Targeted Sirna-Mediated Knockdown of Pro-Apoptotic Genes Delays Adult Retinal Ganglion Cell Death in vivo

Abstract

Purpose: : To assess the efficacy of short interfering RNA (siRNA)-mediated knockdown of pro-apoptotic genes in the survival of adult rat retinal ganglion cells (RGCs) after optic nerve axotomy. We focused on targeted silencing of p53 and its binding partners ASPP1 and ASPP2; and REDD2, a paralogue of RTP801 (REDD1), the latter being known as implicated in hypoxia, ischemia and oxidative stress. Although these targets are known players in the regulation of cell survival and damage; their precise role in RGC death has yet to be characterized.

Methods: : RGCs were labeled by application of the retrograde tracer Fluorogold in the superior colliculus. Optic nerve axotomy was performed one week after retrograde labeling of RGCs. Intraocular injections of siRNAs were performed at the time of axotomy and 1 week later by independent infusion of each siRNA compound into the vitreous chamber of the left eye. A siRNA targeting GFP was used as control. The density of surviving RGCs was quantified at 1 and 2 weeks after optic nerve injury by counting Fluorogold-labeled neurons in 12 standard retinal areas. Delivery of siRNA to RGC was confirmed using Cy3-labeled siRNA and fluorescent microscopy.

Results: : Cy3-siRNA was detected within RGCs as early as 5 hours after intraocular injection. Our data demonstrate that administration of siRNAs against ASPP1, ASPP2 and REDD2 led to striking neuroprotection of axotomized RGCs: ~ 75% of the total RGC population were alive at 1 week after axotomy, and ~25% survived at 2 weeks post-injury, a time when <10% RGCs remained in retinas treated with control siRNA. Intriguingly, treatment with siRNA against p53, which effectively knocks down p53 gene expression, had no effect on RGC survival.

Conclusions: : Our study demonstrates that siRNAs against pro-apoptotic targets can effectively delay RGC death after acute optic nerve injury.

Keywords: apoptosis/cell death • RNAi • neuroprotection