Human Bcl‐2 Protects Against AMPA Receptor‐Mediated Apoptosis

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Abstract
Dysfunctions of the (S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L‐glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor‐mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl‐2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl‐2 gene, the influence of Bcl‐2 on AMPA receptor‐mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1‐100 μM), and cellular injury was analyzed 48 h after insult using phase‐contrast microscopy, a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide viability assay, and DNA staining with 4,6‐diamidino‐2‐phenylindole and Sytox Green. AMPA produced a concentration‐dependent increase in cell death that was significantly attenuated by human Bcl‐2. AMPA (3 μM) increased the number of apoptotic nuclei to 60% of control in wild‐type cultures, and human Bcl‐2 significantly decreased the number of apoptotic nuclei to 30% of AMPA‐treated cultures. Human Bcl‐2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1‐10 nM) and H2O2 (0.1‐30 μM) and where neuronal death was by apoptosis, but not against H2O2‐induced necrosis. Our findings indicate that overexpression of Bcl‐2 in primary cultured neurons protects in an insult‐dependent manner against AMPA receptor‐mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.