Blocking peptidyl arginine deiminase 4 confers neuroprotective effect in the post-ischemic brain through both NETosis-dependent and -independent mechanisms

Feb 19, 2025Acta neuropathologica communications

Blocking an enzyme called PAD4 protects the brain after stroke through both NETosis-related and other pathways

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Abstract

PAD4 levels were significantly elevated in the ischemic core and penumbra of the affected hemisphere at 3-6 and 6-48 hours post-MCAO, respectively.

Citrullination, mediated by PAD4, is associated with the induction of , a process of neutrophil cell death.
NETosis induction, marked by increased citrullinated histone H3, peaks between 48 and 96 hours post-MCAO.
Strong PAD4 induction was primarily observed in neurons during the peak PAD4 expression period of 12-24 hours post-MCAO.
Intranasal administration of the PAD4 inhibitor BB-Cl-amidine significantly reduced infarct volume and improved neurological outcomes at 24 hours post-MCAO.
BBCA treatment suppressed the accumulation of citrullinated proteins in degenerating neurons.
Delayed BBCA administration at 48/72 hours post-MCAO may inhibit NETosis induction at 96 hours post-MCAO.

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Peptidylarginine deiminase 4 (PAD4) is an enzyme that modifies proteins by converting positively charged arginine residues to neutral citrulline residues. This process, termed citrullination, has been known to trigger , a neutrophil cell death pathway involving the release of neutrophil extracellular traps (NETs). Abnormal PAD4 activity and protein citrullination have been linked to various diseases, including those affecting the central nervous system. Herein we investigated the profile of PAD4 expression in an animal model of stroke induced by middle cerebral artery occlusion (MCAO). PAD4 levels were significantly elevated in the ischemic core and penumbra of the affected hemisphere at 3-6 and 6-48 h post-MCAO, respectively. Notably, NETosis induction, indicated by the upregulation of CitH3 (citrullinated histone H3, a NETosis marker), was observed between 48 and 96 h post-MCAO, peaking at 96 h. While PAD4 was present in most brain cell types of sham controls, strong PAD4 induction was primarily observed in neurons during the peak PAD4 induction period (12-24 h post-MCAO). Importantly, intranasal administration of the PAD4 inhibitor BB-Cl-amidine (BBCA) significantly reduced infarct volume and improved neurological and functional outcomes at 24 h post-MCAO, demonstrating a strong protective effect of PAD4 inhibition in ischemic stroke. Staining with an antibody that recognizing citrullinated proteins (F95) revealed an accumulation of these proteins, especially degenerating neurons, however, BBCA treatment significantly suppressed this accumulation in dying neurons. These findings indicate that PAD4-mediated protein citrullination in neurons plays a critical role in promoting ischemic brain damage. Furthermore, delayed administration of BBCA (at 48/72 h post-MCAO) suppresses the NETosis induction observed at 96 h post-MCAO, potentially ameliorating repair processes such as blood vessel regeneration. Collectively, these findings suggest a complex role of PAD4 in cerebral ischemia, with neuroprotective effects (NETosis-independent function) during the acute to subacute period and NETosis-suppressive effects at later time points.

Key numbers

29.2%

Infarct Volume Reduction

Volume of infarcted brain tissue after BBCA treatment compared to untreated controls.

24 h

PAD4 Induction Peak

PAD4 levels peak in the cortical penumbra at this time post-MCAO.

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Blocking peptidyl arginine deiminase 4 confers neuroprotective effect in the post-ischemic brain through both NETosis-dependent and -independent mechanisms

Abstract

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