The interplay of environmental luminance and genetics in the retinal dystrophy induced by the dominant RPE65 mutation

Abstract

SignificanceIn humans, genetic mutations in the retinal pigment epithelium (RPE) 65 are associated with blinding diseases, for which there is no effective therapy alleviating progressive retinal degeneration in affected patients. Our findings uncovered that the increased free opsin caused by enhancing the ambient light intensity increased retinal activation, and when compounded with the RPE visual cycle dysfunction caused by the heterozygous D477G mutation and aggregation, led to the onset of retinal degeneration.

Keywords: RPE65; photoreceptor; retinal degeneration; visual cycle.

Fig. 1.

ERG analysis of retina function in mice maintained under DimL and DayL luminance. (A) Representative dark-adapted single-flash ERG traces at 4 cd⋅s/m² in mice at 9 and 18 mo of age in the indicated genotypes. (B and C) Quantitative evaluation of scotopic response amplitudes of the WT/KI and WT/KO [a-wave (B), b-wave (C)] mice, at 3, 6, 9, 12, 15, and 18 mo of age, expose in DayL and DimL conditions. (D) Representative dark-adapted single-flash ERG recorded over the range of scotopic light intensities, log 0.004, 0.04, 0.4, and 4 cd⋅s/m² in 18-mo-old mice of indicated genotypes. Quantitative evaluation of a-wave (E) and b-wave (F) amplitudes in WT/KI and WT/KO mice maintained under DayL and DimL conditions; x axis values were log-transformed. (G) Representative dark-adapted single-flash ERG traces taken at 40 cd⋅s/m² (mix rod and cone) and photopic 10 cd⋅s/m² at constant background luminance of 10 cd/m² in mice of indicated genotypes at 18 mo of age. Quantitative evaluation of mix cone rod (H) and photopic (I) PR response in WT/KI and WT/KO mice raised in either DimL or DayL conditions. Data are presented as mean ± SEM n = 8 (WT/KI DimL & WT/KO DayL); n = 10 (WT/KO DimL); n = 8 (WT/KI DayL) *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 in one-way ANOVA with Tukey’s post hoc comparison.

Fig. 2.

Delayed rod PR a-wave recovery and reduced isomerase activity in dark-adapted heterozygous RPE65 D477G KI (WT/KI) mice. ERG a-wave recovery was evaluated after exposing dark-adapted mice maintained under DayL and DimL, to light at 1,000 cd/m² for 2 min. Subsequent single-flash ERG at 10 cd⋅s/m² was recorded every 5 min to evaluate the recovery of rod sensitivity in the photobleached mice of indicated genotypes. Percent (%) a-wave recovery was plotted with postbleaching amplitude taken 5 min against initial a-wave dark-adapted amplitude after visual pigment bleach in mice at the ages of 12 mo (A) and 18 mo (B). The dotted line indicates 50% and 100% recovery points on the graphs. (C) Prephotobleach a-wave amplitude was recorded at 10 cd⋅s/m² in dark-adapted mice. This value was taken as 100% recovery. Data are presented as mean ± SEM n = 10 for each group *P < 0.05, **P < 0.01, ***P < 0.001 in one-way ANOVA with Tukey’s post hoc comparison. (D) Representative HPLC chromatograms show separation of retinoids extracted from the eye-cups of 1-y-old DayL-raised mice. Peak 1, retinyl esters; peak 2, 11-cis-ROL. Quantification (Right) of 11-cis-ROL in the extracts showed a significant difference between the two groups. Each point represents data from a single mouse. Data are presented as mean ± SEM n = 8 for each group. Statistical significance was assessed by unpaired t test with Tukey’s post hoc comparison. ***P < 0.001.

Fig. 3.

Retina morphology in 18-mo-old DayL-exposed heterozygous RPE65 D477G KI (WT/KI) mice. (A) Representative images of hematoxylin, eosin, and saffron (H&E)-stained paraffin-embedded eye sections from mice of indicated genotypes. The lower panels (d–f) are retinal sections from three individual DayL-exposed WT/KI mice. Black arrows denote the development ORT in some of the WT/KI-DayL mice. (Scale bars, 400 µm for black, 100 µm for white.) (B) Histological measurement of ONL thickness in mice of indicated genotypes were taken from the optic nerve head (ONH) toward the periphery. Data depicted in spider graph shows a significant difference in ONL thickness only in the DayL-exposed WT/KI retinas, relative to the controls. (Scale bar, 100 µm.) (C) Representative SD-OCT images in live mice eyes of indicated genotypes show reduced TRT in the DayL-exposed WT/KI mice (C, d and e). A-scan shows the en face projection of the acquired retinal OCT volume (14 mm) centered on the optic nerve and illustrating the major blood vessels. B-scans are axial images showing various retinal layers made at the green line marked on the A-scans (C). Red arrow denotes the presence of ORT (C, e). (D) Quantification of TRT. (E) Quantification of individual layer thickness. Data are presented as mean ± SEM. Statistical analysis were performed with one-way ANOVA with Tukey’s post hoc correction. Statistical significance is indicated by **P < 0.01, ***P < 0.001, ^#P < 0.0001. WT/KO DimL, WT/KO DayL, and WT/KI DimL n = 8; WT/KI DayL n = 6. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer.

Fig. 4.

WT/KI mice eye-cups exhibited relative lower protein expression of RPE65 and LRAT compared with WT/KO mice. (A) Representative Western blots for levels of RPE65 and other visual cycle related proteins in mice eye-cups at PD 120. (B) RPE65 levels in the WT/KI, WT/KO, and KI/KI at PD 120 presented as fold-change to WT/WT RPE65. (C) Fold-change of LRAT levels relative to WT/WT. (D–F) Fold-change of other visual cycle protein levels. Each lane contains pooled samples from six eye-cups from three individual mice. n = 3. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, **** or ^#P < 0.0001 in one-way ANOVA with Tukey’s post hoc comparison.

Fig. 5.

D477G variant induces protein aggregation, and increases membrane association and mis-localization of RPE65 in vivo. Sucrose density sedimentation showed the formation of heavier protein aggregates in eye-cup homogenates from mice with indicated genotypes at 4 mo of age. (A) Representative blots with quantification bar graphs in the lower panels. RPE65 signal in each fraction was presented and plotted as percent of total RPE65 intensity. Fraction 1 is of the lowest density, and fraction 13 being the highest and the pellet. Red arrows indicate increased RPE65 signal in the 13th fraction in the WT/KI and KI/KI samples. n = 3. (B and C) Subcellular fractionation of RPE65 in D477G KI mice eye-cups. Fraction purity was analyzed using antibodies against indicated marker proteins. Fibrillarin: nuclear; calnexin: membrane. Representative Western blots (B) and quantification of RPE65 band intensity (C) in the membrane and cytosolic fraction. C, cytosolic; M, membrane; N, nuclear; S, cyto-skeleton. n = 3. (D) Immunofluorescence analyses of RPE65 in eye-cup flat-mounts. Green florescent RPE65 signal (a, e, i, and m) in mice of indicated genotypes. Zo-1 labels RPE cell boundary (b, f, j, and n) and DAPI (blue, c, g, k, and o) labels cell nuclei. (Scale bars, 30 μm for white and 10 μm for red.) (E) Quantification of average intracellular RPE65 signal. Data were presented as mean ± SEM, *P < 0.05, **P < 0.01, or ****P < 0.0001 in one-way ANOVA with Tukey’s post hoc comparison. n = 6 for each mice group.

Fig. 6.

D477G mutant associated with WT-RPE65 protein and predispose RPE65 to accelerated degradation. (A) Co-IP of His-tagged WT-RPE65 and FLAG-tagged D477G-variant showed the mutant to interact with the WT-RPE65. HEK293 cells were either singly or doubly transfected with constructs encoding FLAG-tagged D477G mutant and His-tagged WT-RPE65 protein. Reciprocal co-IP using either the anti-His or anti-FLAG antibody was performed in the above-mentioned transfected cell homogenates. Immunoblotting with either the anti-His or anti-FLAG antibody was used to determine coprecipitation. n = 3. I represents input (total protein extract); F.T. represents flow-through (the leftover unbound to beads antibody–protein fraction); and B represents bound (proteins that are bound by the antibodies and precipitated by the beads). (B) Western blot analysis of RPE65/D477G mutant protein in CHX-treated HEK293 cells. (C) Diagram depicting mean RPE65 degradation curves over 0, 6, and 12 h after the addition of CHX. n = 3. (D) Western blot analysis of RPE65 showing partial degradation arrested in cells coexpressing D477G mutant and WT-RPE65 by treatment with MG132 in the presence of CHX at indicated times. (E) Quantitative evaluation of RPE65 protein levels in cells expressing the indicated constructs and respective treatments. n = 3. All data are presented as means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001 in one-way ANOVA with Tukey’s post hoc comparison.

Fig. 7.

Schematic diagram showing proposed pathogenesis of dominant acting D477G RPE65 in the heterozygote KI mice exposed to DayL luminance. In the RPE of WT/KI mice, the D477G variant associates with WT-RPE65, resulting in RPE65 mislocalization and accelerated degradation of total RPE65. Moreover, abnormal protein aggregates are formed in the presence of the mutant. Under DayL luminance, the increase in demand for chromophore production in the RPE is unfulfilled by the D477G WT/KI RPE, which exhibited decreased RPE65 expression, leading to insufficient 11-cis-RAL regeneration. Both constitutive activation from the unligand opsin in the WT/KI retina and the toxic aggregation of RPE65 in WT/KI RPE together induce retina stress and accentuate the retina pathology. RPE65, retinal pigmented epithelium 65-kDa protein; t-RE, all-trans-retinyl-ester; t-Rol, all-trans-retinol; 11c-Ral, 11-cis-retinal; 11c-Rol, 11-cis-retinol; 11-RDH, 11-cis-retinol dehydrogenase; ?, additional mechanisms responsible for D477G mediated reduced isomerase activity.