期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：11
DOI：10.1073/pnas.2115202119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
In humans, genetic mutations in the retinal pigment epithelium (RPE) 65 are associated with blinding diseases, for which there is no effective therapy alleviating progressive retinal degeneration in affected patients. Our findings uncovered that the increased free opsin caused by enhancing the ambient light intensity increased retinal activation, and when compounded with the RPE visual cycle dysfunction caused by the heterozygous D477G mutation and aggregation, led to the onset of retinal degeneration.
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.
