Abstract
Purpose :
Sector retinitis pigmentosa (RP) is a subset of RP in which one or two retinal quadrants are affected, suggesting environmental influences and the possibility of preventative treatment. Mutations in rhodopsin (RHO) are the primary causes of sector RP, with over a dozen mutations implicated, including P23H and T4K. These mutants have differing pathogenic mechanisms, but both mutants cause light-exacerbated retinal degeneration (RD) in animal models. Here, we investigated two RHO mutants for light-dependent phenotypes that might underlie their associated sector phenotype and explored possible novel mechanisms of RD.
Methods :
We generated transgenic Xenopus laevis expressing human RHO mutants and raised them to 14 days post-fertilization under various lighting conditions. Eyes were enucleated and used for immunohistology or for protein analyses. COS-7 cells were transfected with human RHO cDNA containing RP-associated mutations. Fixed tadpole cryosections and COS-7 cells were labelled with anti-mammalian rod opsin antibodies and counter-labelled with wheat germ agglutinin and Hoechst for confocal microscopy. Solubilized tadpole eyes were analyzed by Western and dot blot.
Results :
We identified two sector RP-associated RHO mutants with increased tendency for multimerization compared to WT and other RHO mutants. Unlike other sector RP mutants we have analyzed, the resulting RD was not reduced by dark rearing. However, the increase in multimerization was light-dependent, with significant differences between cyclic light and dark reared samples that were apparent on Western blots. We found that the multimerization was reversible in darkness, required chromophore binding, and occurred in the rod outer segment. Preventing physiological dimerization of these RHO mutants via the F45L mutation did not affect their trafficking in COS-7 cells.
Conclusions :
We have identified a group of sector RP-associated RHO mutations associated with light-dependent multimerization of RHO. These mutations cause light-independent RD in X. laevis. Thus, light-dependent multimerization is unlikely to be the primary cause of cell death in X. laevis, but could contribute to the sector RP phenotype in humans. Understanding the mechanisms by which environmental influences drive sector RP phenotypes may help to better categorize sector RP patients and could lead to preventative treatments for sector RP.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.