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Sarah Ilona Palko, Nicholas Saba, Megan Rouillard, Paola Bargagna-Mohan, Royce Mohan; PAD4: A Potential Target for Gliosis in Age Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1232. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The post-translational modification called citrullination is increased in human Age-related macular degeneration (AMD) retinas. Citrullination is catalyzed by peptidyl arginine deiminases (PADs). Citrullination becomes increased in the mouse retina after corneal alkali injury, including that of glial fibrillary acidic protein (GFAP) [Molecular Vision 2016, 22, 1137-1155]. We showed that PAD4, specifically, was responsible for increased citrullination of GFAP [Biochemical and Biophysical Research Communications 2017, 487(1), 134-139]. In this study, we have tested whether PAD4 is expressed during gliosis in a laser-injury mouse model of retinal gliosis, emulating AMD.
Four-to six laser lesions were created using the Meridian Merilas 532 nm laser coupled to the Micron III fundus imaging system (Phoenix Research Laboratories). Both male and female 4-6-month old C57Bl/6 mice were used and sacrificed at various times. Tissue cryosections of eyes were collected and stained with antibodies against PAD4 (rabbit polyclonal, Abcam 50247, 1:100 dilution), F95 antibody (citrullination marker, mouse polyclonal, Abcam, 1:200 dilution), and GFAP (chicken polyclonal, Abcam, 1:200 dilution), along with DAPI to mark nuclei. Fluorescent secondary antibodies were applied to stained sections and analyzed by epifluorescence microscopy using Metamorph software.
We found colocalized staining of F95 and GFAP in injured retinas in lesions at early time points, indicating that citrullination was occurring in the Muller Glia. GFAP staining, found at the astrocyte layer in non-injured tissue, stained Muller glial processes increasingly with advancing injury time. In 1 day injured retinas, PAD4 staining was diffuse across inner layers of the retina, but strong colocalized staining of PAD4 and GFAP was observed in the 7-day injured retina in lesion sites initiating from the astrocytic layer. This frames PAD4 as likely being responsible for the increased citrullination during early events in retinal gliosis post laser injury.
PAD4 localization to GFAP filaments in proximity to the nerve fiber layer around lesions suggests activation of the citrullination-gliosis cascade is anatomically regulated from the interior retina despite an injury to the retinal pigment layer. This novel discovery illuminates the need to consider intra retinal communication and how these mechanisms elicit citrullination and retinal gliosis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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