DJ-1 Null Dopaminergic Neuronal Cells Exhibit Defects in Mitochondrial Function and Structure: Involvement of Mitochondrial Complex I Assembly

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O2 consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease.DJ-1是一种与帕金森病（Parkinson's Disease）相关的基因，其编码的蛋白质产物可通过清除胞质活性氧、维持线粒体功能，在细胞稳态中发挥保护作用。然而，目前尚不清楚DJ-1如何调控线粒体功能，以及DJ-1缺失为何会诱导线粒体功能障碍。在既往一项研究中，我们证实DJ-1基因敲除的多巴胺能神经元细胞存在线粒体呼吸链复合物I（Mitochondrial Respiratory Chain Complex I）活性缺陷。在本研究中，我们通过蓝色天然聚丙烯酰胺凝胶电泳（Blue Native-Polyacrylamide Gel Electrophoresis）与二维凝胶分析（2-dimensional Gel Analysis）评估天然复合物状态，以此探究DJ-1在复合物I组装中的作用。基于上述实验结果，我们得出结论：DJ-1基因敲除细胞存在复合物I组装缺陷。伴随复合物I组装异常，DJ-1基因敲除细胞同时出现超复合物（Supercomplex）形成缺陷。已知超复合物的异常形成会损害呼吸链复合物间通道的电子传递流，进而导致线粒体功能障碍。我们采用两种方法研究这些线粒体缺陷：第一种方法通过MitoTracker染色结合共聚焦显微镜（Confocal Microscopy）与电子显微镜，评估线粒体结构缺陷；第二种方法通过检测ATP生成量、氧气消耗量与线粒体膜电位，评估线粒体功能缺陷。最终我们证实，通过腺病毒介导的过表达（adenovirus-mediated overexpression），可逆转DJ-1基因敲除细胞中的组装缺陷以及结构与功能异常，证明了DJ-1对这些线粒体特性的特异性调控作用。由DJ-1突变诱发的上述线粒体缺陷，可能是帕金森病中多巴胺能神经元变性的病理机制之一。