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Jasmine Geathers, Weiwei Wang, Alistair Barber, Jeffrey M. Sundstrom; Fibulin-3 mutation in mice dysregulates RPE cells and causes exosome markers to accumulation in drusen. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5022.
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© ARVO (1962-2015); The Authors (2016-present)
Malattia Leventinese (ML), an autosomal dominant inherited degenerative eye disease, is caused by a point mutation (R345W) in the fibulin-3 (Fib3) gene (efemp1) that results in deposition of sub-retinal pigment epithelium (RPE) drusen. Proteomic studies have revealed that these deposits contain extracellular matrix proteins, including Fib3, and exosome markers such as CD63. Recent studies in our lab have shown that Fib3 is bound to the surface of exosomes, suggesting that it may mediate exosome function. The purpose of the present study was to characterize changes in RPE morphology and drusen accumulation in mice carrying the Fib3-R345W mutation.
Frozen radial cross-sections (10 µm thick) of eyes from wild-type (Fib3+/+), heterozygous (Fib3+/ki) and homozygous (Fib3ki/ki) mice were labelled for Fib3, ApoE, ALIX, and CD63 using immunofluorescence and imaged by confocal microscopy (Leica SP8). Adjacent sections were stained with Periodic Acid Schiff (PAS) and Hematoxylin and Eosin (H&E) and imaged using bright-field microscopy. Electron microscopy was also used to detect subcellular changes in the RPE layer.
The RPE of Fib3+/+ mice in PAS and H&E sections were arranged in a uniform monolayer. In contrast, the RPE of Fib3+/ki and Fib3ki/ki mice were pleomorphic and arranged in multiple layers. The RPE layer of Fib3+/ki and Fib3ki/ki was thicker with more abundant nuclei, suggesting cell proliferation. Discrete ApoE and Fib3 immunofluorescence was identified in sub-RPE patches in all three groups of mice. These lesions appeared to be more abundant in the Fib3+/ki and Fib3ki/ki mice after 6 months of age compared to the Fib3+/+ mice. CD63 immunofluorescence also localized to similar sub-RPE patches in the Fib3+/ki and Fib3ki/ki mice, but not the Fib3+/+ mice, after 18 months of age. ALIX immunofluorescence localized to similar sub-RPE patches in the Fib3ki/ki only.
The results suggest that the Fib3-R345W mutation induces RPE hyperplasia prior to the development of overt sub-RPE drusen formation, and that Fib3-positive aggregates begin to appear after 6 months of age, followed by a similar pattern of CD63 immunoreactivity after 18 months. ApoE and ALIX immunoreactivity also appear after 18 months, suggesting a relationship between exosome marker proteins and Fib3.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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