Abstract
Purpose::
Several studies have demonstrated that light exposure may influence the progression of retinal degeneration in both retinitis pigmentosa (RP) patients and transgenic models of RP. To investigate the mechanisms underlying this effect, we have generated transgenic X. laevis expressing several different N-terminal rhodopsin mutants, and examined the effects of dark rearing on retinal degeneration and rhodopsin biosynthesis.
Methods::
The P23H mutation was introduced into the human, bovine, murine, and X. laevis rhodopsin cDNAs. T4R, T17M, and P23A mutants were introduced into the bovine rhodopsin cDNA. Transgenic X. laevis were reared in either cyclic light or total darkness for fourteen days. Transgenic retinas were analyzed by confocal microscopy and western blot, and the toxic effects and expression levels of the rhodopsins were compared between groups of animals using dot blot assays.
Results::
Amino acid sequence variations in rhodopsins from different species affected the efficiency of folding of the P23H mutants. Bovine P23H rhodopsin showed the greatest capacity for folding and X. laevis P23H rhodopsin showed the least. Moreover, retinal degeneration induced by bovine P23H rhodopsin was completely rescued by dark rearing on the timescale studied. Dark rearing rescued retinal degeneration caused by human P23H rhodopsin to a lesser extent, and had no effect on retinal degeneration caused by X. laevis P23H rhodopsin. Western blot analysis of dark reared retinas revealed that the N-terminus of the mutant P23H rhodopsin molecule was removed by proteolytic cleavage. The extent of rescue from rod death correlated with the extent of proteolytic cleavage (i.e bovine P23H>human P23H>xenopus P23H and dark reared>cyclic reared). Immunolabeling of frozen sections demonstrated that full-length P23H rhodopsins were retained in the ER, while truncated mutant rhodopsin exited the ER and was transported to the rod outer segment. Proteolytic cleavage of rhodopsin did not occur with the other mutants examined.
Conclusions::
Our results suggest a mechanism to explain light sensitivity in human RP patients and transgenic animal models with retinal degeneration induced by the P23H mutation, but not light sensitivity caused by T4R or T17M mutants.
Keywords: retinal degenerations: cell biology • opsins • protective mechanisms