We would like to thank Dr. Harry Orr at the University of Minnesota for his generous gift of transgenic mice. We would also like to thank the following Skidmore College alum for their work performing the preceding experiments: Monica Villegas, Porter Hall, Mitchell Spring, Nicholas Toker, Jenny Zhang, Chloe Larson and Cheyanne Slocum. Furthermore, we would like to thank Skidmore College for funding the development of these methods.

该协议遵循的斯基德莫尔学院IACUC准则小鼠工作。 1.治疗水溶性化合物<ol><li>溶解琥珀酸在笼饮用水0.75毫克/毫升的浓度。注意，在所需的浓度感兴趣的任何水溶性化合物可以在这个阶段被取代。搅拌溶液，以确保该化合物完全溶解。 </li><li>小鼠达到治疗的所需岁以后，与在步骤1.1的溶液更换处理条件组的家笼的饮用水。 </li><li>测量每日两实验组水瓶的重量，以确保两个处理的和对照小鼠饮用的水的量相同。使用这些测量结果计算每笼总药物剂量。继续在整个实验和监测瓶重量的治疗。 </li></ol> 2.足迹分析<ol><li>跑道放置到地板或在密闭室的表。在对与床上用品和食物奖励两旁置箱小幅上升的角度（ 如含糖的谷物）设置了跑道。行跑道的长度与一张白纸。允许受试者对中置箱适应5分钟。 </li><li>除去从主箱主体和油漆左和右后腿，分别以蓝色和红色无毒水性涂料。油漆打印将被用来计算多个输出行为的测量。 </li><li>放置在跑道的开端的主体，并允许该受步行到家庭框。一旦这个问题已经达到了置箱，关闭活门，并允许受回到各自的家笼前呆在家里框内一分钟。清洗，用30％的乙醇在跑道和置箱并更换被褥，奖励食品，并为下一个主题跑道纸张。 </li><li>量化采取匝进展的数目，栅极宽度，步骤的角度，以及数通过根据先前6中所述的分析方法的主题。一个成功的足迹试验被定义为最小的被检体朝向目标框步行5顺序脚印没有转身或停止。 </li></ol> 3.束分析<ol><li>根据与以下6束先前描述6条截面规格设置束装置:梁1 =矩形28毫米宽;梁2 =圆形，直径28毫米;光束3 =矩形12毫米宽;梁4 =圆形，直径为12毫米;梁5 =长方形，6mm宽;梁6 =圆形，直径6毫米。在梁的一端，设置与奖励食物昏暗置箱。 </li><li>培养在梁主体，组装与射束3.装置轻轻地放置在光束相对置箱的端部的主体，并允许该主体在整个行走。允许每个主题最多三个2分钟的试验，以实现成功运行，其定义为穿越波束和在2分钟内进入最后框。在试验之间，让中置箱受试者休息2分钟。在主体之间，清洗，用30％的乙醇在梁和置箱并更换奖励食品中的下一个主题制备。 </li><li>重复步骤3.2，每天一次，共导致的连续3天的训练，未来两天。测试当天，第4天，应遵循顺序培训天。 </li><li>为了测试第4天的主题，第一个组装与梁1设备和步骤3.1的描述设置置箱。放置一个摄像机在装置的前部，并验证充分束装置可以在视频被清晰地捕捉。开始录制，并放置在横梁上对面的家框中的第一个问题，允许最多3 2分钟尝试成功完成审判。在试验间，让受休息中置箱2分钟。 </li><li>继续测试科目上的每个数字顺序六梁，从而开始下梁前须休息中置箱2分钟。主体之间，清洁每个波束并用30％的乙醇置箱和替换奖励食品中为每个主题制剂。 <ol><li>录像带每个试验并记录每束每个受试者试验取得成功的人数如下:"1"表示该主题是成功的第一个试验中，"2"表示主题是成功的第二个试验中，"3"表示主题是成功的第三个和最后一个试验中，和"4"表示对象没有成功的任何三个试验。 </li></ol></li><li>谁是失明的课题实验条件的得分手使用的试验测量footslips录像。定义一个footslip因为这是在下面穿过梁的长度的理论中线照相机浸渍的直接视图后足。从多个得分平均footslip分数。 </li><li>分析的位数uccessful试验和footslips数据的数目，计算出每个基因型和实验条件队列内的平均和标准误差。进行单向ANOVA和多重比较事后分析，以确定是否有治疗和基因型上成功的试验和footslips的数目的数目的效果。 </li></ol> （四）加快转棒<ol><li>适应科目之前，在审判开始试验室10分钟。在此适应期，准备旋转杆装置中，在清洁装置的各泳道，用30％的乙醇。 </li><li>打开转棒软件，并设置启动设置为4转速，加速度设置5分钟，最终速度设置，以40转。这些设置提供来自4的恒定加速度 - 40rpm下在一段5分钟。小鼠可以以40rpm保持在转棒以外的加速期为10分钟的最大游程长度的附加5分钟。 </li><li>后适应，地方主体Øn要在各自的车道旋转杆，而杆4转速进行旋转。一旦所有科目都在各自的车道，开始了第一次审判。记录落使用仪器软件的延迟;二次计时器可以用作备份。 </li><li>试用之后完成，让每一个受其余在各自的车道水库为前下试，防止过度疲劳十分钟。 </li><li>总共四个试验重复步骤4.3和4.4。 </li><li>重复步骤4.3 - 4.5，共连续四天，持续整个试验期间的待遇。完成后，量化延迟脱落旋转杆每科目6每次试验。 </li></ol> 5.小脑提取和固定<ol><li>根据机构IACUC指导原则通过二氧化碳窒息安乐死小鼠。放置受试者入二氧化碳室中，并释放二氧化碳进入腔室。看在步实施安乐死在任何时候都动物和不离开动物无人值守。 </li><li>一旦呼吸已经停止和主题显示了由后足压力没有剩余的疼痛感，去除室内主题，并通过从头骨中间的上皮组织卓越正中切口尾端头骨通过上皮组织的基础。 </li><li>用解剖剪，通过矢状缝到前囟门切断从基部头骨在脊髓。通过每个冠状缝用钝钳脱下额叶，顶叶和interparietal骨前横向切割。 </li><li>使用钝钳骨外侧脱落小脑。使用镊子小脑从丘和脑干中分离并从组织的其余部分将其挤出。分离小脑与镊子左，右半球。 </li><li> 立即将右小脑成液态氮保存HPLC ANA裂解和左小脑成预冷4％低聚甲醛（PFA），用于组织固定。切断主动脉和丢弃根据机构IACUC规则的尸体前收获任何其他感兴趣的组织。 注意:PFA是一种剧毒，易燃，腐蚀性。 </li><li>二十四小时后提取，放置组织成30％蔗糖于4℃多达洗前在磷酸盐缓冲盐水（PBS），其固定在PFA（在PBS中的4％）组织（三次，5分钟/洗涤） 2周。 </li></ol> 6.免疫病理分析<ol><li>附加雪橇切片机的管道到水槽水龙头和温度控制箱。使用温度控制箱，冷却切片机阶段至-25℃。切片厚度旋钮设置为50微米。 注:如果在雪橇切片表盘未达到50微米时，转盘设置为较薄的设置，并通过相应地移位设置相乘的厚度（例如设定对dIAL至10μm和切片为50微米 ）的总厚度之前移位五次。 </li><li>民建联最佳温度切割（OTC）解决方案的一个硬币大小的部分搬上了舞台。场外冻结之前，使用产钳，这样正中矢状切面朝下场外层定位半球。迅速工作，使得半球的横向前端尖朝向叶片，优于阶段轻轻定向钳组织。允许OTC完全冻结。 <ol><li>在图层，添加额外的OTC周围的组织，允许OTC添加到下一层之前完全冻结。在组织和冻结的顶部添加场外的薄层。 </li></ol></li><li>部的组织，等于体重散发到切割刀片而切片。收集使用精细的艺术家的画笔和地方部分为含有1x PBS适当标记的孔板。部分之间，重新设置在切割厚度雪橇切片表盘，如果需要的话，至50微米。 </li><li>在孔板用尿素溶液[0.01M的尿素的PBS]的每个孔取代的PBS并将板在冰上。准备就绪后，每一次揭露抗原表位的免疫标记去除冰和沸腾部分板块中的尿素溶液三次15秒。该步骤可以通过将板的加热块设定为沸腾温度上很容易地完成。每沸腾步骤之间，含凉冰的组织切片板。 注:如果在执行脲，阻断，初级抗体，二级抗体和DAPI步骤在96孔板，在整个使用100μL的体积。如果使用不同大小的孔，相应地调整试剂卷。代替加热块的，微波炉可用于煮沸组织样品中的尿素。在高功率设置了三个15秒的间隔微波炉样本。 </li><li>交的沸腾，洗部分三次，每次除去本解决方案，取代了溶液用1×PBS并搅拌10分钟。 </li><li>块部分用驴血清溶液（3％正常驴血清，0.3％的Triton X-100的PBS）通过在血清中，在室温下轻轻搅动温育组织为一小时。删除驴血清的解决方案。 </li><li>用0.2微克/毫升的抗钙结合蛋白和1标签部分:在驴血清溶液2000抗共济失调蛋白-1抗体（11NQ）16和孵育在4℃下温和搅拌72小时。 </li><li>在4℃（在驴血清溶液稀释500的Alexa Fluor-594抗兔抗体:500的Alexa Fluor-488抗山羊和1 1）培养在适当的二级抗体部分之前用PBS洗部分如在步骤6.5对温和搅拌48小时。 </li><li>洗切片用PBS三倍在步骤6.5之前用DAPI标记细胞核（4&#39;，6-二脒基-2-苯基吲）溶液稀释至0.5微克/毫升的在PBS中的终浓度。 DAPI潜伏期最长不超过10分钟。删除搜索解决方案n和用PBS洗如在步骤6.5。 </li><li>安装组织到2％明胶包被的载玻片用封固剂，以减少光漂白。盖玻片并用指甲油密封。 </li><li>用扫描共聚焦显微镜下找到明光小脑的主要裂缝。用10X UIS2目标，扫描并用DM488 / 559毫微米依次激发DAPI，AlexaFluor-488，并用405nm的二极管激光器，一个488nm的氩气体激光器和一个559纳米二极管激光器，分别AlexaFluor-594信道，在结合二色分光镜。单独使用SDM490和SDM560光束分离器和一个BA575-675带通滤波器，分别收集发射的光。 <ol><li>对于每个通道，构造对于z = 20微米，步长=0.1μm的初级裂隙内的z栈。使用设置有共焦显微镜的后处理软件，以在一个大小标记物加入到最终的图像。 注:另类激光器，过滤器和荧光团可以代替基于availabiliTY。如果AlexaFluor-488或AlexaFluor-594检测弱，磷化镓（磷砷化镓）高灵敏度的探测器可以使用（如果可用）来增强信号。 </li></ol></li></ol> 7.量化分子层的厚度和浦肯野细胞数<ol><li>使用ImageJ，打开彩色主裂的Z堆栈图像并从图像选项卡菜单栏，"颜色"和"分离通道"从颜色中选择"图像"每个图像成它的红色，蓝色和绿色通道分割标签。 </li><li>创建分裂每个信道的最大ž投影。当感兴趣的通道图像是开放的，从菜单栏中选择"图像"，从"节"选项卡中的图像选项卡和"Z-项目&#39;&#39;节&#39;。中的"Z-工程"命令菜单中，选择"最大投影ž"，然后单击"好"。重复每个通道。 </li><li>从日选择"插件"打开细胞计数插件Ë菜单栏，从插件选项卡"分析"，并从分析选项卡"细胞计数"。 注:ImageJ的和ImageJ的细胞计数插件可以在材料和设备的表中提供的网站上下载。 </li><li>通过计数共济失调蛋白-1标记的细胞核沿着一个预先确定的200微米拉伸主裂缝的前部和后部的长度位于量化浦肯野细胞的数目。从菜单栏中选择"图像"从图像选项卡和"门槛"从调整选项卡使用门槛地图，"调整"，使所关心的通道（红色的门槛地图中共济失调-1标有核的情况下， ）。 <ol><li>选择，可以在整个的量化（31-225像素例如）进行归一化的像素范围。从检查的阈值窗口中的"黑后台"框图像丢弃的噪音。图像信号可以被反向为便于计数。 </li></ol></li><li>量化分子层THICkness在使用"拆分频道"和"阈值地图的"工具如在步骤7.1和7.2的绿色通道。利用每个图像为指导的大小标记，随机测量中的每个的两个指定每个主裂缝图像为200微米绵延3分子层的长度。使在浦肯野细胞体到浦肯野树突的前端的基部从浦肯野树突的近端测量。 </li><li>记录浦肯野细胞计数和分子层厚度为平均值加或减标准误差。与多重比较进行单向ANOVA 事后分析，以测试的处理条件或基因型上细胞的数目和树突的长度的影响。 </li></ol> 8.小脑琥珀酸含量治疗后的HPLC分析<ol><li> 分析试样的制备 <ol><li>之前权衡每个收获右侧小脑半球在HPLC分析使用。剁碎半球半部逐个在预冷Dounce匀浆时间和添加的75:25 0.5毫升（按体积）水:甲醇溶液到每个均化半17。 </li><li>采用窄预冷匀浆，严重打破了组织5 DOUNCE招。继续与更广泛的均质精细讳言每个组织样本，运用20 DOUNCE招。 </li><li>转移匀浆预冷的微量离心管。每个样品后，冲洗用0.5mL冰冷的水的均化:甲醇溶液和漂洗添加到离心管中。 </li><li>离心匀浆样品在在25℃下1300×g离心30分钟，并收集在一个干净的微量离心管的上清液。 </li><li>通过安置与10千道尔顿（kDa）的标称分子量极限的再生纤维素过滤器的离心式过滤器单元进行过滤上清液。如果没有立即运行HPLC分析，储存在-80°过滤样本C。 </li></ol></li><li> HPLC仪器和条件 <ol><li>具有至少30厘米的长度和7μm的粒度到高性能液相色谱仪附加一个离子排斥色谱柱。 </li><li>制备和脱气调节到pH为5 1mM的乙酸缓冲液的足够量。 </li><li>设定的为0.8ml /分钟流动相速度的HPLC中。如果使用UV-Vis检测器，选择224纳米的检测器波长。在这些条件下典型的洗脱时间对于琥珀酸为10和12分钟之间。 </li></ol></li><li> 运行和分析的样品和标准 <ol><li>准备一组与0和250ppm的之间的琥珀酸浓度乙酸盐缓冲液流动相的标准。 </li><li>运行每个标准，并使用从每个运行测得的峰面积制成校正曲线。 </li><li>一旦获得准确的标准曲线，运行在乙酸盐缓冲液稀释预先制备的样品。用于测量的精确度更高，可对样品进行琥珀酸已知浓度已经在流动相中稀释被掺入。一式三份运行的所有样本。 </li><li>分析HPLC色谱通过使用校准曲线，然后通过样品体积乘以与由原始样品的质量除以以确定样品的琥珀酸浓度。 </li></ol></li></ol>使用氧电极控制单元小脑线粒体功能9. 离体分析<ol><li>通过首先施加氯化钾一滴创建使用市售系统Clark型电极（50％的KCl重量/体积）在铂阴极溶液，切割1.5厘米的2片商业烟草的卷烟纸和过轻轻放置纸铂阴极。 <ol><li>覆盖阴极和围绕阳极有2.5 平方厘米片聚四氟乙烯（PTFE）纸和紧贴地固定两个MEMBranes与O型圈，确保有膜内没有折叠或气泡。滴50％KCl溶液到周围的井。 </li></ol></li><li>固定预制电极进入试验室，并与空气饱和的水填充。加热腔室至30℃，添加0.8厘米搅拌棒到该溶液中，并使用仪器的软件以70rpm搅拌。通过建立零氧校准室中。这样做，串联还原剂连二亚硫酸钠的几晶体添加到该溶液中。 <ol><li>一旦校准完成，用70％乙醇洗室一次，然后使该腔室，以1.5毫升呼吸缓冲适应（5mM的MgCl 2的，60毫米氯化钾，100mM的甘露糖醇，10mM的前洗净，用去离子水六倍KH 2 PO 4，0.5毫的 Na 2 EDTA，60mM的Tris-盐酸[pH为7.4]）7。 注:连二亚硫酸钠的所有痕迹必须完全在洗涤步骤，尝试测量之前取出呼吸实验过程中的氧浓度。 </li></ol></li><li>轻轻均匀称量小脑半球于0.5mL匀浆缓冲液（0.25M蔗糖，0.5mM的EDTA，50mM的Tris-盐酸[pH为7.4]）。匀浆转移到一个干净的1.5毫升离心管中，并保持冷藏，直到开始试验。 </li><li>匀浆添加到腔室，达到2毫升的最终体积。通过加入毛地黄皂苷或皂苷（5毫克/毫升）的40微升透小脑匀浆;允许匀浆组织孵育10分钟。 注:毛地黄皂苷和皂苷是有毒的，并且温育时间应保持在最低限度。 </li><li>要开始录制的耗氧量，通过激活和使用分别为底物和抑制剂，氧化磷酸化链（每个基板3分钟录音）的抑制确定线粒体功能。 <ol><li>添加使用清洁针具如下基板:10微升2M的谷氨酸[终浓度（FC）0.01 M]。5μL的0.8M的苹果酸[FC 2毫]，20微升的0.5M腺苷二磷酸（ADP）FC的5mM]，1微升1mM的鱼藤酮[FC 0.5μM]，20μL的1M琥珀酸[10毫]，1微升5毫抗霉素A [FC 2.5μM]，1微升0.8M的抗坏血酸[FC 0.4毫]和5微升0.2M的四甲基 - 对苯二胺（TMPD）。 [FC 0.5毫米]在添加基材室要小心，不要引入气泡进入系统。 注:每个底物和抑制剂引起的功能性作用的详细信息在先前公布的协议7中描述。个体剂量 - 反应曲线，可能需要产生，以确定以前没有研究了制剂/组织/种类的底物和抑制剂的最佳浓度。同样地，该协议可以用于组织的先天代谢倾向被优化/物种被研究，例如作为底物偏好。 </li></ol></li><li>一旦数据采集完成后，轻轻一spirate小脑匀浆并清洗用乙醇和去离子水的腔室。一旦清洁，附加小脑匀浆样品可以在不附加的校准，只要在PTFE膜保持完整运行。最终完成，清洁电极用去离子水和乙醇和存储用硅胶或干燥剂后。 注:间同一天运行，填补了室内用去离子水确保膜不会干。 </li><li>使用仪器软件创建的数据表，开始2分钟呼吸导出数据的40分钟后加入底物到电子表格程序。 </li><li>计算每个底物或抑制剂的呼吸速率。由抗坏血酸TMPD衬底加入期间除以呼吸速率的总呼吸正常化的数据。 注:呼吸速率可进一步归一化到组织中的蛋白质含量可以是在神经变性的实验特别期望在该组织中的含量可以变化。这样做，保留50微升样品匀浆（来自步骤9.3）的用于在标准蛋白质测定中使用。 </li></ol>

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作者什么都没有透露。

如果如所述使用这些方法，它们是能够检测和小脑的神经变性疾病的小鼠模型减轻氧化磷酸化介导的线粒体功能障碍。将合并的生物化学和行为测定是用于确定对小脑的神经变性疾病的病理线粒体贡献程度多方面的方法。通过与琥珀酸治疗小鼠，刺激新陈代谢，促进线粒体功能，我们能够显示小脑行为缺陷和小脑变性的救援。 这里提出的方法可以用于测试多种水溶性代谢靶?...

线粒体是三磷酸腺苷（ATP），为细胞能量一种必需的辅酶的关键生产，大多数使用电子传递链通过氧化磷酸化（OXPHOS）产生的线粒体ATP。脑，鉴于其高代谢需求及对氧化磷酸化进行供电的神经活动的依赖，是高度敏感的线粒体功能障碍。其结果是，线粒体功能障碍是在老化过程中触发 1，并在多个神经变性疾病2，3，4的发病机制有关。因此，它遵循线粒体是神经变性吸引力的治疗目标。 在这个协议中，我们已经采用脊髓小脑性共济失调1型（SCA1）作为模型神经退行性疾病线粒体的研究升功能障碍和线粒体靶向疗法的开发。 SCA1是通过在共济失调蛋白-1基因产物触发小脑浦肯野神经元和神经元其他脑区域中的进行性变性一个多聚谷氨酰胺（的polyQ）重复扩张突变引起的。此处所用的转基因小鼠系（指定为SCA1小鼠），它表达了浦肯野细胞特异性启动子的控制下的polyQ突变共济失调蛋白-1基因，允许SCA1 5的浦肯野细胞成分的针对性的分析。 SCA1小鼠进行逐步浦肯野细胞变性和发展共济失调步态6。 线粒体复合物功能障碍和线粒体靶向治疗功效可与分子和行为分析的电池进行评价。线粒体复合物功能障碍是测定体外通过检测在小脑组织内改变氧消耗呼吸测定存在电子传递链底物和抑制剂7。呼吸检测先前已经用于透化组织，线粒体分离物，和全组织7，8，9。它们允许不同形态的数据收集方法，如透射电子显微镜或免疫荧光染色线粒体功能的直接评估。利用整个组织，而不是孤立的线粒体防止在分离过程7可能出现的健康线粒体的偏倚选择。当如图适于协议，所述呼吸检测是在小脑的神经变性疾病状态检测线粒体功能障碍的有价值的方法。 代谢的非特异性活化剂可以用来推断线粒体功能障碍的神经变性diseas的转基因小鼠模型e和在新疗法的发展援助。槲皮素，辅酶Q10和肌酸都被证明改善神经变性疾病的病理的患者，并在神经变性疾病10，11，12，13，14，15，16的动物模型。这里，我们提出了一种新的代谢活化剂，琥珀酸，刺激代谢，促进线粒体功能中的神经变性疾病。以确保该活化剂穿越血脑屏障，高效液相色谱法检测在处理的小鼠17递送至神经组织。 为了评价如琥珀酸代谢靶向水溶性化合物的治疗效果，可以使用行为范例和免疫病理研究的电池。杜E要在小脑的神经变性疾病，足迹跑道试验，梁法和加速旋转杆实验发现，运动协调障碍是用来检测行为病理6，18，19日抢救。这些措施是由一个定义的小脑组织6，20，21的小叶内评估分子层的厚度（定义为浦肯野细胞树突长度）和浦肯野细胞胞体计数补充有小脑细胞构筑的免疫病理评估。在这里，我们提出了诊断和治疗的线粒体功能障碍与代谢有针对性的水溶性化合物多的神经病理学和行为的方法。 我们使用线粒体呼吸体外分析来分析线粒体功能障碍在SCA1 TRANsgenic鼠标。此外，我们表明，疾病的症状和病理学由水溶性线粒体增压琥珀酸改进，还牵连于SCA1疾病进展的线粒体功能障碍。

<table><tbody><tr><td>Adenosine diphosphate</td><td>Sigma Aldrich</td><td>A2754</td><td>ADP</td></tr><tr><td>Ascorbate</td><td>Sigma Aldrich</td><td>A7631</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A2153</td><td>BSA</td></tr><tr><td>4&#039;,6-Diamidino-2-phenylindole</td><td>Sigma Aldrich</td><td>D9542</td><td>DAPI</td></tr><tr><td>Digitonin</td><td>Sigma Aldrich</td><td>D141</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma Aldrich</td><td>D0632</td><td>DTT</td></tr><tr><td>Donkey serum</td><td>Sigma Aldrich</td><td>D9663</td><td></td></tr><tr><td>Glutamate</td><td>Sigma Aldrich</td><td>1446600</td><td></td></tr><tr><td>Malate</td><td>Sigma Aldrich</td><td>6994</td><td></td></tr><tr><td>Mannitol</td><td>Sigma Aldrich</td><td>M4125</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>Potassium-lactobionate</td><td>Bio-Sugars</td><td>69313-67-3</td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td></td></tr><tr><td>Saponin</td><td>Sigma Aldrich</td><td>47036</td><td></td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S3674</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-Tetramethyl-&lt;em&gt;p&lt;/em&gt;-phenylenediamine</td><td>Sigma Aldrich</td><td>T7394</td><td>TMPD</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T9284</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U0631</td><td></td></tr><tr><td>Vectashield mounting medium</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>11NQ antibody (anti-ataxin-1 )</td><td></td><td></td><td>Servadio, &lt;i&gt;et al.&lt;/i&gt; 1995, PMID: 7647801</td></tr><tr><td>Alexa Fluor 488 anti-mouse secondary antibody</td><td>Life Technologies</td><td>A-11015</td><td></td></tr><tr><td>Alexa Fluor 594 anti-rabbit secondary antibody</td><td>Life Technologies</td><td>A-11012</td><td></td></tr><tr><td>Calbindin antibody (goat)</td><td>Santa Cruz</td><td>C-20</td><td></td></tr><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Control transgenic mice</td><td>Harry Orr, Ph.D.</td><td>A02</td><td>Burright, &lt;i&gt;et al.&lt;/i&gt; 1997, PMID: 9217978</td></tr><tr><td>SCA1 mice</td><td>Harry Orr, Ph.D.</td><td>B05</td><td>Burright, &lt;i&gt;et al.&lt;/i&gt; 1997, PMID: 9217978</td></tr><tr><td>Wildtype mice</td><td>The Jackson Laboratory</td><td>001800</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ESM-100L microtome</td><td>ERMA</td><td></td><td>Sledge microtome</td></tr><tr><td>Fluoview FV1200 Confocal Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Glycerol-gelatin slides</td><td>FD Neuro Technologies</td><td>PO101</td><td></td></tr><tr><td>Hamilton syringe</td><td>Sigma Aldrich</td><td>VCAT 80465</td><td></td></tr><tr><td>OXYT1 Oxytherm Electrode Control Unit</td><td>Hansatech Instruments</td><td></td><td></td></tr><tr><td>P.T.F.E. paper</td><td>Cole-Parmer</td><td>UX-08277-15</td><td></td></tr><tr><td>Rotallion Rotarod</td><td>PPP&amp;amp;G</td><td></td><td>contact corresponding author for information</td></tr><tr><td>Ultimate 3000 HPLC</td><td>Dionex</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health</td><td></td><td>&lt;a href=&quot;http://imagej.nih.gov/ij/&quot;&gt;http://imagej.nih.gov/ij/&lt;/a&gt;</td></tr><tr><td>Cell counter plugin (for ImageJ)</td><td>National Institute of Health</td><td></td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij/plugins/cell-counter.html&quot;&gt;http://rsb.info.nih.gov/ij/plugins/cell-counter.html&lt;/a&gt;</td></tr><tr><td>3P&amp;amp;G Rota-Rod v3.3.3 (rotarod software)</td><td>PPP&amp;amp;G</td><td></td><td>contact corresponding author for information</td></tr><tr><td>Phidget21.dll (required for rotarod software)</td><td>DLL-Files.com</td><td></td><td>&lt;a href=&quot;https://www.dll-files.com/phidget21.dll.html&quot;&gt;https://www.dll-files.com/phidget21.dll.html&lt;/a&gt;</td></tr></tbody></table>

treating sca1 mice with water-soluble compounds to non-specifically boost mitochondrial function

线粒体功能障碍起着老化过程，并在神经退行性疾病，包括遗传性的几个小脑萎缩症和小脑进行性变性标记等运动障碍一个显著的作用。该协议的目的是评估在脊髓小脑性共济失调1型（SCA1）线粒体功能障碍，并通过该水溶性化合物琥珀酸评估代谢呼吸药理定位的功效减缓疾病进展。这种方法也适用于其他小脑疾病和可适应的水溶性治疗的宿主。 线粒体呼吸的离体分析被用于检测和量化在线粒体功能疾病相关的变化。遗传证据（未发表的数据），并在SCA1小鼠模型线粒体功能障碍蛋白质组学的证据，我们评价与水溶性代谢增压待遇的功效uccinic酸由该化合物直接溶解到笼子的饮用水。药物的通过血脑屏障的能力可使用高效液相色谱（HPLC）来推导。这些化合物的疗效然后可以使用多种行为范式，包括加速转棒，平衡木试验和足迹分析测试。小脑Cytoarchitectural完整性可以使用检测浦肯野细胞的细胞核和浦肯野细胞树突和胞体免疫荧光法进行评估。这些方法是用于确定线粒体功能障碍和治疗与小脑的神经变性疾病的水溶性化合物的功效健壮的技术。

通过与琥珀酸小脑线粒体的药理学靶向我们能够防止线粒体功能障碍在小脑的神经变性疾病SCA1的小鼠模型。琥珀酸脱氢酶，琥珀酸，所述的典型电子供体中治疗和神经病理学评估的第二周溶于SCA1小鼠家笼饮用水一个月，与行为评估开始以下处理（ 图 1A）。由足迹分析，梁法和加速旋转杆法测定琥珀酸治疗22 SCA1提高小鼠的表现?...

Watch this Scientific Journal Video about Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function at JoVE.com

用水溶性化合物治疗 SCA1 小鼠以非特异性增强线粒体功能医学|视频

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.

治疗SCA1小鼠水溶性化合物，以非特异性升压线粒体功能

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ...

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10.5K 次观看.  Skidmore College. We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases. 

视频: Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

科研

环境科学

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

生物化学

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

发育生物学

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

利用细胞外通量分析探索单个3D微组织球体的线粒体能量代谢

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

癌症研究

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

用于超复合物分析的小组织样品中的线粒体分离

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

作者聚焦：揭示氧化磷酸化系统动力学和线粒体在健康和疾病中的作用

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析 

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

治疗SCA1小鼠水溶性化合物，以非特异性升压线粒体功能

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检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

利用细胞外通量分析探索单个3D微组织球体的线粒体能量代谢

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析

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