Trafficking of membrane-associated proteins to cone photoreceptor outer segments requires the chromophore 11-cis-retinal

Abstract

Lecithin retinol acyl transferase (LRAT) and retinal pigment epithelium protein 65 (RPE65) are key enzymes of the retinoid cycle. In Lrat(-/-) and Rpe65(-/-) mice, models of human Leber congenital amaurosis, the retinoid cycle is disrupted and 11-cis-retinal, the chromophore of visual pigments, is not produced. The Lrat(-/-) and Rpe65(-/-) retina phenotype presents with rapid sectorial cone degeneration, and the visual pigments, S-opsin and M/L-opsin, fail to traffic to cone outer segments appropriately. In contrast, rod opsin traffics normally in mutant rods. Concomitantly, guanylate cyclase 1, cone T alpha-subunit, cone phosphodiesterase 6alpha' (PDE6alpha'), and GRK1 (G-protein-coupled receptor kinase 1; opsin kinase) are not transported to Lrat(-/-) and Rpe65(-/-) cone outer segments. Aberrant localization of these membrane-associated proteins was evident at postnatal day 15, before the onset of ventral and central cone degeneration. Protein levels of cone T alpha and cone PDE6alpha' were reduced, whereas their transcript levels were unchanged, suggesting posttranslational degradation. In an Rpe65(-/-)Rho(-/-) double knock-out model, trafficking of cone pigments and membrane-associated cone phototransduction polypeptides to the outer segments proceeded normally after 11-cis-retinal administration. These results suggest that ventral and central cone opsins must be regenerated with 11-cis-retinal to permit transport to the outer segments. Furthermore, the presence of 11-cis-retinal is essential for proper transport of several membrane-associated cone phototransduction polypeptides in these cones.

Figure 1.

Cone distribution in Lrat^−/− and Rpe65^−/− retinas. A, Flat mount. Lrat^−/− cones were visualized in mice at 4 weeks of age with FITC-conjugated peanut agglutinin (green) binding to the extracellular cone sheaths. Magnification, 20×. B, Bar graph depicting cone density. Cones were counted from five fields per flat-mounted retina, averaged in six mice from each group. Data are expressed as the mean number of cones/mm² ± SEM. Overall cone cell counts did not differ between the two retinoid cycle mutants based on repeated-measures ANOVA followed by Dunnett's post hoc test (p < 0.05), whereas regional differences could be identified using one-way ANOVA followed by pairwise comparison using Tukey's post hoc test (*p < 0.05). White bars, Rpe65^−/− mice; black bars, Lrat^−/− mice; gray bars, WT mice.

Figure 2.

Confocal immunolocalization of M/L-opsin in WT, Lrat^−/−, and Rpe65^−/− cones at postnatal day 15. A–C, M/L-opsin in WT and mutant retinas at P15 ± 1 d. Note that M/L-opsin mislocalizes in part to the inner segment, perinuclear region, and synaptic terminal of mutant cones (B, C, arrows). Immunoreactivity for M/L-opsin in the mutant retinas is mislocalized early (P15) and nearly absent at P30 (data not shown). All retina sections passed through the optic nerve, and in each case, photoreceptors were imaged ventral (inferior) to the nerve where the degeneration was most advanced. Nuclei are contrasted with propidium iodide (red). Scale bar, 10 μm. OS, Outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars and abbreviations are the same for Figures 2, 3, and 5 and supplemental Figures S1–S3 (available at www.jneurosci.org as supplemental material).

Figure 3.

Immunolocalization of peripheral membrane-associated proteins, GC1, and R9AP in P15 WT and Lrat^−/− retinas. P15 littermate WT and Lrat^−/− eyes were coembedded, and central retina sections were probed simultaneously. Sections of P15 WT and Lrat^−/− retinas were probed using anti-PDE6α′ (A, B), anti-cone Tα (C, D), anti-cone Tγ (E, F), anti-CPX III (G, H), anti-GRK1 (I, J), anti-GC1 (K, L), and anti-R9AP (M, N) antibodies. Bright red nuclei in B and H indicate apoptotic photoreceptors. I–L, Central retina sections were probed simultaneously with either monoclonal anti-GRK1 antibody (I, J, green) or anti-GC1 antibody (K, L, green) and polyclonal anti-cone arrestin antibody to identify cones (I–L, red). Colocalization of mCAR with GRK1 (I, yellow at arrows) or GC1 (K, arrows) is evident in WT COSs, whereas in Lrat^−/− sections, COSs are largely devoid of GRK1 (J) and GC1 (L).

Figure 4.

Immunoblot and real-time PCR. A, Summary immunoblot data from P15 WT and Lrat^−/− retina lysates. Blots were probed with antibodies directed against cone PDE6α′, cone Tα, GRK1, PDE6α, or M/L-opsin. Internal loading controls with β-actin were included for each immunoblot. The results shown are representative of several blots using different animals of identical genotypes. Downregulation of cone phototransduction polypeptides is apparent in retina extracts of P15 Lrat^−/− mice. B, Quantitative real-time PCR to determine relative transcript levels of Gnat2 and Pde6c. RNA samples of P15 Lrat^−/−, RPE65−/−, and wild-type retinas were reverse transcribed and amplified using Gnat2-, Pde6c-, and Gapdh-specific primers. Relative mRNA levels, normalized to the fluorescence of the Gapdh standard, are shown as bar graphs. Experiments for each gene were done in parallel and repeated three times.

Figure 5.

Immunocytochemistry of P15 Rpe65^−/− and P20d Rpe65^−/−Rho^−/− retina: 11-cis-retinal administration promotes trafficking of cone opsin and peripheral membrane proteins. Left, Immunocytochemistry of WT and Rpe65^−/− cryosections of 15-d-old retinas. Note that M/L-opsin is mislocalized (F), whereas S-opsin is barely detectable (B) in central, inferior Rpe65^−/− retina. Right, Effect of 11-cis-retinal application on P20 Rpe65^−/−Rho^−/− cones. P20d indicates that mice were kept in the dark after application. Note that in untreated animals, COS antigens are mislocalized or undetectable, whereas in 11-cis-retinal treated animals, COSs are elongated and COS antigens are present. Antibodies tested include the following: anti-S-opsin (A–D); anti-M/L-opsin (E–H); anti-PDE6α′ (I–L); anti-cone Tα (M–P); and anti-GC1 (Q–T). Nuclei are revealed by propidium iodide (red).