New Insights on How Metals Disrupt Amyloid β-Aggregation and Their Effects on Amyloid-β Cytotoxicity

Abstract

Amyloid-␤ protein (A␤) aggregates in the brain to form senile plaques. By using thioflavin T, a dye that specifically binds to fibrillar structures, we found that metals such as Zn(II) and Cu(II) normally inhibit amyloid ␤-aggregation. Another method for detecting A␤, which does not distinguish the types of aggregates, showed that these metals induce a non-␤-sheeted aggregation, as reported previously. Secondary structural analysis and microscopic studies revealed that metals induced A␤ to make non-fibrillar aggregates by disrupting ␤-sheet formation. These non-fibrillar A␤ aggregates displayed much weaker Congo Red birefringence, and in separate cell culture experiments, were less toxic than self ␤-aggregates, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The toxicity of soluble A␤ was enhanced in the presence of Cu(II), which suggests the previously hypothesized role of A␤ in generating oxidative stress. Finally, under an acidic condition, similar to that in the inflammation associated with senile plaques, ␤-aggregation was robustly facilitated at one specific concentration of Zn(II) in the presence of heparin. However, because a higher concentration of Zn(II) virtually abolished this abnormal phenomenon, and at normal pH any concentrations strongly inhibit ␤-aggregation and its associated cytotoxicity, including its anti-oxidative nature we suggest that Zn(II) has an overall protective effect against ␤-amyloid toxicity. Amyloid-␤ protein (A␤)1 is one of the main components of senile plaques, a pathological hallmark of Alzheimer's disease (AD) (1, 2). Although A␤ is undisputedly associated with the pathology of AD, it is still an open question as to what specific aspects of A␤ and its processing are the important variables in the pathophysiology of the disease. For example, fibrillar A␤, but not non-amyloidogenic, amorphous aggregates of A␤, was reported to cause neuronal cell death in primary rat hippocampal cultures (3), and soluble monomeric species of A␤ are relatively nontoxic as compared with fibrillar A␤ (4). Thus, these in vitro studies suggest that the degree of ␤-aggregation is particularly important for neurotoxicity to occur (5-8). However, many controversial results from in vivo studies have been reported concerning the pathological role of plaque formation in AD. Irizarry et al. (9) reported that transgenic (TG) mice expressing human A␤ failed to exhibit neuronal loss despite depositing substantial amounts of A␤. On the other hand, TG mice that express Swedish mutant amyloid precursor protein (APP) formed plaques that were detected by both an anti-APP antibody and a ␤-sheet specific dye (10). Moreover, these APP TG mice also displayed memory deficits. Taken together, these results indicate that, although the plaque assembly process may require further investigation, amyloid ␤-aggregation certainly is an essential event in the pathogenesis of AD.Based on these lines of evidence, the search for a compound that interrupts ␤-aggregation and thus protects agai...

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Attenuated cytotoxicity but enhanced βfibril of a mutant amyloid β‐peptide with a methionine to cysteine substitution

“…Measurements were performed at the excitation and emission wavelength of 450 and 485 nm respectively, which resulted in the optimum detection of amyloid fibrils [24]. To account for background fluorescence, the fluorescence intensity measured from each control solution without Ab peptides was subtracted from that of each solution containing Ab.…”