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Dual druggable genome siRNA and compound screening approaches identify novel modulators of parkin recruitment to mitochondria

Research output: Contribution to journalArticle (Academic Journal)

Original languageEnglish
JournalJournal of Biological Chemistry
DOIs
DateSubmitted - 1 Oct 2019
DateAccepted/In press - 7 Jan 2020
DatePublished (current) - 7 Jan 2020

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.

    Research areas

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

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    Rights statement: This is the final published version of the article (version of record). It first appeared online via ASBMB Publications at https://www.jbc.org/content/295/10/3285. Please refer to any applicable terms of use of the publisher.

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