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

本提案的目的是寻找和研究新的调节剂，这些调节剂参与PINK1/Parkin依赖性去极化线粒体的消除，称为线粒体自噬。散发性帕金森病的病因在很大程度上是未知的。然而，在罕见的单基因形式的疾病的基础上，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PINK1和Parkin突变和失败的患者中，观察到线粒体稳态严重失调。据推测，线粒体功能异常可能导致神经元死亡。因此，对于细胞来说，拥有一个精确的机制来识别功能失调的线粒体是至关重要的，靶向它们进行降解，从而维持一个健康的线粒体网络。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PINK1和Parkin依赖级联去除功能失调的线粒体。PINK1和Parkin稳定在线粒体外膜上，膜电位低，导致泛素附着在这些细胞器上。这作为自噬机制最终消除溶酶体内功能失调的细胞器的信号。在建立的HeLa细胞线粒体去极化和线粒体自噬模型中，经常观察CCCP治疗的2h （Parkin易位）和24h（线粒体自噬）时间点。在过去的几年里，一些基于全基因组siRNA的高含量成像屏幕揭示了许多新的帕金易位调节蛋白。因此，对有丝自噬早期分子过程的认识可以大大扩展。然而，另一个有趣的时间点是线粒体在去极化24小时后完全消除。据我们所知，目前还没有使用基于siRNA的全基因组文库或亚基因组文库对这一时间点的人类细胞进行筛选。我们将建立这样一个筛选有丝分裂的最后一步，我们将确定新的调节蛋白。然后对有前途的调制剂的分子功能进行表征。在这里，我们将使用不同的方法确定线粒体自噬模型的确切功能、定位、相互作用和活性。我们尝试用不同的抑制剂或刺激剂来调节这些功能，以恢复功能失调线粒体的移除。因此，我们期望对线粒体自噬的整个过程有一个更精细的认识，并在线粒体自噬过程中出现新的候选蛋白层。

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.