Isorhodopsin rather than rhodopsin mediates rod function in RPE65 knock-out mice

Abstract

The chromophore of visual pigments is 11-cis-retinal and, thus, in its absence, opsin is not photosensitive and no visual function exists. However, in the RPE65 knockout (Rpe65-/-) mouse, where synthesis of 11-cis-retinal does not occur, a minimal visual response from rod photoreceptors is obtained. We have examined if an alternative pathway exists for cis-retinoid generation in the absence of RPE65. Cyclic-light-reared, 2-month-old Rpe65-/- mice were placed in complete darkness. No exogenous retinoids were administered. After 4 weeks, enhanced a- and b-wave amplitudes were obtained, increasing >10-fold for the a-wave and >3-fold for the b-wave as compared with cyclic-light-reared Rpe65-/- mice. Visual-pigment levels increased to approximately 10 pmol per retina, compared with no measurable pigment for cyclic-light-reared Rpe65-/- mice. The lambdamax of the isolated pigment was 487 nm, characteristic for isorhodopsin. Retinoid extractions confirmed the presence of 9-cis-retinal and the absence of 11-cis-retinal. Once the Rpe65-/- mice were returned to cyclic light, within 48 h the electroretinogram function returned to levels found in Rpe65-/- mice maintained in cyclic light. This dark-mediated pathway is also operational in older animals, because 13-month-old Rpe65-/- mice kept in prolonged darkness (12 weeks) had increased isorhodopsin levels and electroretinogram a- and b-wave amplitudes. These studies demonstrate that a pathway exists in the eye for the generation of 9-cis-retinal that is independent of RPE65 and light.

Fig. 1.

ERG responses in Rpe65^-/- mice increase on dark rearing. (A) Full-field scotopic ERG traces in response to a fixed-light intensity (2.48 photopic m^-2·cd·s) of cyclic-light-reared Rpe65^-/- mice after 1 day (trace a), 1 week (trace b), 2 weeks (trace c), 3 weeks (trace d), and 4 weeks (trace e) of dark rearing. (B) a-waves were plotted on an expanded time base. (C) a-wave amplitude comparisons (n = 22). (D) b-wave amplitude comparisons (n = 22). (E) a-wave amplitude analysis after return of animals to cyclic light (n = 8). Data are presented as means ± SE, the underlines in C-E indicate the time points compared, and ^* indicates a significant difference (one-tailed t test). DA, dark adaptation; CL, cyclic light.

Fig. 2.

Accumulation of endogenous isorhodopsin in long-term dark-adapted Rpe65^-/- mice. Absorption spectra of the endogenous pigment of 2-month-old cyclic-light-reared Rpe65^-/- mice that were then maintained in the dark for 4 weeks (trace i) and of age-matched WT mice with identical light treatment (trace ii) are shown. Spectra represent the difference spectrum of 1% dodecylmaltoside extractions of two retinae before and after bleaching with white light in the presence of NH₂OH (20 mmol/liter). Arrows indicate the respective λ_max. Abs, absorbance; KO, knockout.

Fig. 3.

Retinoid profiles of Rpe65^-/- and WT mice. HPLC chromatograms of retinoid extractions from Rpe65^-/- and WT mice (cyclic-light-reared from birth for 2 months, then dark-reared for 4 weeks, three eyes per sample). (A) Rpe65^-/- mouse retina. (B) Rpe65^-/- mouse RPE fraction. (C) WT mouse retina. (D) WT mouse RPE fraction. (B and D Insets) Full-scale chromatograms. Peaks were identified by comparison to known standards. Peak 1, all-trans-retinyl esters; peak 2, syn-9-cis-retinal oxime; peak 2′, anti-9-cis-retinal oxime; peak 3, syn-11-cis-retinal oxime; peak 3′, anti-11-cis-retinal oxime; peak 4, syn-all-trans-retinal oxime; peak 4′, anti-all-trans-retinal oxime; peak 5, syn-13-cis-retinal oxime; peak 5′, anti-13-cis-retinal oxime. Detection is at 360 nm.

Fig. 4.

Pigment accumulation and ERG a-wave amplitudes. Pigment accumulation and a-wave responses were plotted against time of complete dark adaptation. Data from 2- and 13-month-old animals were combined for this analysis. Pigment accumulation (○) and the increase in a-wave amplitude (•) were found to be linear over time (R = 0.974 and 0.918, respectively).

Fig. 5.

Schematic representation of the generation of 9- and 11-cis-retinals.