Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis

Abstract

RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations.

Significance statement: LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations.

Keywords: Leber congenital amaurosis; Rpe65; chemical chaperone; cone photoreceptor; retina; retinoid visual cycle.

Figure 1.

Ubiquitination-dependent proteasomal degradation of R91W RPE65 in mouse RPE. A, Immunoblot analysis of RPE65 and LRAT in WT and R91W, KI mice eyecups treated with DMSO or the indicated concentrations of MG132 or pepstatin A. Beta-actin served as a loading control. Relative intensities of RPE65 immunoblots were quantified, normalized by the intensities of LRAT, and expressed in the histograms as percentage of RPE65 in DMSO-treated eyecups. Asterisks indicate significant differences between DMSO- and MG132-treated groups (*p < 0.01, **p < 0.005). Error bars show SD (n = 4). B, RPE65 in WT and KI eyecups treated with 20 μm MG132 or DMSO was immunoprecipitated, and the immunoprecipitates were probed with antibodies against ubiquitin (Ub) or RPE65. Polyubiquitinated RPE65 (Ub-RPE65) and monomeric RPE65 (arrowhead) are indicated.

Figure 2.

PSMD13-mediated ubiquitination-dependent degradation of misfolded R91W RPE65. A, Immunoblot analysis of RPE65 and PSMD13 in RPE of WT and KI eyecups transfected with the indicated amounts of PSMD13 siRNA (siPSMD13) or negative control siRNA (siCont, 200 nm). The 26S proteasome non-ATPase regulatory subunit 11 (PSMD11) and LRAT were detected as internal maker for the 19S cap of the proteasome and RPE, respectively. B, C, Relative immunoblot intensities of RPE65 and PSMD13 normalized to LRAT were expressed as fold of RPE65 (B) or percentage of PSMD13 (C) in eyecups transfected with control siRNA. D, Confocal microscopic images of RPE cells in 129S2/Sv mice eyecups transfected with pcDNA or pcDNA encoding EGFP. Nuclei were stained with DAPI. E, Chymotrypsin-like and trypsin-like activities of the proteasomes in RPE of WT and KI mice eyecups transfected with 200 nm PSMD13 siRNA or control siRNA. F, Immunoblot analysis of RPE65, PSMD13, and the indicated proteins in WT and KI eyecups transfected with pRK5 or pRK5 encoding PSMD13. G, H, Relative immunoblot intensities of RPE65 and PSMD13 normalized to LRAT in F. I, Chymotrypsin-like and trypsin-like activities of the proteasomes in RPE of WT and KI mice eyecups transfected with pRK5 or PSMD13 construct (pPSMD13). J, RPE65 in WT and KI eyecups transfected with siPSMD13 or siCont was immunoprecipitated and probed with an Ub antibody. K, Immunoblot analysis of RPE65 and PSMD13 in RPE of WT and KI eyecups maintained at 37°C or 30°C after transfecting pRK5 or pPSMD13. L, Relative immunoblot intensities of RPE65 and PSMD13 (bottom) in the KI mice eyecups in K. Asterisks indicate statistically significant differences between test and control groups (*p < 0.05, **p < 0.005). Error bars indicate SD (n = 4).

Figure 3.

PBA increased the isomerase activity and chromophore synthesis in KI mice. A, Immunoblot analysis of RPE65 in RPE of 5-week-old KI mice injected intraperitoneally with the indicated amounts of PBA or saline for 3 weeks, starting at P14. Histogram shows relative immunoblot intensities of RPE65 in PBA-treated mice versus control mice. B, Representative HPLC chromatograms of retinoid isomerase assays of RPE from WT and KI mice treated with 50 mg PBA or saline. The peaks of 11-cis-retinol (11cROL) are indicated by arrows. Histograms show relative isomerase activities of WT and R91W RPE65 in PBA-treated versus saline-treated mice. C, Amounts of atRE in WT and the indicated mutant mice eyes. D, Amounts of the indicated ocular retinoid (11cRAL, 11-cis-retinal; atRAL, all-trans-retinal; 9cRAL, 9-cis-retinal and atROL, all-trans-retinol) in KI mice treated with saline or PBA. Asterisks indicate statistically significant differences between PBA- and saline-treated KI mice (p < 0.01). Error bars indicate SD (n = 6).

Figure 4.

Increase in membrane-association and colocalization of R91W RPE65 with LRAT in KI mice treated with PBA. A, Immunoblot analyses of RPE65 and cadherin-1 (CDH1) in membrane fractions of RPE from 10-week-old WT and KI mice treated with 50 mg PBA or saline for 8 weeks. Histograms show relative immunoblot intensities of RPE65 in the membrane fractions. B, Immunohistochemistry showing increase in colocalization of R91W RPE65 with LRAT in RPE of KI mice treated with PBA. Arrows indicate colocalization areas. Scale bar, 20 μm. C, Representative immunoblot analysis showing increase of R91W RPE65, but not LRAT, in RPE of KI mice treated with PBA for 8 weeks. Histogram shows relative expression levels of RPE65 and LRAT in WT mice and KI mice treated with saline or PBA. Asterisks indicate significant differences between PBA- and saline-treated KI mice (p < 0.005). Error bars indicate SD (n = 4).

Figure 5.

PBA attenuated S-opsin mislocalization in KI mice. A, Representative S-opsin immunoreactivity in retinal sections of WT and KI mice treated with PBA (50 mg/kg) or saline. ONL, Outer nuclear layer; INL, inner nuclear layer. Scale bar, 20 μm. B, Percentage of S-opsin (S-op) mislocalization estimated by dividing S-opsin immunofluorescence in the OPL by the sum of immunofluorescence in OPL and OSs. Note the decrease in S-opsin mislocalization in KI mice after administering PBA. C, Immunoblot analysis of S-opsin in retinas of WT and KI mice treated with PBA or saline. Histogram shows relative expression levels of S-opsin in PBA- or saline-treated KI mice versus saline-treated WT mice. Asterisks indicate significant differences between PBA- and saline-treated KI mice (p < 0.007). Error bars indicate SD (n = 4).

Figure 6.

Prolonged cone survival in KI mice treated with PBA. A, Low-magnification images of immunohistochemistry for CAR on retinal sections of 10-week-old WT and KI mice treated with PBA or saline for 8 weeks. ON, Optic nerve. Scale bar, 500 μm. B, High-magnification images showing the superior and inferior central regions highlighted in A. Scale bar, 20 μm. C, Counts of CAR-positive cone OS in the superior and inferior retinas from the indicated mice. D, Quantitative immunoblot analysis of CAR in WT mice, as well as KI and rd12 mice treated with PBA or saline for 8 weeks. Asterisks indicate significant differences between PBA- and saline-treated KI mice (p ≤ 0.01). Note that CAR expression levels in rd12 mice treated with PBA are similar to those in rd12 mice treated with saline. Error bars indicate SD (n = 4).

Figure 7.

PBA reduced M-cone degeneration in KI mice. A, Representative M-opsin immunoreactivity in the superior retinas of 10-week-old WT and KI mice treated with PBA or saline. Scale bar, 20 μm. B, Total numbers of M-cones in the superior and inferior retinal sections of WT and KI mice treated with PBA or saline. C, Immunoblot analysis showing increase of M-opsin in KI mice treated with PBA. Asterisks indicate significant differences between PBA- and saline-treated KI mice (p ≤ 0.01). Error bars indicate SD (n = 4).

Figure 8.

PBA improved cone visual function in KI mice but not in rd12 mice. A, Representative photopic ERG in 10-week-old WT and KI mice treated with PBA or saline for 8 weeks. ERG responses were elicited with the indicated flash intensities of white light under a rod-saturating background light. B, Amplitudes of photopic ERG b-waves, evoked with the indicated achromatic light flashes, in WT and KI mice treated with PBA or saline. C, Representative ERG responses of S-cones in 10-week-old WT, KI, and rd12 mice treated with PBA or saline for 8 weeks. Data were acquired using the indicated flash intensities of 360 nm UV light under a rod-saturating background light. D, Amplitudes of S-cone b-waves, evoked with the indicated UV light flashes, in KI and rd12 mice treated with PBA or saline. Asterisks indicate statistically significant differences between PBA- and saline-treated KI mice (p < 0.01). Note the similar b-wave amplitudes in PBA- and saline-treated rd12 mice. Error bars indicate SD (n = 7).