Human cone photoreceptor dependence on RPE65 isomerase

Abstract

The visual (retinoid) cycle, the enzymatic pathway that regenerates chromophore after light absorption, is located primarily in the retinal pigment epithelium (RPE) and is essential for rod photoreceptor survival. Whether this pathway also is essential for cone photoreceptor survival is unknown, and there are no data from man or monkey to address this question. The visual cycle is naturally disrupted in humans with Leber congenital amaurosis (LCA), which is caused by mutations in RPE65, the gene that encodes the retinoid isomerase. We investigated such patients over a wide age range (3-52 years) for effects on the cone-rich human fovea. In vivo microscopy of the fovea showed that, even at the youngest ages, patients with RPE65-LCA exhibited cone photoreceptor loss. This loss was incomplete, however, and residual cone photoreceptor structure and function persisted for decades. Basic questions about localization of RPE65 and isomerase activity in the primate eye were addressed by examining normal macaque. RPE65 was definitively localized by immunocytochemistry to the central RPE and, by immunoblotting, appeared to concentrate in the central retina. The central retinal RPE layer also showed a 4-fold higher retinoid isomerase activity than more peripheral RPE. Early cone photoreceptor losses in RPE65-LCA suggest that robust RPE65-based visual chromophore production is important for cones; the residual retained cone structure and function support the speculation that alternative pathways are critical for cone photoreceptor survival.

Fig. 1.

Photoreceptor layer thickness in the central retinas of a normal child and two children with RPE65-LCA. (A–C) Horizontal and vertical OCT scans with the ONL highlighted in blue and labeled on the left. In the scans from P6 and P8, the white line indicates the lower limit of normal retinal thickness. (D) ONL thickness along the horizontal and vertical meridians in RPE65-LCA patients (n = 11; ages 3–14 years). Data from P7 (line without symbols) have been previously published (22). Gray regions represent normal limits (mean ± 2 SD; n = 10; ages 4–16 years). Temp, temporal; Sup, superior.

Fig. 2.

Prolonged survival of residual central cones after early loss in RPE65-LCA patients. (A–C) Comparison of mean central ONL thickness along horizontal and vertical meridians in three age groups of RPE65-LCA (symbols) and normal (gray regions) individuals. Error bars and the extent of the gray regions represent mean ± 2 SE. Subject ages for RPE65-LCA patients/normals were as follows: group 1, 3–14 years/4–16 years; group 2, 18–28 years/19–28 years; group 3, 31–52 years/33–63 years. (D) Average ONL thickness of the foveal region (central cross extending to 0.6 mm) as a function of age in patients (black symbols) and normal controls (gray symbols). The 95% prediction interval of linear regression fit to the normal data (gray lines) and linear regression fit to the patient data (black line) are shown. (E) Average sRBI of the foveal region as a function of age in patients and normal controls. The 95% prediction interval of linear regression fit to the normal data (gray lines) is shown.

Fig. 3.

Persistent cone visual function in RPE65-LCA means persistent chromophore availability. (A) (Upper) Dark-adapted psychophysical sensitivities to chromatic and achromatic stimuli presented across the horizontal meridian in young adult RPE65-LCA patients. Sensitivities to chromatic stimuli are shown on a common axis of radiometric equivalence, and sensitivities to achromatic stimuli are vertically shifted to match the blue stimulus results. Mean normal dark-adapted rod sensitivity to 500-nm stimulus and dark-adapted cone sensitivity to 650-nm stimulus during the cone plateau are shown (gray lines). (Lower) Comparison of the difference in chromatic sensitivity at each locus (symbols) to predicted difference for rod or cone mediation (dashed lines) based on spectral sensitivities of normal rod- and cone-mediated vision. Physiological blind spot is shown as a hatched bar. Temp, temporal; Nas, nasal. (B) Best-corrected visual acuity for the better-seeing eye in patients with RPE65 mutations, showing the range of function in young patients and worsening of acuity after the third decade of life. HM, hand motions; LP, light perception. Dashed line corresponds to 20/20 (log of the minimal angle of resolution, logMAR, 0.0) acuity. (C) Instability of fixation determined under retinal visualization in RPE65-LCA as it relates to visual acuity. Normal results are shown (gray symbols); error bars indicate ±SD.

Fig. 4.

RPE65 localization and RPE65-dependent isomerase activity and its distribution in the macaque eye. (A) Double-fluorescence immunolabeling of foveal rod outer segments (green) and cones (red) with rhodopsin and cone arrestin antibodies, respectively, shows normal lamination of the central retina. RPE, retinal pigment epithelium; IS, inner segments; OS, outer segments; ONL, outer nuclear layer; HFL, Henle's fiber layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Double-fluorescence immunolabeling of a monkey fovea with RPE65 (green) and R/G opsin (red) antibodies shows no expression of RPE65 in cones. Nuclei were stained with DAPI (blue), and Nomarski differential interference contrast microscopy optics was used. (C) Immunolabeling with RPE65 antibody (green) is only seen in the RPE. (Inset) Overexposed image does not show any RPE65 labeling in the fovea other than in the RPE. (Scale bars: A–C, 40 μm.) (D) HPLC separation of nonpolar retinoids extracted from the isomerization reaction mixture. RPE microsomes were isolated from a single eye, and the isomerization reaction was carried out as described in Materials and Methods. (E) Isomerization activities in different regions of the eye. T, temporal; S, superior; M, macula; I, inferior; N, nasal. (F) Immunoblot analysis of RPE65 expression levels in 5-mm biopsy punches taken from different areas of the eye. Lines 1–5 represent RPE microsomes dissected from temporal, superior, macula, inferior, and nasal parts of an eye, respectively. Line 6 is a human recombinant-tagged RPE65 expressed in Sf9 cells.