Study of the retinoid visual cycle in RPE65 knockout mouse.

摘要

RPE65, an abundant protein in retina pigment epithelium, plays a critical role in the retinoid visual cycle as an isomerohydrolase, which is essential for the conversion from all-trans retinoid to 11- cis retinoid. In this dissertation, the Rpe65 -/- mouse was used as a model for studies of both the retinoid visual cycle and the role of this protein in the eye.; This study proved that a retinal isomer, 9-cis retinal, accumulates in eyes through a RPE65 and light-independent pathway and forms a functional pigment isorhodopsin in Rpe65-/- mice.; The isolated rods from dark-reared Rpe65-/- mice were shown to have a greater circulating current and sensitivity than those from the cyclic light-reared Rpe65-/- mice. In Rpe65-/- mice, the spectral sensitivity of the isolated rods indicated that the rod responses were indeed generated by isorhodopsin and their sensitivities were found to correlate with the pigment levels. Retinal morphology indicated that the retinal degeneration is slower in dark-reared Rpe65-/- animals compared with the cyclic light-reared Rpe65-/- animals. These results confirmed that photoreceptor degeneration in Rpe65-/- mice results from opsin activation.; Interestingly, the Rpe65-/- mice with varied pigmentation were shown to present different rates of 9-cis retinal accumulation. The 9-cis retinal accumulated two-fold faster in tan than in agouti Rpe65-/- mice. This tan Rpe65-/- mouse is expected to be a useful model in elucidation of the 9-cis retinal metabolic pathways.; Surprisingly, in Rpe65-/- mice, evidence showed that the rods degenerate slower than expected. Significant amounts (20%) of opsin in Rpe65-/- animals were phosphorylated, even in the darkness. By studying RK-/-Rpe65 -/- mice, the rhodopsin kinase was shown to be responsible for the opsin phosphorylation, and this opsin phosphorylation partially protected the photoreceptors against degeneration, presumably by inhibiting the constitutively activated opsin.; In summary, it is the first time that endogenous 9-cis retinal has been identified in the mouse retina. These studies will provide the foundation for further understanding of 9-cis retinal metabolism in the retina, which may be useful for clinical treatment of patients affected with 11-cis retinal metabolism defects. Additionally, the opsin phosphorylation study demonstrated the important role of RK in the prevention of retinal degeneration.