Role of AMP-Dependent Kinase in the Function of Retinal Pigment Epithelium

Abstract

Introduction: : Purpose : Oxidative stress at the level of retinal pigment epithelium (RPE) is considered to be an important factor in the pathogenesis of both forms of age related macular degeneration. AMP dependent kinase (AMPK) is a key energy sensor for the cell, especially in stress conditions where AMP levels rise. When activated AMPK switches off energy consuming pathways and activates energy producing processes. Moreover, AMPK has been linked to the maintenance of cellular polarity, a vital feature for the proper function of RPE. The aim of the current study was to investigate the role of AMPK in the function of RPE.Methods : Twelve eyes of six mice were included in the study. RPE cells were isolated from 6-8 weeks old male wild-type C57Bl/6 mice. Briefly, animals were sacrificed and eyes were enucleated. A circular incision was made around the ora serrata of each eye and the lens and neural retina were carefully removed. Intact sheets of RPE cells were peeled and protein (n=3) was extracted. Same procedure was followed for mRNA isolation (n=3). The level of protein expression of AMPK was evaluated by Western blot analysis using antibodies specific for the catalytic isoforms of AMPK -α1 and α2-. Furthermore, quantitative real time PCR was used for the evaluation of mRNA expression of the two AMPK isoforms.Results : Both catalytic isoforms of AMPK are being expressed by the RPE, as confirmed by Western Blot analysis and quantitative real time PCR. AMPKα1 catalytic isoform is significantly (p<0.005) expressed to more extent than α2 isoform.Conlusions : We demonstrate here that AMPK is expressed by the murine RPE. Interestingly, the α1 isoform is expressed at higher levels compared to α2 isoform. Further experiments will define the molecular role of AMPK in the function of RPE cells, as well as in AMD pathogenesis.

Keywords: retinal pigment epithelium • stress response • photoreceptors