Abstract
The amyloid beta peptide (Abeta) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer's disease. However, the nature of the toxic Abeta species and its precise mechanism of action remain to be determined. It has been reported that the methionine residue at position 35 has a pivotal role to play in the toxicity of Abeta. We examined the effect of mutating the methionine to valine in Abeta42 (AbetaM35V). The neurotoxic activity of AbetaM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Abeta42. AbetaM35V binds Cu2+ and produces similar amounts of H2O2 as Abeta42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AbetaM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Abeta and copper in a lipid environment as shown by EPR analysis, which indicated that the valine substitution made the peptide less rigid in the bilayer region with a resulting higher affinity for the bilayer. Circular dichroism spectroscopy showed that both Abeta42 and AbetaM35V displayed a mixture of alpha-helical and beta-sheet conformations. These findings provide further evidence that the toxicity of Abeta is regulated by binding to neuronal cells.
Highlights
Genetic evidence derived from early onset cases of Alzheimer’s disease (AD)1 indicated that the metabolism of the amyloid  peptide (A) is clearly linked to the pathogenesis of this disease [1]
Because AM35V is potently toxic to cells at 5 M after 96 h of treatment, we examined the cell viability of the cortical neurons at shorter treatment times of 24, 48, and 72 h (Fig. 1B)
When we treated our cortical neuronal cell cultures for a longer period of time (96 h compared with 24 h [24, 25]), we showed that Met(O)A caused significant decreases in cell viability, and this was rescued by catalase and attenuated by the metal chelator clioquinol [26]
Summary
Genetic evidence derived from early onset cases of Alzheimer’s disease (AD)1 indicated that the metabolism of the amyloid  peptide (A) is clearly linked to the pathogenesis of this disease [1]. To determine whether the interaction between A and copper in a lipid environment is altered for AM35V, we obtained a series of EPR spectra of 16NPS in LUVs containing 0.05 M PBS pH 6.9, and after adding 0.03 mol of peptide/lipid of both A42 and AM35V coordinated with 0.3 mol equivalents of Cu2ϩ per mole of peptide.
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