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Derek Stuart Welsbie, Katherine L. Mitchell, Jessica Kim, Scott E. Martin, John A Fuller, Zhiyong Yang, Yan Ge, Eugen Buehler, Cindy Berlinicke, Donald J Zack; A whole-genome, RNAi-based screen identifies activating transcription factor 2 (ATF2) genotropic activity as a key mediator of retinal ganglion cell death. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2593.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a leading cause of irreversible blindness worldwide. Vision loss is caused by the axonal injury and death of retinal ganglion cells (RGCs), the projection neurons that process and transmit vision from the retina to the brain. In order to identify, in a comprehensive and unbiased manner, genes that could serve as novel targets for neuroprotective glaucoma therapy, we performed the first genome-wide RNAi-based screen in primary RGCs.
For this new screen, we developed a high-throughput method for knocking down gene expression in primary RGCs with small interfering RNA oligonucleotides (siRNAs) and coupled it with a quantitative assay of RGC survival. Using this approach, we screened an arrayed library of 52,728 siRNAs, providing three-fold coverage of the mouse genome, for the ability to promote the survival of axotomized RGCs. 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. After 72 hours of culture, viability was measured with a plate reader using an ATP-based luminescence assay.
Amongst the top genes whose siRNAs promoted survival was activating transcription factor 2 (ATF2), known to be regulated by c-Jun N-terminal kinases (JNKs) - key mediators of RGC cell death. In other cell types, ATF2 has been shown to trigger cell death through both mitochondrial and nuclear/transcriptional pathways. To distinguish between these, we took advantage of mice carrying an allele of ATF2 (ATF2f/f) in which the DNA-binding domain is flanked by loxP sites. In response to the P1 bacteriophage Cre recombinase, the genotropic function of ATF2 is selectively disrupted. RGCs were isolated from ATF2f/f mice and treated with adenovirus expressing Cre or a GFP control. Deletion of the DNA-binding domain resulted in robust survival, similar to ATF2 knockdown. Using these same mice, we were also able to validate our in vitro findings in the mouse optic nerve crush model of axonal injury.
These results suggest that ATF2’s genotropic function may be a critical link between the dual-leucine zipper kinase/JNK signaling cascade and the pro-apoptotic transcriptional response in axotomized RGCs.
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