High Content genomweiter RNAi Screen der Parkin abhängigen Eliminierung von depolarisierten Mitochondrien

01.09.2016 bis 31.08.2019

The aim of this study is to find new components which are involved in the PINK1/Parkin dependent elimination of depolarized mitochondria, termed mitophagy. Within distinct forms of Parkinson’s disease (PD) the failure of the monogenetic recessive gene products PINK1 and Parkin contributes to a severe deregulation of the mitochondrial homeostasis. PD patients, who carry mutations in the PINK1 or Parkin gene, show altered mitochondria morphology, reduced mitochondrial membrane potential, respiratory chain defects and enhanced oxidative stress. Thus, it is crucial for the cell to possess a precise mechanism to identify non-functional mitochondria targeting them for degradation and hence maintain a healthy mitochondrial network.
The failure of mitochondria function can be simulated in a cell culture model via uncoupling the mitochondrial membrane potential using the protonophore CCCP. This starts a cascade to remove non-functional mitochondria. First, PINK1 is stabilized on the outer mitochondrial membrane with low membrane potential. Second, Parkin and components of the ubiquitin proteasome system are recruited to these mitochondria. Third, proteins of the outer mitochondrial membrane like the mitofusins 1 and 2 (Mfn1, Mfn2), the subunits of the translocase of the outer membrane 20 and 70 (TOM20, TOM70) and the voltage dependent anion channel 1 (VDAC1) are ubiquitinated in a Parkin dependent manner. Fourth, p62, an autophagic adaptor binds the ubiquitin chains on mitochondria and recruits the lipidated form of LC3, a component of the autophagic machinery. Fifth, the mitochondria are surrounded by an autophagic membrane which subsequently fuses with the lysosomes to form the autophagolysosomes. Finally, the non-functional mitochondria are degraded within the lysosomes as whole organelles.
In a well established HeLa cell model of mitochondrial depolarization and mitophagy the 2h and 24h time point of CCCP treatment were frequently investigated. A key step of the process is the stabilization of PINK1 and the recruitment of Parkin to mitochondria. This occurs typically within two hours (2h) after depolarization. In the last years several whole genome siRNA based high-content imaging screens revealed numerous new regulatory proteins of Parkin translocation and greatly extended the knowledge of the molecular process of mitophagy. However, the other interesting time point is the complete elimination of mitochondria after 24 hours (24h) of depolarization. To our knowledge, a screen of this time point using a siRNA based whole genome or sub-library was not performed up to date. Such a screen would additionally reveal new players participating in mitophagy beyond the well investigated Parkin translocation. Thus, we expect a more refined view on the whole process of mitophagy and create a complete new layer of potential druggable candidate proteins in the process of mitophagy.