An S-opsin Knockin Mouse (F81Y) Has Reduced Expression That Is Restored By (exogenous) 11-cis Retinal

Abstract

Purpose: : To investigate the mechanisms of reduced S-opsin expression in Opn1sw^F81Y/F81Y mice

Methods: : We created a knockin mouse with a point mutation (F81Y) in S-opsin that shifted the (_max) from 360 to 425 nm, near that of human S-opsin, but unexpectedly resulted in its reduced expression. The following methodology was applied in parallel to WT and Opn1sw^F81Y/F81Y littermates to investigate the mechanisms for the reduced S-opsin expression. Opsin expression was probed with qRT-PCR and quantitative Western blot analysis. Electron microscopy was applied to fixed retinal tissue to assess cone morphology. Cone function was assessed with single cone recordings (sunction pipette recording). Immunohistochemistry methods were employed to investigate S-opsin distribution throughout the cone cell. Immuno-precipitation was performed in order to probe association of S-opsin with ER associated degradation (ERAD) machinery. WT and Opn1sw^F81Y/F81Y littermate pairs were injected with 11-cis retinal or vehicle on alternate days for a 10 day period.

Results: : Our results indicate that S-opsin expression in the outer segments of Opn1sw^F81Y/F81Y mice was reduced to 30-50% of the WT level, with no more than a 15% decrease in transcription. Despite a significant decrease of S-opsin expression, Opn1sw^F81Y/F81Y mouse cones were morphologically normal and responded to light with normal kinetics and the predicted shift in _max. Mistrafficking of the mutant opsin from ER/golgi to other cellular compartments does not account for its reduced expression. Our immunohistochemistry experiments show a substantial percentage (~25%) of total S-opsin signal outside of the outer segment compartment, with comparable fractions in each compartment in both Opn1sw^F81Y/F81Y and WT mice. However, F81Y S-opsin showed an increased association with components of ERAD, ER degradation-enhancing alpha-mannosidase-like 1 (EDEM1) and valosin-containing protein (p97/VCP). The S-opsin in Opn1sw^F81Y/F81Y mice was restored to WT levels in littermate mouse pairs receiving exogenous 11-cis retinal.

Conclusions: : Despite reduced opsin expression, Opn1sw^F81Y/F81Y mouse cone photoreceptors are healthy. However our results indicate that the mutant S-opsin is less efficient at attaining a properly folded mature form in the ER, and a large fraction of nascent S-opsin is degraded by ERAD. Our 11-cis retinal supplementation results suggest that chromophore binding of nascent cone opsins is critical for efficient biosynthesis and export from the ER.

Keywords: opsins • photoreceptors • retinal degenerations: cell biology