Abstract
Purpose :
15% of non-syndromic retinitis pigmentosa (RP) cases belong to a subclass called sector RP. Sector RP is defined by the diseased phenotype being restricted to one or two quadrants of the retina. The most common causes of sector RP are mutations in RHODOPSIN (RHO), where over a dozen mutations have been associated with sector RP including T4K and P23H. Previously, we have shown that altered lighting conditions can modulate retinal degeneration (RD) in transgenic Xenopus laevis models of sector RP. Here, we investigated sector RP-associated RHO mutations for changes in RHO localization in rod cells and RD under different lighting regimes.
Methods :
X. laevis expressing human RP-associated RHO mutants under control of the X. laevis rod opsin promoter were generated by restriction enzyme-mediated integration. Transgenes were based on the human RHO cDNA. Tadpoles were raised for 14 days in either constant light, cyclic light, or complete darkness. Eyes were enucleated and used for histology or for protein level analyses through western and dot blots using mammalian rod opsin antibodies.
Results :
We found variable degrees of inner segment (IS) RHO retention associated with RP-associated mutations. Most sector RP-associated RHO mutations did not affect RHO trafficking to the outer segment (OS); however, a third of the mutations studied caused significant IS retention of RHO. RD in animals with OS-localizing RHO mutants (including L31Q and T58R) was mitigated when the animals were dark reared. RD in animals with intermediate RHO localization patterns (significantly different than wildtype but with some OS localization, including S22R and D190G) was not prevented by dark rearing.
Conclusions :
Localization of mutant RHO in the IS suggests an ER stress-related cell death mechanism while RHO mutants with localization indistinguishable from wildtype is likely indicative of a photoactivation-induced mechanism. The majority of mutations that we examined did not mislocalize to the IS and therefore likely act via a mechanism originating in the OS; however, mutants that localize to both the IS and OS could have multiple cell death mechanisms occurring simultaneously. Classification of RHO mutations using this system may identify mutations that are most likely to benefit from ER stress-alleviating therapies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.