The Cys214-->Ser mutation in peripherin/rds causes a loss-of-function phenotype in transgenic mice

Abstract

P/rds (peripherin/retinal degeneration slow) is a photoreceptor-specific membrane glycoprotein necessary for outer segment disc morphogenesis. Mutations in P/rds are associated with different blinding diseases. A C214S (Cys214-->Ser) missense mutation has been shown to be the cause for a late-onset form of ADRP (autosomal dominant retinitis pigmentosa) in humans. In the present study, we generated transgenic mice expressing P/rds with the C214S mutation and crossed them into rds mutant mice to elucidate the mechanism underlying the pathology of ADRP. Although an ample amount of transgene message was formed in C214S retinas from all transgenic lines, only a trace amount of the mutant protein was detected by Western blotting and immunoprecipitation. C214S mice on the wild-type or rds+/- backgrounds exhibited no signs of negative effects of the mutation on retinal structure or function, suggesting a loss-of-function phenotype. This phenotype is further supported by the absence of outer segment formation in the C214S mice on the rds-/- background. In contrast, expression of C214S protein in the inner retinal cells of transgenic mice or in COS cells resulted in the formation of a substantial amount of mutant protein, signifying a possible photoreceptor-specific regulation of P/rds. These results provide evidence that the loss-of-function phenotype seen in C214S transgenic mice shows a disease progression that correlates with ADRP patients carrying the same mutation, indicating that the C214S mutation on one allele of P/rds results in haploinsufficiency.

Figure 1. Generation and expression of the C214S transgene

(A) Diagram of the mutation and transgene construct. The hIRBP promoter directs expression to rods and cones; rds cDNA contains the C214S mutation and the P341Q modification; SV40 poly A signal allows for recognition of transgene expression. (B) Northern-blot analysis of whole retinal RNA extracts (20 μg/sample) taken from 20 days old mice of indicated genetic backgrounds of non-transgenic controls (lanes 1–3) and transgenic littermates (lanes 4–6). Two different probes were hybridized to the membrane: a cDNA probe that recognizes both endogenous and transgenic message, and an SV40 poly A probe that only recognizes the transgenic message. The 28 S band was included as a control for loading. This blot is representative for three independent experiments.

Figure 2. C214S protein expression, localization and interaction with Rom-1

(A) Western-blot analysis of retinal extracts (20 μg/sample) from 20–30 days old mice that were subjected to a reducing SDS/PAGE (10% polyacrylamide). Lanes 1–6 show a side-by-side comparison of transgenic retinas on the wild-type and rds backgrounds relative to non-transgenic retinas on the same backgrounds. The blot was probed with anti-P/rds-CT, anti-Rom-1-CT and anti-actin antibodies. (B) Western blot of P/rds and Rom-1 immunoprecipitates of retinal extracts (20 μl of sample from the 50 μl eluate/lane). In lanes 1–3, 200 μg of whole retinal protein extract was loaded into the reaction mixture. Lane 4 contained 300 μg of protein, while lanes 5 and 6 contained 400 and 500 μg of protein respectively. The blot was probed with the same anti-P/rds-CT and anti-Rom-1-CT antibodies that were used for immunoprecipitation. (C, D) Immunohistochemical analysis of non-transgenic and C214S retinas on wild-type, rds^+/− and rds^−/− backgrounds using anti-P/rds-CT and 1D4 (C) and the monoclonal antibody (3B6) and the polyclonal anti-opsin (D). NMP retina in (D) was used as a positive control for 3B6. Two different anti-opsin antibodies, monoclonal 1D4 (C) and a polyclonal IgG (D), were used as correlative, photoreceptor-specific reagents to label the outer and inner segments of the rod.

Figure 3. Morphology of C214S retinas is indistinguishable from controls on the same rds genetic backgrounds

Retinas taken from (A) 1 month old mice and (B) 6 months old mice. Retinal sections are representative of retinas from four different animals. The left panels of both (A) and (B) represent non-transgenic retinas, whereas the right panels represent C214S animals bred into the corresponding backgrounds (+/+, rds^+/− or rds^−/−). (C) Electron microscopy of rod OSs from non-transgenic retinas (left panel) and C214S retinas in the indicated genetic backgrounds (right panel). Scale bar, 4 μm.

Figure 4. The C214S mutation causes a loss-of-function phenotype

Dark-adapted (left panels in A and B) and light-adapted (right panels in A and B) ERG amplitudes from 1 month old (A) and 6 months old (B) C214S and non-transgenic mice on different rds backgrounds. ERG values are an average of 8–10 eyes for each genotype.

Figure 5. Expression of C214S protein in non-photoreceptor cells

(A) Northern-blot analysis of the 550 mouse line with the C214S transgene. Lanes 1–6 show the comparison of wild-type and rds RNA transcripts with the C214S RNA transcripts from the 550 line in the corresponding genetic backgrounds. (B) Immunohistochemical analysis of wild-type and C214S retinas from line 550 on the rds^−/− background at 1 month of age. The OS, inner segment (IS), outer nuclear layer (ONL) and inner nuclear layer (INL) are indicated. The C214S retina does not have any OSs, due to the rds^−/− background. Sections are representative of four different retinas per genotype. (C) Western blot of retinal extracts (20 μg/lane) from wild-type (lane 1) and C214S animals in the rds^−/− background at different ages (lanes 2–5; P, post-natal. Ages in days.) that were run on a 10% reducing gel. The blot was probed with anti-P/rds-CT and anti-actin antibody. (D) Western blot of P/rds immunoprecipitates (IP) of retinal extracts (20 μl of sample from the 50 μl eluate). Whole retinal extract (200 μg) was added to each reaction mixture (lanes 1–6). The blot was probed with anti-P/rds-CT and anti-Rom-1-CT antibody. The IgG was included as a control for loading.

Figure 6. Protein analysis of C214S and wild-type P/rds expressed in COS cells

(A) Western blot of P/rds from wild-type retinal extract (5 μg in lane 1) and COS cell extracts (30 μg in lanes 2–4). The control is extracted from COS cells transfected with the pcDNA3.1 vector alone. (B) Immunocytochemistry of COS cells transiently transfected with either wild-type rds cDNA (pcDNA3.1-WT) or C214S rds cDNA (pcDNA3.1-C214S). The inset is a picture of a single cell without the DAPI staining. Pictures are representative of five different fields for three separate experiments.