Role of glycine-33 and methionine-35 in Alzheimer's amyloid β-peptide 1-42-associated oxidative stress and neurotoxicity

Recent theoretical calculations predicted that Gly33 of one molecule of amyloid β-peptide (1-42) (Aβ(1-42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Aβ(1-42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Aβ(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Aβ(1-42) was substituted by Val (Aβ(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Aβ(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Aβ(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Aβ(1-42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-α-phenylnitrone (PBN). While Aβ(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Aβ(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Aβ(1-42G33V) does not form fibrils nearly as well as Aβ(1-42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Aβ(1-42) and that contribute to the peptide's toxicity in Alzheimer's disease brain.