Mortiboys Heather, Thomas Kelly Jean, Koopman Werner J H, Klaffke Stefanie, Abou-Sleiman Patrick, Olpin Simon, Wood Nicholas W, Willems Peter H G M, Smeitink Jan A M, Cookson Mark R, Bandmann Oliver
Academic Neurology Unit, Medical School, University of Sheffield, Sheffield, United Kingdom.
Ann Neurol. 2008 Nov;64(5):555-65. doi: 10.1002/ana.21492.
There are marked mitochondrial abnormalities in parkin-knock-out Drosophila and other model systems. The aim of our study was to determine mitochondrial function and morphology in parkin-mutant patients. We also investigated whether pharmacological rescue of impaired mitochondrial function may be possible in parkin-mutant human tissue.
We used three sets of techniques, namely, biochemical measurements of mitochondrial function, quantitative morphology, and live cell imaging of functional connectivity to assess the mitochondrial respiratory chain, the outer shape and connectivity of the mitochondria, and their functional inner connectivity in fibroblasts from patients with homozygous or compound heterozygous parkin mutations.
Parkin-mutant cells had lower mitochondrial complex I activity and complex I-linked adenosine triphosphate production, which correlated with a greater degree of mitochondrial branching, suggesting that the functional and morphological effects of parkin are related. Knockdown of parkin in control fibroblasts confirmed that parkin deficiency is sufficient to explain these mitochondrial effects. In contrast, 50% knockdown of parkin, mimicking haploinsufficiency in human patient tissue, did not result in impaired mitochondrial function or morphology. Fluorescence recovery after photobleaching assays demonstrated a lower level of functional connectivity of the mitochondrial matrix, which further worsened after rotenone exposure. Treatment with experimental neuroprotective compounds resulted in a rescue of the mitochondrial membrane potential.
Our study demonstrates marked abnormalities of mitochondrial function and morphology in parkin-mutant patients and provides proof-of-principle data for the potential usefulness of this new model system as a tool to screen for disease-modifying compounds in genetically homogenous parkinsonian disorders.
在帕金森蛋白敲除的果蝇和其他模型系统中存在明显的线粒体异常。我们研究的目的是确定帕金森蛋白突变患者的线粒体功能和形态。我们还研究了在帕金森蛋白突变的人体组织中，受损的线粒体功能是否有可能通过药物挽救。
我们使用了三组技术，即线粒体功能的生化测量、定量形态学以及功能连接的活细胞成像，以评估纯合或复合杂合帕金森蛋白突变患者成纤维细胞中的线粒体呼吸链、线粒体的外形和连接性，以及它们的功能性内部连接。
帕金森蛋白突变细胞的线粒体复合物I活性和与复合物I相关的三磷酸腺苷生成较低，这与线粒体分支程度较高相关，表明帕金森蛋白的功能和形态学效应是相关的。在对照成纤维细胞中敲低帕金森蛋白证实，帕金森蛋白缺乏足以解释这些线粒体效应。相比之下，敲低50%的帕金森蛋白，模拟人类患者组织中的单倍剂量不足，并未导致线粒体功能或形态受损。光漂白后荧光恢复测定表明线粒体基质的功能连接水平较低，在鱼藤酮暴露后进一步恶化。用实验性神经保护化合物治疗可挽救线粒体膜电位。
我们的研究证明了帕金森蛋白突变患者中线粒体功能和形态存在明显异常，并为这种新模型系统作为筛选遗传性帕金森病中疾病修饰化合物的工具的潜在有用性提供了原理验证数据。
