A dual druggable genome-wide siRNA and compound library screening approach identifies modulators of parkin recruitment to mitochondria

Helen L Scott, Nicola Buckner, Francesc Fernandez-Albert, Elisa Pedone, Lorena Postiglione, Gongyu Shi, Nicholas Allen, Liang-Fong Wong, Lorenzo Magini, Lucia Marucci, Gregory O’Sullivan, Sarah Cole, Justin Powell, Peter Maycox, James B Uney

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Abstract

Genetic and biochemical evidence points to an association between mitochondrial dysfunction and Parkinson’s disease (PD). PD-associated mutations in several genes have been identified and include those encoding PTEN-induced putative kinase 1 (PINK1) and parkin. To identify genes, pathways, and pharmacological targets that modulate the clearance of damaged or old mitochondria (mitophagy), here we developed a high-content imaging-based assay of parkin recruitment to mitochondria and screened both a druggable genome-wide siRNA library and a small neuroactive compound library. We used a multi-parameter principal component analysis and an unbiased parameter-agnostic machine-learning approach to analyze the siRNA-based screening data. The hits identified in this analysis included specific genes of the ubiquitin proteasome system, and inhibition of ubiquitin-conjugating enzyme 2 N (UBE2N) with a specific antagonist, Bay 11-7082, indicated that UBE2N modulates parkin recruitment and downstream events in the mitophagy pathway. Screening of the compound library identified kenpaullone, an inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3), as a modulator of parkin recruitment. Validation studies revealed that kenpaullone augments the mitochondrial network and protects against the complex I inhibitor MPP+. Finally, we used a microfluidics platform to assess the timing of parkin recruitment to depolarized mitochondria and its modulation by kenpaullone in real time and with single-cell resolution. We demonstrate that the high-content imaging-based assay presented here is suitable for both genetic and pharmacological screening approaches, and we also provide evidence that pharmacological compounds modulate PINK1-dependent parkin recruitment.
Original languageEnglish
Pages (from-to)3285-3300
Number of pages16
JournalJournal of Biological Chemistry
Volume295
DOIs
Publication statusPublished - 6 Mar 2020

Keywords

  • mitochondria
  • parkin
  • ubiquitin
  • mitophagy
  • Parkinson’s
  • kenpaullone

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