11–Cis Retinal Corrects Cone Opsin Mislocalization in Rpe65–/– Mice

Abstract

Abstract: : Purpose: RPE65 is required for the generation of 11–cis retinal, the chromophore for rod and cone opsins. Cones in the Rpe65^–/– retina degenerate rapidly, eliminating most cones by P28. Administration of 11–cis retinal has been shown to partially prevent cone loss if administed by P14. This project was designed to investigate whether cone functionality can be increased upon supplying 11–cis retinal. Methods: Rpe65^–/–::Rho^–/– mice were used to remove any interference of rods and compared to wild–type (wt) mice. Pups were injected intraperitoneally with 11–cis retinal for a total of four times, starting at P10, and maintained in complete darkness. At P25, cone function was assessed using single–flash and flicker ERGs; cone survival was determined immunohistochemically with cone–specific antibodies; and estimates of cone pigment were obtained by quantitative RT–PCR. Results: (1) At P25 a minute cone electroretinogram could be elicited from Rpe65^–/–::Rho^–/– mice after careful averaging.. Cone density was drastically reduced and cone opsin mRNAs were down–regulated ∼4–fold when compared to wt mice. Confocal microscopy using cone opsin specific antibodies showed that in the remaining cones the cone apoproteins were mislocalized and transport to the outer segments (OS) was impaired. (2) 11–cis retinal injections initiated at P10 preserved cones anatomically and cone opsin mRNAs were increased when compared to un–injected littermates. Consequently, cone b–wave amplitudes reached amplitudes of 135±18 compared with 210±40 µV for the wt. After retinal administration, the cone opsins were found to localize predominantly to the cone OS. Conclusions: These results indicate that in the absence of 11–cis retinal, cones may degenerate due to opsin mislocalization. The ligand appears to be required during cone opsin synthesis as a prerequisite for successful trafficking to the outer segment. Thus, the results described here may have important consequences for treating cone degenerations.

Keywords: opsins • retinoids/retinoid binding proteins • photoreceptors