Tat-NR2B9c Prevents Excitotoxic Neuronal Superoxide Production

Abstract

The Tat-NR2B9c peptide has shown clinical efficacy as a neuroprotective agent in acute stroke. Tat-NR2B9c is designed to prevent nitric oxide (NO) production by preventing postsynaptic density protein 95 binding to N-methyl-D-aspartate (NMDA) receptors and neuronal nitric oxide synthase; however, PSD-95 is a scaffolding protein that also couples NMDA receptors to other downstream effects. Here, using neuronal cultures, we show that Tat-NR2B9c also prevents NMDA-induced activation of neuronal NADPH oxidase, thereby blocking superoxide production. Given that both superoxide and NO are required for excitotoxic injury, the neuroprotective effect of Tat-NR2B9c may alternatively be attributable to uncoupling neuronal NADPH oxidase from NMDA receptor activation. Keywords: DNA damage; glutamate; ischemia; nitric oxide; oxidative stress; stroke INTRODUCTION Brain ischemia leads to acute cell death, in part through sustained activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDAr) and the resulting production of nitric oxide (NO) and superoxide. 1,2 While neither NO or superoxide alone is highly reactive or toxic, in combination they form peroxynitrite and related free radical species that avidly react with lipids, proteins, DNA, and other cell components to cause excitotoxic cell death. 1,2 Postsynaptic density protein 95 (PSD-95) serves as a scaffolding protein for the clustering of synaptic receptors, ion channels, and associated signaling proteins. 3 It binds both to neuronal NO synthase (nNOS) and to the NR2B subunits of NMDAr. 4 Targeted disruption of these binding interactions with a Tat-conjugated peptide comprising the nine C-terminal residues of NR2B blocks NMDA-induced NO production. This peptide, termed 'Tat-NR2B9c' or 'NA-1' also blocks excitotoxic neuronal death, reduces acute ischemic cell death in animal stroke models, and has shown efficacy as a neuroprotective agent in clinical stroke. 2,5,6 Given the crucial role of NO in excitotoxic injury, the disruption of NMDAr/nNOS interaction is a likely mechanism for the neuroprotective effects observed with Tat-NR2B9c. However, superoxide production by NADPH oxidase (NOX2) is equally required for peroxynitrite formation and for excitotoxic and ischemic cell death. 1,2,7-9 These factors raise the possibility that disruption of NMDAr/NOX2 interactions might also (or alternatively) contribute to the mechanism of this promising stroke therapeutic. The present studies confirm that Tat-NR2B9c blocks NMDA-induced superoxide formation by a mechanism that prevents phosphorylation of the NOX2 p47 phox subunit. Journal of Cerebral Blood

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

“…Interestingly, the TAT-fused arginine-rich NADPH oxidase complex inhibitor peptide gp91ds-TAT (CSTRIRRQL-TAT-NH2; net charge with NH2 group +12) inhibited neuronal NMDA induced superoxide production to a greater extent than TAT-NR2B9c, while the TAT-fused non arginine-rich scrambled peptide (CSFNSYELGSLCY-TAT) did not. The Chen et al (2015) study also showed that TAT-NR2B9c did not inhibit NMDA induced neuronal calcium influx.…”

“…In a recent study (Chen et al, 2015), TAT-NR2B9c was shown to inhibit neuronal NMDA induced superoxide production by blocking the activation of the membrane-bound NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) complex. It was proposed that the TAT-NR2B9c peptide by inhibiting the PSD-95/ NR2B9c interaction blocked PSD-95 adaptor protein APPL1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) coupling with PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase), thereby inhibiting PKCζ (protein kinase C) to phosphorylate the NADPH oxidase subunit protein p46 phox , which is required for activation of the complex.…”

“…Recently, it was reported that TAT-NR2B9c reduces synaptically induced neuronal superoxide production (Chen et al, 2015) by disrupting a signalling cascade initiated by NR2B/PSD-95 coupling and activation of the NADPH oxidase membrane complex. However, it is more likely that the TAT-NR2B9c peptide through its endocytosis-inducing properties acts by reducing the levels of neuronal NMDA receptors and/or the NADPH oxidase membrane complex, thereby also reducing superoxide production.…”

Beneficial Effects of a CaMKIIα Inhibitor TatCN21 Peptide in Global Cerebral Ischemia

“…Our results showed that tatCN21 mediated neuroprotection was accompanied by decreased membrane PSD95 aggregation, implying that decreased PSD95 membrane localization after GCI could contribute to the neuroprotective properties of tatCN21. Indeed, previous study showed that disruption of membrane aggregation of PSD95 and PSD95-NR2B binding following ischemic stroke conferred neuroprotection (Chen et al , 2015). Further studies should investigate whether tatCN21 affects the association of NR2B and PSD95 at the synaptic membrane, as well as other PSD95 associated contributors to ischemic injury.…”

Efficacy of the PSD95 inhibitor Tat-NR2B9c in mice requires dose translation between species

“…10,11 An important downstream consequence is that Tat-NR2B9c attenuates ROS induced by NMDAR activity. 12,13 The preclinical efficacy of Tat-NR2B9c as a stroke neuroprotectant has been validated by us and others in several stroke models in rats. 2,4,10,[14][15][16] and in comprehensive studies in non-human primates that provided strong evidence of efficacy.…”