Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates

Abstract

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca+2-regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma.Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage.

Peptidylarginine Deiminases as Mediators of Microvesicular Release - Novel Therapeutic Interventions

“…Previous studies on in vivo models of CNS damage showed that PAD-upregulation and increased protein deimination lead to vast neuronal death which was preventable by pharmacological PAD inhibition, significantly reducing neuroinflammatory responses and histone H3 deimination, which has implications in gene regulation [23,[33][34][35]. In addition we have observed increased PAD expression during disease progression in tau mutation mouse models compared to age-matched controls [36].…”

“…Administration of one dose of Cl-Am (80 mg/kg) straight after injury and up to two hours post-injury, in a chick-model of spinal cord damage, significantly reduced neuronal cell death, tissue loss and histone H3 deimination, compared to non-treated control injuries [32]. In two different mouse models of neonatal hypoxic ischaemic encephalopathy (HIE), firstly where permanent left carotid occlusion was followed with severe hypoxia (8% oxygen for 60 min), or secondly with 30 min hypoxia in combination with infection (as mimicked by LPS stimulation), one dose of Cl-Am (30 mg/kg) straight after hypoxia, or 10 min after LPS stimulation and again straight after hypoxia, significant reduction was observed in microglial activation, histone H3 deimination and cell death in all affected brain regions [33]. Interestingly, EV release has previously been associated with cerebral hypoxia induced by acute ischaemic stroke [93,94] while increased protein deimination has also been detected in the pathology of traumatic brain injury [95].…”

“…Previous work in two animal models of acute CNS damage has demonstrated that pharmacological pan-PAD inhibition is neuroprotective in vivo [32][33][34]. Administration of one dose of Cl-Am (80 mg/kg) straight after injury and up to two hours post-injury, in a chick-model of spinal cord damage, significantly reduced neuronal cell death, tissue loss and histone H3 deimination, compared to non-treated control injuries [32].…”

“…PAD4 is also linked with oestrogen receptor target gene activity via histone tail deimination [67]. In spite of lacking a classic nuclear translocation site such as found in PAD4, both PAD2 and PAD3 have also been localized and detected in the nucleus [32,33,56,68]. In cancer cells, PAD2, which is the most widely expressed isozyme in the body [20], has been shown to deiminate histone H3 and play a role in gene regulation [30,56,69,70].…”

“…Although some target proteins have been described, most deiminated proteins remain to be identified. We have, using proteomic analysis of deiminated proteins in the injured CNS [32,33,36], identified several proteins with neurodegenerative implications including neuroinflammation, perivascular drainage of Aβ, and the SNARE proteins, which are involved in EV release [139,140]. In pilot studies, using iPSC neuronal models derived from fibroblasts from patients [37] carrying valosin-protein containing mutations VCPR155C and VCPR191Q we have observed significantly increased pan-protein deimination compared to control (non-mutation carrying) neurones, with significant increases in histone H3 deimination in VCPR155C carrying neurones [36].…”