Citrullination of glial intermediate filaments is an early response in retinal injury

PURPOSE: A hallmark of retinal gliosis is the increased detection and modification of the type III intermediate filament (IF) proteins vimentin and glial fibrillary acidic protein (GFAP). Here, we investigated vimentin and GFAP in Müller glia in a mouse model of alkali injury, focusing on the posttranslational modification of citrullination. METHODS: Mice were injured by corneal exposure to 1.0 N NaOH, and eyes were enucleated at different time points following injury. The levels of soluble and cytoskeletal forms of IF proteins and citrullination were measured using western blot analysis. Citrullinated GFAP was identified by immunoprecipitation followed by two-dimensional (2D) isoelectric focusing–polyacrylamide gel electrophoresis (IEF-PAGE) western blotting using a specific antibody that recognizes citrullinated GFAP. Vimentin, GFAP, and citrullinated proteins were localized in the retina by immunohistochemistry (IHC). Drug treatments were investigated in retinal explant cultures of posterior eyecups obtained from mouse eyes that were injured in vivo. RESULTS: Detection of GFAP in injured retinas increased over a period of 1 to 7 days, showing increased levels in both soluble and cytoskeletal forms of this IF protein. The global level of citrullinated proteins was also induced over this period, with low-salt buffer extraction showing the most abundant early changes in citrullination. Using IHC, we found that GFAP filaments assembled at Müller glial end feet, growing in size with time through the inner layers of the retina at 1–3 h postinjury. Interestingly, over this early time period, levels of soluble citrullinated proteins also increased within the retina, as detected by western blotting, coincident with the localization of the citrullinated epitopes on growing GFAP filaments and existing vimentin filaments by 3 h after injury. Taking advantage of the in vivo injury model to promote a robust gliotic response, posterior eyecups from 7-day postinjured eyes were treated in explant cultures with the peptidyl arginine deiminase inhibitor Cl−amidine, which was found to reduce global citrullination. Surprisingly, the detection of injury-induced high-molecular-weight GFAP species containing citrullinated epitopes was also reduced by Cl−amidine treatment. Using a low dose of the potent type III IF drug withaferin A (WFA), we showed that Cl−amidine treatment in combination with WFA reduced global protein citrullination further, suggesting that GFAP may be a key component of pathological citrullinated targets. CONCLUSIONS: Our findings illuminate citrullination as a potential novel target for trauma-induced retinal gliosis. We also propose that strategies for combining drugs targeting type III IFs and citrullination may potentiate tissue repair, which is an idea that needs to be validated in vivo.