Overexpression of the nuclear factor kappaB inhibitor A20 is neurotoxic after an excitotoxic injury to the immature rat brain

Abstract

Background: The zinc finger protein A20 is an ubiquitinating/deubiquitinating enzyme essential for the termination of inflammatory reactions through the inhibition of nuclear factor kappaB (NF-kappaB) signaling. Moreover, it also shows anti-apoptotic activities in some cell types and proapoptotic/pronecrotic effects in others. Although it is known that the regulation of inflammatory and cell death processes are critical in proper brain functioning and that A20 mRNA is expressed in the CNS, its role in the brain under physiological and pathological conditions is still unknown.

Methods: In the present study, we have evaluated the effects of A20 overexpression in mixed cortical cultures in basal conditions: the in vivo pattern of endogenous A20 expression in the control and N-methyl-d-aspartate (NMDA) excitotoxically damaged postnatal day 9 immature rat brain, and the post-injury effects of A20 overexpression in the same lesion model.

Results: Our results show that overexpression of A20 in mixed cortical cultures induced significant neuronal death by decreasing neuronal cell counts by 45±9%. In vivo analysis of endogenous A20 expression showed widespread expression in gray matter, mainly in neuronal cells. However, after NMDA-induced excitotoxicity, neuronal A20 was downregulated in the neurodegenerating cortex and striatum at 10–24 hours post-lesion, and it was re-expressed at longer survival times in reactive astrocytes located mainly in the lesion border. When A20 was overexpressed in vivo 2 hours after the excitotoxic damage, the lesion volume at 3 days post-lesion showed a significant increase (20·8±7·0%). No A20-induced changes were observed in the astroglial response to injury.

Conclusions: A20 is found in neuronal cells in normal conditions and is also expressed in astrocytes after brain damage, and its overexpression is neurotoxic for cortical neurons in basal mixed neuron–glia culture conditions and exacerbates postnatal brain excitotoxic damage.

Keywords:

• Postnatal brain injury
• Excitotoxicity
• Gene therapy
• Non-viral modular vector
• A20
• Nuclear factor kappaB
• Neurodegeneration

We would like to thank Miguel A. Martil and Lola Mulero for their excellent technical help. This work was supported by a FI fellowship from the Generalitat de Catalunya (HP) and BFU2008-04407 and ACI2009-0919 (Ministry of Science and Innovation, Spain). AV received an ICREA Academia award.