Photoreceptor Disc Enclosure Is Tightly Controlled by Peripherin-2 Oligomerization

Abstract

Mutations in the PRPH2 gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened "disc" membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous "whorls" instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENT The light-sensitive photoreceptor outer segment contains a stack of flattened "disc" membranes that are surrounded, or "enclosed," by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

Keywords: cilium; outer segment; peripherin; photoreceptor; photoreceptor disc; retina.

Figure 1.

C150S/+ and Y141C/+ photoreceptors have an increased number of open discs at the outer segment base. A, Representative low-magnification TEM images of tannic acid/uranyl acetate-stained retinas of WT, rds/+, C150S/+, and Y141C/+ mice at P16. Scale bar, 2 µm. B, Representative high-magnification TEM images of photoreceptors from WT, rds/+, C150S/+, and Y141C/+ mice at P16. Scale bar, 0.5 µm. C, Quantification of the number of darkly stained open discs at the rod outer segment base in retinas obtained from WT, rds/+, C150S/+, and Y141C/+ mice. Each data point represents a single outer segment. For each genotype, three retinas were analyzed with at least 18 outer segments analyzed per retina. One-way ANOVA with post hoc Tukey's multiple comparisons analysis revealed statistically significant increases in the number of open discs in the C150S/+ (p < 0.0001) and Y141C/+ (p < 0.0001) combined samples and a statistically significance decrease in the rds/+ (p = 0.0371) combined samples. *p < 0.05. ****p < 0.0001. D, High-magnification TEM image of a photoreceptor from a Y141C/+ mouse at P16 showing an extraordinarily high number of open discs. Scale bar, 0.5 µm.

Figure 2.

The C150S and Y141C heterozygous mutations affect PRPH2 supramolecular organization without causing a significant reduction in the PRPH2 expression level. A, B, A single Western blot probed for both PRPH2 and ROM1 after protein separation by SDS-PAGE under reducing conditions. Each sample contained 10 µg of lysate obtained from eyecups of WT, rds/+, C150S/+, or Y141C/+ collected at P16. The amount of PRPH2 and ROM1 was quantified on the same blot using densitometry of at least four independent samples, normalized to β-actin as a loading control and plotted in the graph on the right relative to WT. One-way ANOVA with Dunnett's multiple comparisons post hoc test revealed a statistically significant reduction in PRPH2 for rds/+ (**p = 0.0096) but not C150S/+ or Y141C/+ eyecups (p = 0.9999 and p = 0.9346, respectively), while there was no statistical significance in ROM1 levels. C, D, A single Western blot probed for PRPH2 and ROM1 performed after protein separation by SDS-PAGE under nonreducing conditions. Each sample contained 10 µg of lysate obtained from eyecups of WT, rds/+, C150S/+, or Y141C/+ collected at P16. Under these conditions, PRPH2 and ROM1 run as abnormal high order complexes (H, >250 kDa), dimers (D, ∼75 kDa), and monomers (M, ∼37 kDa). Protein amount in each band was quantified using densitometry of three independent samples and plotted in the graph on the right. Two-way ANOVA with Dunnett's multiple comparisons post hoc test revealed that more PRPH2 exists as a monomer than dimer in C150S/+ eyecups (monomer, p < 0.0001; dimer, p < 0.0001) as well as smaller changes in both rds/+ (monomer, p = 0.0243; dimer, p = 0.0243) and Y141C/+ eyecups (monomer, p < 0.0001, dimer, p = 0.0002), while there was no statistical significance in any differences in ROM1 complexes. The high order complex that includes both PRPH2 and ROM1 was only observed in Y141C/+ eyecups.

Figure 3.

Velocity sedimentation analysis of PRPH2 supramolecular organization in rds/+, C150S/+, and Y141C/+ retinal lysates. A, Lysates obtained under nonreducing conditions from eyecups of WT, rds/+, C150S/+, and Y141C/+ mice at P16 were subjected to velocity sedimentation on 5%-20% sucrose gradients. Twelve fractions were collected with fraction #1 corresponding to 20% sucrose and fraction #12 to 5% sucrose. Proteins from each fraction were subjected to reducing SDS-PAGE and Western blotting for PRPH2. B, Quantification of PRPH2 in each fraction was performed using densitometry of three independent lysates and normalized to the total PRPH2 content across all fractions. Two-way ANOVA with Dunnett's multiple comparisons post hoc test revealed a statistically significant change in PRPH2 content of two fractions from C150S/+ lysates: a reduction in high order oligomers in fraction #1 (*p = 0.0124) and a corresponding increase in smaller PRPH2 complexes in fraction #5 (*p = 0.0156). C, Proteins obtained from fraction #1 of three independently sedimented WT and Y141C/+ samples were separated by SDS-PAGE under nonreducing conditions and Western blotted for PRPH2. Under these conditions, PRPH2 runs predominantly as a dimer (D, ∼75 kDa) with very few monomers (M, ∼37 kDa). Abnormal, high order complexes (H, >250 kDa) exist only in the Y141C/+ sample.

Figure 4.

C150S/+ mice exhibit a stepwise progression of defects in disc enclosure leading to the formation of membranous whorls. A–G, Representative TEM images of tannic acid/uranyl acetate-stained retinas of C150S/+ mice at P16. Outer segment structural defects ranged from slightly overgrown open discs to membranous whorls. On occasion, overgrown membrane structures extend laterally across the outer segment giving the appearance of open discs in the middle of the outer segment (E). Scale bars, 0.5 µm.

Figure 5.

Y141C/+ mice exhibit a stepwise progression of defects in disc enclosure leading to the formation of membranous whorls. A–G, Representative TEM images of tannic acid/uranyl acetate-stained retinas of Y141C/+ mice at P16. Outer segment structural defects ranged from slightly overgrown open discs to membranous whorls. Scale bars, 0.5 µm.