Overexpression of Norrin in the RPE of Transgenic Mice Protects Against Light-Induced Photoreceptor Damage

Abstract

Purpose: : Norrin is a secreted protein that controls capillary formation in the developing retina and acts via the Wnt/ß-catenin pathway. We tested if Norrin has neuroprotective effects on photoreceptors that are independent from its effects on vascular development.

Methods: : Transgenic Rpe65-Norrin mice that express norrin under the control of the Rpe65 promoter were developed and analyzed by Northern/Western blotting, and by immunohistochemistry. Wnt/ß-catenin signaling was investigated by analyzing ß-catenin, and by LacZ staining of mixed Rpe65-Norrin/TOP-Gal Wnt reporter mice. Apoptotic cell death was analyzed by TUNEL-labeling and histone ELISA. Outer nuclear layer thickness was measured on semithin sections. Retinal structure and function were tracked in vivo by SLO, OCT and ERG. Rhodopsin regeneration after bleaching was determined by measuring rhodopsin levels at different time points in darkness.

Results: : Retinae of Rpe65-Norrin mice showed a distinct increase in the amounts of Norrin, but otherwise expressed no obvious phenotype. Levels of retinal ß-catenin were higher, as was the intensity of LacZ staining in Rpe65-Norrin/TOP-Gal mice indicating activation of the Wnt/ß-catenin pathway. 30 hours after light-induced damage (white light, 5000 lux for 1 h), retinae of Rpe65-Norrin mice showed significantly fewer TUNEL-positive cells as compared to wild-type littermates, an effect that was supported by histone ELISA. 7 and 14 days after damage, the outer nuclear layer was significantly thicker in Rpe65-Norrin mice than in controls, indicating an increase in photoreceptor survival. By ERG, retinal function was significantly better in Rpe65-Norrin mice as compared to wild-type littermates. The effects on photoreceptor survival could be partially blocked by adding Dickkopf-1, an inhibitor of the Wnt/ß-catenin pathway. Metabolic rhodopsin regeneration was delayed in Rpe65-Norrin mice when compared to wild-type littermates.

Conclusions: : Transgenic overexpression of Norrin via the RPE protects photoreceptors from light-induced apoptotic cell death, indicating a neuroprotective role of Norrin. This effect might is mediated, at least partially, through the Wnt/ß-catenin pathway, and may act via an inhibition of metabolic rhodopsin regeneration.

Keywords: neuroprotection • photoreceptors • transgenics/knock-outs