Fluorescent reporter of Caenorhabditis elegans Parkin: Regulators of its abundance and role in autophagy-lysosomal dynamics

Roman Vozdek; Bingying Wang; Kathy H Li; Peter P Pramstaller; Andrew A Hicks; Dengke K Ma

doi:10.12688/openreseurope.14235.2

Fluorescent reporter of Caenorhabditis elegans Parkin: Regulators of its abundance and role in autophagy-lysosomal dynamics

Open Res Eur. 2023 Sep 15:2:23. doi: 10.12688/openreseurope.14235.2. eCollection 2022.

Authors

Roman Vozdek¹, Bingying Wang², Kathy H Li³, Peter P Pramstaller¹, Andrew A Hicks¹, Dengke K Ma²

Affiliations

¹ Institute for Biomedicine, Eurac Research, Affiliated institute of the University of Lübeck, Bolzano, 39100, Italy.
² Cardiovascular Research Institute and Department of Physiology, University of California San Francisco, San Francisco, CA, 94158, USA.
³ Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, 94158, USA.

Abstract

Background: Parkin, which when mutated leads to early-onset Parkinson's disease, acts as an E3 ubiquitin ligase. How Parkin is regulated for selective protein and organelle targeting is not well understood. Here, we used protein interactor and genetic screens in Caenorhabditis elegans ( C. elegans) to identify new regulators of Parkin abundance and showed their impact on autophagy-lysosomal dynamics and alpha-Synuclein processing. Methods: We generated a transgene encoding mCherry-tagged C. elegans Parkin - Parkinson's Disease Related 1 (PDR-1). We performed protein interactor screen using Co-immunoprecipitation followed by mass spectrometry analysis to identify putative interacting partners of PDR-1. Ribonucleic acid interference (RNAi) screen and an unbiased mutagenesis screen were used to identify genes regulating PDR-1 abundance. Confocal microscopy was used for the identification of the subcellular localization of PDR-1 and alpha-Synuclein processing. Results: We show that the mCherry::pdr-1 transgene rescues the mitochondrial phenotype of pdr-1 mutants and that the expressed PDR-1 reporter is localized in the cytosol with enriched compartmentalization in the autophagy-lysosomal system. We determined that the transgenic overexpression of the PDR-1 reporter, due to inactivated small interfering RNA (siRNA) generation pathway, disrupts autophagy-lysosomal dynamics. From the RNAi screen of putative PDR-1 interactors we found that the inactivated Adenine Nucleotide Translocator ant-1.1/hANT, or hybrid ubiquitin genes ubq-2/h UBA52 and ubl-1/h RPS27A encoding a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively, induced PDR-1 abundance and affected lysosomal dynamics. In addition, we demonstrate that the abundant PDR-1 plays a role in alpha-Synuclein processing. Conclusions: These data show that the abundant reporter of C. elegans Parkin affects the autophagy-lysosomal system together with alpha-Synuclein processing which can help in understanding the pathology in Parkin-related diseases.

Keywords: C. elegans; Parkin; Parkinson’s disease; RNA interference; Synuclein; autophagy; genetic screen.

Grants and funding

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 844497. This research was also partly funded by the Department of Educational Assistance, University and Research of the Autonomous Province of Bolzano, Italy, through a core funding initiative to the Institute for Biomedicine.