Effects of NADPH Oxidase Inhibitors and Mitochondria-Targeted Antioxidants on Amyloid β 1-42 -Induced Neuronal Deaths in Mouse Mixed Cortical Cultures

Abstract

The Amyloid beta peptide (Abeta) is a main component of senile plaques in Alzheimer's disease. Currently, NADPH oxidase (NOX) and mitochondria are considered as primary sources of ROS induced by Abeta. However, the contribution of NOX and mitochondria to Abeta-induced ROS generation has not been well defined. To delineate the relative involvement of NOX and mitochondria in Abeta-induced ROS generation and neuronal death in mouse cortical cultures, we examined the effect of NOX inhibitors, apocynin and AEBSF, and the mitochondria-targeted antioxidants (MTAs), mitotempol and mitoquinone, on Abeta-induced ROS generation and neuronal deaths. Cell death was assessed by measuring lactate dehydrogenase efflux in bathing media at 24 and 48 hrs after exposure to Abeta1-42. Abeta1-42 induced dose- and time-dependent neuronal deaths in cortical cultures. Treatment with 20 microM Abeta1-42 markedly and continuously increased not only the DHE fluorescence (intracellular ROS signal), but also the DHR123 fluorescence (mitochondrial ROS signal) up to 8 hrs. Treatment with apocynin or AEBSF selectively suppressed the increase in DHE fluorescence, while treatment with mitotempol selectively suppressed the increase in DHR123 fluorescence. Each treatment with apocynin, AEBSF, mitotempol or mitoquinone significantly attenuated the Abeta1-42-induced neuronal deaths. However, any combined treatment with apocynin/AEBSF and mitotempol/mitoquinone failed to show additive effects. These findings indicate that 20 microM Abeta1-42 induces oxidative neuronal death via inducing mitochondrial ROS as well as NOX activation in mixed cortical cultures, but combined suppression of intracellular and mitochondrial ROS generation fail to show any additive neuroprotective effects against Abeta neurotoxicity.