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

• Applicant: Dr. Sven Geisler
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2016 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 288418809

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.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Shushant Jain