Objective: We aimed to identify genome-wide regulators of the PINK1-Parkin pathway using a novel flow cytometry reporter, endogenously tagged MFN2, as a readout of the endogenous PINK1-Parkin activity.

Background: Mutations in PINK1 and PRKN (encoding Parkin) cause Parkinson’s disease (PD), the second most common neurodegenerative disorder. These proteins are responsible for regulating a mitochondrial specific autophagy (mitophagy) pathway.  Disruptions in this mitochondrial quality control mechanism have been associated with PD. Traditional strategies to assess PINK1-Parkin activation focus on monitoring mitophagy. However, this approach lacks sensitivity in the setting of physiologic Parkin concentrations. We took an orthogonal approach of monitoring degradation of a preferred Parkin substrate, MFN2, which is robustly degraded via the proteosome following ubiquitination by Parkin.

Method: We performed a genome-wide CRISPRi screen utilizing a pooled dual-guide library in HEK293 cells expressing dCas9-ZIM3 with endogenously tagged MFN2-HaloTag. Cells were left untreated or treated with the mitochondrial uncoupler CCCP to activate the PINK1-Parkin pathway. Samples were sorted via fluorescence activated cell sorting to collect the low and high MFN2-expressing cells from each condition. Finally, genomic DNA was extracted followed by next generation sequencing to determine guide counts from each population.

Results: Flow cytometry and immunoblotting verified that MFN2 levels were decreased following activation of endogenous Parkin in a PINK1-dependent manner. As expected, the results of the screen indicate that PINK1 and Parkin were among the gene perturbations required for MFN2 degradation. Other top hits included the E2 required for Parkin ubiquitin charging, VCP and its chaperones for MFN2 removal, proteasomal subunits for its degradation, and key components of the mitochondrial integrated stress response pathway. Additionally, we identified transcription factors that control the gain of the PINK1-Parkin mitophagy pathway.

Conclusion: To our knowledge we completed the first endogenous screens of the PINK1-Parkin mitophagy pathway utilizing a MFN2-Halo reporter. This approach will provide insight into regulators of this pathway that may have been overlooked in previous screens utilizing exogenous Parkin, identifying potential targets for treatment of neurodegenerative disorders like PD.

« Back to 2024 International Congress

MDS Abstracts - https://www.mdsabstracts.org/abstract/a-novel-single-cell-reporter-identifies-regulators-of-the-endogenous-pink1-parkin-mitochondrial-quality-control-pathway/