A Dominant Mutation in Rpe65, D477G, Delays Dark Adaptation and Disturbs the Visual Cycle in the Mutant Knock-In Mice

Abstract

RPE65 is an indispensable component of the retinoid visual cycle in vertebrates, through which the visual chromophore 11-cis-retinal (11-cis-RAL) is generated to maintain normal vision. Various blinding conditions in humans, such as Leber congenital amaurosis and retinitis pigmentosa (RP), are attributed to either homozygous or compound heterozygous mutations in RPE65. Herein, we investigated D477G missense mutation, an unprecedented dominant-acting mutation of RPE65 identified in patients with autosomal dominant RP. We generated a D477G knock-in (KI) mouse and characterized its phenotypes. Although RPE65 protein levels were decreased in heterozygous KI mice, their scotopic, maximal, and photopic electroretinography responses were comparable to those of wild-type (WT) mice in stationary condition. As shown by high-performance liquid chromatography analysis, levels of 11-cis-RAL in fully dark-adapted heterozygous KI mice were similar to that in WT mice. However, kinetics of 11-cis-RAL regeneration after light exposure were significantly slower in heterozygous KI mice compared with WT and RPE65 heterozygous knockout mice. Furthermore, heterozygous KI mice exhibited lower A-wave recovery compared with WT mice after photobleaching, suggesting a delayed dark adaptation. Taken together, these observations suggest that D477G acts as a dominant-negative mutant of RPE65 that delays chromophore regeneration. The KI mice provide a useful model for further understanding of the pathogenesis of RP associated with this RPE65 mutant and for the development of therapeutic strategies.

Figure 1

Generation of the D477G KI mouse model. A: The D477G targeting strategy is shown as a schematic representation. The targeting construct is designed to span 5.7 kb upstream of the point mutation (LA) and 1.7 kb downstream of the Neo cassette (RA). The Neo cassette was inserted in the intron region between exons 13 and 14. FRT-flanked Neo cassette in the targeted allele is deleted by FRT-FLP recombination, thereby generating a mutant allele containing the point mutation in exon 13 (asterisks). B: A PCR strategy was used to screen for the positive clones. LAN1 and A2 primer pair targeting the RA yielded a 2.18-kb product. WT genomic DNA was used as a negative control, and an individual clone (before reconfirmation) was used as a positive control. C: The PCR-confirmed clones were further validated by Southern blotting. DNA digested with ScaI or MfeI was hybridized with a probe against the Neo cassette. Corresponding signals from ScaI and MfeI were detected at 12.5 and 9 kb, respectively. D: Genotyping PCR using primers F1 and R1 flanking the FRT site after Flp-mediated deletion of the Neo cassette produced 549- and 448-bp products, each representing targeted and WT allele, respectively. Extra 101 bp present in targeted allele comprises 34-bp-long FRT sequence and 67-bp-long remnants from the Neo cassette. E: Sequence confirmation of the point mutation at codon 477. c.1430G>A is confirmed by sequencing the PCR amplicon obtained with a primer pair encompassing the region of the point mutation. +Ve, positive; F, forward; HET, heterozygous; HYB, hybrid; KI, knock in; LA, left homology arm; R, reverse; RA, right homology arm; WT, wild type.

Figure 2

Reduced RPE65 levels in D477G KI mice. A: Relative levels of the RPE65 mRNA in the eyecups of these mice were analyzed by real-time PCR. Representative values are normalized to mRNA levels of a housekeeping gene (Gapdh). B: Eyecups containing sclera, choroid, and RPE were dissected and homogenized. The eyecup homogenates from wild-type (WT/WT), the heterozygous KI (WT/KI), and the homozygous KI (KI/KI) mice were then subjected to Western blot analysis with an antibody against RPE65 (PETLET). β-Actin was used as loading control. Densitometry was used for semiquantification. C: Immunofluorescence labeling of RPE65 on the retinal cross sections. RPE65 is shown in red, and nucleus is shown in blue (DAPI). Error bars represent SDs from the triplicates within the experiment (A). n = 3 per group (A); n = 4 per group (B). ^∗∗∗P < 0.001 (t-test). Scale bar = 50 μm (C). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; KI, knock in; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium; WT, wild type.

Figure 3

Analysis of opsin expression and localization. A: Retinal cross sections from 3.5-month-old WT/WT and WT/KI mice were immunolabeled with antibodies against short-wavelength cone opsin (S-opsin, yellow), midwavelength cone opsin (M-opsin, red), or rhodopsin (green). Nuclei were counterstained with DAPI (blue). B: Light microscopic images of the retinal cross sections from 13-month-old WT/WT and WT/KI mice. C: Transmission electron microscopy images of 13-month-old WT/WT and WT/KI. An unusual accumulation of oil droplet–like organelles in the RPE of WT/KI mice are indicated by arrowheads. Scale bars: 50 μm (A); 40 μm (B); 10 μm (C). GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; KI, knock in; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium; WT, wild type.

Figure 4

Visual function assessment by electroretinography (ERG): Mice were fully dark adapted overnight before ERG. A: Scotopic ERG measurement (0.8 cd·s/m²) on WT/WT and WT/KI mice at 3, 4, and 5 months of age. B: Maximal ERG response (40 cd·s/m²) of these mice. C: Photopic ERG measurements (40 cd·s/m² with background of 10 cd/m²) of these mice. Top panels indicate averaged waveforms from each genotype at indicated ages, and bottom panels are the corresponding histograms of A- and B-wave values. Data are given as means ± SD (A–C, bottom panels). n = 12 (A–C, WT/WT); n = 8 (A–C, WT/KI). KI, knock in; WT, wild type.

Figure 5

Retinoid profile and isomerase activity in D477G KI mice. A: High-performance liquid chromatography (HPLC) retinoid profile analysis on the fully dark-adapted eyeballs from WT/WT and WT/KI mice. Both 11-cis-retinal (11-cis-RAL) and all-trans-retinyl ester (all-trans-RE) levels were measured. B: Isomerase assay using the eyecups from indicated mice. Levels of the generated 11-cis-retinol were measured by HPLC. The eyecup homogenates used for isomerase assay were examined by Western blot analysis to ensure the comparable amount of the total protein. Data are given as means ± SEM (A and B). n = 4 (A); n = 6 (B). ^∗P < 0.05 (t-test). KI, knock in; WT, wild type.

Figure 6

Analysis of visual chromophore regeneration. A: Double-flash electroretinography (ERG) on WT/WT and WT/KI mice. Fully dark-adapted mice were subjected to single flash (4 cd·s/m²), followed by test flashes (40 cd·s/m2) at various delay times. Representative waveforms from each genotype are shown. B: Normalized amplitude of A-wave from WT/WT and WT/KI mice are shown. A-wave amplitude generated from each test flash was normalized by a fully dark-adapted A-wave amplitude. C: Dark-adaptation recovery ERG examining the A-wave value. Mice in indicated groups were bleached at 1000 cd/m² for 2 minutes, followed by 60 minutes of rest in the dark. A single-flash ERG response at 10 cd·s/m² was acquired every 5 minutes. Representative waveforms from each genotype are shown. Black and red traces are before and after bleach ERG responses, respectively. Response from each time point is depicted in varying shades of gray. D: Averaged recovering A-wave amplitudes of WT/WT and WT/KI mice are shown. Data were fitted using a nonlinear regression curve with previously published formula. Data are given as means ± SEM (B and D). n = 6 (B, WT/WT); n = 8 (B, WT/KI); n = 10 (D, WT/WT); n = 16 (D, WT/KI). ^∗P < 0.05, ^∗∗∗P < 0.001 (t-test). KI, knock in; WT, wild type.

Figure 7

High-performance liquid chromatography (HPLC) analysis on 11-cis-RAL regeneration after photobleaching. HPLC retinoid profile analysis to quantify 11-cis-retinal (A) and all-trans-retinyl ester (all-trans-RE; B) in WT/WT, WT/KI, and WT/KO mice. Mice were subjected to 30 minutes of bleach in a light box with 5000 lux fluorescent light, followed by recovery in the dark for 15 or 30 minutes. The eyes were dissected and homogenized in the dark for HPLC analysis. Data are given as means ± SEM (A and B). n = 4. ^∗P < 0.05, ^∗∗∗P < 0.001 (t-test). KI, knock in; KO, knockout; WT, wild type.