Ferro Austin, Carbone Emily, Marzouk Evan, Siegel Asher, Nguyen Donna, Polley Kailen, Hartman Jessilyn, Frederick Kimberley, Ives Stephen, Lagalwar Sarita
Neuroscience Program, Skidmore College.
Chemistry Department, Skidmore College.
J Vis Exp. 2017 Jan 22(119):53758. doi: 10.3791/53758.
Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies. Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.
线粒体功能障碍在衰老过程以及神经退行性疾病中发挥着重要作用，这些疾病包括几种遗传性脊髓小脑共济失调以及其他以小脑进行性退化为特征的运动障碍。本实验方案的目的是评估1型脊髓小脑共济失调（SCA1）中的线粒体功能障碍，并评估通过水溶性化合物琥珀酸对代谢呼吸进行药物靶向治疗以减缓疾病进展的疗效。这种方法适用于其他小脑疾病，并且可以适用于许多水溶性疗法。线粒体呼吸的体外分析用于检测和量化线粒体功能中与疾病相关的变化。基于SCA1小鼠模型中线粒体功能障碍的遗传学证据（未发表数据）和蛋白质组学证据，我们通过将这种化合物直接溶解到笼内饮用水中来评估水溶性代谢增强剂琥珀酸的治疗效果。可以使用高效液相色谱法（HPLC）推断该药物通过血脑屏障的能力。然后可以使用多种行为范式来测试这些化合物的疗效，包括加速转棒试验、平衡木试验和足迹分析。可以使用检测浦肯野细胞核以及浦肯野细胞树突和胞体的免疫荧光测定法来评估小脑的细胞结构完整性。这些方法是确定小脑神经退行性疾病中线粒体功能障碍以及水溶性化合物治疗效果的可靠技术。
