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Derek S. Welsbie, Scott E. Martin, Katy L. Mitchell, Yan Ge, Zhiyong Yang, Donald J. Zack; High-throughput siRNA Screening in Primary Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3484.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is defined by the death of retinal ganglion cells (RGCs), the neurons that transmit vision from the retina to the brain. Current therapies all act by lowering the intraocular pressure (IOP). Unfortunately, lowering IOP can produce undesirable side effects and/or be difficult to achieve. Lacking are neuroprotective agents that directly interfere with the cell death process in RGCs. To identify such agents, we created a high-throughput drug target screening platform using RNA interference in primary mouse RGCs. We hypothesized that gene targets whose knockdown by small interfering RNAs (siRNAs) promotes survival would represent potential drug targets.
Primary mouse RGCs were isolated by immunopanning dissociated retinal cells from P0-4 pups using an antibody against the RGC surface antigen Thy1.1. Cells were transfected with siRNA using the NeuroMag reagent (Oz Biosciences). After 72 hours of culture in neutrophin-depleted media, viability was measured with a plate reader using CellTiter-Glo luminescence or with automated fluorescent imaging and calcein AM-staining.
A limited subset of the Ambion Silencer druggable library (2112 siRNAs covering 704 genes), including mostly kinases and G-protein coupled receptors, was screened in primary RGCs. Since no survival-promoting siRNAs were previously known, the Qiagen All-Stars #3 proprietary toxic siRNA mix served as the positive control for siRNA-mediated survival change. With this, screening achieved a Z-factor of 0.65. Moreover, duplicate screens using the same library had a correlation of determination coefficient (R2) of 0.63. Amongst the top survival-promoting hits was the endothelin receptor type B, previously shown to be important in RGC death. As a complementary approach, the same data set was probed for receptors whose knockdown promoted cell death (i.e. hypothesizing that the ligands may be neuroprotective). Amongst the most toxic receptor siRNAs were estrogen receptor alpha, melanocortin 2 receptor and cholecystokinin B receptor. Satisfyingly, estrogen receptor signaling is known to be neuroprotective to RGCs.
High-throughput siRNA screening in primary mouse RGCs can be used to identify potential neuroprotective agents. Ongoing efforts include the confirmation of our current hits using mouse models of optic neuropathy and an expanded, whole-genome siRNA screen.
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