Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations

Abstract

Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-cis-retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its retinal cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore during rod outer segment development.

FIGURE 1.

Retinal phenotype of humans with adRP caused by the P23H opsin gene mutation. A, extent of the kinetic visual field (for the larger V-4e stimulus, left, and smaller I-4e, right) is shown as a function of age for 19 patients. Longitudinal data from a subset of the patients are shown as symbols connected by lines. Fields that are normal (>90%) are shown with unfilled symbols, and fields that are abnormal with dark gray symbols; light gray symbols refer to those patients with normal V-4e but abnormally reduced I-4e fields. Inset (right), three representative patients showing a full visual field (P1), altitudinal (superior field) loss (P2), and a small central island with a residual island of nasal field function (P3). B, extent of visual field constriction for the I-4e stimulus shows a faster progression rate compared with that measured with the V-4e stimulus in the subset of patients with longitudinal data. Presumably, visual field constriction in all individuals progresses with an invariant exponential rate (7%/year for V-4e and 18%/year for I-4e) after a variable age of disease onset, which is estimated from the average of the intercepts fit to log-linear data for each target. C, retinal topography of rod sensitivity loss (upper row) and cone sensitivity loss (lower row) demonstrating mild (n = 2; ages 15 and 22), intermediate (n = 5; ages 18–54), and severe (n = 6; ages 30–82) stages of this disease. Rod loss was measured with a 500-nm stimulus (dark-adapted) and cone loss with a 600-nm stimulus (light-adapted). Color changes delineate the 50th percentile contour for a given level of sensitivity loss (specified in log units on the color scale; sc, scotoma). Maps are shown as visual fields of the right eye. S, I, N, and T refer to superior, inferior, nasal, and temporal visual field, respectively. D, retinal laminar architecture of a normal control subject (age 31) and P4 (age 18) imaged with optical coherence tomography along the vertical meridian from the fovea extending 6.5 mm into the superior retina. White rectangle represents location of the LRP shown on the right (black trace). Correspondence between local reflectivity changes on the LRPs and anatomical laminae of ONL, rod, and cone outer segments are shown. The thickness of the laminae was quantified at three neighboring locations (centered at 1.7, 2.3, and 2.9 mm from the fovea in the superior retina) in three patients (P4, P3, and P2) and compared with results in normals (error bars ±2 S.D.). Significant differences from normal are marked with an asterisk. Note the superior retinal region in P4 with remaining ONL showing loss of the signal originating from the connecting cilium between inner and outer segments (arrow).

FIGURE 2.

Two-dimensional model of rhodopsin, targeting strategy, and molecular characterization of P23H knock-in mice. A, two-dimensional model of human and mouse rhodopsin. The 18 residues that are not conserved between human and mouse are indicated with white and bold black letters, respectively. Residues of transmembrane helices are indicated by open circles. Lys²⁹⁶, indicated by white filled circle, makes a Schiff base linkage with 11-cis-retinal. The RefSeq data base accession numbers for the presented proteins are NP_000530.1 and NP_663358.1 for human and mouse opsin, respectively, and the two-dimensional model was modified from Ref. . Amino acid sequence of mouse and human opsin are well conserved including Pro²³. The P23H mutation (indicated by an arrow) is located on the extracellular or intradiscal face of the protein near the Asn¹⁵ N-linked glycosylation site. B, targeting strategy for P23H opsin knock-in mice. Top, mouse opsin consists of five exons (E1–5). The opsin enhancer region 2 kb upstream of the opsin gene is indicated by gray line. 2nd row, the targeting vector contained 6.4 kb of 5′ and 1.9 kb of 3′ homologous sequence with C to A transversion at codon 23 (A inside arrowhead). Vector backbone sequences are indicated by hatched lines, and β-lac stands for the β-lactamase gene. 3rd row, as a result of homologous recombination, codon 23 in exon 1 was changed to CAC and the flippase recognition target (FRT)-loxP flanked neomycin resistance gene (Neo) was inserted after exon 1. Bottom row, neomycin was removed from targeted locus by cre-loxP recombination. By this knocking-in method, we generated mice with a point mutation at codon 23, which encode the P23H opsin. This mouse lacks the neomycin cassette but has a 174-bp vector backbone insertion in intron between exon 1 and exon 2. C, chromatogram of opsin cDNA from a P23H/+ mouse. The cDNA sequence chromatograms of two clones from one PND 26 P23H/+ mouse retina confirm the presence of both mutant and WT mRNAs. D, genotyping. PCR primers were designed to amplify the intron where the 174 bp of vector backbone sequence was inserted (Fig. 2B, bottom). The WT allele produced 399 bp and the P23H opsin allele produced 573 bp of PCR products. The negative control displayed no template contamination. Identities of PCR products were confirmed by sequencing (supplemental data 2). E, cDNA ratios of the P23H opsin to WT opsin. A total of 139 opsin cDNA clones isolated from four heterozygous mice (mouse ID 129, 131, 132, and 141, PND 26) were sequenced and used to identify ratios of P23H and WT opsin to subtotal clone numbers in each mouse. There was no statistically significant difference between P23H and WT opsin ratios (p = 0.62 between WT and P23H).

FIGURE 3.

Progressive retinal degeneration in P23H/+ mice. A, plastic sections of retinas from WT (+/+), heterozygous (P23H/+), and homozygous (P23H/P23H) mice of differing PND ages. Sections were stained with toluidine blue. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; and GC/NFL, ganglion cell/nerve fiber layer. Arrows represent surviving rod nuclei in a homozygous P23H/P23H mouse. B, data showing the number of nuclei per column in genetically different mice. Cryosections from WT, P23H/+, and P23H/P23H mice of differing PND ages were used for quantification. Data were derived from three eyes of three or five eyes of five mice. P23H/+ mice show age-dependent progressive retina degeneration, which is more severe in the ventral retina compared with the dorsal retina. P23H/P23H mice lost most of their photoreceptor cells by PND 63.

FIGURE 4.

Rods and cones in P23H/+ mice. Cryo-sections from PND 35 WT and P23H/+ mice from the same litter were double-stained with opsin (1D4) antibody and lectin PNA. Images labeled with same marker were captured under identical conditions. Significant shortening of ROS was observed in P23H/+ mice, but the number of cone photoreceptor cells labeled by PNA was not reduced by this age. RPE, retinal pigmented epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC/NFL, ganglion cells/nerve fiber layer; DIC, differential interference contrast.

FIGURE 5.

Rod and cone nuclei numbers and images in P23H/+ mice. A, average numbers of rod and cone nuclei in 200-μm lengths of WT and P23H/+ mouse retina were determined at PND 63. Compared with WT littermates, the number of rod photoreceptor nuclei in P23H/+ mouse was almost half, whereas the numbers of cone nuclei were virtually the same. B, plastic sections of retinas from PND 63 WT and P23H/+ mice were stained with toluidine blue. Rod and cone photoreceptor nuclei were distinguishable by the shape of their chromatin; cone nuclei are indicated by arrows. We also identified dead rod photoreceptor nuclei that had lost clumps of chromatin (indicated by arrowheads). C, TEM images reveal dead photoreceptor cell nuclei (arrowheads) in both WT and P23H/+ mice. IS, inner segment; OLM, outer limiting membrane; and ONL, outer nuclear layer.

FIGURE 6.

ERG responses in P23H/+ mice. Full field ERG responses of P23H/+ and WT (C57BL/6) mice were recorded under scotopic (A and B) and photopic (C) conditions. Compared with WT control mice, rod photoreceptor cell-evoked a- and b-wave amplitudes under scotopic conditions were reduced in P23H/+ mice at all analyzed ages, and the extent of this abnormality increased with age (A and B). Cone photoreceptor cell-dependent b-wave amplitudes under photopic conditions in P23H/+ mice were same as in WT mice at PND 40–41 but then decreased with age (C). Rod photoreceptor cell function was reduced to about half that of WT at PND 40–41 and decreased even more with age. Cone function was close to WT at PND 40–41, but reduced to about 80% of WT at PND 70, and then it decreased with age as well.

FIGURE 7.

P23H opsin, glycosylation and ratio to WT opsin protein. A, retinal homogenates from PND 23 WT, P23H/+, and P23H/P23H mice were labeled with N-terminal (B6–30) and C-terminal (1D4) opsin antibodies. All lanes were loaded with 10 μg of homogenate. Opsin monomers (∼35 kDa) produced double bands due to differences in glycosylation. The molecular mass of opsin monomers shifted down to ∼30 kDa after treatment with PNGase F. Double bands were due to incomplete deglycosylation. No glycosylation difference was observed between WT and P23H/+ opsin. No detectable signal was observed in retinal homogenates from P23H/P23H mice. B, retinal homogenates from PND 28 mice were labeled with B6-30 and anti-GFP antibodies. Left panel, in homozygous GFP-tagged human opsin knock-in mice (hrhoG/hrhoG), strong bands were observed for the monomer of GFP-tagged human opsin (∼70 kDa) and its dimer (∼160 kDa). A band of human opsin with a truncated GFP tag (∼ 40 kDa) was also seen that was distinguishable from the P23H opsin band (∼35 kDa) observed in lane of P23H/hrhoG; a heterozygous hrhoG and P23H knock-in mouse. Two μg of protein were loaded. Middle panel, different amounts of retinal homogenate from P23H/hrhoG mice were loaded, from left to right: ×1, 0.175 μg; ×10, 1.75 μg; and ×100, 17.5 μg. A faint GFP-tagged human opsin monomer band was detected in lane ×1 (∼70 kDa, thick black arrow) and very faint P23H opsin monomer band was detected in lane ×10 (∼35 kDa, open arrow). The P23H band was obvious in lane ×100 (open arrow) where a fainter truncated GFP tagged human opsin monomer band was also seen (∼40 kDa, narrow arrow). Right panel, 2 μg of retinal homogenate from a P23H/hrhoG mouse labeled with anti-GFP antibody produced a single broad band at ∼70 kDa confirming the identity of GFP-tagged human opsin. C, immunoblots of 2 μg of protein samples treated with Endo H and PNGase F were resolved on 12% SDS-polyacrylamide gels (upper panels) and 10% gels (lower panels). Left panel, retinal homogenate from a WT mouse (C57BL/6J, 5-month-old) labeled with 1D4 antibody. Both WT opsin dimer and monomer were shifted to lower molecular weights after either Endo H- or PNGase F treatment. Middle panel, retinal homogenate from a P23H homozygous mouse (PND 10) labeled with 1D4 antibody evidenced a similar shift. Right panel, retinal homogenate from a P23H homozygous mouse (PND 10) labeled with B6-30 antibody. Arrowheads in upper panels indicate P23H opsin dimers resolved in 12% gels. Arrowheads in lower panels show P23H opsin dimers resolved in 10% gels. Open arrow, P23H opsin monomer. Differing from WT opsin (left), PNGase F-treated P23H opsin shifted only ∼4 kDa relative to Endo H-treated retinal homogenate. This ∼4-kDa difference arose from two N-acetylglucosamines left at the glycosylation sites. Thus, glycosylation of P23H opsin is Endo H-sensitive.

FIGURE 8.

Subcellular localization of opsin in P23H/+ mice. Retinal cryosections from a PND 35 WT mouse (+/+) and P23H/+ mouse of the same litter were double-stained with antibodies against opsin (1D4) and calreticulin or opsin (1D4) and prominin 1. The 1D4 antibody reacted with both WT and P23H opsin, localizing in the ROS of both WT and P23H/+ mice (1st and 2nd row) and illustrating the markedly shortened angular ROS in P23H/+ animals. The ER marker calreticulin localized to the RPE, inner segments, and ONL (left panel, 3rd row) and did not co-localize with the 1D4 signal (left panel, merged). Prominin 1 localized to both rod and cone OS (right panel, 3rd row). In ROS, prominin 1 accumulated at the base of OS as suggested by the punctuate signals in both WT and P23H/+ mice. The 1D4 signal did colocalize with the prominin 1 signal (right panel, merged). Thus, even though the ROS in P23H/+ mice were shorter, their subcellular localization of opsin was same as in WT mouse. RPE, retinal pigmented epithelium; OS, outer segment; IS, inner segment; OLM, outer limiting membrane; ONL, outer nuclear layer; and DIC, differential interference contrast.

FIGURE 9.

Rod outer segment structures in WT and P23H/+ mice. Transmission electron micrographs of ROS from WT (A and A′) and P23H/+ (B–F, B′ and C′) mice. Images were taken from PND 63 littermates. Disorganization of ROS was observed in all three analyzed P23H/+ mice (B, C, and D) as perpendicularly placed discs (arrows) or elongated discs (arrowheads). C, E, and F retinal images were obtained from one mouse to show disorganization at the incisure (C) and base of ROS (E) and both types of disorganization in a single OS (F). Lower magnification images of A–C are shown in A′–C′. Arrows correspond to perpendicularly placed discs. Arrowheads, perpendicularly placed elongated discs. Open arrowheads, vesicles. Open arrow in C, an incisure.

FIGURE 10.

Effect of genetic depletion of 11-cis-retinal production on retinal degeneration. A, whole retinal images obtained from PND 36 P23H/+ and P23H/+Lrat^−/− littermate mice. Plastic sections were made from dorsal-ventral axis cut and stained with toluidine blue. Both mice were heterozygous with respect to the L450M variation of RPE65 (RPE65^450L/450M). The ONL, observed as a purple line in the P23H/+ mouse retina (left panel), was almost gone and only retained near the cilial marginal zone (CMZ) in the P23H/+Lrat^−/− mouse (right panel; indicated by curved line). B, high magnification retinal images shown in A and Rho^+/−Lrat^−/−mice at PND 28–30. Images were taken from the midpoint between optic nerve (ON) and ciliary marginal zone. Genetic deletion of 11-cis-retinal production promoted retinal degeneration in the P23H/+ mouse at PND 36 but not in Rho^+/−/Lrat^−/− mouse at PND 28–30. In the P23H/+Lrat^−/− mouse, however, the ROS were almost undetectable, and the number of photoreceptor nuclei was dramatically reduced compared with a littermate that had 11-cis-retinal (P23H/+). C, statistical data of the number of photoreceptor nuclei per ONL column in P23H/+Lrat^−/−. Cryosections from PND35 P23H/+Lrat^−/− RPE65^450L/450L− mice were used for quantification. Data were derived from three eyes of three mice. P23H/+Lrat^−/− mice showed severe retinal degeneration, and numbers of photoreceptor nuclei per column were similar to those in the P23H/P23H mouse (Fig. 3B, PND 35).

FIGURE 11.

Subcellular localization and protein levels of opsin after genetic deletion of 11-cis-retinal production in Lrat^−/− mice. A, cryosections of retina from P23H/+Lrat^−/− mice were subjected to immunohistochemical analyses with antibodies against opsin (1D4) and calreticulin at PND 35. Even though massive photoreceptor degeneration is seen, subcellular localization of opsin was normal. B, retinal homogenates from mice at PND 14 were labeled with N-terminal (B6-30) opsin antibodies. All lanes were loaded with 2 μg of homogenate. hrhoG/+ denotes the sample from a heterozygous GFP-tagged human knock-in opsin (hrhoG) and endogenous WT opsin mouse. P23H/hrhoG, heterozygous hrhoG and P23H mouse. P23H/hrhoG Lrat^−/−; a heterozygous hrhoG and P23H knock-in mouse lacking lecithin-retinol acyltransferase. GFP-tagged human opsin (∼70 kDa) is indicated by an arrow. Opsin monomers (∼35 kDa) are indicated by an open arrow. Top panel, 4-min exposure was used to analyze the ratio between P23H opsin or WT opsin and GFP-tagged human opsin. Lower panel, 1-s exposure was used to analyze the signal intensity of GFP-tagged human opsin in each lane. The P23H protein level was significantly lower than GFP-tagged human opsin even though 11-cis-retinal production was genetically depleted. DIC, differential interference contrast. RPE, retinal pigmented epithelium; OS, outer segment; IS, inner segment; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer.