这项研究得到了来自山东省杰出科学家 (JQ201421) 的赠款和自然科学基金 (81371226) 赠款的资助。

1. 线粒体的分离
注: 所有解决方案和设备应预换热器 0-4 摄氏度, 并保持在冰上。
<ol><li>种子约 3 x 106 SH-SY5Y 细胞每10厘米细胞培养皿。高糖 Dulbecco 修饰鹰培养基中培养细胞, 10% 胎牛血清, 青霉素 (100 U/毫升)-链霉素 (100 µg/毫升)。在包含 5% CO2的孵化器中保持37摄氏度。 注意: 每个隔离检测都需要至少 1-2 x 107 SH-SY5Y 单元格。</li>
	<li>取出培养基, 在10厘米细胞培养皿中用大约1毫升的冰冷 PBS 冲洗细胞3次。</li>
	<li>在10厘米细胞培养皿中加入新的1毫升冰冷 PBS, 将黏附细胞刮成新的1.5 毫升管。离心机在 800 x g 10 分钟在4摄氏度。</li>
	<li>在离心过程中, 准备5毫升线粒体隔离缓冲 (250 毫米蔗糖, 10 毫米 HEPES (4-(2-羟乙基)-1-哌嗪磺酸), 10 毫米氯化钾, 1.5 毫米氯化镁2, 1 毫米 EDTA) 补充 50 ul 蛋白酶抑制剂库存溶液 (100x 浓度; 1 无 EDTA 片剂溶于500µL 的 ddH2O)。</li>
	<li>取出上清液, 并用重悬1毫升线粒体隔离缓冲颗粒。在冰上孵化1.5 毫升管30分钟。</li>
	<li>溶解在冰上手动向上和向下的玻璃均质机 (15-25 冲程) 悬浮细胞。 注意: 确保细胞均匀时没有气泡。用显微镜观察完整的细胞和裸露的细胞核, 并确认 > 90% 细胞破损发生。</li>
	<li>将匀浆转化为1.5 毫升管, 离心机在 1000 x g 处, 5 分钟到4摄氏度, 去除碎片 (细胞核和剩余完好的细胞)。</li>
	<li>将上清液转移到新的1.5 毫升管和离心机在 1000 x g 5 分钟4°c。</li>
	<li>将上清液转移到新管和离心机在 1.4万 x g 15 分钟4°c。</li>
	<li>并用重悬1毫升线粒体隔离缓冲液去除含有线粒体的颗粒。</li>
	<li>离心溶液为15分钟在 1.4万 x g 4 °c 沉淀线粒体。</li>
	<li>所产生的线粒体颗粒必须保持在冰和悬浮 50-100 ul 氯化钾介质 (125 毫米氯化钾, 2 毫米 K2HPO4, 1 毫米氯化镁2, 20 毫米 HEPES, 5 毫米谷氨酸, 5 毫米苹果酸, 2 微米鱼藤酮, pH 7.4) 根据颗粒体积前任何驴哎。</li>
	<li>使用5的线粒体溶液 ul 测定线粒体蛋白浓度通过标准的评估测试, 测量 OD562 使用一个板读取器13。</li>
</ol>2. 线粒体 Ca2 +保留容量的测定
<ol><li>准备氯化钾介质, 并在实验前将 Ca2 +结合绿色荧光染料添加到最终浓度为0.5 微米。</li>
	<li>在含0.5 微米 Ca2 +的氯化钾介质中转移1毫升的分离线粒体 (0.4 毫克/毫升), 将绿色荧光染料结合到每个6井板中。</li>
	<li>孵化线粒体和 Ca2 +-结合绿色荧光染料在6井板室温保护, 从环境光1分钟. 添加 4 ul 整除数20毫米 CaCl 2 解决方案 (20 毫米 CaCl 2, 127 毫米氯化钾, 1 毫米氯化镁 2 , 20 毫米 HEPES, 5 毫米谷氨酸, 5 毫米苹果酸, pH 7.4) 到每个6井板井使用自动分配设置引入 200 nmol Ca2 +/镁线粒体蛋白。</li>
	<li>使用荧光光谱仪监测每3秒的荧光变化为2分钟, 激发波长为 506 nm, 发射波长为 531 nm。在软件控制的读数 (图 1) 之间, 板上的震动为 600 rpm 3 秒。</li>
	<li>添加一个额外的 4 ul 整除20毫米 CaCl2解决方案, 每个井和监测荧光变化每3秒为2分钟. 重复此步骤, 直到荧光随时间增加。 注意: 通过增加的记录荧光指示的添加的 Ca2 + , 线粒体面临挑战。当 mPTP 打开时, 荧光随时间增加。</li>
	<li>解释添加的 ca2 +的总金额, 直到 mPTP 作为 Ca2 +保留容量打开为止。</li>
</ol>3. 钙2 +诱导的线粒体肿胀的测定
<ol><li>在实验前准备氯化钾介质。<ol>
<li>将氯化钾介质中1毫升的分离线粒体 (0.4 毫克/毫升) 转移到每个6井板中。</li>
	</ol>
</li>
	<li>添加 10 ul 20 毫米 CaCl2水溶液 (500 nmol/镁线粒体蛋白) 成线粒体蛋白, 引发线粒体肿胀。</li>
	<li>记录由软件控制的微板块读取器上每3秒 540 nm 的吸光度降低10分钟 (图2)。荧光的变化是由溶质流入线粒体内膜引起的。 注: 540 nm 的吸收量减少, 相当于肿胀的线粒体。如果阅读器没有观察到吸收的减少, 可以采用更长的阅读间隔或阅读时间。</li>
</ol>

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太阳监督实验。李伟进行了实验。陈张和 x. 孙写了这篇论文。

在这里, 我们描述了一个简单有效的线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验的协议。
对于线粒体的隔离, 要确保所有的材料和管子都在冰上, 特别是当细胞均匀化时。0.25% 胰蛋白酶-EDTA 也可用于分离皿中的细胞5分钟, 在37摄氏度。15-25 冲程后, 必须观察显微镜下的完整细胞膜, 避免线粒体膜的断裂。然而, 我们获得的分离的线粒体是粗线粒体和纯?...

线粒体是主要的细胞器官, 以产生近95% 的 ATP 在哺乳动物细胞中使用氧化磷酸化。据报道, 通过线粒体、adp 和无机磷酸盐的存在, micromolar 的 Ca2 +的螯合浓度可以用来 phosphorylate adp 合成 ATP1。当胞浆 Ca2 +的浓度超过阈值时, 线粒体可以快速吸收 Ca2 +并缓慢地将其流出。因此, 功能线粒体可以流入增加胞浆 Ca2 +。与参与氧化磷酸化无关, 线粒体 Ca2 +也参与胞浆钙信号和激活线粒体凋亡机制, 诱导开放的 mPTP 在内线粒体膜2。人们普遍认识到, 胞内 Ca 的异常海拔2 +可以通过打开 mPTP3来诱发线粒体大面积肿胀。因此, 本协议旨在通过量化线粒体 Ca2 +保留容量和 Ca2 +-触发线粒体肿胀来评估线粒体功能。
Ca2 +保留容量是衡量线粒体吸收胞浆钙的能力。线粒体可以缓冲胞浆游离钙和调节钙依赖的细胞过程中的钙吸收形式的非活性沉淀。受损的 Ca2 +保留容量发生在与能源限制有关的压力现象中, 甚至神经退行性疾病4,5。分离的线粒体或 digitonin-透细胞可用于识别 Ca2 +保留容量, 并且由具有额外谷氨酸和苹果酸的孤立线粒体积累 Ca2 +的提升能力不受线粒体隔离过程6。滴定量的 digitonin 应用于 permeabilize 不同细胞类型的血浆膜。ca 的 hexapotassium 盐2 +-结合绿色荧光染料, 一个 Ca2 +敏感细胞-impermeant 可见光兴奋指示器, 已被广泛使用的7。Ca2 +-结合绿色荧光染料是一种低亲和力指示器, 用于表明胞内游离 Ca2 +浓度在长时间 (5 分钟) 刺激8中继续上升。该方法比放射性过滤技术灵敏度低, 但大大简化。额外的 Ca2 +提升了钙绿色荧光, 线粒体的 Ca2 +吸收将荧光返回到基线。连续添加了 ca2 + , 直到线粒体未能摄取 extramitochondrial Ca2 + 9。荧光微板块读取器可用于连续报告钙绿色荧光。
在 ca2 +累积之后, 线粒体偏振光, 将 Ca2 +释放到介质中, 并开始膨胀。Ca2 +触发的线粒体肿胀用于指示 mPTP 开口。电子显微镜和光吸收率降低 540 nm 可用于测量 Ca2 +触发的线粒体肿胀10,11。线粒体体积可以直接确定的前角光散射12, 其中吸收减少反映线粒体基质的被动肿胀。
在这里, 我们说明了检查线粒体 Ca2 +保留容量和 Ca2 +触发线粒体肿胀从 SH-SY5Y 细胞分离的线粒体的方法。

<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

氧化磷酸化生产 ATP 是线粒体的主要功能。高真核生物中的线粒体也参与胞浆 Ca2 +缓冲, 线粒体中的 ATP 生产可以通过 intramitochondrial 自由 Ca2 +浓度进行介导。Ca2 +保留容量可以被视为线粒体在线粒体基质中保留钙的能力。累积胞内 Ca2 +导致内线粒体膜通透性, 称为线粒体通透性过渡孔 (mPTP) 的开口, 导致分子量小于 1.5 kDa 的分子泄漏。Ca2 +触发的线粒体肿胀用于指示 mPTP 开口。在这里, 我们描述了两种检测, 以检查 ca2 +保留容量和 ca2 +触发线粒体肿胀在孤立的线粒体。添加了一定数量的 Ca2 +后, 所有步骤都可以在一天内完成, 并由微板块读取器记录。因此, 这两种简单而有效的检测方法可以用来评估与 Ca2 +相关的线粒体功能。

Ca2 +保留容量的代表性结果: 结果以荧光值表示。随着 Ca2 + (200 nmols/毫克线粒体蛋白) 的额外脉冲, 荧光量在基线上增加了两个 mPTP 开口。5微米 Bongkrekate (BKA), 一抑制剂的 ca2 +诱导 mPTP 开放, 或1微米苍术苷二 (ATR), 一个激活剂的ca2 + 诱导 mPTP 开放, 增加了孤立的线粒体。添加的 ca2 +的总量, 直到在基线荧光以上的双增量指示的 ...

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

线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验

本协议的目的是描述一种检查 ca2 +保留容量和 ca2 +触发的 SH-SY5Y 细胞线粒体肿胀的方法逐步。

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

Research

JoVE Journal

Neuroscience

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

11.1K 次观看.  Qilu Hospital of Shandong University. 本协议的目的是描述一种检查 ca2 +保留容量和 ca2 +触发的 SH-SY5Y 细胞线粒体肿胀的方法逐步。

视频: 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.

在单个细胞中的线粒体DNA复制的可视化EDU信号放大

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

科研

神经科学

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.

神经调节和线粒体运输:在较长的时间里，海马神经元的实时成像

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.

活大鼠皮层神经元线粒体膜电位和活性氧的测定

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.

对检测方法果蝇行为

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.

在脑血管内皮细胞生物能量功能的评价

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.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

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.

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

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.

用于线粒体疾病建模的人脑类器官的生成

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.

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

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.

使用遗传编码的Ca2 + 指示剂（GECI）对星形胶质细胞和神经元中的线粒体Ca2 + 摄取进行成像

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