Down-regulation of apoptosis mediators by RNAi inhibits axotomy-induced retinal ganglion cell death in vivo

Abstract

Transection of the optic nerve induces an apoptotic degeneration of retinal ganglion cells (RGC) in the rat retina. The immediate early gene c-Jun, the proapoptotic Bcl-2 family member Bax and the apoptosome constituent Apaf-1 have been shown previously to play major roles in the induction or execution of the apoptosis cascade. In this study we have designed and generated short interfering RNAs (siRNAs) against c-Jun, Bax and Apaf-1, which were injected into the optic nerve stump in order to inhibit axotomy-induced apoptosis. siRNAs were first tested in vitro to ensure silencing efficiency. In vivo, a clear neuronal localization of Cy3-labelled siRNA could be visualized in retinal flat mounts. Retinas that were injected with anti-Apaf-1- and anti-c-Jun-siRNA showed significantly more surviving RGC than non-injected or anti-EGFP-injected controls (approximately 2- to 3-fold, respectively). Anti-Bax-siRNA-injected retinas showed a trend towards an increased RGC number (not significant). Regulation of target proteins in situ could be visualized by immunohistochemical stainings. We conclude that (i) c-Jun and Apaf-1 play major roles in the apoptotic cascade of RGC and may represent useful targets for antiapoptotic strategies in RGC in vivo, and (ii) injection of siRNAs into the optic nerve stump is a new method to down-regulate target genes specifically in RGC.

Kv1.1 and Kv1.3 channels contribute to the degeneration of retinal ganglion cells after optic nerve transection in vivo

“…Retinas were fixed 24 h later. Retrograde transport to the RGC somata from the cut nerve stump has been well characterized 8,24,26,38,39 and, as expected, essentially all RGCs were labeled with siRNA or Fluorogold (Figure 3a). (ii) For the remaining experiments, RGCs were retrograde labeled with Fluorogold by briefly placing a 2-mm 3 piece of Gelfoam soaked in 5 ml of 3% Fluorogold (false-colored green) against the freshly cut optic nerve stump.…”

“…Transcript expression was monitored for several genes known to have important roles in RGC death after axotomy. 5,[6][7][8]26,27 Quantitative real-time reverse transcriptase-PCR (qRT-PCR) on whole retinas was conducted at 7 days after axotomy, a time when RGC apoptosis is well underway but before these cells are lost. 3,8 First, mRNA expression in naive healthy retinas was compared with axotomized control retinas that had been treated with control siRNA against firefly luciferase.…”

“…A similar siRNA injection technique has been used to knockdown apoptosis-related proteins and transcription factors. 26 As a control treatment throughout these studies, an irrelevant, Cy3-labeled siRNA directed against a nonmammalian protein, firefly luciferase (Upstate, Charlottesville, VA, USA) was injected. The Kv-channel-specific siRNAs (Table 1) were synthesized using in vitro transcription procedures (Silencer siRNA kit, Ambion, Austin, TX, USA) according to the manufacturer's protocol.…”

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

“…The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6,7,[13][14][15] . Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve [16][17][18][19] or injecting vectors into their target the superior colliculus 10 . This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia.…”

Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders

“…Sectioning of the optic nerve results in massive apoptosis of retinal ganglion cells and is felt to be relevant to the gradual apoptosis of retinal ganglion cells that occurs due to high intraocular pressure in glaucomatous eyes. 23 Lingor et al 24 used siRNAs directed against c-Jun, apoptotic protease-activating factor-1 (Apaf-1), or Bax to investigate their role in axotomy-induced apoptosis of retinal ganglion cells. They used a clever strategy of injecting siRNA into the proximal stump of the optic nerve, and using Cy-3-labeled siRNA they demonstrated that this technique results in specific transduction of retinal ganglion cells.…”