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Alexander V Kolesnikov, Chloe Potter, David Razafsky, Andrew Hughes, Joseph C Corbo, Didier Hodzic, Vladimir J Kefalov; Substitution of Rod for Cone Opsin Increases the Resistance of M-Cones to Chromophore Deficiency-Induced Degeneration. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Leber Congenital Amaurosis (LCA) is a severe retinal dystrophy causing child blindness. Mutations in retinoid isomerase RPE65 account for up to 16% of LCA cases and lead to early loss of cones. The mechanisms of this disease are poorly understood. Cone pigments are generally less stable and rely on the supply of visual chromophore. We tested the hypothesis that the low stability of cone opsin makes mammalian cones susceptible to degeneration under conditions of sustained chromophore deprivation in the absence of RPE65.
We generated a novel knockin mouse model expressing rod pigment in cones instead of their native M-opsin (RhoKI). Cone expression of rhodopsin was confirmed in RhoKI-Rho-/- animals by IHC and further quantified by qPCR. To induce chromophore deficiency, we generated RhoKI-Rpe65-/-Gnat1-/- mice and their Rpe65-/-Gnat1-/- controls lacking rod signaling. We then compared their cone numbers and M-cone transretinal ERG responses (with exogenously added 9-cis-retinal) at the ages of 1 or 5 months.
The total level of rhodopsin mRNA in RhoKI-Rho-/- retinas was 15-fold lower than that of native M-cone pigment in control (Rho-/-) mice. This caused corresponding desensitization of M-cones in RhoKI animals. However, their maximal cone response amplitude and their amount of cone transducin were normal. Rpe65-/-Gnat1-/- mice had rapidly progressing degeneration of nearly all ventral and most dorsal cones. Notably, there was a substantial preservation of central-dorsal cones in 5-month-old RhoKI-Rpe65-/-Gnat1-/- animals. Furthermore, RPE65-deficient cones expressing rhodopsin had a 2-fold higher maximal response and 10-fold higher sensitivity than age-matched controls. Following a chromophore treatment, the overall physiological function of RhoKI-Rpe65-/-Gnat1-/- cones was only 2–2.5-fold lower than in RhoKI-Gnat1-/- animals with normal chromophore supply. This is in stark contrast to Rpe65-/-Gnat1-/- cones whose sensitivity was several orders of magnitude lower than that in Gnat1-/- mice.
Cones expressing rhodopsin are viable and have robust phototransduction. The substitution of cone M-opsin with even small amount of more stable rhodopsin increases the survival of cones under conditions of severe chromophore deficiency. This opens the potential for respective gene-therapy based approach for treating LCA and related retinal pathologies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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