Omega-3 Fatty Acids Supplementation: Therapeutic Potential in a Mouse Model of Stargardt Disease
Ekatherine ProkopiouPanagiotis KolovosMaria KalogerouAnastasia NeokleousOrthodoxia NicolaouKleitos SokratousKyriacos KyriacouTassos Georgiou
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To evaluate the therapeutic effects of omega-3 (ω3) fatty acids on retinal degeneration in the ABCA4-/- model of Stargardt disease when the blood level of arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is between 1 and 1.5.Eight-month-old mice were allocated to three groups: wild type (129S1), ABCA4-/- untreated, and ABCA4-/- ω3 treated. ω3 treatment lasted 3 months and comprised daily gavage administration of EPA and docosahexaenoic acid (DHA). Blood and retinal fatty acid analysis was performed using gas chromatography to adjust the blood AA/EPA ∼1 to 1.5. Eyecups were histologically examined using transmission electron microscopy and confocal microscopy to evaluate lipofuscin granules and the photoreceptor layer. Retinal N-retinylidene-N-retinylethanolamine (A2E), a major component of retinal pigment epithelium lipofuscin, was quantified using liquid chromatography and tandem mass spectrometry, in addition to retinal proteomic analysis to determine changes in inflammatory proteins.EPA levels increased and AA levels decreased in the blood and retinas of the treatment group. Significantly less A2E and lipofuscin granules were observed in the treatment group. The thickness of the outer nuclear layer was significantly greater in the treatment group (75.66 ± 4.80 μm) than in the wild-type (61.40 ± 1.84 μm) or untreated ABCA4-/- (56.50 ± 3.24 μm) groups. Proteomic analysis indicated lower levels of complement component 3 (C3) in the treatment group, indicative of lower complement-induced inflammatory response.Three months of ω3 supplementation (AA/EPA ∼1-1.5) reduces A2E levels, lipofuscin granules, and C3 levels in the ABCA4-/- mouse model of Stargardt disease, consistent with slowing of the disease.Keywords:
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Abstract Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4 −/− mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4 −/− mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.
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Stargardt disease, also known as juvenile macular degeneration, occurs in approximately one in 10,000 people and results from genetic defects in the ABCA4 gene. The disease is characterized by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE) of the eye and by vision loss. No cure or treatment is available. Although lipofuscin is considered a hallmark of Stargardt disease, its mechanism of formation and its role in disease pathogenesis are poorly understood. In this work we investigated the effects of long-term administration of deuterium-enriched vitamin A, C20-D3-vitamin A, on RPE lipofuscin deposition and eye function in a mouse model of Stargardt's disease. Results support the notion that lipofuscin forms partly as a result of the aberrant reactivity of vitamin A through the formation of vitamin A dimers, provide evidence that preventing vitamin A dimerization may slow disease related, retinal physiological changes and perhaps vision loss and suggest that administration of C20-D3-vitamin A may be a potential clinical strategy to ameliorate clinical symptoms resulting from ABCA4 genetic defects. Stargardt disease, also known as juvenile macular degeneration, occurs in approximately one in 10,000 people and results from genetic defects in the ABCA4 gene. The disease is characterized by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE) of the eye and by vision loss. No cure or treatment is available. Although lipofuscin is considered a hallmark of Stargardt disease, its mechanism of formation and its role in disease pathogenesis are poorly understood. In this work we investigated the effects of long-term administration of deuterium-enriched vitamin A, C20-D3-vitamin A, on RPE lipofuscin deposition and eye function in a mouse model of Stargardt's disease. Results support the notion that lipofuscin forms partly as a result of the aberrant reactivity of vitamin A through the formation of vitamin A dimers, provide evidence that preventing vitamin A dimerization may slow disease related, retinal physiological changes and perhaps vision loss and suggest that administration of C20-D3-vitamin A may be a potential clinical strategy to ameliorate clinical symptoms resulting from ABCA4 genetic defects.
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Stargardt 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.SD 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.Histological 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.The 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.
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Significance Recessive Stargardt macular degeneration (STGD1) and a subset of cone–rod dystrophies are caused by mutations in the Abca4 gene. The ABCA4 protein is a flippase in photoreceptor cells that helps eliminate retinaldehyde, a toxic photoproduct of vision. Here we found that ABCA4 is additionally present in the retinal pigment epithelium (RPE) of mice at approximately 1% of its abundance in the neural retina. Genetically modified mice that express ABCA4 in RPE but not in photoreceptor cells showed partial rescue of both the lipofuscin accumulation and photoreceptor degeneration observed in Abca4 −/− mice and in STGD1 patients. These observations suggest that ABCA4 in the RPE prevents photoreceptor degeneration in Abca4 −/− mice and possibly in STGD1 patients.
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To investigate fundus autofluorescence (AF) characteristics in the Abca4(-/-) mouse, an animal model for AMD and Stargardt disease, and to correlate findings with functional, structural, and biochemical assessments.Blue (488 nm) and near-infrared (790 nm) fundus AF images were quantitatively and qualitatively analyzed in pigmented Abca4(-/-) mice and wild type (WT) controls in vivo. Functional, structural, and biochemical assessments included electroretinography (ERG), light and electron microscopic analysis, and A2E quantification. All assessments were performed across age groups.In Abca4(-/-) mice, lipofuscin-related 488 nm AF increased early in life with a ceiling effect after 6 months. This increase was first paralleled by an accumulation of typical lipofuscin granules in the retinal pigment epithelium (RPE). Later, lipofuscin and melanin granules decreased in number, whereas melanolipofuscin granules increased. This increase in melanolipofuscin granules paralleled an increase in melanin-related 790 nm AF. Old Abca4(-/-) mice revealed a flecked fundus AF pattern at both excitation wavelengths. The amount of A2E, a major lipofuscin component, increased 10- to 12-fold in 6- to 9-month-old Abca4(-/-) mice compared with controls, while 488 nm AF intensity only increased 2-fold. Despite pronounced lipofuscin accumulation in the RPE of Abca4(-/-) mice, ERG and histology showed a slow age-related thinning of the photoreceptor layer similar to WT controls up to 12 months.Fundus AF can be used to monitor lipofuscin accumulation and melanin-related changes in vivo in mouse models of retinal disease. High RPE lipofuscin may not adversely affect retinal structure or function over prolonged time intervals, and melanin-related changes (melanolipofuscin formation) may occur before the decline in retinal function.
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Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease.
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Abstract Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of granular pigmented organelles found in the retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin granules in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study the disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4 -/- mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess the melanosome density in vivo , whereas the AF images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize the lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased number of melanosomes in the RPE of Abca4 -/- mice and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo in the two mouse strains.
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