TNFα potentiates glutamate neurotoxicity by inhibiting glutamate uptake in organotypic brain slice cultures: neuroprotection by NFκB inhibition

Glutamate and the proinflammatory cytokine, tumor necrosis factor alpha (TNFα), have been suggested to contribute to neurodegenerative diseases. We investigated the interaction of TNFα and glutamate on neuronal cell death using fluorescence propidium iodide uptake in rat organotypic hippocampal-entorhinal cortex (HEC) brain slice culture that maintains the cytoarchitecture of the intact brain. Time course and concentration studies indicate that glutamate produced significant neuronal cell death in all four brain areas examined, for example, entorhinal cortex, hippocampal CA1 and CA3 fields, and dentate gyrus. TNFα alone at concentration of 20 ng/ml caused little or no detectable neuronal cell death, however, when combined with submaximal glutamate (3.3 mM), TNFα significantly increased and accelerated glutamate neurotoxicity. TNFα potentiation of glutamate neurotoxicity is blocked by NMDA receptor antagonists but not by AMPA antagonists CNQX and NBQX. Studies directly measuring [14C]-glutamate uptake in HEC slices indicate that TNFα dose-dependently inhibited glutamate uptake. Further, inhibitors of glial glutamate transporters potentiated glutamate neurotoxicity similar to TNFα. The antioxidant butylated hydroxytoluene (BHT) and the NFκB inhibitor PTD-p65 peptide inhibit NFκB activation and TNFα potentiation of glutamate neurotoxicity. BHT prevented the inhibition of TNFα on glutamate transport in HEC slices and also blocked nuclear translocation of NFκB subunit p65. These data indicate that TNFα and glutamate can act synergistically to induce neuronal cell death. TNFα potentiation of glutamate neurotoxicity through the blockade of glutamate transporter activity may represent an important mechanism of neurodegeneration associated with neuroinflammation.