The relationship between opsin overexpression and photoreceptor degeneration.

PURPOSE: To characterize the process by which overexpression of normal opsin leads to photoreceptor degeneration. METHODS: Three transgenic mouse lines were generated that express different levels of an opsin with three amino acid modifications at the C terminus. These modifications created an epitopic site that can be readily distinguished from the endogenous protein using a bovine opsin-specific antibody. Evidence of degeneration associated with opsin overexpression was provided by anatomic studies and electroretinogram (ERG) recordings. Western blot analysis was used to confirm the production of the transgenic opsin, and an enzyme-linked immunosorbent assay (ELISA) was used to determine the amounts of opsin overexpressed in each line. Immunocytochemistry was used to determine the cellular localization of transgenic opsin. Amounts of 11-cis retinal were determined by extraction and high-performance liquid chromatography (HPLC). RESULTS: Opsin expression levels in the three lines were found to be 123%, 169%, and 222% of the level measured in nontransgenic animals, providing direct correlation between the level of transgene expression and the severity of the degenerative phenotype. In the lower expressing lines, ERG a-wave amplitudes were reduced to less than approximately 30% and 15% of normal values, whereas responses of the highest expressing line were indistinguishable from noise. In the lowest expressor, a 26% elevation in 11-cis retinal was observed, whereas in the medium and the high expressors, 11-cis retinal levels were increased by only 30% to 33%, well below the 69% and 122% increases in opsin levels. CONCLUSIONS: The overexpression of normal opsin induces photoreceptor degeneration that is similar to that seen in many mouse models of retinitis pigmentosa. This degeneration can be induced by opsin levels that exceed by only approximately 23% that of the normal mouse retina. Opsin overexpression has potential implications in retinitis pigmentosa.

Retinoid kinetics in eye tissues of VPP transgenic mice and their normal littermates.

PURPOSE: VPP mice, which possess a mutant transgene for opsin (V20G, P23H, P27L), exhibit a progressive rod degeneration that resembles one form of human autosomal dominant retinitis pigmentosa. In the present study the association of the development of VPP rod degeneration with abnormal operation of the retinoid visual cycle was examined. METHODS: Dark-adapted VPP mice and normal littermates were anesthetized and the pupils dilated. One eye of each animal was illuminated for 2 minutes; the other eye was shielded from the light and served as a control. Each animal was then dark adapted for a defined period (0-300 minutes) and killed. Retinoids contained in the retina, retinal pigment epithelium (RPE), and extracellular medium were recovered by means of formaldehyde-, isopropanol- and ethanol-based extractions and analyzed by high-performance liquid chromatography. RESULTS: Total amounts of retinoid recovered from unilluminated eyes of 2-month-old normal and VPP mice were 425 +/- 90 picomoles per eye and 115 +/- 33 picomoles per eye, respectively (mean +/- SD). Relative distributions of retinoids within normal and VPP eyes were similar. In normal and VPP animals, illumination for 2 minutes produced a similar immediate reduction in the molar percent of total retinoid represented by 11-cis retinal in the retina (average reduction of 34% and 28% in normal and VPP animals, respectively) and a similar transient increase of all-trans retinal in the retina. In both groups the decline of all-trans retinal was accompanied by an increase in total retinyl ester. In normal and VPP animals, a period of approximately 40 minutes or more preceded initiation of the recovery of 11-cis retinal in the retina, and the time course of this recovery was generally similar to that for the decline of retinyl ester. The overall dark-adaptation period required for half-completion of 11-cis retinal recovery was approximately 150 minutes. In neither group did illumination produce a substantial peak of all-trans retinol in the retina. CONCLUSIONS: The evident approximately fourfold reduction of total retinoid in the eyes of 2-month-old VPP mice is consistent with histologic and electroretinographic abnormalities determined in previous studies. Despite this marked abnormality in retinoid content, retinoid cycling in the VPP is remarkably similar to that in normal littermates. The data place constraints on the functional consequences of any abnormality in retinoid processing that may be present at this stage of the VPP rod degeneration.