Targeting Peptidylarginine Deiminases in Neurons and Astrocytes in Central Nervous System Injury-Effects of Pan-PAD Inhibitor Cl-Amidine in an Oxygen-Glucose Deprivation Model of Ischaemia (OGD/R) and LPS Stimulation In Vitro

  • Int J Mol Sci. 2026 Jun 5;27(11):5118. doi: 10.3390/ijms27115118.
Dina Ahmed  1 ,  Stephen J Getting  1 ,  Maria Ashioti  1 ,  Sigrun Lange  1
Affiliations
  • 1. School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK.
PMID: 42278641 DOI: 10.3390/ijms27115118
Abstract

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 central nervous system (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, indicating roles for several PAD isozymes in acute CNS injury. Astrocyte cells showed the most prominent PAD4 detection, with significantly lower levels of PAD1, PAD2, PAD3 and PAD6, while differentiated SH-SY5Y neuronal cells showed the highest detection of PAD3, followed by PAD2 and PAD1, as well as strong PAD6 positivity, but negligible PAD4 detection. Histone H3 citrullination was significantly reduced in response to Cl-amidine treatment in both cell types, indicating changes in histone H3-dependent events in CNS injury. Cl-amidine treatment modulated key neuronal (beta-3 tubulin) and astrocytic (GFAP) markers and also reduced inflammatory cytokine IL-6 levels in astrocytes following 4 h OGD/R in conjunction with LPS stimulation. This study indicates roles for several PAD isozymes, with differing prominence in neurons and astrocytes, and emphasises the potential for pharmacological PAD inhibitor treatment in CNS injury.

Keywords
Cl-amidine; astrocyte; central nervous system (CNS); deimination/citrullination; hypoxia; ischaemia; neuron; oxygen–glucose deprivation; peptidylarginine deiminases (PADs); regeneration.
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