Mou Yongchao, Mukte Sukhada, Chai Eric, Dein Joshua, Li Xue-Jun
Department of Biomedical Sciences, University of Illinois College of Medicine Rockford; Department of Bioengineering, University of Illinois at Chicago.
Department of Biomedical Sciences, University of Illinois College of Medicine Rockford.
J Vis Exp. 2020 Feb 9(156). doi: 10.3791/60548.
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 "MultiKymograph", "Bioformat importer", and "Macros" 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.
神经元为支持其功能对高能量有强烈需求。在人类神经元中已观察到沿轴突的线粒体运输受损，这可能在各种疾病状态下导致神经退行性变。尽管在活体人类神经中检查线粒体动力学具有挑战性，但此类范例对于研究线粒体在神经退行性变中的作用至关重要。本文描述了一种用于分析源自人类诱导多能干细胞（iPSC）的前脑神经元轴突中线粒体运输和线粒体形态的方案。使用成熟方法将iPSC分化为端脑谷氨酸能神经元。用MitoTracker CMXRos对神经元的线粒体进行染色，并使用配备细胞培养箱的活细胞成像显微镜捕获轴突内的线粒体运动。使用带有“MultiKymograph”、“Bioformat importer”和“Macros”插件的软件分析延时图像。生成线粒体运输的动态图像，并从动态图像中读取顺行和逆行方向的平均线粒体速度。关于线粒体形态分析，使用ImageJ获得线粒体长度、面积和纵横比。总之，该方案允许对沿轴突的线粒体运输进行表征并分析其形态，以促进神经退行性疾病的研究。
