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RNAi Screen Finds Regulators of Parkin, Suggests Possible Therapeutic Targets

NEW YORK (GenomeWeb News) – Using genome-wide RNAi screens, Richard Youle at the US National Institute of Neurological Disorders and Stroke and his colleagues have identified a bevy of regulators of parkin, a gene that is closely linked with Parkinson's disease, as they reported in the online, early edition of Nature yesterday.

Mitochondrial dysfunction has increasingly been linked to the development of neurodegenerative diseases like Parkinson's. Some Parkinson's disease cases have been linked to parkin, whose protein typically acts as a sort of label on damaged mitochondria that marks them for destruction by the cell's lysosomes. However, some mutations in the parkin gene prevent that tagging process from happening, which then leads to the buildup of dysfunctional mitochondria in the cell.

Youle and his colleagues turned to RNAi screens to uncover other genes whose proteins assist parkin and its associated protein PINK1, in the tagging operation and identified at least four.

"We discovered a network of genes that may regulate the disposal of dysfunctional mitochondria, opening the door to new drug targets for Parkinson's disease and other disorders," Youle, an investigator at NINDS, said in a statement.

According to NINDS, Parkinson's disease, which is marked by tremors, is both chronic and progressive, and has no cure, though there are therapies that treat the symptoms of the disease.

To find other genes involved in the process, the researchers constructed a HeLa cell line that stably expressed GFP-parkin as well as a mitochondrial-targeted red fluorescent protein, which would identify siRNAs that deplete mitochondria.

Drawing on two diverse libraries, they performed a genome-wide RNAi screen in that cell line. After the cells, which were in 384-well plates, were treated with siRNA, the researchers chemically depleted the mitochondrial membrane potential to imitate the pathological condition of the disease.

The researchers determined the degree of parkin translocation in the cells through high-content microscopy image analysis.

Some 24 candidate genes overlapped between the two screens.

After factoring in possible off-target and housekeeping gene effects, the researchers selected 106 candidate genes for validation, of which 67 appeared to inhibit parkin translocation with two additional siRNA reagents and based on qRT-PCR assays.

One validated gene, TOMM7, makes up part of the protein translocase of the outer mitochondrial membrane, and when the researchers knocked down TOMM7 in the HeLa cells, there was a decrease in GFP-parkin translocation as well as the expected decrease of TOMM7 mRNA. Additionally, while a TOMM7 knock-out cell line exhibited a lack of TOMM7, the levels of PINK1 remained unchanged.

However, after CCCP treatment, which induces parkin translocation, full-length PINK1 did not accumulate in the cells. Based on that and successive experiments of radiolabeled PINK1, the researchers concluded that a main function of TOMM7 is to transport and keep PINK1 at the surface of damaged mitochondria.

Additionally, in human induced pluripotent stem cell-derived neurons, the researchers knocked down TOMM7 using lentiviral short hairpin RNA. Similar to the other cells, a decrease in TOMM7 was associated with a decline in PINK1 accumulation after mitochondrial depolarization. TOMM7, the researchers reported, likely recruits parkin to the mitochondria through stabilizing PINK1 in neurons that express tyrosine hydroxylase.

HSPA1L and BAG4, meanwhile, appeared to be positive and negative co-regulators of parkin function, respectively. Knocking down HSPA1L decreased parkin translocation while knocking BAG4 down led to its increase, an effect they confirmed through TALEN editing.

Knocking down both genes, though, diminished the phenotype, the researchers noted, indicating that a balance of the two proteins was needed to regulate parkin translocation.

Additionally, SIAH3 looks to be a negative regulator of PINK1 stabilization. Its knockdown did not increase the levels of PINK1 mRNA or protein before mitochondrial damage, though it did after CCCP treatment.

"These genes work like quality control agents in a variety of cell types, including neurons," Youle added. "The identification of these helper genes provides the research community with new information that may improve our understanding of Parkinson's disease and other neurological disorders."

The screening data from this study, the researchers noted, have been included in PubChem.

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