Regeneration of Photopigment Is Enhanced in Mouse Cone Photoreceptors Expressing RPE65 Protein

Abstract

As cone photoreceptors mediate vision in bright light, their photopigments are bleached at a rapid rate and require substantial recycling of the chromophore 11-cis retinal (RAL) for continued function. The retinal pigment epithelium (RPE) supplies 11-cis RAL to both rod and cone photoreceptors; however, stringent demands imposed by the function of cones in bright light exceed the output from this source. Recent evidence has suggested that cones may be able to satisfy this demand through privileged access to an additional source of chromophore located within the inner retina. In this study, we demonstrate that the protein RPE65, previously identified in RPE as the isomerohydrolase of the RPE-retinal visual cycle, is found within cones of the rod-dominant mouse retina, and the level of RPE65 in cones is inversely related to the level in the RPE. The light sensitivity of cone ERGs of BALB/c mice, which had an undetectable level of cone RPE65, was enhanced by ~3-fold with administration of exogenous chromophore, indicating that the cones of these animals are chromophore deficient. This enhancement with chromophore administration was not observed in C57BL6 mice, whose cones contain RPE65. These results demonstrate that RPE65 within cones may be essential for the efficient regeneration of cone photopigments under bright light conditions.

RPE65 Is Present in Human Green/Red Cones and Promotes Photopigment Regeneration in anIn VitroCone Cell Model

“…To investigate if RPE65 is localized within human cones, two polyclonal antibodies raised against distinct epitopes of RPE65 were used that have been previously shown to exhibit high specificity in mouse (Redmond and Hamel, 2000; Tang et al, 2011) and chicken (Moiseyev et al, 2008) (see Table 1). We evaluated the specificities of both antibodies for human RPE65 in the RPE, as this is the region of the eye where the protein is widely accepted to be localized and to function.…”

“…Evidence first arose from the discovery of mRNA in salamander cones (Ma et al, 1998) and the protein in mammalian cones through immunohistochemistry (IHC) (Znoiko et al, 2002). We recently localized RPE65 to the outer segment (OS) region of the mouse cone and found that mouse strains with lower amounts of cone RPE65 were less efficient at regenerating cone photopigment than those with higher levels (Tang et al, 2011). …”

Evolution of Mammalian Opn5 as a Specialized UV-absorbing Pigment by a Single Amino Acid Mutation

“…These antibodies were affinity-purified prior to use by Protein Purify Ltd. The primary antibodies used in this study included guinea pig anti-chick Opn5m antibody (7) and rabbit anti-RPE65 anti-body (a gift from Dr. Rosalie K. Crouch, Medical University of South Carolina) (15). We performed Western blotting analysis using the antibodies and confirmed that the anti-mouse and human/monkey Opn5m antibodies recognized the recombinant proteins of full-length mouse and human Opn5m expressed in HEK293 cells, respectively (Fig.…”

Complementation Test ofRpe65Knockout and Tvrm148

“…8); furthermore, many remaining cones are not functional at that age in Rpe65 À/À mice, although they can be restored to activity by reintroduction of 11-cis-retinal. 55 The source and identity of chromophore for rod photoreceptors in T À/À mice, though, is unclear at this time. However, Rpe65 À/À mice may generate and utilize 9-cis-retinal to form isorhodopsin.…”