Abstract
Purpose :
15% of non-syndromic retinitis pigmentosa (RP) cases belong to a subclass called sector RP. Sector RP is defined by the localization of the disease phenotype to certain quadrants of the retina, usually the inferior. Mutations in RHODOPSIN (RHO) are the primary cause of sector RP, with well-characterized mutations residing in the N-terminal domain (T4K, T17M, and P23H). Dark rearing animals expressing RHO containing sector RP-associated mutations (including T4K, T17M, and P23H) changes the protein’s stability, mitigating the retinal degeneration (RD) phenotype; however, many other sector RP-associated RHO mutations have not been investigated in this way. We investigated sector RP-associated mutations outside the N-terminus (T58R and D190G) and an N-terminal mutation that does not interfere with a glycosylation site (L31Q).
Methods :
We generated transgenic Xenopus laevis by injecting wildtype (WT) oocytes with a mixture of WT sperm nuclei and linearized plasmids containing a human RHO transgene (either WT or containing missense mutations) under control of a X. laevis rod opsin promotor. Tadpoles were raised in either cyclic light or constant dark for 14 days before euthanization. Tadpole eyes were enucleated and were either cryosectioned and immunolabelled using anti-mammalian rhodopsin antibodies for confocal microscopy or analysed for rhodopsin levels using a dot blot assay.
Results :
We observed mitigated RD in the dark rearing condition for animals expressing the L31Q or T58R RHO transgenes. However, D190G RHO-expressing animals failed to display mitigated RD in the dark rearing condition. Localization of the transgenic rhodopsin in the three mutations was primarily to the outer segment; however, D190G RHO animals display more inner segment localization compared to WT, L31Q, or T58R.
Conclusions :
We investigated three sector RP-associated RHO mutations that produced rhodopsin which primarily localized to the outer segment. Similar to previously described models of sector RP, two of these mutations result in RD that is reduced when animals are reared in complete darkness. The localization pattern and the RD of L31Q RHO and T58R RHO mimic the patterns previously seen in T4K RHO and T17M RHO X. laevis. However, RD caused by D190G RHO was not alleviated by dark rearing, suggesting that the origin of the sector RP phenotype in D190G RHO patients may be novel.
This is a 2021 ARVO Annual Meeting abstract.