Citrullination is an important protein posttranslational modification (PTM) caused by an irreversible modification of the arginine residues on proteins to the non- coded citrulline residues by the peptidyl arginine deiminase enzymes (PADs). Citrullination is a permanent/irreversible change in numerous critical protein targets, and citrullinated proteins have been detected as disease-specific biomarkers in several major inflammatory diseases, such as rheumatoid arthritis, and multiple sclerosis (MS). Targeting PADs with small molecule inhibitors has been shown to be effective in reducing disease burden in several preclinical models. We have recently demonstrated that citrullination is induced in the mouse retina by alkali injury. Of particular interest is the discovery that activated Muller glia, and retinal astrocytes displayed elevated levels of citrullinated soluble glial fibrillary acidic protein (GFAP). Furthermore, inhibition of citrullination alters expression characteristics of soluble GFAP, and linked PAD's activity to retinal gliosis. We discovered that these increases in the citrullination pathway are found during chronic gliosis induced by severe alkali injury, but reversed in milder alkali burn model. Interestingly, activated Muller glia selectively expressed PAD4, which is responsible for GFAP citrullination, and inhibition of PAD4 reduces soluble GFAP expression. In this R21 grant proposal, we will test the hypothesis that PAD4-driven citrullination controls the chronic gliotic response in the laser injury model of age-related macular degeneration (AMD).
In specific Aim 1, we will investigate the dynamics of PAD4 expression during acute versus chronic gliosis, and PAD4's association with overexpression of citrullination, and GFAP's modification(s) in different injury paradigms.
In Aim 2, we will investigate the consequence of inhibiting PAD4 enzymatic activity as well as causing PAD4 deficiency in astrocytes/glial cells through conditional target knockout strategy, and investigate how the inhibition/absence of this critical enzyme affects injury response. This approach that focuses on PAD4 will help establish whether PAD4 is druggable in vivo in injury states and for the first time lay down the foundation to probe this druggable target in conditions, such as AMD, that cause irreversible blindness.
