The R345W Fibulin-3 mutation causes RPE dysfunction in mice and induces UPR and EMT markers in vitro

Abstract

Purpose : 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) deposits and hyper-pigmentation. The purpose of this study was to determine the underlying mechanisms of the R345W-induced dysregulation in C57/BL6J mice.

Methods : Frozen radial cross-sections (10 µm thick) of eyes from wild-type (WT) heterozygous (Fib3^+/ki) and homozygous (Fib3^ki/ki) mice were labelled for Fib3 using immunofluorescence and imaged by confocal microscopy (Leica SP8). ARPE-19 cells were infected with R345W Fib3 constructs with Gaussia Luciferase tags using lentivirus. Migration of the cells was determined by scratch assays and qPCR was used to quantify epithelial-mesenchymal transition (EMT), ER-stress, and unfolded protein response (UPR) marker expression.

Results : The RPE of WT mice in the cross-sections were arranged in a uniform monolayer. In contrast, the RPE of Fib^3+/kiand Fib3^ki/kimice were pleomorphic and arranged in multiple layers in the absence of overt sub-RPE deposits. The RPE layer of Fib3^+/kiand Fib3^ki/kiwas thicker with more abundant nuclei, suggesting cell proliferation. These lesions appeared to be more abundant in the Fib3^+/kiand Fib3^ki/kimice after 6 months of age and had more intense Fib3 immunoreactivity compared to the WT mice. R345W Fib3 significantly increased the scratch assay recovery rate of ARPE-19 cells compared to the WT (p<0.01). The Fib3-R345W mutation also significantly elevated the mRNA levels of EMT, ER-stress and UPR related markers in R345W-Fib3 cells compared to WT (p<0.01).

Conclusions : The results suggest that the Fib3-R345W mutation induces RPE cell proliferation prior to the development of significant deposit formation and causes overexpression or accumulation of the Fib3 protein. Upregulation of EMT-, ER stress-, and UPR-markers suggests that the R345W Fib3 mutation induces a dramatic change in RPE phenotype, which may contribute to the pathology observed in the mice.

This is a 2020 ARVO Annual Meeting abstract.