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Nestor Mas Gomez, Emily Dutrow, Simone Iwabe, Frank Stefano, Kathleen Boesze-Battaglia, Gustavo Aguirre, Karina Guziewicz; Bestrophinopathy Large Animal Model Shows Abnormal Accumulation of Lipofuscin in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1704.
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BEST1 gene encodes for bestrophin-1, an integral membrane protein localized basolaterally in the RPE. BEST1 mutations are responsible for a group of inherited retinal disorders known as bestrophinopathies. A major pathological hallmark of these diseases is lipofuscin overload within the RPE cell monolayer. The main lipofuscin component, fluorophore A2E, has been shown to cause accumulation of free and esterified cholesterol in RPE cells. The aim of this study was to investigate whether canine multifocal retinopathy 1 (cmr1), a large animal model for human bestrophinopathies, demonstrates this extensive buildup of lipofuscin, and whether these accumulations correlate with increased levels of cholesterol in the cmr1-affected RPE.
cmr1 disease progression was monitored in vivo by cSLO/SD-OCT using NIR and FAF modes. Retinal cryosections were obtained from control and dogs with advanced disease selected for autofluorescence spectra study, and lipofuscin granules were quantified in comparison to WT control. Sections were excited at 406nm and emission spectra were recorded from 406-731nm. Cholesterol was localized and quantified by filipin binding. For cholesterol and A2E correlation studies, sections were excited at 406nm and images were collected at 420-440nm and 470-570nm, respectively. The percentage of co-localization was determined as Pearson coefficient. Data analysis was performed with the 4.0 Elements software of the Nikon A1R laser scanning confocal microscope.
Both the cmr1-affected and the WT RPE accumulated autofluorescencent debris with age; however, cmr1 tissue showed an abnormal buildup and 2.5 fold increase (p<0.001) in lipofuscin granules compared to the control. The emission spectrum for lipofuscin granules peaked at 523nm correlating with the emission peak of A2E. Increased free cholesterol levels were observed in cmr1 samples, and Pearson coefficient analysis showed 0.67 ± 0.02 co-localization between filipin and A2E. In addition, an accumulation of uncharacterized material was found in the subretinal space between photoreceptors and the RPE apical membrane.
The cmr1 RPE pathology resembles that of human bestrophinopathies. The model will be important for characterizing the molecular mechanisms of BEST1-related retinopathies, and thus, benefit studies on AMD and Stargard's disease.
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