Expression of peptidylarginine deiminase 4 in an alkali injury model of retinal gliosis

- Retinal gliosis is associated with increased citrullination during injury.
- PAD4 expression is induced in Muller glia and localizes along GFAP filaments.
- PAD4 inhibition reduces protein citrullination and GFAP protein levels.

Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p < 0.05), whereas Pad2 levels did not change significantly (p > 0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.

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