Human Bcl-2 protects against AMPA receptor-mediated apoptosis

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionat e (AMPA) subtype of ionotropic receptor for the brain's major excitatory ne urotransmitter, L-glutamate, occur in various neurological conditions. We h ave previously demonstrated that AMPA receptor-mediated excitotoxicity occu rs by apoptosis and here examined the influence of the expression of cell d eath repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortic al cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compare d with that seen with staurosporine and H2O2. At day 6 cultures were expose d to AMPA (0.1-100 mu M), and cellular injury was analyzed 48 h after insul t using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe nyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino -2-phenylindole and Sytox Green. AMPA produced a concentration-dependent in crease in cell death that was significantly attenuated by human Bcl-2. AMPA (3 mu M) increased the number of apoptotic nuclei to 60% of control in wil d-type cultures, and human Bcl-2 significantly decreased the number of apop totic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided sig nificant neuroprotection against neuronal injury induced by tow concentrati ons of staurosporine (1-10 nM) and H2O2 (0.1-30 mu M) and where neuronal de ath was by apoptosis, but not against H2O2-induced necrosis. Our findings i ndicate that overexpression of Bcl-2 in primary cultured neurons protects i n an insult-dependent manner against AMPA receptor-mediated apoptosis, wher eas protection was not seen against more traumatic insults. This study prov ides new insights into the molecular therapeutics of neurodegenerative cond itions.