Direct evidence of oxidative injury produced by the Alzheimer's beta-amyloid peptide (1-40) in cultured hippocampal neurons.
The beta-Amyloid peptide (A beta) is hypothesized to mediate the neurodegeneration 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 electron paramagnetic resonance (EPR) spectroscopy that A beta (1-40) generates free radicals in solution. It was therefore suggested that A beta radicals can attack cell membranes, initiate lipoperoxidation, damage membrane proteins, and compromise ion homeostasis resulting in neurodegeneration. To evaluate this hypothesis, the ability of A beta to induce neuronal oxidation, changes in calcium levels, enzyme inactivation, and neuronal death were compared with the ability of A beta 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 A beta to produce free-radicals was determined using EPR with the spin-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 intensity of radical generation by A beta and neurotoxicity. The highest neuronal oxidation and toxicity was seen at a time when A beta was capable of generating the most intense radical signal. Furthermore, little oxidation and toxicity was seen when cultures were treated with freshly dissolved A beta, which did not generate a detectable radical signal. These data are consistent with the hypothesis that free-radical-based oxidative damage induced by A beta contributes to the neurodegeneration of Alzheimer's disease.
Harris
M E
ME
Department of Pharmacology, University of Kentucky College of Medicine, Lexington 40536, USA.
Hensley
K
K
Butterfield
D A
DA
Leedle
R A
RA
Carney
J M
JM
D016229:Amyloid beta-Peptides / Q000633:toxicity*; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D002118:Calcium / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D004578:Electron Spin Resonance Spectroscopy; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005609:Free Radicals; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D006624:Hippocampus / Q000166:cytology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000032:analysis; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013113:Spin Labels
D016229:Amyloid beta-Peptides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005609:Free Radicals; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D013113:Spin Labels; C078592:amyloid beta-protein (1-40); C092727:biotin-4-amidobenzoic hydrazide; C029569:diacetyldichlorofluorescein; D001710:Biotin; D005974:Glutamate-Ammonia Ligase; D002118:Calcium