Disulfide-containing High Mobility Group Box-1 promotes N-methyl-D-aspartate receptor function and excitotoxicity by activating Toll-Like Receptor 4-dependent signaling in hippocampal neurons.

Aims: Using primary cultures of mouse hippocampal neurons, we studied the molecular and functional interactionsbetween high mobility group box-1 (HMGB1) and the N-methyl-d-aspartate receptor (NMDAR), twoproteins playing a key role in neuronal hyperexcitability. By measuring NMDA-induced calcium (Ca2+) increasein neuronal somata and neurotoxicity as functional read-out parameters, we explored the role of the redox stateof HMGB1, the receptor involved, and the molecular signaling underlying its interactions with postsynapticNMDAR. We also investigated whether HMGB1 redox state affects its proconvulsive effects in mice. Results:Nonoxidizable HMGB1 with a triple cysteine-to-serine replacement (3S-HMGB1) was ineffective on NMDAresponse. Conversely, the disulfide-containing form of HMGB1 dose dependently enhanced NMDA-inducedCa2+ increase in neuronal cell bodies. This effect was prevented by BoxA, a competitive HMGB1 antagonist, andby Rhodobacter sphaeroides lipopolysaccharide (LPS-RS), a toll-like receptor 4 (TLR4) selective antagonist, andit was abrogated in neurons lacking TLR4 while persisting in the absence of receptor for advanced glycation endproducts (RAGE). TLR4 and NMDAR subunit 1 (NR1) and 2B (NR2B) were colocalized in neurons. DisulfideHMGB1 effect on NMDA-induced Ca2+ influx was prevented by 3-O-methylsphingomyelin (3-O-MS) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine, (PP2) selective inhibitors of neutral sphingomyelinaseand Src-family Tyr kinases, respectively. Disulfide HMGB1, but not 3S-HMGB1, increased Tyr1472phosphorylation of the NR2B subunit of the NMDAR, which is known to increase Ca2+ channel permeability.Similarly, disulfide HMGB1 increased NMDA-induced neuronal cell death in vitro and enhanced kainate-inducedseizures in vivo. Innovation and Conclusion: We describe a novel molecular neuronal pathway activatedby HMGB1 that could be targeted in vivo to prevent neurodegeneration and seizures mediated by excessiveNMDARs stimulation.