RPE cell specific loss of ERK1/2 leads to retinal degeneration.

Abstract

Purpose : ARMD patients exhibit accumulation of lipofuscin granules as well as phagolysosomes. One of the major components of lipofuscin A2E, regulate ERK1/2 pathway, which is required for the maintenance and proliferation of RPE cells. In order to understand the function of ERK1/2 in the RPE cells, RPE specific knockout of Erk1/2 was carried out.

Methods : Erk1^(-/-)/Erk2^(fl/fl) (DKO) mouse were crossed with a mice expressing RPE-Cre in a doxycycline dependent manner, to obtain [RPE-Cre-Erk1^(-/-)/Erk2^(lx/lx) (Cre-DKO)]. Two months post-birth, the mice were injected with Doxycycline. Non-invasive analyses were performed and eyes enucleated for immunohistochemical and biochemical analyses.

Results : On phenotypic level, fundus analysis of Cre-DKO mice showed macular depigmentation and neovascularization. Electroretinogram (ERG) analysis combined with the retinoids measurement showed dysfunctional vision as well as significant decrease in the retinoids content both in the RPE cells and retina. Optical coherence tomography (OCT) analysis confirmed the retinal structural disorganization.
Immunohistochemical analyses demonstrated photoreceptor (PR) loss and decrease in outer nuclear layer (ONL) and inner nuclear layer (INL) to more than half of the original. The decrease in ONL was first due to the loss of cone photoreceptors as investigated by immunostaining, western blot & QPCR analysis. Homogeneity and structure of RPE cells was rapidly modified in Cre-DKO mice as shown by phalloidin staining and electron microscopy analysis. To decipher the molecular mechanism, we investigated the levels of various RPE cells markers. Loss of ERK1/2 led to specific decrease of retinal pigment epithelium-specific protein 65kDa (RPE65) with mislocalization of lecithin retinol acyltransferase (LRAT). This decrease was dependent on the presence of an AP-1 site in RPE65 promoter region and mutation of this site abrogated the decrease.

Conclusions : Specific decrease of ERK1/2 in RPE cells lead to a rapid disorganization of RPE cells, a decrease of retinoids levels, a dowregulation of RPE65 and a mislocaization of LRAT, and finally to retinal degeneration. Here, we show for the first time, an upstream regulator of RPE65, ERK1/2, which is able to directly regulate this RPE marker at transcriptional level.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.