The interplay of environmental luminance and genetics in the retinal dystrophy induced by the dominant RPE65 mutation

The retinol isomerase retinal pigment epithelium (RPE) 65 is a key enzyme in the visual cycle that regenerates the chromophore, 11-cis-retinal, required for vision in all vertebrates. Mutations in RPE65 are associated with blinding diseases. D477G (C.1430G > A) is the only known RPE65 variant to cause autosomal dominant retinitis pigmentosa. Previously, we reported that heterozygous D477G knockin (KI) mice maintained in dim-light conditions exhibited delayed visual chromophore regeneration and slowed recovery of photoreceptor sensitivity following visual pigment photobleaching. However, heterozygous D477G KI mice did not manifest detectable decline in full-field electroretinography (ERG) or in abnormal retinal structure, relative to the WT mice. In the present study, when maintained under the physiological relevant light intensity (2,000 lx), the heterozygous D477G KI mice displayed degenerative retina features, including reduced scotopic ERG amplitudes and thinning of the retinal layers, recapitulating that observed in human patients. In the same retinas, we also detected increased free opsin levels and up-regulated GFAP expression. Molecularly, we found reduced RPE65 and LRAT (lecithin: retinol acyltransferase) levels, decreased retinol isomerase activity, and altered subcellular localization and membrane association of RPE65 in the RPE of heterozygous D477G KI mice. Moreover, the D477G mutant, both in vivo and in vitro, formed protein complexes with WT-RPE65, leading to a reduction in RPE65 protein stability, which could not be completely rescued by the addition of MG132, a proteasome inhibitor of ubiquitin-dependent protein degradation. Altogether, our findings uncovered the dominant-negative nature of the D477G mutation and highlighted the importance of the light environment in the mechanism of its pathogenicity.