Insights into the mechanisms of macular degeneration associated with the R172W mutation in RDS

Abstract

Mutations in the photoreceptor tetraspanin gene peripherin-2/retinal degeneration slow (PRPH2/RDS) cause both rod- and cone-dominant diseases. While rod-dominant diseases, such as autosomal dominant retinitis pigmentosa, are thought to arise due to haploinsufficiency caused by loss-of-function mutations, the mechanisms underlying PRPH2-associated cone-dominant diseases are unclear. Here we took advantage of a transgenic mouse line expressing an RDS mutant (R172W) known to cause macular degeneration (MD) in humans. To facilitate the study of cones in the heavily rod-dominant mouse retina, R172W mice were bred onto an Nrl(-/-) background (in which developing rods adopt a cone-like fate). In this model the R172W protein and the key RDS-binding partner, rod outer segment (OS) membrane protein 1 (ROM-1), were properly expressed and trafficked to cone OSs. However, the expression of R172W led to dominant defects in cone structure and function with equal effects on S- and M-cones. Furthermore, the expression of R172W in cones induced subtle alterations in RDS/ROM-1 complex assembly, specifically resulting in the formation of abnormal, large molecular weight ROM-1 complexes. Fundus imaging demonstrated that R172W mice developed severe clinical signs of disease nearly identical to those seen in human MD patients, including retinal degeneration, retinal pigment epithlium (RPE) defects and loss of the choriocapillaris. Collectively, these data identify a primary disease-causing molecular defect in cone cells and suggest that RDS-associated disease in patients may be a result of this defect coupled with secondary sequellae involving RPE and choriocapillaris cell loss.

Figure 1.

R172W protein is expressed and properly localized on the Nrl^−/− background. (A) Retinal extracts were harvested at P30 from the indicated genotypes. Shown are representative WBs probed with RDS-CT, ROM1-CT and actin antibodies (loading control) from reducing SDS-PAGE. (B) Quantification of WB data. Protein levels measured densitometrically and normalized to actin then to the mean WT value on each blot. N = 6 retinas per genotype, shown are means ± SEM. **P < 0.01, ***P < 0.001 by one-way ANOVA with Bonferroni's post hoc comparison. (C and D) IF was performed on P30 retinal sections using mAB 3B6 (green, C and D, for R172W RDS), and either RDS-CT (red, C, R172W and WT RDS), S-opsin (red, D, left) or M-opsin (red, D, right). Sections were counterstained with DAPI (blue). Images are single planes from a confocal stack. N = 3–4 eyes per genotype. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; R, rosette. Scale bars: 25 µm (C), and 10 µm (D).

Figure 2.

R172W and ROM-1 properly traffic to the OS. (A) Frozen retinal sections from transgenic and non-transgenic animals were immunolabeled with ROM1-2H5 (green) and RDS-CT (red) antibodies. (B). Sections were labeled with the inner segment marker Na⁺K⁺ ATPase (ATPase, green) and either RDS-CT or ROM1-CT (red). N = 3 eyes per genotype. Abbreviations as in Figure 1. Scale bar: 10 µm.

Figure 3.

R172W causes a dominant defect in cone function. Full-field spectral photopic ERG was performed on P30 R172W transgenic mice or age-matched non-transgenic (Non-T) controls. Shown are maximum photopic b-wave amplitudes from mice in the rod-dominant (A) or cone-dominant (Nrl^−/−, B) background. ERGs were measured in response to green (left, M-cones) or UV (right, S-cones) light. N = 8–12 mice per genotype, shown are means ± SEM. **P < 0.01, ***P < 0.001 by one-way ANOVA with Bonferroni's post hoc comparison.

Figure 4.

R172W leads to dominant defects in cone OS ultrastructure. Eyes from the indicated genotypes were collected and fixed at P30 for histology at the light (A) and EM (B) levels. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; IS, inner segment; OS, outer segment. Scale bars: 25 µm (A) and 1 µm (B). N = 3 eyes per genotype.

Figure 5.

R172W does not alter the distribution of RDS complexes. Retinal extracts collected at P30 from transgenic or non-transgenic controls in the rod-dominant (A) or cone-dominant (Nrl^−/−, B) background were fractionated on continuous 5–20% non-reducing sucrose gradients. Fractions were separated on reducing SDS-PAGE and blots were probed for RDS or ROM-1. Graphs show the amount of RDS (solid line) or ROM-1 (dashed line) in each fraction as a percent of total immunoreactivity. Plotted are means ± SEM. Peak fractions for molecular weight markers in the gradient are shown with arrows: apoferritin (443 kD), beta-amylase (200 kD), alcohol dehydrogenase (150 kD), bovine serum albumin (66 kD) and carbonic anhydrase (29 kD). Two retinas were pooled for each extraction/fractionation and three to six independent experiments were performed per genotype.

Figure 6.

R172W leads to the formation of abnormal ROM-1 complexes in the Nrl^−/− background. Retinal extracts from the non-reducing gradients shown in Figure 5 were separated on non-reducing SDS-PAGE. Blots were probed with RDS (A) or ROM-1 (B). Graphs show the amount of RDS/ROM-1 found as monomer (dashed line, A and B), dimer (solid line, A and B) or in the case of ROM-1, an abnormal large MW complex (dotted line, B) as a percent of total immunoreactivity. Plotted are means ± SEM. Peak fractions for molecular weight markers in the gradient are shown with arrows as in Figure 5. Two retinas were pooled for each extraction/fractionation and three six independent experiments were performed per genotype.

Figure 7.

R172W binds to ROM-1 in cones. (A) Retinal extracts underwent reciprocal co-IP with RDS-CT (top) or ROM1-CT (bottom). Shown are reducing SDS-PAGE/WBs on the bound fraction (input and unbound fractions are found in Supplementary Material, Fig. S4) probed with RDS-CT or ROM1-CT. (B) Extracts underwent IP with RDS-CT and non-reducing SDS-PAGE/WBs are shown (probed with RDS-CT or ROM-1 mAB 2H5). I, input; B, bound; U, unbound. Two retinas were pooled for each IP and three independent experiments were performed per genotype.

Figure 8.

R172W causes RPE atrophy and degeneration of the choriocapillaris. R172W mice (A) or age-matched non-transgenic controls (WT: B, rds^+/−: C) underwent fundus imaging (left column) and FA (right column) at 2 months of age. (A) Images from three different R172W/rds^−/− eyes to illustrate the varied phenotype in this background. Images in the left column are from the same eye as those in the right column. Yellow arrows in (A) show fundus abnormalities and red arrows show shadows of retinal vasculature in a different imaging plane. (D–F) Shown are fluorescein angiograms (right) and histological sections (left) from the indicated genotypes at 5 months of age. White arrows point to small vessels of the choriocapillaris and white arrowheads show RPE vacuoles. N = 6/8 eyes per genotype. Scale bar, 10 µm. Sc, sclera; Ch, choroid; RPE, retinal pigment epithelium; ChC, choriocapillaris.

Figure 9.

R172W/rds^−/− eyes exhibit pan-retinal degeneration and RPE defects. (A) Shown are representative 40× micrographs captured from the indicated region from eyes harvested at 5 months of age. (B) Shown is the quantification of the thickness of the ONL from sections as in (A). N = 4–5 eyes/group. *P < 0.05, *** P < 0.001. (C) Shown are higher magnification sections of the RPE in the region near the optic nerve head (ONH). Observe vacuoles in the RPE. Scale bar, 10 µm. Ch, choroid; RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.