Abstract
Purpose :
Muller glia (MG) are the principal drivers of reactive gliosis and can cause subretinal fibrosis. We previously demonstrated that citrullination catalyzed by peptidyl-arginine deiminase 4 (PAD4) is both an acute and chronic post-translational modification seen in reactive MG in human and age-related macular degeneration (AMD) models. Modeling a therapeutic approach, we compared acute and delayed onset of PAD4 deficiency using glial-specific conditional PAD4 knockout (PAD4cKO) mice to investigate PAD4 in the laser injury model. We also employed the JR5558 genetic model of spontaneous retinal degeneration to study citrullination and the potential of therapeutic PAD4 inhibition.
Methods :
The laser injury model was employed in C57Bl6 and PAD4cKO mice (GFAP-Cre/ERT2 mice crossed with Padi4flox/flox mice). PAD4cKO and litter mates (Padi4flox/flox, control) were given tamoxifen to temporally delete the Pad4 gene in glial cells either at the time of injury or 15 days post-injury (dpi). JR5558 mice (Crb1rd8/Jak3m1J) spontaneously develop retinal lesions. Cl-amidine, a PAD inhibitor was provided by intraperitoneal injections on alternate days to JR5558 pups for a 30-day period and retinal lesions were characterized by fundoscopy. Immunostaining of retinal sections was used to detect GFAP, citrullination, PAD4, and fibronectin.
Results :
In the laser injury model, acute PAD4cKO showed significantly reduced retinal gliosis, hypercitrullination and fibrosis (P<0.0001; P<0.0001; P<0.0001) at 30 dpi. Interestingly, delayed PAD4cKO also showed significantly reduced gliosis and hypercitrullination (P<0.001; P<0.001). In JR5558 mice lesion-associated gliotic MG reveal significantly increased hypercitrullination at 2 and 4 months of age (P=0.0182; P=0.0011) with temporal subretinal fibrosis also becoming significant (P=0.0347; P=0.0011). Cl-amidine treated JR5558 mice are currently being pursued for target validation follow-up studies.
Conclusions :
Early and delayed PAD4 abrogation was found to be effective in the laser injury model suggesting a therapeutic window in which PAD4 inhibition reduces retinal pathology. Manifesting similar retinal lesions and hypercitrullination, the JR5558 line differs because the aggressive pathology is driven by chronic structural changes affecting both MG and neurosensory photoreceptors. Thus, the importance of understanding the underlying cellular deficit(s) with each model will be key to illuminating PAD4 as a therapeutic target.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.