DIRECT EVIDENCE OF OXIDATIVE INJURY PRODUCED BY THE ALZHEIMERS BETA-AMYLOID PEPTIDE (1-40) IN CULTURED HIPPOCAMPAL-NEURONS

The beta-Amyloid peptide (A beta) is hypothesized to mediate the neuro degeneration seen in Alzheimer's disease, Recently, we proposed a new hypothesis to explain the toxicity of A beta based on the free-radical generating capacity of A beta. We have recently demonstrated using el ectron paramagnetic resonance (EPR) spectroscopy that A beta (1-40) ge nerates free radicals in solution. It was therefore suggested that A b eta radicals can attack cell membranes, initiate lipoperoxidation, dam age membrane proteins, and compromise ion hameostasis resulting in neu rodegeneration. To evaluate this hypothesis, the ability of A beta to induce neuronal oxidation, changes in calcium levels, enzyme inactivat ion, and neuronal death were compared with the ability of A beta to pr oduce free-radicals. Using hippocampal neurons in culture, several met hods for detection of oxidation were utilized such as the conversion o f 2,7-dichlorofluorescin to 2,7-dichlorofluorescein, and a new fluores cence microscopic method for the detection of carbonyls, The ability o f A beta to produce free-radicals was determined using EPR with the sp in-trapping compound N-tert-butyl-alpha-phenylnitrone. Consistent with previous studies, we found that preincubation of A beta increased the toxicity of the peptide. There is a strong correlation between the in tensity of radical generation by A beta and neurotoxicity. The highest neuronal oxidation and toxicity was seen at a time when A beta was ca pable of generating the most intense radical signal, Furthermore, litt le oxidation and toxicity was seen when cultures were treated with fre shly dissolved A beta, which did not generate a detectable radical sig nal, These data are consistent with the hypothesis that free radical b ased oxidative damage induced by A beta contributes to the neurodegene ration of Alzheimer's disease. (C) 1995 Academic Press, Inc.