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RNA Interference

RNAi Screen for Genes Involved in the Recruitment of Parkin to Damaged Mitochondria

Parkinson’s disease is a movement disorder characterized by tremor, stiffness of limbs, slow movement, and impaired balance and coordination. The symptoms result from death of cells in the brain that make a chemical important for movement. Studies suggest that this cell death may be caused by damage to a structure within cells crucial for cell functioning: the mitochondria. Soon after mitochondria are damaged, a protein called Parkin helps repair them and prevents cell death. The investigators will screen for small interfering RNAs (siRNAs), which selectively and systematically inhibit gene activity, to find genes that enable Parkin to respond to mitochondrial injury. The information gained from this study could help scientists find drugs that enhance this repair response and better treat neurodegenerative diseases such as Parkinson’s.

NIH scientists used RNA interference to find genes that interact with parkin (green), a protein that tags damaged mitochondria (red). Mutations in parkin are linked to Parkinson’s disease and other mitochondrial disorders. (NINDS Photo/Youle Laboratory)

NIH scientists used RNA interference to find genes that interact with parkin (green), a protein that tags damaged mitochondria (red). Mutations in parkin are linked to Parkinson’s disease and other mitochondrial disorders. (NINDS Photo/Youle Laboratory)

Scientific Synopsis

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson's. Following mitochondrial damage and the loss of mitochondrial membrane potential, the proteins PINK1 and Parkin coordinate a rapid response that can attenuate cell death. Given that enhancing this response may help elude the consequences of mitochondrial dysfunction, genes associated with the localization of Parkin to damaged mitochondria could serve as important drug targets. Unfortunately, the exact mechanism of this response is poorly understood. The aim of this study is to use genome-wide siRNA screens to identify genes involved in the recruitment of Parkin to damaged mitochondria. Screening has yielded many candidates and a number of these have been extensively validated by the Youle lab at the National Institute of Neurological Disorders and Stroke. These efforts will provide a better understanding of mitochondrial quality control, and may inform on therapeutic targets for neurodegenerative diseases such as Parkinson’s.

Lead Collaborators

National Institute of Neurological Disorders and Stroke, Bethesda, Maryland
Richard Youle, Ph.D.

George Washington University, Washington, D.C.
Wenge Zhu, Ph.D.

Public Health Impact

These efforts will provide a better understanding of mitochondrial quality control and may inform on therapeutic targets for neurodegenerative diseases such as Parkinson’s.

Publication

Hasson, S.A., Kane, L.A., Yamano, K., et al. High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy. Nature, Nov. 24, 2013.

Outcomes

These studies have implicated numerous genes in the recruitment of Parkin to damaged mitochondria. Follow-up studies have further validated several genes, including TOMM7, which was found to be essential for stabilizing PINK1 on the outer mitochondrial membrane following damage.

Project Details

Hela cells stably expressing GFP-Parkin and a mitochondrial-targeted red fluorescent protein are transfected with siRNA duplexes in 384-well plates. After a 48 hour siRNA knockdown, the proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is added to each well to induce rapid mitochondrial depolarization. Following bulk mitochondrial depolarization in all cells, GFP-Parkin translocation to damaged mitochondria is allowed to progress for 2.5 hours before cells are fixed. The degree of Parkin translocation to mitochondria is then assessed by high-content acquisition and analysis.

Screen for Genes Involved in the Recruitment of Parkin to Damaged Mitochondria

Schematic representation of the workflow involved in a small interfering RNA (siRNA) screen to find genes involved in parkin translocation from damaged mitochondria. Workflow steps include culturing cells in a 384-well plate, transfecting the cells with siRNAs, treating with an inducer of mitochondrial damage, fixing the cells, and imaging and quantifying parkin translocation.

Left image thumbnail: GFP-Parkin is translocated to the site of damaged mitochondria. Right image thumbnail: Translocation is blocked through siRNA knockdown of PINK1.

Last updated: 06-25-2015
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