No abstract

Aggregation of the amyloid-b (Ab) peptide into amyloid plaques is a characteristic feature of Alzheimer's disease neuropathogenesis.We and others have previously demonstrated delayed Ab aggregation as a consequence of oxidizing a single methionine residue at position 35 . Here, we examined the consequences of Met-35 oxidation on the extremely aggregation-prone peptides Ab1-42 and Ab1-40Arctic with respect to proto¢bril and oligomer formation as well as neurotoxicity. Size exclusion chromatography and mass spectrometry demonstrated that monomer/dimers prevailed over larger oligomers after oxidizing Met-35, and consequently proto¢bril formation and aggregation of both Ab1-42 and Ab1-40Arctic were delayed.The oxidized peptides completely lacked neurotoxic e¡ects in cortical neuronal cultures under these conditions, in contrast to the neurotoxic properties of the unoxidized peptides. We conclude that oxidation of Met-35 signi¢cantly attenuates aggregation of Ab1-42 and Ab1-40Arctic, and thereby reduces neurotoxicity. IntroductionSoluble amyloid-b (Ab) oligomers are currently suspected to be the major neuropathogens active in Alzheimer's disease (AD) [1,2]. Cognitive decline has been correlated with cerebral levels of soluble Ab in AD patients [2] and neurotoxic properties have been attributed to small Ab oligomers [3] as well as larger oligomers, that is protofibrils [4].Whether monomeric Ab induces neurotoxicity is not unambiguous, largely because of the technical difficulties in preparing pure monomeric Ab solutions at relevant concentrations and maintaining them in a cell culture environment. For instance, the literature is conflicting on whether low molecular weight Ab (a preparation consisting largely of monomers and possibly smaller oligomers [5]) is nontoxic [6] or toxic [4].We have demonstrated previously that oxidation of the single methionine residue at position 35 (Met-35) attenuates Ab1-40 trimer formation. This finding provides us with a useful tool to evaluate the neurotoxicity of low molecular weight Ab primarily consisting of monomers and dimers. Therefore, we compared the aggregation and neurotoxic properties of Ab1-42 and Ab1-40Arctic (E22G) in their reduced and oxidized form. The latter peptide is produced by carriers of the Arctic amyloid precursor protein mutation, and spontaneously generates high levels of protofibrils in vitro [1]. Methodsb-Amyloid peptides and reagents Synthetic Ab peptides were purchased from Biosource (Camarillo, California, USA). For the size exclusion chromatography (SEC) experiments, Ab1-42 and Ab1-40Arctic peptides were Met-35 oxidized according to our previously published protocols using 2.7% H 2 O 2 [7]. For all other experiments, oxidized AbMet-35 was purchased from Biosource where oxidation had been carried out using dimethylsulfoxide. All Met-35 oxidized and unoxidized peptides were checked for their correct identity by sequence analysis, and mass determination using mass spectrometry. All reagents and antibodies were purchased from SigmaAldrich (St. Louis, Missou...

Several proteins and peptides that can convert from ␣-helical to ␤-sheet conformation and form amyloid fibrils, including the amyloid ␤-peptide (A␤) and the prion protein, contain a discordant ␣-helix that is composed of residues that strongly favor ␤-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but A␤ apparently lacks such interactions. The helical propensity of the A␤ discordant region (K 16 LVFFAED 23 ) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the ␣-helical content of A␤(12-28) and reduces aggregation and fibril formation of A␤(1-40), A␤(12-28), A␤(12-24), and A␤(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on A␤ secondary structure or fibril formation. These data suggest that in free A␤, the discordant ␣-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.