INHIBITION OF PROTEIN ARGININE DEIMINASES IMPROVES COGNITION AND REDUCES NEUROPATHOLOGICAL CHANGES IN MOUSE MODELS OF ALZHEIMER’S DISEASE

Abstract

Inflammation has been correlated to Alzheimer's disease (AD) and a better understanding of its mechanisms may help to develop new therapies for AD. We have recently demonstrated that neutrophils infiltrate the brain and produce neutrophil extracellular traps (NETs) in subjects with AD and mice with AD-like disease. Furthermore, inhibition of neutrophil trafficking has therapeutic effect in animal models of AD. Recent studies have shown that protein arginine deiminases (PADs) mediate NET formation and have a role in innate immunity responses. PAD inhibitors have demonstrated remarkable efficacy in numerous inflammatory diseases including rheumatoid arthritis, lupus, ulcerative colitis, multiple sclerosis and atherosclerosis. The aim of our study was to test the potential therapeutic effect of PAD inhibition in AD models. BB-Cl-amidine, a pan-PAD inhibitor, and GSK199, a PAD4 specific inhibitor were used to study the role of PADs in vitro and in vivo. The role of PADs in rapid adhesion in vitro was assessed in adhesion assays on integrin ligands. Epifluorescence intravital microscopy and two-photon microscopy were performed to study the role of PADs in neutrophil adhesion in vivo. 5xFAD mice, which develop amyloid pathology, and 3xTg-AD mice, which develop both amyloid and tau pathology, were used to test the effect of PAD inhibition on disease. Treatment of neutrophils with PAD inhibitors reduced integrin-dependent rapid adhesion triggered by chemoattractants and TNF. PAD inhibition also reduced rolling interactions and drastically inhibited firm adhesion in inflamed brain microcirculation. Moreover, two-photon microscopy performed in the brain of AD mice showed a drastic reduction of adhesion and intravascular crawling of neutrophils treated with BB-Cl-amidine compared to untreated cells, suggesting that PADs control neutrophil trafficking in the AD brain. Notably, treatment with BB-Cl-amidine of 3xTg-AD and 5xFAD mice led to a significant restoration of memory in Y maze and contextual fear conditioning behavioral tests when compared to control animals. Neuropathological studies showed that treatment with BB-Cl-amidine reduced microglial activation and amyloid deposition compared to control animals. PAD inhibition interferes with neutrophil trafficking and ameliorates cognitive impairment and neuropathological changes in animal models of AD, suggesting PADs represent new therapeutic targets in AD.