Loss of cone photoreceptors caused by chromophore depletion is partially prevented by the artificial chromophore pro-drug, 9-cis-retinyl acetate

Abstract

Inactivating mutations in the retinoid isomerase (RPE65) or lecithin:retinol acyltransferase (LRAT) genes cause Leber congenital amaurosis (LCA), a severe visual impairment in humans. Both enzymes participate in the retinoid (visual) cycle, the enzymatic pathway that continuously generates 11-cis-retinal, the chromophore of visual pigments in rod and cone photoreceptor cells needed for vision. We investigated human RPE65-LCA patients and mice with visual cycle abnormalities to determine the impact of chronic chromophore deprivation on cones. Young patients with RPE65 mutations showed foveal cone loss along with shortened inner and outer segments of remaining cones; cone cell loss also was dramatic in young mice lacking Rpe65 or Lrat gene function. To selectively evaluate cone pathophysiology, we eliminated the rod contribution to electroretinographic (ERG) responses by generating double knockout mice lacking Lrat or Rpe65 together with an inactivated rod-specific G protein transducin gene (Gnat1-/-). Cone ERG responses were absent in Gnat1-/-Lrat-/- mice which also showed progressive degeneration of cones. Cone ERG responses in Gnat1-/-Rpe65-/- mice were markedly reduced and declined over weeks. Treatment of these mice with the artificial chromophore pro-drug, 9-cis-retinyl acetate, partially protected inferior retinal cones as evidenced by improved ERGs and retinal histochemistry. Gnat1-/- mice chronically treated with retinylamine, a selective inhibitor of RPE65, also showed a decline in the number of cones that was ameliorated by 9-cis-retinyl acetate. These results suggest that chronic lack of chromophore leads to progressive loss of cones in mice and humans. Therapy for LCA patients should be geared toward early adequate delivery of chromophore to cone photoreceptors.

Figure 1.

Foveal cone morphology of young LCA patients with RPE65 mutations. (A) Cross-sectional scans along the horizontal meridian of the central retina of a normal child (upper panel) and three children with LCA due to RPE65 mutations (lower panels). Arrows and brackets indicate ONL, outer nuclear layer; OLM, outer limiting membrane; IS, inner segment; OS, outer segment. (B) Mean foveal ONL, IS and OS thickness in a group of young subjects with normal vision (ages 5–15 years) and in eight young patients with RPE65–LCA (ages 6–20 years). Mean values for the parameters from the RPE65–LCA group are also shown (Avg). Error bars represent ± 2 SD.

Figure 2.

Histological and functional evaluation of cone photoreceptor cells in Gnat1^−/−Rpe65^−/− and Gnat1^−/−Lrat^−/− mice. (A and B) Cone photoreceptor cells were stained with PNA (green, cone outer segments) and DAPI (red, nuclei) and representative images of immunohistochemical (IHC) sections from the second zone from the center (Z2) in the superior and inferior retina are shown. Cone photoreceptor cells in both the superior and inferior retina of 4-week-old (4 w) Gnat1^−/−Lrat^−/− mice (A, upper panel) and Gnat1^−/−Rpe65^−/− mice (B, upper panel) were slightly decreased. However, severe cone photoreceptor cell death was observed in the inferior retinas of both double knockout strains at 8 weeks of age, even though cone photoreceptor cells in the superior retina were still maintained (A and B, lower panels). ONL, outer nuclear layer; OS, outer segment. Bar indicates 10 µm. (C and D) Populations of cone photoreceptor cells were quantified in 4-, 6- and 8-week-old mice at four zones in the superior and inferior retina, respectively, and age-related changes in these cone cell numbers are shown. Cones were markedly reduced in both strains between 4 and 6 weeks of age, most prominently in the inferior retina when compared with both those in 4-week-old Gnat1^−/− mice, and those in 4-week-old double knockout mice of both strains (P < 0.0001). (E and F) Scotopic single flash ERG responses were obtained from Gnat1^−/−Lrat^−/− mice and Gnat1^−/−Rpe65^−/− mice at 4, 6 and 8 weeks of age and compared with 4-week-old Gnat1^−/− mice. ERG b-wave amplitudes are plotted. ERG responses were severely reduced in both double knockout strains when compared with those of Gnat1^−/− mice. No responses were obtained from Gnat1^−/−Lrat^−/− mice at these sequential ages; only the data obtained from 4 weeks of age are plotted (E). Gnat1^−/−Rpe65^−/− mice showed markedly diminished but measurable responses at higher light intensities (F, P < 0.0001). Bars indicate SDs, n = 4–7.

Figure 3.

Histological evaluation of cone photoreceptor cells in Gnat1^−/−Lrat^−/− and Gnat1^−/−Rpe65^−/− mice after 9-cis-retinyl acetate (9cRAc) treatment. Gnat1^−/−Lrat^−/− and Gnat1^−/−Rpe65^−/− mice were treated with 9-cis-retinyl acetate (from P10 to P21, 1.0 µg/g body weight; from P22 to P56, 50 µg/g body weight; see Materials and Methods for regimen) and cone photoreceptor cells were quantified immunohistochemically at 6 and 8 weeks (6–8 w) of age by PNA (green, outer segments) and DAPI (red, nuclei) staining. Representative images from Z2 in the superior and the inferior retina of 8-week-old 9-cis-retinyl acetate-treated and vehicle-treated Gnat1^−/−Lrat^−/− mice (A) and Gnat1^−/−Rpe65^−/− mice (B) are shown. Cone photoreceptor cells were better preserved in retinas of 9-cis-retinyl acetate-treated mice (upper panels) than in vehicle-treated controls (lower panels). Populations of cone photoreceptor cells also were significantly greater, especially in the inferior retina of both strains when compared with vehicle-treated controls at 6 and 8 weeks of age (Gnat1^−/−Lrat^−/− mice, C; Gnat1^−/−Rpe65^−/− mice, D). ONL, outer nuclear layer; OS, outer segment. For (A and B) bars indicate 10 µm. For (C and D) bars indicate SDs. n = 4–7, P < 0.0001 for inferior retina.

Figure 4.

Functional evaluation of cone photoreceptor cells in Gnat1^−/−Rpe65^−/− and Gnat1^−/−Lrat^−/− mice after 9-cis-retinyl acetate (9cRAc) treatment. ERG responses were obtained in 4-, 6- and 8-week-old Gnat1^−/−Rpe65^−/− and Gnat1^−/−Lrat^−/− mice on the day following the last day of treatment with 9-cis-retinyl acetate (from P10 to P21, IP injection of 1.0 µg/g body weight; from P22 to P56, gavage with 50 µg/g body weight; see Materials and Methods for regimen). (A) Representative traces of scotopic single flash ERG recordings are shown for Gnat1^−/− and for both strains of 4-week-old double knockout mice either treated with 9-cis-retinyl acetate or vehicle solution. Comparison of these recordings shows that both double knockout strains had reduced responses relative to the Gnat1^−/− mouse and that treatment with 9-cis-retinyl acetate partially preserved ERG responses in both double knockout strains. (B) ERG b-wave amplitudes indicate that treatments with 9-cis-retinyl acetate improved responses in Gnat1^−/−Lrat^−/− mice (left panel) and Gnat1^−/−Rpe65^−/− mice (right panel) at 4, 6 and 8 weeks of age. No b-wave responses were detected in vehicle-treated Gnat1^−/−Lrat^−/− mice at 4, 6 and 8 weeks of age (only data from 4-week-old Gnat1^−/−Lrat^−/− mice are presented). Gnat1^−/−Rpe65^−/− mice showed some residual responses during the experimental period. Bars indicate SDs; n = 4–7, P < 0.0001.

Figure 5.

Cone photoreceptor cell degeneration due to chromophore deficiency in Gnat1^−/− mice is prevented by 9-cis-retinyl acetate (9cRAc) administration. To determine whether the cone photoreceptor cell death observed in Gnat1^−/−Lrat^−/− and Gnat1^−/−Rpe65^−/− mice might be induced by 11-cis-retinal deficiency, a phenotype mimicking this condition was produced in Gnat1^−/− mice by administration of the retinoid cycle inhibitor, retinylamine. Four-week-old Gnat1^−/− mice were gavaged with retinylamine (Ret-NH₂)(250 µg/g body weight) followed 6 h later by strong light bleaching (500 cd/m² for 30 min) on day 1 and maintained in the dark for a week (from days 1–7); the same regimen was repeated during the following week (from days 8–14). Cone photoreceptor cell death and function were evaluated by viewing retinal histology (PNA, green for cone photoreceptors; DAPI, red for nuclei), counting cone cell numbers, and recording ERGs on day 14. To determine the effects of artificial chromophore replenishment on cone photoreceptor cells, mice undergoing the 2 week retinylamine/bleaching chromophore depletion regimen were supplemented with 9-cis-retinyl acetate gavaged at 50 µg/g body weight three times a week (day 2, 4, 6, 9, 11 and 13). Cone photoreceptor immunohistochemistry, populations and function were then evaluated on day 14. No significant effects on cone photoreceptor immunohistochemistry (A and B, bar indicates 10 µm), population (G) or ERG responses (H) were observed after light exposure in Gnat1^−/− vehicle-treated control mice, whereas chromophore deprivation by retinylamine gavage and light exposure induced drastic cone photoreceptor degeneration in both the inferior and the superior retina (C, D, G ***, P < 0.0001) of Gnat1^−/− mice. Retinal dysfunction in these animals was also evidenced by ERG recordings (H ***, P < 0.0001). However, cone photoreceptor immunohistochemistry, populations and function were maintained in 9-cis-retinyl acetate-treated Gnat1^−/− mice comparably to vehicle-treated control animals (E, F, G, H). OS, outer segments; ONL, outer nuclear layer. Bars indicate SDs; n =3–6/each group.