Peptidyl arginine deiminase 4 deficiency protects against subretinal fibrosis by inhibiting Müller glial hypercitrullination

Abstract

Retinal scarring with vision loss continues to be an enigma in individuals with advanced age‐related macular degeneration (AMD). Müller glial cells are believed to initiate and perpetuate scarring in retinal degeneration as these glial cells participate in reactive gliosis and undergo hypertrophy. We previously showed in the murine laser‐induced model of choroidal neovascularization that models wet‐AMD that glial fibrillary acidic protein (GFAP) expression, an early marker of reactive gliosis, increases along with its posttranslational modification citrullination. This was related to increased co‐expression of the citrullination enzyme peptidyl arginine deiminase‐4 (PAD4), which also colocalizes to GFAP filaments. However, whether such hypercitrullination in Müller glial drives fibrotic pathology has remained understudied. Here, using male and female C57Bl6 mice subjected to laser injury, we investigated in a temporal study how citrullination impacts GFAP and PAD4 dynamics. We found that high molecular weight citrullinated species that accumulate in Müller glia corresponded with dynamic changes in GFAP and PAD4 showing their temporal redistribution from polymeric cytoskeletal to soluble protein fractions using immunostaining and western blot analysis. In conditional glial‐specific PAD4 knockout (PAD4cKO) mice subjected to laser injury, there was a stark reduction of citrullination and of polymerized GFAP filaments. These injured PAD4cKO retinas showed improved lesion healing, as well as reduced fibronectin deposition in the subretinal space at 30 days. Taken together, these findings reveal that pathologically overexpressed PAD4 in reactive Müller glia governs GFAP filament dynamics and alters their stability, suggesting chronic PAD4‐driven hypercitrullination may be a target for retinal fibrosis.

Unveiling Drivers of Retinal Degeneration in RCS Rats: Functional, Morphological, and Molecular Insights

“…Recently, peptidyl arginase deiminase 4 (PAD4) was identified in reactive gliosis during retinal degeneration [20]. Our RNA-Seq identified neutrophil degranulation and NETosis as enriched pathways in the microglial dataset.…”

CX3CL1/CX3CR1 Signaling Mediated Neuroglia Activation Is Implicated in the Retinal Degeneration: A Potential Therapeutic Target to Prevent Photoreceptor Death

“…Glial fibrillary acid protein (GFAP) is a typical marker of retinal gliosis, and its expression increases with its post-translational modification of citrullination. 37 At P1, the GFAP immunostaining was distributed throughout the retina, forming the internal limiting membranes (ILMs) and external limiting membranes (ELMs). However, these Müller cells were activated to produce glial filaments at P7, and the distribution range of GFAP gradually extended to ONL.…”

Wnt5a/β-catenin-mediated epithelial-mesenchymal transition: a key driver of subretinal fibrosis in neovascular age-related macular degeneration

“…The underlying mechanisms of subretinal fibrosis secondary to nAMD are complicated and remain incompletely elucidated. Extensive literature corroborated the involvement of additional factors, such as pericyte-myofibroblast transition (PMT) [ 53 ], EndMT [ 54 ], activated microglia [ 55 ], macrophages [ 56 ] and Müller glia [ 57 ] in the laser-induced CNV mouse model, which contribute to the accumulation of differentiated myofibroblasts and deposition of ECM, ultimately leading to fibrosis formation in nAMD. Therefore, the contribution of Wnt5a or Wnt/β-catenin in the MT of other cell types during subretinal fibrosis remains to be elucidated.…”