PAD4 in the Accelerated Mouse Model of AMD and Human AMD

Abstract

Purpose : Human AMD retinas undergo citrullination, a post-translational modification catalyzed by peptidyl arginine deiminases (PADs). Here we have assessed PAD4 expression in the laser-induced mouse model of gliosis, as well as, in human wet-AMD eyes.

Methods : Human donor eyes (n=4), procured within 8 hours of death, were dissected in buffered formalin and then the posterior eye cup imaged using SD-OCT (Spectralis; Heidelberg Engineering) as described (Pang et al., Ophthalmology 2015). 8.2 mm, 7 line scans on macular crossing the optic disc were obtained. 30° x 30° autofluorescence fundus images were obtained with 488 nm argon blue laser and averaged from 9 images. 8 mm tissue punches of the macula were collected and cryopreserved for immunostaining. Mouse retinas were subjected to 6 laser burns (250 mW, 100 ms) focused on the RPE to elicit focal gliosis. Mouse eyes were analyzed at different times post injury. Cryosections from human and mouse tissues were stained with antibodies for PAD4, citrullination (F95) and GFAP and examined by confocal microscopy. Mouse retinas were sequentially extracted in low and high salt buffers to collect soluble and cytoskeletal intermediate filament proteins, and both protein fractions subjected to western blot (WB) analysis.

Results : In lasered eyes, GFAP and citrullinated proteins were co-localized at 7 and 16 days post-injury in reactive astrocytes and Muller Glia. WB analysis showed significant increases in citrullinated GFAP, PAD4 and high molecular weight citrullinated proteins in soluble fraction (P<0.05), with PAD4 levels also increased in the cytoskeletal fraction (P<0.05) of injured retinas. PAD4 immunostaining showed co-localization with GFAP in astrocytes and Muller cells, with staining reaching the outer plexiform layer. In human male and female wet-AMD retinas, increased PAD4 and GFAP expression was observed in ganglion cell layer astrocytes and Muller cells compared to their age-matched controls. PAD4 staining in the female AMD eye was stronger than that in the male eye, and it was also elaborated in a greater area (outer nuclear layer).

Conclusions : PAD4 localizes to reactive astrocyte-Muller glia in laser lesions revealing tight regulation of citrullination in the gliosis response. Human AMD retinas also show elevated levels of PAD4, in part, in astrocytic and Muller cells. Thus, PAD4 may potentially be driving citrullination in AMD, and could potentially also serve as an AMD biomarker.

This is a 2020 ARVO Annual Meeting abstract.