RPE miR-204 or -211 KNOCK OUT ALTERS CELL MORPHOLOGY AND FUNCTION

Abstract

Purpose : miRNA-204 and -211 are highly enriched in retinal pigment epithelium (RPE) relative to the adjacent retina and choroid. These two miRNAs play a fundamentally important role in maintaining epithelial phenotype and RPE physiology (Wang, F., Zhang, C., et al., 2010). In order to better understand the role of these miRNAs, we specifically deleted miR-204 or 211 in postnatal mouse RPE. In these conditional KO models, we are analyzing the underlying cell signaling networks that mediate changes in RPE structure and function.

Methods : Floxed miR-204 (& 211) KO mice were generated by homologous recombination in germline mice and verified by PCR and Southern blotting. Knockout of target microRNAs in RPE was achieved by crossing the floxed alleles into the VMD2-CRE line. Loss of miR-204 or -211 expression in RPE was verified by TaqMan® microRNA assay. Ocular structure was evaluated by fundus examination using OCT (Heidelberg Spectralis HRA+OCT) and histology using both light and electron microscopy. RPE morphology was also studied by employing in - house developed software (REShAPE) for quantitative morphological analysis of ZO-1 distribution in RPE flat mounts. Retinal or RPE physiological changes were assessed by retinal regular ERG or direct coupled (DC)-ERG recordings, respectively.

Results : In the eye of RPE specific miR-204 or -211 KO mice, gross anatomical structures appeared normal (6 weeks to 1 year). However, RPE apical processes were significantly altered. During the first 12 months, fundus autofluorescence images showed increasing hyper-auto fluorescent spots near the RPE/choroid. These structural changes were associated with an increased variation in RPE cell size and morphology. The retinal ERG responses (a- and b- waves) were unchanged, but the c - wave and Fast Oscillation (FO) responses of the DC - ERG were significantly reduced.

Conclusions : The present experiments indicate that miR-204 and -211 in RPE are crucial for maintaining the integrity of apical membrane structure and RPE cell morphology and physiology. These KO mice thus provide a model for the analysis of barrier function and the miRNA - dependent cell signaling pathways that mediate RPE function.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.