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Peter H. Tang, Lee Wheless, Jian-Xing Ma, Rosalie K. Crouch; Rpe65 Is Present Within Human Cone Photoreceptors And Is Required For Efficient Cone Visual Pigment Regeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3642. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have suggested that RPE65, the retinoid isomerase of the classic visual cycle, is present within cones of various animal species. In the present study, we evaluate the presence of RPE65 within cones of the rod-dominant retina, including human and mice, and investigate its possible role within cones.
Antibodies targeting residues 150-164 (PETLET) or 473-486 (DALEED) of bovine RPE65 were developed. Donor human retinas were obtained from two patients (11 & 21 years). To generate mice with varying levels of RPE65 in the RPE, we crossed C57BL6 mice with BALB/c or Rpe65-/- mice. Immunoblots were performed to verify antibody specificity and to quantify RPE65. Immunohistochemistry techniques were utilized to localize and quantify the amount RPE65 within cones. Mice were treated with either 9-cis retinal or saline and then dark-adapted for 12 hours before electroretinography recordings were performed.
Both PETLET and DALEED antibodies showed similar RPE65 staining within the outer segments of human cones, and this pattern was reflected in mouse cones. Across the various mouse strains/lines, an inverse relationship in the amount of RPE65 was observed between cones and the RPE. Chromophore treatment enhanced UV-cone responses in mice with low or undetectable cone RPE65, but had no effect in mice with high cone RPE65. The rod responses were unchanged.
Antibodies with high affinity and specificity for RPE65 are essential to detect the protein within cones. Our data show RPE65 is present within human cones and mouse is an appropriate model. Cones with low or undetectable RPE65 appear to be partially chromophore deprived, suggesting that RPE65 within cones is essential to maintain fully regenerated visual pigments.
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