The Y141C knockin mutation in RDS leads to complex phenotypes in the mouse

Abstract

Mutations in the photoreceptor-specific gene peripherin-2 (PRPH-2, also known as retinal degeneration slow/RDS) cause incurable retinal degeneration with a high degree of phenotypic variability. Patient phenotypes range from retinitis pigmentosa to various forms of macular and pattern dystrophy. Macular and pattern dystrophy in particular are associated with complex, poorly understood disease mechanisms, as severe vision loss is often associated both with defects in the photoreceptors, as well as the choroid and retinal pigment epithelium (RPE). Since there is currently no satisfactory model to study pattern dystrophy disease mechanisms, we generated a knockin mouse model expressing an RDS pattern dystrophy mutation, Y141C. Y141C mice exhibited clinical signs similar to those in patients including late-onset fundus abnormalities characteristic of RPE and choroidal defects and electroretinogram defects. Ultrastructural examination indicated that disc formation was initiated by the Y141C protein, but proper sizing and alignment of discs required wild-type RDS. The biochemical mechanism underlying these abnormalities was tied to defects in the normal process of RDS oligomerization which is required for proper RDS function. Y141C-RDS formed strikingly abnormal disulfide-linked complexes which were localized to the outer segment (OS) where they impaired the formation of proper OS structure. These data support a model of pattern dystrophy wherein a primary molecular defect occurring in all photoreceptors leads to secondary sequellae in adjacent tissues, an outcome which leads to macular vision loss. An understanding of the role of RDS in the interplay between these tissues significantly enhances our understanding of RDS-associated pathobiology and our ability to design rational treatment strategies.

Figure 1.

Genetic knockin of the Y141C mutation in RDS does not have an impact on RDS expression levels. The Y141C mutation was introduced into exon 1 of the RDS gene in order to drive properly regulated expression of the Y141C-RDS allele (A). Total RNA was isolated from the retinas of mice of the indicated genotypes at P30 and subjected to qRT-PCR for RDS and the housekeeping gene HPRT (B). Total RNA was also subjected to northern blotting using radiolabeled RDS cDNA as a probe (C). 28S and 18S ribosomal RNA are marked for size reference and 28S is shown as loading control. Levels of RDS protein in total retinal extracts were analyzed from indicated genotypes by WB followed by visualization using antibodies specific to RDS, ROM-1 and actin (D). Band densities were normalized to actin and set relative to WT: RDS (E), ROM-1 (F). Shown are mean ± SEM. N = 3 (B and C) and 8 (E and F) independent samples. ***P < 0.001 by one-way ANOVA with Bonferroni's post hoc comparison.

Figure 2.

Y141C-RDS traffics to the OS. Retinal sections at P30 from the indicated genotypes were labeled with antibodies against RDS (green A–C) and Na⁺K⁺ATPase (red, A), ROM-1 (red, B), rhodopsin (red, top C) or S-opsin (red, bottom C). Arrows and insets indicate regions of co-localization between RDS and opsins. OS, outer segments; IS, inner segment; ONL, outer nuclear layer. Scale bar: 20 µm.

Figure 3.

Y141C-RDS is able to support limited OS function. The ability of the rds^Y/+ and the rds^Y/Y animals to support OS function was assessed using scotopic (rod) and photopic (cones) ERG at P30 (A and B) and at P180 (C). Wave forms were prepared by averaging the values from all available ERGs of the indicated genotypes at P30 and showing the mean (black) ± SEM (gray shading) (A). The amplitude of maximum scotopic a- and b-waves, and maximum photopic b-waves, is plotted as mean ± SEM at P30 and P180 (B and C). N = 4–14 animals for each genotype/age. ***P < 0.001 by one-way ANOVA with Bonferroni's post hoc comparison.

Figure 4.

Expression of Y141C-RDS leads to photoreceptor degeneration. Representative light microscopic images from retinal sections at P30 (A) and P180 (B) are shown. OS length was quantified and plotted as mean ± SEM (C). Rhodopsin protein levels were assessed by WB and quantified. (D) ONL thickness was assessed in central retinal sections at increasing distances from the optic nerve head at P30 (E) and P180 (F). Plotted are means ± SEM from N = 3 eyes/genotype/age. RPE, retinal pigment epithelium; OS, outer segments; IS, inner segment; ONL, outer nuclear layer. Scale bars: 5 µm.

Figure 5.

Fundus photographs of rds^Y/+ show retinal flecking at P180. Fundoscopic examination was performed at the indicated ages and genotypes. At P30 no significant pigmentation defects were observed in any of the genotypes when compared with WT (A). At P180, rds^+/− fundus still appeared normal; however, yellow flecks were apparent throughout the fundus of the rds^Y/+, rds^−/− and rds^Y/Y (arrows show examples of this widespread flecking, B). Left and right columns of (B) come from the same eye, left column is brightfield, and right column shows fundus autofluorescence. N = 4–11 eyes/genotype/age.

Figure 6.

Abnormal complexes of both RDS and ROM-1 are observed by WB. Non-reducing SDS–PAGE/WBs were prepared from total retinal extracts in the presence of NEM and probed with antibodies specific to RDS (A, top), ROM-1 (A, middle) and actin (A, bottom). Note that while RDS runs as a monomer and a dimer in both the WT and rds^+/− retinal extracts, in both the rds^Y/+ and the rds^Y/Y abnormal high-molecular-weight complexes were observed when probed with antibodies against RDS and with ROM-1 (arrows). The same samples were also analyzed by reducing SDS–PAGE/WB (A, right). Only monomers were observed when samples were prepared under reducing conditions. Quantification of relative levels of each RDS and ROM-1 complex is shown for reference, N = 4 independent samples (B). Reciprocal co-IPs confirm that RDS/ROM-1 interactions were maintained in retinal extracts from animals carrying the Y141C-RDS mutation (C). FT, flow through (unbound); Pre, preclear, N = 3.

Figure 7.

Y141C-RDS leads to formation of abnormal high-molecular-weight oligomers. To analyze RDS/ROM-1 complexes under non-denaturing conditions, whole retinal extracts were prepared and run on a continuous 20% (fraction 1) to 5% (fraction 12) non-reducing sucrose gradient. Fractions were collected and analyzed using non-reducing SDS–PAGE/western blotting with antibodies specific for RDS (top) and ROM-1 (bottom). The position that molecular weight markers appear when using this protocol is listed above the fraction number for reference. In the rds^Y/+ a full complement of normal RDS monomer and dimer were seen; however, abnormal high-molecular-weight complexes are observed in the heavier fractions as well as the pellet (arrowheads, middle). The rds^Y/Y lacks normal higher-order complexes (dimers in high-density fractions) of RDS which are replaced by very distinct high-molecular-weight complexes in fractions 1–3 and in the pellet (arrowheads, right). N = 3–4 independent experiments/genotype.

Figure 8.

Abnormal disulfide-linked complexes are formed in the presence of the Y141C mutation. Pictorial representation of velocity sedimentation data from Figure 7. RDS: black, ROM-1: white, Y141C-RDS: grey. Striped molecules could be either WT ROM-1 or Y141C-RDS (white and grey) or WT RDS or ROM-1 (black and white). Complexes that appear abnormal by non-reducing SDS–PAGE are underlined.

Figure 9.

The Y141C-RDS protein is unable to support normal OS ultrastructure. Transmission electron microscopy was performed on eyes from the indicated genotypes at P30 (A and B). Y141C-RDS-expressing photoreceptors appear to have a significant improvement in overall OS structure (arrowhead) when compared with appropriate rds^+/− controls, while rds^Y/Y exhibits only small highly malformed OSs (arrow). Higher magnification images reveal that despite increases in overall organization, Y141C-RDS-expressing retinas still are characterized by OS discs which lack proper orientation and sizing (B). Interestingly, in the rds^Y/Y a large number of abnormal membranous vesicular structures appear to surround the flattened discs (A and B, asterisks). OS, outer segments; IS, inner segments. Scale bars: 5 µm in (A) and 500 nm in (B). N = 3 eyes/genotype.

Figure 10.

Y141C-RDS and ROM-1 accumulate in a distinct region from rhodopsin. Retinal thin sections collected at P30 from the indicated genotypes were IG labeled with antibodies for RDS (A), ROM-1 (B) and rhodopsin (C) and secondary antibodies conjugated to 10 nm gold particles. Both ROM-1 and RDS localize properly to the rim regions of OS discs in the WT, rds^Y/+ and the rds^Y/Y(arrows). In the rds^Y/Y, the abnormal membranous vesicles are enriched for RDS and ROM-1 and contain relatively less rhodopsin labeling (asterisks). CC, connecting cilium. Scale bar: 500 nm.