Targeting Peptidylarginine Deiminases in Neurons and Astrocytes in Central Nervous System Injury—Pro-Regenerative Effects of Cl-Amidine in an Oxygen-Glucose Deprivation Model of Ischaemia (OGD/R) In Vitro

Peptidylarginine deiminases (PADs) are a family of five isozymes (PAD1-4, PAD6) in humans, with PAD2, 3 and 4 associated with the central nervous system. PAD mediated post-translational citrullination/deimination of target proteins contributes to pathobiological processes, including in the CNS, where the potential of PAD inhibitor treatment has been reported. This study aimed to identify PAD-dependent pro-regenerative responses in neuronal and astrocytic cells respectively, using human cellular in vitro models to assess the therapeutic effects of pan-PAD, PAD2- and PAD4- isozyme specific inhibitors in an oxygen-glucose deprivation/reperfusion model of ischaemia (OGD/R) at different time windows (30 min, 1 h and 4 h) in conjunction with scratch injury and LPS stimulation. Key findings suggest that pan-PAD inhibitor Cl-amidine promotes CNS regeneration through enhancing wound healing of both neuronal and astrocytic cells, including with a significant inhibition of PAD2 and PAD3 in the neuronal cells, while protective effects in the astrocytic cells indicate involvement of PAD2 and PAD4. His-tone H3 citrullination was significantly reduced in response to Cl-amidine treatment in both cell types, indicating changes in epigenetic regulation and immune-responses via NETosis in CNS injury. Cl-amidine treatment modulated key neuronal (beta-3 tubulin) and astrocytic (GFAP) markers and significantly reduced inflammatory cytokine IL-6 levels in astrocytes following 4 h OGD/R in conjunction with LPS. This study emphasizes the potential for pharmacological PAD inhibitor treatment in CNS injury, including future PAD3 inhibitor development to target neuronal injury.