Direct evidence of oxidative injury produced by the Alzheimer's β-Amyloid peptide (1-40) in cultured hippocampal neurons

The β-Amyloid peptide (Aβ) is hypothesized to mediate the neurodegeneration seen in Alzheimer's disease. Recently, we proposed a new hypothesis to explain the toxicity of Aβ based on the free-radical generating capacity of Aβ. We have recently demonstrated using electron paramagnetic resonance (EPR) spectroscopy that Aβ (1-40) generates free radicals in solution. It was therefore suggested that Aβ radicals can attack cell membranes, initiate lipoperoxidation, damage membrane proteins, and compromise ion homeostasis resulting in neurodegeneration. To evaluate this hypothesis, the ability of Aβ to induce neuronal oxidation, changes in calcium levels, enzyme inactivation, and neuronal death were compared with the ability of Aβ to produce free-radicals. Using hippocampal neurons in culture, several methods for detection of oxidation were utilized such as the conversion of 2,7-dichlorofluorescin to 2,7-dichlorofluorescein, and a new fluorescence microscopic method for the detection of carbonyls. The ability of Aβ to produce free-radicals was determined using EPR with the spintrapping compound N-tert-butyl-α-phenylnitrone. Consistent with previous studies, we found that preincubation of Aβ increased the toxicity of the peptide. There is a strong correlation between the intensity of radical generation by Aβ and neurotoxicity. The highest neuronal oxidation and toxicity was seen at a time when Aβ was capable of generating the most intense radical signal. Furthermore, little oxidation and toxicity was seen when cultures were treated with freshly dissolved Aβ, which did not generate a detectable radical. signal. These data are consistent with the hypothesis that free-radical-based oxidative damage induced by Aβ contributes to the neurodegeneration of Alzheimer's disease.