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John Wizeman, Anthony P Nicholas, Royce Mohan; Induction of Citrullination during Pathological Retinal Gliosis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2272.
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© ARVO (1962-2015); The Authors (2016-present)
Gliosis is an insidious process underlying a majority of retinal diseases. While the upregulation of the type III intermediate filaments (IFs) vimentin (vim) and the glial fibrillary acidic protein (GFAP) are hallmark features of gliosis, the mechanisms of gliosis are poorly characterized. IFs undergo several post-translational modifications, including citrullination. Citrullination is increased in glaucoma patients, but the direct impact of citrullination on gliosis is unknown. Here we identify gliosis-related citrullination in the retina and investigate the possible contribution of IFs to hypercitrullination.
The DBA/2J mouse model of glaucoma and control C57BL/6 mice were used to investigate onset of citrullination in glaucoma. Vim-deficient (Vim-KO) and WT 129SvEV were subjected to a corneal injury with 1N NaOH. Injured and uninjured eyes were enucleated after 3 to 7 days. Eyes were then frozen down for analysis by immunohistochemistry with GFAP, vim and F95 (citrullinated proteins) antibodies, or posterior eye cups were put into explant culture for 1 to 3 days as a novel method for inducing hypercitrullination. Retinal proteins were extracted as soluble and insoluble fractions. Samples were subjected to western blot analysis for GFAP, vim and F95 expression.
DBA/2J mice accumulate citrullinated proteins by 8 months of age, while citrullination was not detected in age-matched C57BL/6 mice by western blot. Müller glia express citrullinated filaments in DBA2/J mice. There was no difference in GFAP expression between strains. Vim-KO mice exhibit a pattern of hypercitrullination at 1 year of age that was not found in young mice or in WT controls. With injury, WT eyes displayed increased expression of citrullinated proteins in the retina compared to uninjured retinas. Injured eyes exhibit hypercitrullination and a corresponding GFAP overexpression in explant cultures.
Our results suggest that citrullination is a pathological process underlying gliosis in the retina. Significantly, increased citrullination occurs in the DBA/2J model of glaucoma, suggesting that it could be an early trigger for pathological gliosis. The injury-explant model revealed a molecular switch from citrullination to hypercitrullination. Hypercitrullination was also observed in uninjured aged Vim-KO mice, suggesting that IFs may play a role in the regulation of citrullination in the retina.
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