Genome-wide high-content siRNA screens for regulators of Parkin translocation in selective mitophagy (Ambion Library)
An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson's (1). Recent studies of the PD-associated genes PINK12 and PARK2 (Parkin) (3) suggest that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria (4-7). Following the loss of mitochondrial more ..
High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy
An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson's (1). Recent studies of the PD-associated genes PINK12 and PARK2 (Parkin) (3) suggest that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria (4-7). Following the loss of mitochondrial membrane potential, the kinase Pink1 and the E3-ubiquitin ligase Parkin coordinate a rapid proteasomal (8) and autophagic (mitophagy) (6) response that may attenuate cell death (9-11). Given that enhanced mitophagy may ameliorate the deleterious consequences of mitochondrial dysfunction (12-14), we hypothesized that genes regulating Parkin translocation would be useful drug targets for increasing mitochondrial quality control. However, the mechanism of Parkin recruitment following Pink1 accumulation on the outer mitochondrial membrane is too poorly understood to target, and may involve a host of coordinated events. To identify genes important for the Pink1-dependent recruitment of Parkin to damaged mitochondria, we conducted two diverse genome-wide siRNA screens with high-throughput automation. Our screening approach took advantage of the well-characterized phenotype of Parkin accumulation on depolarized mitochondria in cells (6). Cells stably expressing GFP-Parkin and a mitochondrial-targeted red fluorescent protein were transfected with siRNA duplexes in 384-well plates. After a 48 hour siRNA knockdown, the proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to each well to induce rapid mitochondrial depolarization. Following bulk mitochondrial depolarization in all cells within each well, GFP-Parkin translocation to the damaged mitochondria was allowed to progress for 2.5 hours and then cells were fixed. The degree of Parkin translocation to the mitochondria of each cell was assessed by high-content analysis. Primary screening yielded gene candidates involved in an array of diverse cellular processes that were validated in confirmatory assays.
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Parkin, PARK2, PINK1, mitophagy, autophagy, CCCP, mitochondrial damage, Parkinson's disease, neurodegeneration, siRNA, RNAi screen, HTS, genome-wide, functional genomics, assay, high content screening, 384 well, National Institutes of Health, National Center for Advancing Translational Sciences, NIH Center for Translational Therapeutics, National Institute of Neurological Disorders and Stroke
† RNAi Target.
Data Table (Concise)