Downregulation of cone-specific gene expression and degeneration of cone photoreceptors in the Rpe65-/- mouse at early ages.

PURPOSE: RPE65 is essential for the generation of 11-cis retinal. Rod photoreceptors in the RPE65-knockout (Rpe65(-/-)) mouse are known to degenerate slowly with age. This study was designed to examine cone photoreceptors and the expression of cone-specific genes in the Rpe65(-/-) mouse. METHODS: Gene expression changes were identified by microarray and confirmed by real-time RT-PCR. Cone photoreceptors were stained by peanut agglutinin (PNA) lectin in the flatmounted retina. The 9- or 11-cis retinal was supplied by intraperitoneal injections. RESULTS: The short-wavelength (SWL) cone opsin mRNA was markedly decreased at 2 weeks of age, whereas the decrease in the middle-wavelength (MWL) cone opsin mRNA occurred relatively later in age. In contrast, the rhodopsin mRNA level did not show any significant change at all the ages analyzed. Consistent with the cone opsin changes, the cone transducin alpha-subunit mRNA decreased at both 4 and 8 weeks of age, whereas again the rod transducin alpha-subunit did not show any significant change. Rpe65(-/-) mice showed significant cone loss in both the central and ventral retina between 2 and 3 weeks of age. Administration of 9- or 11-cis retinal to Rpe65(-/-) mice 2 weeks of age increased cone density by twofold in these areas. CONCLUSIONS: In the Rpe65(-/-) mouse, the expression of cone-specific genes is downregulated and is accompanied by cone degeneration at early ages. Early administration of 9- or 11-cis retinal can partially prevent cone loss, suggesting that the absence of 11-cis chromophore may be responsible for the early cone degeneration.

A novel Xenopus SWS2, P434 visual pigment: structure, cellular location, and spectral analyses.

PURPOSE: The purpose of this study was to clone and characterize the green rod pigment in Xenopus laevis. METHODS: The cDNA for the Xenopus "green rod" pigment was cloned and sequenced from Xenopus retina mRNA by reverse transcription polymerase chain reaction and the 5' end cloned by rapid amplification of the cDNA ends. The cellular localization of the Xenopus opsin was determined by immunolabeling of flat-mounted retinas using a specific antibody against this opsin. Spectral properties of the expressed protein were determined by absorption spectroscopy using recombinant pigment. RESULTS: A novel Xenopus opsin cDNA containing a full-length coding region has been cloned and sequenced. The deduced amino acid sequence predicts a protein of 362 amino acids, forming 7 hydrophobic helices. Sequence analysis indicates that it belongs firmly to the SWS2 class of visual pigments and has 89%, 80%, and 75% amino acid sequence identity with bullfrog, tiger salamander, and newt SWS2 pigments, respectively. Staining of Xenopus retina with a Xenopus SWS2 opsin-specific polyclonal antibody demonstrated that the SWS2 pigment is expressed in green rods. After expression in COS cells, reconstitution with 11-cis retinal, and purification, the SWS2 pigment exhibits an absolute absorption maximum of 434 nm Thus, the name "SWS2, P434" was assigned for this opsin. The pigment decays rapidly in hydroxylamine in the dark, unlike the red rod pigment, rhodopsin. CONCLUSIONS: A novel green rod opsin cDNA has been cloned and sequenced from the retina of adult Xenopus laevis, which encodes a protein belonging to the SWS2 group of opsins. The expressed opsin possesses cone-opsin-like properties although it was identified only in the Xenopus green rod cells.

Identification of the RPE65 protein in mammalian cone photoreceptors.

PURPOSE: The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium (RPE). Previous studies have identified the RPE65 mRNA in salamander cones, but not in rods. The purpose of the present study was to determine whether RPE65 is expressed at the protein level in mammalian cones, as well as in those of amphibians. METHODS: The specificity of the anti-RPE65 antibody was demonstrated by Western blot analysis. RPE65 cellular localization was determined using immunohistochemistry on flatmounted retinas and retinal sections. RESULTS: RPE65 protein was detected in cones in flatmounted retinas of the mouse, rabbit, and cow, in addition to Xenopus laevis. The morphology and location of labeled cones in the retina were confirmed by double staining of mouse retina sections with the anti-RPE65 antibody and peanut agglutinin (PNA) lectin, which is known to label both types of cones in mouse. The double staining in the flatmounted retinas demonstrated that RPE65 was expressed in both types of the cones in the mouse retina. Under the same double-labeling conditions, however, cones in homozygous RPE65-knockout mouse were labeled by PNA lectin, but not by the anti-RPE65 antibody, indicating that the protein recognized by the anti-RPE65 antibody is encoded by the RPE65 gene rather than by another homologous gene. No RPE65 was detected in rods of any of the species tested. CONCLUSIONS: RPE65 is expressed in mammalian cones, but not in rods. These results provide further support for physiological observations that cones may have an alternative retinoid cycle.