Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.

Understanding the structural and assembly dynamics of the amyloid β-protein (Aβ) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Aβ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform “scanning PICUP.” Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Aβ40) or 42 (for Aβ42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil → α/β → β transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr1-substituted homologues of Aβ40 and Aβ42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr1]Aβ40 fibrils. Substitution of Aβ40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Aβ40 and Aβ42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr1) to alter Aβ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Aβ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.

Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of A beta 42 are believed to be the main cause of AD; therefore, disruption of A beta oligomerization is a promising approach for developing therapeutics for AD. Formation of A beta 42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of A beta 42 may get incorporated into oligomers of A beta 42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using A beta fragments with the general formula A beta(x-42) (x = 28-39). A cell viability screen identified A beta(31-42) as the most potent inhibitor. In addition, the shortest peptide, A beta(39-42), also had high activity. Both A beta(31-42) and A beta(39-42) inhibited A beta-induced cell death and rescued disruption of synaptic activity by A beta 42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of A beta 42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with A beta 42 to form heterooligomers. Thus, A beta(31-42) and A beta(39-42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure-activity approach.

An increasing body of evidence suggests that soluble assemblies of amyloid proteins are the predominant neurotoxic species in many amyloid-related diseases. Consequently, the focus of research on pathologic mechanisms underlying amyloidoses has shifted from amyloid fibrils to oligomers. Biophysical characterization of oligomers is difficult due to their metastable nature. The most popular experimental method for detection of oligomers has been SDS-PAGE. However, we provide experimental evidence that SDS-PAGE is not a reliable method for characterization of amyloid protein oligomers and discuss alternative approaches. In addition, we discuss how inconsistent nomenclature has obfuscated our understanding of the process and products of protein assembly. The goals of this paper are to identify pitfalls associated with the methods and language used to study protein oligomers and to provide alternatives, thereby facilitating successful elucidation of the mechanisms controlling amyloid protein oligomer assembly and toxicity.