Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window

Abstract

Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter.

Objective: With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol.

Methods: We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD).

Results: The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation. Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20μg/kg and 200μg/kg) or resveratrol (6800μg/kg) individually. However, the administration of lowest doses of MS-275 (2μg/kg) and resveratrol (68μg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-xL promoter and decreased at the Bim promoter.

Conclusion: Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window.

Keywords: HDAC inhibitors; MCAO; OGD; RelA acetylation.