシンプルで効果的なアッセイを採用して、Ca2 +を評価できる-関連するミトコンドリアの機能。

Ca2 +保持能力の代表の結果: 結果は、蛍光値として表現されました。Ca2 + (200 nmols/mg ミトコンドリア蛋白質)、mPTP 開口基準上増蛍光付加パルス。5 μ M Bongkrekate (BKA)、Ca2 +の阻害剤-mPTP が開いたり、1 μ M atractyloside (ATR)、Ca2 +の活性化を誘導-mPTP オープニングを誘発、ミトコン ドリアにしました。合計追加 Ca2 +ベースライン蛍光上ダブルの増加によって示される mPTP オープニングまでの量は、Ca2 +保持能力と解釈されました。我々 のデータあったミトコンドリア Ca2 +保持容量 BKA 治療で ATR グループよりもはるかに高かった。これらのデータは、ミトコンドリア Ca2 +保持能力 (図 3) の適当な測定を確認しました。
Ca2 +の代表結果-誘発ミトコンドリアの膨潤。 結果は 540 で吸光度の変動率として表された CaCl2の添加後 10 分の期間の間に初期の吸光度と nm。Ca2 + (500 nmol/mg ミトコンドリア蛋白質)、540 で監視吸光度低下の添加によるトリガー nm にミトコンドリアの腫れが示されています。BKA や ATR の治療、較正曲線 BKA できます ATR mPTP (図 4) を開くを容易にすることができます中に開く mPTP を抑制することを示す初期の吸光度と比較してわずかなあるいは急激な減少を示した。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56236/56236fig1.jpg"> 図 1: ミトコンドリアの Ca2 +の保持の能力分析のソフトウェア設定します。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56236/56236fig2.jpg"> 図 2: ミトコンドリア Ca2 +ソフトウェアの設定-ミトコンドリアの膨潤アッセイをトリガーします。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56236/56236fig3.jpg"> 図 3: Ca2 +保持能力の測定。余分なミトコンドリア Ca2 +分離ミトコンドリアのクマリンを用いて測定した Ca2 +-5 μ M bongkrekate (BKA)、1 μ M atractyloside (ATR)、または空白のコントロール (コン) の存在下で緑の蛍光色素を結合します。BKA、ATR、コンと分離ミトコンドリアによる Ca2 +保持の痕跡を測定した 506 nm (Ex) と 531 nm (Em) マイクロ プレート リーダーに。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56236/56236fig4.jpg"> 図 4: Ca2 +の測定-ミトコンドリアの膨潤を誘起します。Ca2 +-誘導ミトコンドリアの膨潤 SH SY5Y 5 μ M bongkrekate (BKA)、1 μ M atractyloside (ATR)、または空白のコントロール (コン) の存在下では 540 で初期の吸光度の比率低下較正曲線として表現された nm。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56236/56236fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

ミトコンドリアの Ca2 +保持容量測定と Ca2 +-ミトコンドリアの膨潤アッセイをトリガー

このプロトコルが Ca2 +保持能力と Ca2 +を調べる方法を説明することを目的 - トリガー SH SY5Y 分離ミトコンドリアの膨潤ミトコンドリア細胞ステップバイ ステップ。

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

mitochondrial-ca2-retention-capacity-assay-ca2-triggered

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

11.1K ビュー.  Qilu Hospital of Shandong University. このプロトコルが Ca2 +保持能力と Ca2 +を調べる方法を説明することを目的 - トリガー SH SY5Y 分離ミトコンドリアの膨潤ミトコンドリア細胞ステップバイ ステップ。

ビデオ: Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

EDU信号増幅による個別細胞におけるミトコンドリアDNAの複製の可視化

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

神経科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

ニューロモデュレーションとミトコンドリア輸送：長時間にわたって海馬ニューロンのライブイメージング

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

ライブラット皮質ニューロンのミトコンドリア膜電位と活性酸素種の定量

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

アッセイのメソッドショウジョウバエ行動

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

脳血管内皮細胞での生体エネルギー機能の評価

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sod1 突然変異体蛋白質集計における細胞の高いコンテンツ定量化のための分析法の開発

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

遺伝性痙性対麻痺におけるヒト人工多能性幹細胞由来ニューロンにおけるミトコンドリア輸送と形態学の解析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

ミトコンドリア病モデルのためのヒト脳オルガノイドの生成

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Ex Vivoのレティナル組織サンプルにおけるミトコンドリア呼吸と解糖の測定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

遺伝子組み換えCa2+ 指標(GEC)を用いたアストロサイトとニューロンのイメージングミトコンドリアCa2+ 取り込み

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

ミトコンドリアの Ca^{2 +}保持容量測定と Ca^{2 +}-ミトコンドリアの膨潤アッセイをトリガー

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