Abstract
Purpose::
Visual transduction is quenched when light-activated rhodopsin (Rh*) is phosphorylated and binds arrestin. Defects in rhodopsin phosphorylation result in excessive signaling and cause retinal degeneration. We have created a mutant high-affinity arrestin that binds to both activated phosphorylated rhodopsin (P-Rh*) and Rh*. We examined the morphology of rod photoreceptors in several lines of transgenic mice expressing these mutants at different levels on arrestin knockout (AKO) background.
Methods::
The transgenic mice were raised in normal cyclic light, and morphology of their retinas was examined at 8 and 16 weeks. Arrestin expression was measured by quantitative Western blot and normalized to rhodopsin expression. The thickness of the outer nuclear layer (ONL) and the length of rod outer segments (OS) were used to determine the state of the photoreceptors in mice expressing wild type (WT) and mutant phosphorylation-independent arrestin at 10%, 50%, and 240% of endogenous levels. The rate of photoreceptor apoptosis was measured as the proportion of TUNEL-positive nuclei in the ONL.
Results::
Rods in AKO mice lose their OS by two months. Retinal morphology of mice expressing mutant arrestin at the level of 10% (relative to WT) was indistinguishable from WT for at least four months, expression at 50% also protected the OS. The expression of mutant at 240% induced rapid retinal degeneration with high incidence of apoptosis, although surviving rods (in sharp contrast to AKO mice) retained their OS for at least four months. The highest ratio of apoptotic nuclei in ONL was reached at ~ 7 weeks, and by 16-17 weeks their ONL was only 2-3 nuclei thick. Increasing co-expression of wild type arrestin (transgene on AKO, Arr+/-, and Arr+/+ backgrounds) progressively decreased the rate of photoreceptor apoptosis and improved the morphology of ONL, indicating that WT arrestin protects photoreceptors from degeneration induced by the mutant.
Conclusions::
Low expression levels (10% and 50%) of the transgene are sufficient to maintain photoreceptor health, whereas high expression induces rapid retinal degeneration through apoptosis of photoreceptor cells. The phosphorylation-independent nature of the transgene, rather than its expression levels per se, is responsible for the degeneration. Our study may have implications for certain forms of retinitis pigmentosa, where mutations in rhodopsin that cause the formation of stable rhodopsin-arrestin complexes induce photoreceptor degeneration.
Keywords: photoreceptors • protein structure/function • signal transduction