Apoptosis induced via AMPA-selective glutamate receptors in cultured murine cortical neurons

We have investigated the mechanisms of cell death induced by long-term exposure to the glutamate receptor agonist (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate [(S)-AMPA]. Using primary cultures of pure neurons (95%) grown in serum-free conditions, we found that 24-h exposure to (S)-AMPA (0.01-1,000 microM) induced concentration-dependent neuronal cell death (EC50 = 3 +/- 0.5 microM) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. (S)-AMPA induced a delayed cell death with DNA fragmentation occurring in approximately 50% of cells at concentrations between 100 and 300 microM detected using terminal transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6-diamidino-2-phenylindole, a fluorescent DNA binding dye. Cell death induced by (S)-AMPA was attenuated by the AMPA receptor-selective antagonist LY293558 (10 microM) and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM), yielding EC50 values of 73 +/- 5 and 265 +/- 8 microM, respectively, and was unaffected by the NMDA receptor antagonist MK-801 (10 microM). The number of apoptotic nuclei induced by 300 microM (S)-AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL-positive at 24 h. In addition, cycloheximide (0.5 microg/ml) also inhibited (S)-AMPA-induced DNA fragmentation and cell death. Our results show that long-term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA-sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.