作者感谢巴西机构 Coordenação de Aperfeiçoamento de Pesquisa Pessoal de Nível Superior （CAPES EPIDEMIAS 09 #88887.505377/2020）。P.M. （#88887.512821/2020-00） 和 TD （#88887.512883/2020-00） 是研究奖学金获得者。

我们使用了来自布卢明顿果蝇种群中心（ID号：34749）的菌株w1118（白色）和w[*] Pink1[B9]/FM7i，P{w[+mC]=ActGFP}JMR3（称为Pink1B9）（FlyBase ID：FBgn0029891）。在这项研究中，将雄性黑腹果蝇PINK1B9-null突变体与来自w1118菌株的雄性黑腹果蝇进行比较，后者用作对照组（遗传背景）。其他参数必须与呼吸测量实验同时进行分析，以确保果蝇具有正确的基因型（Pink1B9/Y），例如胸部畸形和运动问题，这些在pink1B9突变果蝇24,25,26中得到了很好的描述。
1. 动物和住房
<ol><li>将苍蝇保存在含有10mL标准饮食（酵母1.73%，大豆粉0.9%，玉米粉7.3%，琼脂0.5%，玉米糖浆7.6%和丙酸0.48%）的玻璃瓶中，温度和湿度恒定（分别为25°C和60%），光/暗循环为12小时：12小时。</li>
	<li>对于每个实验，使用两只苍蝇在闭合后1-3天。 注： 将 PINK1B9-null 突变的处女雌性苍蝇与 w1118 雄性杂交，以获得基因型为 Pink1B9/Y 的 F1 雄性。</li>
</ol>2. 样品制备
<ol><li>如 表1所述准备MiR05缓冲液。</li>
	<li>麻醉冰上的苍蝇并将它们转移到微量离心管（每管两只苍蝇）。</li>
	<li>在每个试管中，加入 200 μL 冷冻的 MiR05 缓冲液并使果蝇匀浆。手动匀浆，施加温和的压力（大约4-6次杵的冲程）以防止线粒体的崩解。</li>
</ol>3. 极谱法氧传感器的高分辨率呼吸测量校准
注：OROBOROS 腔室的总体积约为 2 mL。需要校准以确保氧通量接近 0 pmol 才能开始测定。
<ol><li>开始校准，向腔室中加入 2 mL MiR05。用塞子盖住腔室，不要留下气泡;然后，小心地拉动塞子以形成一个气泡。</li>
	<li>打开 OROBOROS Dat.Lab 软件。在模块温度字段中输入25°C的温度，然后单击连接到Oxygraph-2k（图1A）。</li>
	<li>单击并选择要保存校准的文件夹，然后单击 “保存”。</li>
	<li>当打开一个新窗口时，命名实验和样品（如果校准，只需输入名称 校准），然后单击 保存。</li>
	<li>转到 “布局” 菜单，然后选择第一个选项： 01 Calibration Exp. Gr.3 - Temp ，如 图 1B 所示。</li>
	<li>等待程序重定向到校准布局，等到红线显示标准直线，点在 0 pmol 左右。然后，当红线正好为零时，选择两个 点：一个用于 腔室A ，另一个用于 腔室B （图1C）。 注意：所选点的氧通量（红线）必须在 0 左右。<ol><li>要标记两个腔室的点，请选择屏幕右侧的 O2 浓度 ，选择标记点，然后复制相对于该点的 温度 和 气压 值。将这些值粘贴到下面的框中，然后单击屏幕右下角的 Calibrate and Copy to Clipboard ，如 图 2 所示。</li>
		<li>对两个 腔室 （A 和 B）执行此过程，然后转到 “文件 ”菜单并选择 “保存并断开连接”。 注意： 校准过程完成后，程序会将用户重定向到主屏幕，以便软件准备好开始 HRR 测试。对于此测试，我们将使用 SUIT 协议。</li>
	</ol></li>
</ol>4. SUIT协议
<ol><li>校准完成后，取下塞子并打开腔室。</li>
	<li>按此顺序用 100% 乙醇、70% 乙醇和蒸馏水清洗塞子（握住不接触样品的尖端）和腔室。 注意：对每个样品更换重复该过程。</li>
	<li>在200μL缓冲液中匀浆苍蝇，使用微量移液器将所有样品内容物置于腔室中，并将塞子重新定位在腔室中，注意不要产生气泡。 注意：如果形成气泡，轻轻取下塞子并加入 100 μL MIR05 缓冲液。</li>
	<li>单击 “布局” 菜单，然后选择“ 05 Flow by Corrected Volume ”选项，如 图 1B 所示。</li>
	<li>等待重定向到新窗口。单击 “保存 ”以选择要保存实验的文件夹。</li>
	<li>等待另一个新窗口打开。将空间中的实验命名为 Experimental Code。然后，在“样品”字段中将每个样品命名为各自腔室中的 A 和 B，并将字段“单位”设置为“单位”。 注意：也可以选择其他单位，例如， 百万个细胞 或 毫克。</li>
	<li>考虑到该协议使用两只苍蝇（2个单位）并且腔室容积为2mL，在 浓度 字段中，定义 每mL1 个（图1D）。 注意：在这里，样本量按苍蝇数量归一化;然而，这种归一化可以通过样品的蛋白质含量、线粒体 DNA 定量或柠檬酸合酶的活性来执行。</li>
	<li>当氧通量信号（红线）稳定在正值上时，通过底物、抑制剂和解偶联剂的滴定开始 HRR 方案（图 3）。所有后续试剂和设备均见 表1。<ol><li>加入洋地黄皂苷 （5 μg/mL） 以透化线粒体膜。</li>
		<li>加入丙酮酸（5mM），苹果酸（2mM）和脯氨酸（10mM）底物，等待氧通量增加并稳定。要标记每次添加试剂的事件，请按键盘上的 F4 键并输入每个试剂 的名称 。</li>
		<li>加入ADP（5mM）耦合线粒体呼吸链，等待氧通量的增加和稳定。</li>
		<li>加入琥珀酸盐（10mM）并等待氧通量的增加和稳定。</li>
		<li>加入寡霉素（2.5μM）以抑制ATP合酶并寻找氧通量的降低。</li>
		<li>等到红线稳定下来，并使用滴度为0.25μM的羰基-4-（三氟甲氧基）苯腙氰化物（FCCP）解耦线粒体电子转移，直到达到最大耗氧量，红线的上升证明了这一点。稳定氧通量后，开始添加每种复合物的抑制剂，一次一个。</li>
		<li>加入鱼藤酮（0.5μM），一种复合物I抑制剂。等待氧通量的降低和稳定后再添加下一个抑制剂。</li>
		<li>加入丙二酸酯（5mM），一种复合物II抑制剂。等待氧通量的降低和稳定后再添加下一个抑制剂。</li>
		<li>最后，加入抗霉素（2.5μM），一种复合物III抑制剂。等待氧通量下降，然后增加和稳定氧通量。 注意：氧通量应稳定在正值，但低于线粒体蛋白复合物最后一次抑制的值。</li>
	</ol></li>
	<li>在分析结束时，使用微量移液器除去腔室的所有内容物。必要时储存在-20°C以备将来分析。</li>
</ol>5. 数据分析
<ol><li>使用适当的统计软件包进行数据分析。</li>
	<li>执行 t 检验以评估两组之间的差异。对于涉及多种处理的情况，使用方差分析 （ANOVA） 作为统计检验 27。</li>
	<li>从软件生成的图形中提取 O2 通量值。OXPHOS CI 是指添加 ADP 后 O2 通量的值。OXPHOS CII 是通过减去 OXPHOS CI&CII - OXPHOS CI 获得的。OXPHOS CI&CII是加入琥珀酸盐后提取的O2 助焊剂的值。ETS CI&CII是添加FCCP后的O2 通量值。ETS CI = FCCP - 鱼藤酮和 ETS CII = 鱼藤酮 - 丙二酸。</li>
	<li>将 ATP 合成计算为 OXPHOS （P） 和 LEAK （L） （P - L） 之间的差值28。</li>
	<li>通过计算比率 OXPHOS/LEAK 来确定呼吸控制比 （RCR），其中 RCR = P/L.11</li>
</ol>

分析果蝇的线粒体生理学和生物能量学

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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=Accessory+subunits+are+integral+for+assembly+and+function+of+human+mitochondrial+complex+I.">Accessory subunits are integral for assembly and function of human mitochondrial complex I.</a> Nature. 538 (7623), 123-126 (2016).</li><li>Senyilmaz, D., 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=Regulation+of+mitochondrial+morphology+and+function+by+stearoylation+of+TFR1.">Regulation of mitochondrial morphology and function by stearoylation of TFR1.</a> Nature. 525 (7567), 124-128 (2015).</li><li>Poddighe, S., 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=Mucuna+pruriens+(Velvet+bean)+rescues+motor,+olfactory,+mitochondrial+and+synaptic+impairment+in+PINK1B9+Drosophila+melanogaster+genetic+model+of+Parkinson's+disease.">Mucuna pruriens (Velvet bean) rescues motor, olfactory, mitochondrial and synaptic impairment in PINK1B9 Drosophila melanogaster genetic model of Parkinson's disease.</a> PloS One. 9 (10), e110802 (2014).</li><li>Pesta, D., Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+respirometry:+OXPHOS+protocols+for+human+cells+and+permeabilized+fibers+from+small+biopsies+of+human+muscle.">High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle.</a> Methods Mol Biol (Clifton, N.J). 810, 25-58 (2012).</li><li>Winer, L. S. P., Wu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+glycolytic+and+oxidative+substrate+flux+of+cancer+cells+in+a+microplate.">Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate.</a> PloS One. 9 (10), e109916 (2014).</li><li>Plitzko, B., Loesgen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+oxygen+consumption+rate+(OCR)+and+extracellular+acidification+rate+(ECAR)+in+culture+cells+for+assessment+of+the+energy+metabolism.">Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in culture cells for assessment of the energy metabolism.</a> Bio Protoc. 8 (10), e2850 (2018).</li><li>Wu, M., 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=Multiparameter+metabolic+analysis+reveals+a+close+link+between+attenuated+mitochondrial+bioenergetic+function+and+enhanced+glycolysis+dependency+in+human+tumor+cells.">Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.</a> Am J Physiol. Cell Physiol. 292 (1), C125-C136 (2007).</li><li>Divakaruni, A. S., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+for+the+analysis,+standardization+and+interpretation+of+oxygen+consumption+measurements.">A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements.</a> Nat Metab. 4 (8), 978-994 (2022).</li><li>Bingol, B., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitophagy:+PINK1,+Parkin,+USP30+and+beyond.">Mechanisms of mitophagy: PINK1, Parkin, USP30 and beyond.</a> Free Radic Biol Med. 100, 210-222 (2016).</li></ol>

作者声明没有竞争利益。

HRR 是一种强大的技术，用于研究 黑腹果蝇 和其他生物体的线粒体呼吸和能量代谢。它提供了对线粒体功能的详细和定量评估，使研究人员能够深入了解细胞的生物能量学。此处介绍的方案描述了使用SUIT协议在 黑腹果蝇中评估线粒体呼吸链功能和特定线粒体复合物的活性。SUIT方案涉及系统地操纵各种底物、解偶联剂和抑制剂，以检查线粒体呼吸的不同方面。
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线粒体是控制重要功能的细胞器，包括细胞凋亡调节、钙稳态和参与生物合成途径。通过拥有自主的遗传物质，它们能够为细胞维持和修复过程做出贡献。它们的结构包含电子传递链和氧化磷酸化，这两者都对细胞能量至关重要 1,2,3。特别是，能量控制是通过氧化磷酸化 （OXPHOS） 产生三磷酸腺苷 （ATP） 来实现的2。能量代谢紊乱伴线粒体功能受损发生在细胞存活和死亡中 4,5，通常与多种人类病理学（如癌症）和神经退行性疾病（如帕金森病 （PD）3,6）有关。
帕金森病是一种慢性、进行性和神经系统疾病。这种疾病的主要原因是脑细胞的死亡，特别是在黑质中，黑质负责产生控制运动的神经递质多巴胺 6,7,8。最早将帕金森综合征与线粒体功能障碍联系起来的观察是在 1988 年，在使用抑制呼吸链复合物 I9 的毒素的实验模型中。
目前，有几种方法可以评估线粒体功能障碍 10,11,12,1 3;然而，与传统方法相比，高分辨率呼吸测量法 （HRR） 具有卓越的灵敏度和优势13,14。例如，HRR方案允许评估整个线粒体呼吸链功能或特定线粒体复合物的活性14,15。线粒体功能障碍可以在完整细胞、分离的线粒体甚至体外10、11、13、14 中进行评估。
线粒体功能障碍与许多病理和生理过程密切相关。因此，使用遗传和实验上可处理的模型系统研究线粒体生理学和生物能量学非常重要。在这方面，对果蝇果蝇（Drosophila melanogaster）的研究有几个优点。该模型与人类共享基本的细胞特征和过程，包括使用 DNA 作为遗传物质、共同细胞器以及参与发育、免疫和细胞信号传导的保守分子通路。此外，果蝇具有生命周期快、易于维护、成本低、通量高、伦理问题少等特点，是解剖复杂细胞过程的宝贵工具16,17,18,19,20。
此外，PTEN诱导的推定激酶1（pink1）基因的同源物在黑腹果蝇中表达。它通过线粒体自噬过程在去除受损线粒体方面起着至关重要的作用8.在人类中，该基因的突变使个体易患与线粒体功能障碍相关的常染色体隐性遗传家族性 PD 8,21,22,23。因此，果蝇是一种强大的动物模型，用于研究帕金森病的病理生理学和筛选以线粒体功能障碍和生物能量学为重点的候选药物。因此，本工作解释了如何使用 HRR 技术在 OROBOROS 中使用底物-解偶联剂-抑制剂-滴定 （SUIT） 方案分析黑腹果蝇 PD 模型中的线粒体功能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A5285</td>
			<td>Adenosine 5&#8242;-diphosphate sodium sal (CAS number 72696-48-1); &#8805;95%; molecular weight = 501.31 g/mol.</td>
		</tr>
		<tr>
			<td>&#193;gar</td>
			<td>Kasv</td>
			<td>K25-1800</td>
			<td>For bacteriologal use</td>
		</tr>
		<tr>
			<td>Antimycin-A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>Antimycin A from Streptomyces sp. (CAS number 1397-94-0); molecular weight&#160; 540 g/mol;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td>Bovine Serum Albumin (CAS number 9048-46-8); pH 7,0 &#8805; 98%</td>
		</tr>
		<tr>
			<td>Datlab software</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20700</td>
			<td>Software for data acquisition and analysis</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>D 5628</td>
			<td>CAS number 11024-24-1</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila melanogaster strain w[*] Pink1[B9]/FM7i, P{w[+mC]=ActGFP}JMR3</td>
			<td></td>
			<td></td>
			<td>Obtained from Bloomington Drosophila stock center</td>
		</tr>
		<tr>
			<td>Drosophila melanogaster strain&#160;w1118</td>
			<td></td>
			<td></td>
			<td>Obtained&#160; from the Federal University of Santa Maria</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E8145</td>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (CAS number 13638-13-3); &#8805;97%; molecular weight =468.28 g/mol</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td>Carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone&#160; (CAS number 370-86-5); &#8805;98% (TLC), powder&#160;</td>
		</tr>
		<tr>
			<td>GraphPad Prism version 8.0.1.</td>
			<td></td>
			<td></td>
			<td>Software for data acquisition and analysis</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9); &#8805;99,5% (titration), cell cultured tested; molecular weight = 238.30 g/mol</td>
		</tr>
		<tr>
			<td>High-resolution respirometer Oxygraph O2K</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>10022-02</td>
			<td>Startup O2K respirometer kit</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379</td>
			<td>Monopotassium phosphate (CAS number 7778-77-0); Reagente Plus, molecular weigt = 136.09 g/mol</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>211473</td>
			<td>Potassium hydroxide (CAS number 1310-58-3); ACS reagent, &#8805;85%, pellets</td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma-Aldrich</td>
			<td>M1296</td>
			<td>Malonic acid (CAS number 141-82-2); 99%, molecular weight = 104.06 g/mol). A solution is pH adjusted to approximately 7.0.</td>
		</tr>
		<tr>
			<td>Malic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M1000</td>
			<td>(S)-(&#8722;)-2-Hydroxysuccinic acid (CAS number 97-67-6); &#8805;95% ; molecular weight = 134.09 g/mol</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>2-(N-Morpholino)ethanesulfonic acid (CAS number 4432-31-9); &#8805;99% (titration); molecular weight = 195.24 g/mol</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Magnesium chloride (CAS number 7786-30-3); anhydrous, &#8805;98%, molecular weight = 95.21 g/mol</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>O2K-Titration Set</td>
			<td>Oroboros Instruments, Innsbruck, Austria</td>
			<td>20820-03</td>
			<td>Hamilton syringes with different volumes</td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>O 4876</td>
			<td>Oligomycin from&#160;Streptomyces diastatochromogenes (CAS number&#160; 1404-19-9); &#8805;90% total oligomycins basis (HPLC)</td>
		</tr>
		<tr>
			<td>Pistil to homogenization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>Sigma-Aldrich</td>
			<td>P0380</td>
			<td>L-Proline (CAS number 147-85-3); powder; 99%; molecular weight = 115.13 g/mol</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td>Sodium pyruvate (CAS number 113-24-6), &#8805;99%; molecular weight = 110.04 g/mol</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td>Rotetone (CAS number 83-79-4); &#8805;95%, molecular weight 394.42 g/ mol</td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma-Aldrich</td>
			<td>S 2378</td>
			<td>Sodium succinate dibasic hexahydrate (CAS number 6106-21-4); &#8805;99%</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck</td>
			<td>107,651,000</td>
			<td>Sucrose for microbiology use (CAS number 57-50-1)</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td>CAS number 107-35-7</td>
		</tr>
	</tbody>
</table>

analyzing mitochondrial function in a drosophila melanogaster pink1b9-null mutant using high-resolution respirometry

神经退行性疾病，包括帕金森病 （PD），和细胞紊乱，如癌症，是一些破坏能量代谢并损害线粒体功能的疾病。线粒体是控制能量代谢和细胞存活和死亡的细胞过程的细胞器。出于这个原因，评估线粒体功能的方法可以为病理和生理过程中的细胞状况提供重要的见解。在这方面，高分辨率呼吸测量 （HRR） 方案允许评估整个线粒体呼吸链功能或特定线粒体复合物的活性。此外，研究线粒体生理学和生物能量学需要遗传和实验上可处理的模型，例如 果蝇黑腹果蝇。
该模型具有多种优点，例如与人体生理学的相似性、生命周期快、易于维护、成本效益高、高通量能力以及将伦理问题降至最低。这些属性共同确立了它作为解剖复杂细胞过程的宝贵工具。本工作解释了如何使用 果蝇黑腹 果蝇 PINK1B9-null 突变体分析线粒体功能。 pink1 基因负责编码 PTEN 诱导的推定激酶 1，通过一个被认为是线粒体自噬的过程，这对于从线粒体网络中去除功能失调的线粒体至关重要。该基因的突变与常染色体隐性遗传性早发性家族性 PD 有关。该模型可用于研究与帕金森病病理生理学有关的线粒体功能障碍。

Mitochondria are cellular organelles that control important functions, including apoptotic regulation, calcium homeostasis, and participation in biosynthetic pathways. By possessing autonomous genetic material, they are capable of contributing to cellular maintenance and repair processes. Their structure houses the electron transport chain and oxidative phosphorylation, both crucial for cellular energy1,2,3. In particular, energy control is achieved through adenosine triphosphate (ATP) production via oxidative phosphorylation (OXPHOS)2. Disruption of energy metabolism with impairment of mitochondrial functions occurs both in cell survival and death4,5, frequently associated with a wide range of human pathologies, such as cancer, and neurodegenerative diseases such as Parkinson&#39;s Disease (PD)3,6.
PD is a chronic, progressive, and neurological disorder. The primary cause of this disease is the death of brain cells, especially in the substantia nigra, which are responsible for the production of the neurotransmitter dopamine, which controls movement6,7,8. The earliest observation that linked Parkinsonism to mitochondrial dysfunction was made in 1988, in experimental models using toxins that inhibit the respiratory chain Complex I9.
Currently, there are several methods to evaluate mitochondrial dysfunction10,11,12,13; however, compared to conventional approaches, high-resolution respirometry (HRR) presents superior sensitivity and advantages13,14. For example, HRR protocols allow the evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes14,15. Mitochondrial dysfunctions can be assessed in intact cells, isolated mitochondria, or even ex vivo10,11,13,14.
Mitochondrial dysfunctions are closely associated with many pathological and physiological processes. It is therefore important to study mitochondrial physiology and bioenergetics using genetically and experimentally tractable model systems. In this regard, research on Drosophila melanogaster, the fruit fly, has several advantages. This model shares fundamental cellular characteristics and processes with humans, including the use of DNA as genetic material, common organelles, and conserved molecular pathways involved in development, immunity, and cell signaling. In addition, fruit flies have a rapid life cycle, easy maintenance, low cost, high throughput, and fewer ethical concerns, thus constituting an invaluable tool for dissecting complex cellular processes16,17,18,19,20.
Furthermore, a homolog of the PTEN-induced putative kinase 1 (pink1) gene is expressed in D. melanogaster. It plays a crucial role in the removal of damaged mitochondria through the process of mitophagy8. In humans, mutations in this gene predispose individuals to an autosomal recessive familial form of PD associated with mitochondrial dysfunction8,21,22,23. Consequently, the fruit fly is a powerful animal model for studies on the pathophysiology of PD and screening of drug candidates focusing on mitochondrial dysfunction and bioenergetics. Therefore, the present work explains how to analyze mitochondrial function in a model of PD from D. melanogaster using the HRR technique in the OROBOROS with the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

作者聚焦：使用黑腹果蝇探索线粒体功能和化学毒性 

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

Our research uses Drosophila melanogaster as an experimental model to understand mitochondrial function in disease and chemical toxicity. Some questions we want to answer are, how does PINK-1 mutation affect mitochondrial function and how is this linked to Parkinson's Disease? How can we revert, or avert, the mitochondrial dysfunction associated with this mutation?High-resolution respirometry requires specialized training for precise operation and data analysis. In addition, the system we used doesn't allow working with several samples simultaneously and is not educated for high-throughput analysis. Since it's only possible to run two samples at the same time, the experimental design must be well delineated.Our findings indicate progressive impairment of the mitochondrial function we face in PINK1-Null mutants, which is characterized by decrease in OXPHOS CI link, and ETS CI&CII link, and a metabolic metabolic shift in ATP production from oxidative to glycolytic in pathways. These findings are closely related to Parkinson's Disease pathophysiology. One of the key points of high-resolution respirometry is its ability to provide direct and accurate measurements of oxygen consumption, allowing for detailed analysis of mitochondrial function and cellular metabolism.Also, it's versatile. It can be used for wide range of sample types, including isolated mitochondria. Considering that mitochondrial dysfunction is a common feature of neurodegenerative diseases, such as Parkinson's disease, in the future, we will focus on investigating how mitochondrial function and energy elevation could be important targets for therapies directed to neurodegenerative diseases.

在这里，我们发现，与对照果蝇相比，PINK1 B9 无效果蝇在 PINK1B9 无效果蝇中，OXPHOS CI （P = 0.0341） 和 OXPHOS CI&II （P = 0.0392） 状态下的 O2 通量降低（图 4）。这一结果在我们小组29,30 的先前研究结果中也观察到。
CI 和 CII 是电子传输系统 （ETS） 的关键组成部分，其中 CI 负?...

Watch this Scientific Journal Video about Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster at JoVE.com

在这里，我们提出了一种高分辨率呼吸测量方案，用于分析 PINK1B9-null 突变果蝇的生物能量学。该方法使用底物-解偶联剂-抑制剂滴定 （SUIT） 方案。

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

我们的研究使用黑腹果蝇作为实验模型来了解疾病中的线粒体功能和化学毒性。我们想要回答的一些问题是，PINK-1突变如何影响线粒体功能，这与帕金森病有什么关系？我们如何恢复或避免与这种突变相关的线粒体功能障碍？高分辨率呼吸测量需要专门的培训才能进行精确操作和数据分析。此外，我们使用的系统不允许同时处理多个样品，也没有接受过高通量分析的教育。由于只能同时运行两个样品，因此必须明确描述实验设计。我们的研究结果表明，我们在 PINK1-Null 突变体中面临的线粒体功能逐渐受损，其特征在于 OXPHOS CI 连接和 ETS CI&CII 连接减少，以及 ATP 产生的代谢代谢转变从氧化到糖酵解途径。这些发现与帕金森病的病理生理学密切相关。高分辨率呼吸测量的关键点之一是它能够提供直接和准确的耗氧量测量，从而可以详细分析线粒体功能和细胞代谢。此外，它是多功能的。它可用于多种样品类型，包括分离的线粒体。考虑到线粒体功能障碍是神经退行性疾病（如帕金森病）的共同特征，未来我们将重点研究线粒体功能和能量升高如何成为针对神经退行性疾病的治疗的重要靶点。

analyzing-mitochondrial-function-drosophila-melanogaster-pink1b9-null

Research

JoVE Journal

Biochemistry

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

614 次观看.  Federal University of Santa Maria. 我们的研究使用黑腹果蝇作为实验模型来了解疾病中的线粒体功能和化学毒性。我们想要回答的一些问题是，PINK-1突变如何影响线粒体功能，这与帕金森病有什么关系？我们如何恢复或避免与这种突变相关的线粒体功能障碍？高分辨率呼吸测量需要专门的培训才能进行精确操作和数据分析。此外，我们使用的系统不允许同时处理多个样品，也没有接受过高通量分析的教?...

视频: 作者聚焦：使用黑腹果蝇探索线粒体功能和化学毒性 

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

科研

生物化学

Calibrating Polarographic Oxygen Sensors for High-Resolution Respirometry Analysis in Drosophila melanogaster

Calibrating Polarographic Oxygen Sensors for High-Resolution Respirometry Analysis in Drosophila melanogaster  

To begin, add two milliliters of MIRO five buffer to the Oroboros chamber. Cover the chamber with a stopper without introducing air bubbles. Then carefully pull the stopper to form a single air bubble.Open the Oroboros DatLab software in the block temperature field. Enter a temperature of 25 degrees Celsius and click on Connect to Oxygraph-2k. Click and select the folder where the calibration is to be saved.And click on save. When a new window opens, name the experiment and the samples. Then click on save.Next, go to the layout menu and choose the first option one, calibration experiment. Graph three, temp. When the red line shows a standard straight line with points around zero picomole, select two points, one for chamber A and the other for chamber B.Select oxygen concentration on the right of the screen.Select the marked point and copy the temperature and barometric pressure values relative to the point. Paste these values in the boxes below, and click on calibrate and copy to clipboard. Then go to the file menu and select save and disconnect.

校准用于黑腹果蝇高分辨率呼吸测量分析的极谱法氧传感器

Substrate-Uncoupler-Inhibitor-Titration for Mitochondrial Respiration Analysis in Drosophila melanogaster

Substrate-Uncoupler-Inhibitor-Titration for Mitochondrial Respiration Analysis in Drosophila melanogaster  

After calibrating polarographic oxygen sensors, remove the stoppers and open the chambers. Wash the stoppers and chambers with 100%ethanol, 70%ethanol, and distilled water. Transfer the flies into a microcentrifuge tube.Add 200 microliters of chilled MiR05 buffer into the tube and homogenize the flies by applying gentle pressure with four to six strokes of the pestle. Using a micropipette, transfer the homogenized fly suspension into the chamber. Cover the chamber with a stopper without introducing air bubbles.Next, click on the layout menu and select Option 5, Flow by Corrected Volume. Another new window will open. Here, in the space, experimental code, name the experiment.In the sample field, name each sample in its respective chamber A and B.Then set the unit field to Unit and in the concentration field, define the chamber volume as one per milliliter. To start the high-resolution respirometry, ensure that the oxygen flux signal is steady on the positive value. Add digitonin to permeabilize the mitochondrial membrane.Then add pyruvate, malate, and proline substrates until the oxygen flux increases and stabilizes. Press the F4 key on the keyboard and enter the name of the reagent to mark the event. Add ADP to couple the mitochondrial respiratory chain, and wait for the increase and stabilization of the oxygen flux.Then add succinate and measure the oxygen flux. TO inhibit ATP synthase, add oligomycin and examine for a decrease in oxygen flux. After the red line stabilizes, uncouple the mitochondrial electron transfer using 0.25-micromolar FCCP until maximum oxygen consumption is reached, demonstrated by the rise of the red line.Next, add rotenone complex I inhibitor and wait for the decrease and stabilization of the oxygen flux. Then add malonate complex II inhibitor and measure the oxygen flux. Finally, add antimycin complex III inhibitor and wait for the decrease, followed by the increase and stabilization of the oxygen flux.Click on the GraphPad Prism software to extract the oxygen flux values from the graphs. OXPHOS CI refers to the value of oxygen flux after adding ADP. Then use the given formula to calculate OXHPOS CII.Using the following equation, calculate the ETS CI and CII oxygen values for the uncouples and inhibitors. Then calculate the ATP synthesis as the difference between the OXPHOS and LEAK. Finally, calculate the OXPHOS by LEAK ratio to determine the respiratory control ratio.The oxygen flux in both OXHPOS CI and OXPHOS CI and CII states was significantly reduced in PINK1 B9 null flies. compared to control flies. Additionally, the oxygen flux in ETS CI, ETS CII, and ETS CI and CII state were lower in PINK1 B9 null flies, indicating an impaired electron transfer system compared to the control flies.The disruption in the flux of electrons affects the OXPHOS process, leading to reduced ATP synthesis in the PINK1 B9 null flies. The reduction in the respiratory control ratio of null flies suggests mitochondrial uncoupling, indicating that the mitochondria were less efficient at utilizing oxygen and producing ATP.