Ultrastructural alterations in the retinal pigment epithelium and photoreceptors of a Stargardt patient and three Stargardt mouse models: indication for the central role of RPE melanin in oxidative stress

Abstract

BackgroundStargardt disease (SD) is characterized by the accumulation of the age-pigment lipofuscin in the retinal pigment epithelium (RPE) and subsequent neuroretinal degeneration. The disease leads to vision loss early in life. Here, we investigate age-dependent ultrastructural changes in three SD mouse models: albino Abca4-/- and pigmented Abca4-/- and Abca4-/-.Rdh8-/- mice. Since we found indications for oxidative stress primarily in albino SD mice, we tested RPE melanin for its antioxidative capabilities.MethodsSD mouse eyes were investigated by light, fluorescence and electron microscopy and were compared to the respective albino and pigmented wild type mice and to a human donor SD eye. To confirm the role of RPE melanin in scavenging oxidative stress, melanin from S. officinalis as a standard and porcine RPE were tested for their capability to quench superoxide anions.ResultsHistological alterations indicative of oxidative stress and/or lysosomal dysfunction were present in albino Abca4-/- and Abca4-/-.Rdh8-/- mice. Retinal damage, such as inner segment rupture and pyknotic or free photoreceptor nuclei in the subretinal space and RPE vacuolization were exclusively found in albino Abca4-/- mice. Shortened and disorganized photoreceptor outer segments and dead RPE cells were found in albino Abca4-/- and Abca4-/-.Rdh8-/- mice, with earlier onset in albino Abca4-/- mice. Undegraded phagosomes and lipofuscin accumulation were present in the RPE of all three SD strains, but numbers were highest in Abca4-/-.Rdh8-/- mice. Lipofuscin morphology differed between SD strains: (melano-)lipofuscin granules in pigmented Abca4-/- mice had a homogenous electron density and sharp demarcations, while lipofuscin in albino Abca4-/- mice had a flocculent electron density and often lacked a surrounding membrane, indicating loss of lysosomal integrity. Young Abca4-/-.Rdh8-/- mice showed (melano-)lipofuscin granules with homogenous electron density, while in aged animals granules with flocculent electron density predominated. Both strains of pigmented SD mice had melanolipofuscin clusters as found in the human SD eye. Like melanin from S. officinalis, porcine RPE melanin can also quench superoxide anions.DiscussionThe presented pathologies in albino Abca4-/- and Abca4-/-.Rdh8-/- mice suggest oxidative stress and/or lysosomal dysfunction within the RPE. Since albino Abca4-/- mice have the earliest onset and severest damage and as absence of melanin and also melanin turnover with age are known to diminish RPEs anti-oxidative properties, we assume that RPE melanin plays a role in SD related damages. A lack of pathology in pigmented Abca4-/- mice due to lower stress levels as compared to the Abca4-/-.Rdh8-/- mice underlines this hypothesis. It is also supported by the finding that RPE melanin can quench superoxide anions. We therefore suppose that RPE melanin is important in retinal health and we discuss its role as an oxidative stress scavenger.

Fundus autofluorescence, spectral‐domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model

“…In rodents, RPE cells in the central retina contain a higher level of lipofuscin and a lower level of melanin than in the peripheral retina . The RPE degeneration induced by blue light was mainly located in the central area of eyes in pigmented Abca4 −/− mice, consistent with the spatial distribution of phototoxic RPE lipofuscin and RPE melanin that is known to have antioxidative properties . Based on our research, we conclude that lipofuscin accumulated in the RPE is the main mediator of photodamage in Abca4 −/− mice.…”

“…The peri-macular tissue from a 72-year-old patient with late-stage STGD1-like phenotype was investigated for ex vivo retinal AF. RPE cells of the peri-macular tissue were packed with pigment granules, including lipofuscin and melanolipofuscin stained by toluidine blue but devoid of melanosomes ( Figure 6D), as previously shown by Taubitz et al 24 Figure 6E,F show hyper lipofuscin-AF and hyper NIR-AF of pigment granules in RPE cells, respectively. The overlay yellow color in the merged fluorescence image demonstrated that most pigment granules were hyperautofluorescent at both excitation wavelengths (white arrows).…”

“…The peri-macular tissue of a 72-year-old human donor with a clinical diagnosis of Stargardt disease was obtained from the Foundation Fighting Blindness (Columbia, MD, USA), as previously described. 24 In the meantime, a mutation in the peripherin-2 (PRPH2) gene (c.629>G, p. (Pro210Arg)) was identified in the donor. Therefore, the patient has now been characterized in this study as an STGD1-like patient.…”

“…The pigmented Abca4 −/− mouse, the first animal model for Stargardt disease, is characterized by delayed dark adaption, delayed clearance of all‐trans‐retinal after partial photobleaching using white light, and marked accumulation of A2E and lipofuscin granules in RPE cells. However, with the absence of retinal degeneration, this mouse model manifests rather the pathology of the early stage of Stargardt disease . Considering the deleterious effect of excessive blue light and lipofuscin in the retina, studying clinical ophthalmic photography, retinal function, and histology in blue light‐irradiated pigmented Abca4 −/− mice may provide insights into developing a phenotype of the advanced stage of Stargardt disease and age‐related macular degeneration (AMD).…”

“…However, with the absence of retinal degeneration, this mouse model manifests rather the pathology of the early stage of Stargardt disease. 23,24 Considering the deleterious effect of excessive blue light and lipofuscin in the retina, studying clinical ophthalmic photography, retinal function, and histology in blue light-irradiated pigmented Abca4 −/− mice may provide insights into developing a phenotype of the advanced stage of Stargardt disease and age-related macular degeneration (AMD).…”