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

Abstract

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.

Compartmentalized citrullination in Muller glial endfeet during retinal degeneration

“…To test whether PAD4 is responsible for MG hypercitrullination, we selectively ablated Padi4 gene expression by crossing GFAP-Cre ERT2 with Padi4 flox/flox mice and administered tamoxifen to induce PAD4 deficiency in glial cells (PAD4 conditional knockout mice [PAD4cKO]) ( SI Appendix ). Immunostaining of retinas from control and PAD4cKO mice subjected to laser injury ( SI Appendix ) revealed loss of citrullinated GFAP (cit-GFAP) in PAD4cKO retinas compared to control injured retinas as detected by a site-specific (R270 and R416) anticitrullinated GFAP antibody ( 4 ) ( Fig. 1 O and P ).…”

“…Muller glia (MG) are the principal reactive cells of the retina that respond to stress via a process known as reactive gliosis ( 1 ). The posttranslational modification (PTM) citrullination ( 2 ) of glial fibrillary acidic protein (GFAP) can occur in reactive MG, where citrullination driven by peptidyl arginine deiminase-4 (PAD4) expression is closely regulated early during gliosis in a mouse model of corneal alkali injury ( 3 , 4 ). The conversion of arginine residues on GFAP could promote its depolymerization ( 5 ), or potentially, citrullinated GFAP could also be cleaved ( 6 ), and citrullinated fragments may serve as autoimmunogens ( 7 ).…”

Latent Sensitization in a Mouse Model of Ocular Neuropathic Pain

“…Alkali burn to the ocular surface has been widely used to study corneal neovascularization, wound healing, and inflammation 18,26–28. Tissue injury and remodeling beyond the ocular surface including retinal and optic nerve gliosis also have been reported after alkali injury to the cornea 26,28–31. Although alkali burn causes corneal nerve damage and significant pain in patients, little is known about the long-term effects of alkali burn injury to the oculo-trigeminal sensory system 32.…”

Neurochemical regulation of the expression and function of glial fibrillary acidic protein in astrocytes

“…In mouse eyes, retina and choroid express PAD2 and PAD4 transcripts. PAD4 inhibition reduces injury‐induced citrullination and soluble GFAP protein levels in organ cultures (Wizeman & Mohan, ). It was also reported that dibutyryl‐cAMP significantly increased PAD expression and enzyme activity in a dose‐dependent manner, which was inhibited by PKA inhibitor KT5720 in human astrocytoma U‐251MG cells (Masutomi et al, ).…”