High-content genome-wide RNAi screen of the Parkin dependent elimination of depolarized mitochondria

该建议的目的是发现和研究参与PINK 1/Parkin依赖性消除去极化线粒体的新调节剂，称为线粒体自噬。散发性帕金森病的原因在很大程度上是未知的。然而，基于罕见的单基因形式的疾病，人们试图深入了解帕金森病的分子疾病进展。例如，在单基因隐性基因产物PINK 1和Parkin突变和失败的患者中，观察到线粒体稳态的严重失调。预计这种功能障碍的线粒体可能导致神经元死亡。因此，细胞必须拥有一种精确的机制来识别功能失调的线粒体，靶向它们进行降解，从而维持健康的线粒体网络。线粒体功能的失效可以在细胞培养模型中通过使用质子载体CCCP解偶联线粒体膜电位来模拟。这启动了PINK 1和Parkin依赖性级联反应，以清除功能失调的线粒体。PINK 1和Parkin稳定在具有低膜电位的线粒体的线粒体外膜上，这导致泛素附着在这些细胞器上。这作为自噬机制的信号，最终消除溶酶体内功能失调的细胞器。在已建立的线粒体去极化和线粒体自噬的HeLa细胞模型中，经常研究CCCP处理的2 h（帕金易位）和24 h（线粒体自噬）时间点。近年来，基于全基因组siRNA的高容量成像筛选发现了许多新的Parkin易位调控蛋白。因此，线粒体自噬早期的分子过程的知识可以大大扩展。然而，另一个有趣的时间点是去极化24小时后线粒体完全消除。据我们所知，迄今为止尚未使用基于siRNA的全基因组文库或子文库在该时间点的人细胞中进行筛选。我们将为线粒体自噬的最后一步建立这样的筛选，我们将鉴定新的调节蛋白。然后将表征有前途的调节剂的分子功能。在这里，我们将使用不同的方法确定线粒体自噬模型中的确切功能、定位、相互作用和活性。我们试图用不同的抑制剂或刺激剂来调节这些功能，以恢复功能失调的线粒体的去除。因此，我们期望对线粒体自噬的整个过程有一个更精细的看法，并在线粒体自噬过程中出现一个新的候选蛋白质层。

The aim of this proposal is to find and to investigate new modulators which are involved in the PINK1/Parkin dependent elimination of depolarized mitochondria, termed mitophagy. The causes of sporadic Parkinsons disease are largely unknown. However, on the basis of rare monogenetic forms of the disease one try to gain insight into the molecular disease progression of Parkinsons Disease. For example in patients with mutations and failure of the monogenetic recessive gene products PINK1 and Parkin, a severe deregulation of the mitochondrial homeostasis was observed. It is expected, that such malfunctioned mitochondria may contribute to the death of neurons. Thus, it is crucial for the cell to possess a precise mechanism to identify dysfunctional mitochondria, targeting them for degradation and hence maintain a healthy mitochondrial network. The failure of mitochondrial function can be simulated in a cell culture model via uncoupling the mitochondrial membrane potential using the protonophore CCCP. This starts a PINK1 and Parkin dependent cascade to remove dysfunctional mitochondria. PINK1 and Parkin are stabilized on the outer mitochondrial membrane of mitochondria with low membrane potential which leads to attachment of ubiquitin on these organelles. This serves as signal for the autophagic machinery to finally eliminate the dysfunctional organelles within the lysosomes. In an established HeLa cell model of mitochondrial depolarization and mitophagy the 2h (Parkin translocation) and 24h (mitophagy) time point of CCCP treatment were frequently investigated. In the last years several whole genome siRNA based high-content imaging screens revealed numerous new regulatory proteins of Parkin translocation. Thus, the knowledge of the molecular processes early in mitophagy could be greatly extended. However, the other interesting time point is the complete elimination of mitochondria after 24 hours of depolarization. To our knowledge, a screen in human cells of this time point using a siRNA based whole genome library or a sub-library was not performed up to date. We will establish such a screen for the final step of mitophagy and we will identify new regulatory proteins. Then the molecular function(s) of promising modulators will be characterized. Here, we will determine the exact function, the localization, the interactions and the activity in the mitophagy model using different methods. We try to regulate these functions with different inhibitors or stimulators to restore the removal of dysfunctional mitochondria. Thus, we expect a more refined view on the whole process of mitophagy and a new layer of candidate proteins in the process of mitophagy would emerge.