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V. J. Kefalov, P. H. Tang, R. K. Crouch, A. V. Kolesnikov; Is RPE65 Involved in the Mouse Retina Visual Cycle?. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1116.
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© ARVO (1962-2015); The Authors (2016-present)
It has been suggested that RPE65, a rod visual cycle retinoid isomerase, is also expressed in cone photoreceptors of various mammalian species including mouse. The lack of RPE65 leads to cone opsin mislocalization and rapid cone degeneration. Cone function can be partially preserved by injecting exogenous 9-cis retinal. We are using this approach to investigate a possible role of RPE65 in the cone-specific retina visual cycle.
To facilitate cone recordings, we crossed RPE65 knockout (Rpe65-/-) mice with rod transducin alpha (Gnat1-/-) knockout mice that lack rod signaling. We then repeatedly injected pups at P10, P16, and P21 with Matrigel loaded with 9-cis retinal. The animals were kept in constant darkness during the injections period. Using transretinal ERG recordings, we obtained cone responses (a-wave) from isolated P23-P25 mouse retinas. We compared dark-adapted photosensitivity and kinetics of recovery of sensitivity following a full M-cone pigment bleach in cones from isolated Rpe65-/-/Gnat1-/- and control Gnat1-/- retinas.
Mice treated with 9-cis retinal exhibited increased M-opsin immunolocalization within the cone outer segments compared to untreated group indicating efficient chromophore delivery. However, cone sensitivity of Rpe65-/-/Gnat1-/- retinas was still up to 20-fold lower than in Gnat1-/- controls, yet several times higher than in uninjected littermates. Surprisingly, the kinetics of cone responses was greatly retarded in double KO mice (time-to-peak at 250-300 ms as compared to 70-80 ms in Gnat1-/- mice and much slower response termination). On the other hand, rod sensitivity in retinoid-injected Rpe65-/- mice increased by up to 2 log units and reached WT levels. Interestingly, the kinetics of cone sensitivity recovery following a bleach was significantly slower in Rpe65-/-/Gnat1-/- compared to control Gnat1-/- retinas, with time constants of 330 s and 70 s, respectively.
Our preliminary results may indicate that deletion of RPE65 leads to substantial decrease of mouse cone sensitivity, even in the presence of exogenously delivered chromophore, as well as to a significantly slower recovery following bright illumination. Further experiments will be required to confirm the possible function of RPE65 in the retina visual cycle. Our preliminary results indicate normal cone response kinetics in Rpe65-/-/Rho-/- mice which could be used as alternative model to address this question.
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