Abstract
Purpose: :
The RPE65 gene knockout (Rpe65–/–) mouse lacks isomerohydrolase activity in the visual cycle and lacks of 11–cis retinal, the chromorphore for rod and cone visual pigments. It has been suggested that the Rpe65–/– mouse is a model for vitamin A deficiency. The purpose of this study was to compare the phenotypes of the Rpe65–/– mouse with that in mice with vitamin A deficiency.
Methods: :
The mice (129sv) were fed with a vitamin A–deprived diet for 6 months. The age–matched control mice and Rpe65–/– mice were maintained under normal vitamin A–containing diet. The isomerohydrolase and LRAT activities were measured using an in vitro enzymatic activity assay. Endogenous retinoid profiles were analyzed by HPLC in mouse eyecup homogenates. Photoreceptor–specific gene expression was determined using real–time RT–PCR. Cone degeneration was determined by cone–specific staining and counting cones in the flat–mounted retina. The structure of photoreceptor outer segment was compared using SEM.
Results: :
The mice with vitamin A deficiency had lower levels of endogenous retinyl ester and 11–cis retinoids, compared with control 129sv mice, whereas RPE65 gene knockout resulted in the over–accumulation of retinyl ester and lack of 11–cis retinoids in the eyecup. In vitro enzymatic activity assay showed that vitamin A deprivation decreased the LRAT activity, but Rpe65–/– mice showed an unchanged LRAT activity. In the vitamin A deficient mice, the decreased expression of the MLW cone opsin preceded the decrease of the SLW cone opsin, whereas in the Rpe65–/– mice, the SLW cones degeneration occurred earlier than that of the MLW cones. SEM showed the outer segments of photoreceptors were disorganized but lengths unchanged in the vitamin A deficient mice, while the outer segments in the Rpe65–/– mice shorten significantly, but were not disorganized structurally, compared with the aged–matched wt mice.
Conclusions: :
The Rpe65–/– mouse has many different changes in retinoid profile, enzymes in the visual cycle and cone photoreceptor degeneration, compared to the vitamin–deficient mouse.
Keywords: retinal degenerations: hereditary • vitamin A deficiency • retinoids/retinoid binding proteins