Peptidylarginine deiminase (PAD4) expression and citrullination levels in normal human and mouse retinas and in murine models of late- (Sod1^-/-) and early-onset (Tyro3^-/-, Axl ^-/-, Mertk -/- or TAM mice) retinal degeneration

Abstract

Purpose: PADs are immunomodulating enzymes that deiminate arginine residues through an irreversible post-translational process called citrullination. Of five mammalian PADs known, PAD2 was believed to be the main retinal PAD. Here we show that also PAD4, a prominent treatment target in autoimmune diseases, is expressed in human and mouse retina, and present evidence that PAD4 expression and citrullination levels are aberrantly high early on in Sod1-/- mice (a model of late-onset, AMD-like retinopathy) and TAM mice [a model of early-onset retinitis pigmentosa (RP)].

Methods: Retinas from normal human donor tissues and from wild-type (WT) mice were extracted for SDS-PAGE and probed using an antibody against PAD4 by Western blot (WB). Eyes from WT, Sod1+/-, Sod1-/- and TAM mice were cryosectioned and labeled by immunohistochemistry (IHC) using anti-PAD4 and F95 anti-citrullinated peptide antibodies. Sod1-/- mice examined at age 6-10 months, before the onset of the retinal degenerative phenotype. TAM mice were examined at age 9 weeks, when degeneration is severe. Age-matched WT and Sod1+/- mice were used as controls.

Results: WBs showed a ~75 kDa band consistent with the MW of PAD4 in both macular and peripheral human retina tissue, in WT and in Sod1+/- mouse retinas. IHC showed labeling of the ganglion cell layer (GCL), inner (INL) and outer nuclear layers (ONL). PAD4 and citrullination levels were already mildly increased in pre-phenotype stage Sod1-/- retinas. PAD4 expression and citrullination were markedly increased and deranged, in a clustered pattern, in the ONL of 9-wk TAM retinas, with aberrant reactivity localization also to the inner/outer segment remnants.

Conclusions: In addition to PAD2, we show PAD4 expression in human and murine retinas. PAD4 expression and citrullination levels are abnormally high before phenotype development in a mouse AMD model and in the advanced stages of an RP model. This suggests an important role of PAD4-mediated intraretinal inflammation in retinal degenerative diseases that could further contribute to disease severity and progression. Thus, as in systemic diseases, PAD4 may represent a valuable treatment target also in retinal degenerations like AMD and RP, and may also have a role in inflammatory and/or autoimmune retinal diseases.