NADPH Oxidase Mediates β-Amyloid Peptide-Induced Neuronal Death in Mouse Cortical Cultures.

β-Amyloid peptide (Aβ) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aβ-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aβ-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aβ, a fragment of Aβ with an equivalent neurotoxic effect. Aβ induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aβ increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aβ increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aβ. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aβ-induced neuronal death.