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.optical coherence tomography ͉ retinal degeneration ͉ retinoid cycle ͉ Leber congenital amaurosis ͉ gene therapy V ertebrate retinas have two types of photoreceptors: rods for the perception of dim light and cones for the perception of bright light. Light absorbed by visual pigments of photoreceptors triggers a series of enzymatic reactions known as phototransduction (1). The light-capturing part of the visual pigment, the chromophore, must then be regenerated. The pathway for regeneration of rhodopsin (the rod visual pigment) is known as the visual (retinoid) cycle (2, 3) and involves both rod cells and the adjacent retinal pigment epithelium (RPE). All-trans-retinal is reisomerized to 11-cis-retinal by a reaction in the RPE that requires the retinal isomerase, retinal pigment epithelium-specific 65-kDa protein (RPE65), and lecithinretinol acyltransferase (LRAT) activities. Functional visual pigment is then reformed by combining the 11-cis-chromophore with the photoreceptor opsin apoprotein (4). Molecular knowledge of the visual cycle has increased as the genes encoding the enzymes of this pathway have been elucidated (2, 5, 6).Decades ago, cone pigment regeneration was proposed to be RPE cell-independent (7, 8). More recently, cone-dominant ground squirrel and chicken retinas have been shown to exhibit retinoid isomerase activity that is distinct from RPE65 (5, 9, 10). Murine double knockout experiments, however, provide evidence that RPE65 and the RPE are essential for cone function (11...

Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics

“…Normal retina and patient 2 are at Ϸ5 mm superior retina, patient 1 is at Ϸ5 mm inferior retina. Schematics of photoreceptors and RPE are overlaid (yellow) and to the right of the actual optical images; these are derived from cynomolgus monkey histology (3,28). INL, inner nuclear layer; PR-IS and PR-OS, photoreceptor inner and outer segments.…”

“…The role(s) played by RPE65 in providing chromophore for cone function and/or survival remains unclear and controversial (1,3,40,(43)(44)(45)(46)(47)(48)(49). Restoration of cone-as well as rod photoreceptor-based visual function by subretinal AAV gene therapy in the canine model of RPE65-LCA (4,8), together with the existence of remnant cone function in patients with untreated RPE65-LCA (3), suggested a potential to improve cone function in patients undergoing gene therapy.…”

“…In vivo microscopy of the human retina was performed with optical coherence tomography (OCT) as published (2,3,51). A Fourier-domain OCT system (RTVue-100; Optovue) was used for data acquisition, and postacquisition processing of data was performed with custom programs (MatLab 6.5; MathWorks).…”

“…Visual field testing was performed with kinetic and static perimetry as published (3,(23)(24)(25)(26)(27)52). For kinetic perimetry, white (318 cd⅐m Ϫ2 ) targets of Goldmann sizes III (patient 1) or V (patients 2 and 3) were used on a 10 cd⅐m Ϫ2 achromatic background.…”

“…In RPE65 deficiency, photoreceptor cells do not regenerate their visual pigment and vision is not sustained. Retinal anatomy also degenerates, but not entirely (2,3).…”