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Beatrice M. Tam, Syed M. Noorwez, Shalesh Kaushal, Orson L. Moritz; Eliminating Chromophore Binding Via Vitamin A Deprivation Results In Different Outcomes In Two Transgenic X. Laevis Models Of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1815.
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We have developed a number of transgenic X. laevis models of RP based on expression of rhodopsin mutants. Several of these models demonstrate significant exacerbation of retinal degeneration when exposed to light. We have hypothesized that this could be due either to destabilization of the mutant rhodopsin upon loss of chromophore binding during biosynthesis, or to instability of the active conformation of mutant rhodopsin in the photoreceptor outer segment. In this study, we compared the effects of vitamin A deprivation and rhodopsin activation on our light sensitive X. laevis models of RP.
Transgenic X. laevis F1 tadpoles expressing either P23H rhodopsin or T4K rhodopsin were reared on diets either containing or deficient in vitamin A. Tadpoles were initially reared in constant dark for 3 weeks and then exposed to cyclic light for an additional week. Retinas of transgenic X. laevis were analyzed by dot blot for total and transgenic rhodopsin, and by confocal microscopy to assess severity of retinal degeneration and protein localization. The effects of light exposure on the stability of recombinantly expressed mutant rhodopsins was also tested in in vitro assays.
Retinal degeneration in transgenic tadpoles expressing P23H and T4K rhodopsin is exacerbated by light exposure and can be largely eliminated by rearing the animals in the dark. When animals were dark reared and simultaneously deprived of vitamin A, retinas expressing P23H opsin degenerated, indicating a toxic effect of vitamin A deprivation, while those expressing T4K opsin did not degenerate. Moreover, after three weeks of vitamin A deprivation, light exposure no longer induced retinal degeneration in animals expressing T4K opsin. In in vitro assays, light exposed T4K rhodopsin was unable to re-bind chromophore.
Our results demonstrate that the mechanisms underlying light-exacerbated retinal degeneration are different for two models of RD. In the case of P23H rhodopsin, vitamin A acts as a pharmacological chaperone that increases the stability of the mutant protein, thereby reducing its toxicity. In contrast, in the case of T4K rhodopsin vitamin A is detrimental, as it facilitates activation and destabilization of the mutant protein. These results suggest that vitamin A supplementation may be contra-indicated for a subset of RP patients.
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