Genetic ablation of PAD4 in the Accelerated Mouse Model of AMD Results in Reduced Citrullinated Products

Abstract

Purpose : Protein citrullination is catalyzed by the peptidyl arginine deiminases (PADs). PAD4 has emerged as an important biomarker in mouse and human wet-AMD (IOVS June 2020, Vol.61, 3689). Here we have investigated the contribution of PAD4 to pathological citrullination in the mouse model of wet-AMD, as well as in human wet-AMD maculae.

Methods : PAD4 homozygous deficient (PAD4 KO) mice were generated by breeding PAD4^flox/flox mice with GFAP-Cre^ERT2 mice and progeny treated with tamoxifen. PAD4 KO mice and litter mates were subjected to focal laser burns of the retinal pigment epithelium using the Meridian Merilas 532 nm laser coupled to the Micron III fundus imaging system. Mouse eyes were cryosectioned and stained with antibodies against F95 antibody (citrullination) and glial fibrillary acidic protein (GFAP). Mouse retino-choroidal tissue was lysed in RIPA buffer for western blot (WB) analyses. Human donor AMD eyes (n=2) and age-matched normal eyes (n=2; male and female) were subjected to SD-OCT, as described (Pang et al., Ophthalmology 2015). A circular 8 mm retino-choroidal button centered at the macula was cryosectioned and stained with antibodies to PAD4, GFAP and citrullinated GFAP (CTGF-1221) and analyzed by confocal microscopy.

Results : PAD4 KO 7-day-laser injured mice showed decreased citrullination of specific species, as well as high molecular weight species compared to injured litter mates in WB analysis. PAD4 KO mice also showed reduced filamentous citrullinated protein in Muller glia compared to wildtype mice. In human female AMD maculae, the CTGF-1221 antibody recognizing citrullinated arginine (R270 and R416) on GFAP showed increased positive staining in the astrocyte layer, as well the inner nuclear layer. The male AMD retina also showed modest increase of citrullinated-GFAP. The citrullinated-GFAP species are in the cell body in both layers and have decreased filamentous structure compared to age matched controls.

Conclusions : Increased retinal citrullination in the mouse AMD model is driven by Muller cell expression of PAD4. Providing proof of the importance of this enzyme, PAD4 genetic ablation results in reduced citrullination. Human wet-AMD maculae that show increased PAD4 expression revealed corresponding increased citrullinated GFAP species. These findings illuminate PAD4 as an important druggable target in AMD and its attenuation for pathological citrullination.

This is a 2021 ARVO Annual Meeting abstract.