Peptidyl arginine deiminase‐4: A Gliosis‐Associated Target for Age‐related Macular Degeneration
Authors: Saba Nicholas John , Palko Sarah Ilona , Bargagna‐Mohan Paola , Mohan Royce 
Published: June 2021

Citrullination is a protein posttranslational modification (PTM) involving the irreversible modification of arginine residues on proteins to citrulline. Citrullinated proteins have recently been recognized as biomarkers in several major autoimmune and inflammatory diseases, such as rheumatoid arthritis and the central nervous system disease multiple sclerosis. Moreover, targeting the peptidyl arginine deiminases (PADs), the enzymes responsible for protein citrullination, with small molecule inhibitors has also been shown to be effective in reducing disease burden in several preclinical models. We recently reported that mice subjected to corneal chemical injury elicits retinal gliosis, wherein activated Muller glia displayed elevated levels of citrullinated glial fibrillary acidic protein (GFAP) (Mol Vision 2016; 22:1137–1155). Inhibition of PAD activity using a small molecule inhibitor potently decreased expression of citrullinated targets, identifying PADs as potential druggable targets for retinal pathology. Chemical injury to the cornea causes inner retinal pathology, and hence, initiates GFAP polymerization at Muller glial end feet. We found that the isozyme PAD4 was induced and also localized along glial processes (BBRC, 2017 487;134–139). As retinal gliosis, including altered citrullinated proteins, have been reported in tissue from age‐related macular degeneration (AMD) patients, we explored the idea that PAD4 may be responsible for the altered citrullinated proteome in AMD. Here we have exploited a laser injury model of AMD in mice, where laser ablation of 50 micrometer regions in the retinal pigment epithelium (RPE) causes focal lesions. We employed this model to determine if PAD4 expression is co‐regulated with retinal gliosis, and to also determine the localization of PAD4. Our findings show GFAP expression is induced as early as 1‐day post injury, as evidenced by western blot analysis. Time course studies using immunohistochemical analysis to examine polymerization of GFAP filaments show that this process initiates at the end feet of Muller glia and progresses into longer filaments that stain in the outer layers of the retina. Surprisingly, PAD4 distribution overlapping with GFAP reveals this polarized staining pattern despite the focal injury activating gliosis occurs in the RPE. Our findings reveal for the first time a previously unrecognized feature about the polarized distribution of PAD4 in retinal pathology, and enlists the hypothesis that a possible signal relay from the outer retina to the inner retina drives these events. Towards future studies testing this hypothesis, three questions can be asked. One, how are increased calcium levels required for PAD4 activity transmitted proximally to the Muller end feet? Two, because the retina is accessible for non‐invasive optical imaging, can this localization of PAD4 help to inform about early retinal pathology using PAD4‐targeted biomarker probes? Three, because PAD4 inhibitors are currently available, can this newly discovered localization within the inner retina be exploited for delivery of precision medicine for AMD?Support or Funding InformationJohn A. and Florence Mattern Solomon Endowed Chair in Vision Biology and Eye Diseases; Connecticut Bioscience Innovation Fund; NIH R01 EY016782This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.