Section of Clinical and Molecular Neurogenetics at the Department of Neurology, University of Lübeck, 23562 Lübeck, Germany.
J Biol Chem. 2013 Jan 25;288(4):2223-37. doi: 10.1074/jbc.M112.391680. Epub 2012 Dec 4.
Mutations in the E3 ubiquitin ligase Parkin and the mitochondrial PTEN-induced putative kinase 1 (PINK1) have been identified to cause autosomal recessive forms of familial Parkinson disease, with PINK1 functioning upstream of Parkin in a pathway important for the maintenance of mitochondrial function and morphology. Upon the loss of the mitochondrial membrane potential, Parkin translocates to mitochondria in a PINK1-dependent manner to ubiquitinate mitochondrial proteins. Parkin-mediated polyubiquitination of outer mitochondrial membrane (OMM) proteins recruits the ubiquitin- and LC3-binding adaptor protein p62 to mitochondria and induces mitophagy. Although previous studies examined mitophagy in established cell lines through overexpression approaches, there is an imperative to study the role of endogenous Parkin and PINK1 in human-derived and biologically relevant cell models. Here, we demonstrate in human primary fibroblasts and induced pluripotent stem-derived neurons from controls and PINK1 mutation carriers that endogenous levels of Parkin are not sufficient to initiate mitophagy upon loss of the mitochondrial membrane potential, caused by its (self-)ubiquitination, followed by degradation via the ubiquitin proteasome system. Next, we showed differential PINK1-dependent, Parkin-mediated ubiquitination of OMM proteins, which is Parkin dose-dependent and affects primarily OMM proteins of higher molecular mass. In contrast to the situation fibroblasts, we did not detect mitophagy in induced pluripotent stem-derived neurons even upon overexpression of Parkin. Taken together, our data demonstrate that mitophagy differs between human non-neuronal and neuronal cells and between "endogenous" and "Parkin-overexpressing" cellular models.
E3 泛素连接酶 Parkin 和线粒体 PTEN 诱导的假定激酶 1（PINK1）的突变已被确定为常染色体隐性家族性帕金森病的致病原因，PINK1 在维持线粒体功能和形态的途径中位于 Parkin 的上游。在线粒体膜电位丧失时，Parkin 以 PINK1 依赖的方式易位到线粒体，泛素化线粒体蛋白。Parkin 介导的外膜（OMM）蛋白多泛素化募集泛素和 LC3 结合衔接蛋白 p62 到线粒体，并诱导自噬。尽管先前的研究通过过表达方法研究了已建立的细胞系中的自噬，但有必要在人类来源的和具有生物学相关性的细胞模型中研究内源性 Parkin 和 PINK1 的作用。在这里，我们在对照和 PINK1 突变携带者的人原代成纤维细胞和诱导多能干细胞衍生神经元中证明，在由其自身泛素化引起的线粒体膜电位丧失后，内源性 Parkin 水平不足以引发自噬，随后通过泛素蛋白酶体系统降解。接下来，我们显示了 OMM 蛋白的 PINK1 依赖性、Parkin 介导的泛素化的差异，这是 Parkin 剂量依赖性的，主要影响较高分子量的 OMM 蛋白。与成纤维细胞的情况相反，即使过表达 Parkin，我们也没有在诱导多能干细胞衍生神经元中检测到自噬。总之，我们的数据表明，人非神经元和神经元细胞之间以及“内源性”和“Parkin 过表达”细胞模型之间的自噬存在差异。
