Formation of amyloid oligomers and fibrils underlies the pathogenesis of a number of neurodegenerative diseases such as Alzheimer's. One mechanism of action by which Aβ aggregates cause neuronal toxicity is through interactions with cellular membranes. Aβ aggregates have been shown to disrupt membrane integrity via pore formation, membrane thinning, or lipid extraction. At the same time, lipid membranes also affect the rate of Aβ aggregation and remodel pre-formed Aβ fibrils. Here we show that Aβ42 globulomers, a type of well-characterized and stable Aβ oligomers, convert to amyloid fibrils in the presence of DOPC liposomes. Electron paramagnetic resonance studies show that the fibrils converted from Aβ42 globulomers adopt the same structure as fibrils formed directly from monomers. Our results suggest that the interactions between Aβ oligomers and cellular membranes are dynamic. By converting Aβ oligomers to fibrils, the lipid membrane can reduce the membrane-disrupting activities caused by these oligomers. Modulation of Aβ-membrane interactions as a therapeutic strategy should take into account the dynamic nature of these interactions.

Formation of Aβ oligomers and fibrils plays a central role in the pathogenesis of Alzheimer's disease. There are two major forms of Aβ in the brain: Aβ42 and Aβ40. Aβ42 is the major component of the amyloid plaques, but the overall abundance of Aβ40 is several times that of Aβ42. In vitro experiments show that Aβ42 and Aβ40 affect each other's aggregation. In mouse models of Alzheimer's disease, overexpression of Aβ40 has been shown to reduce the plaque pathology, suggesting that Aβ42 and Aβ40 also interact in vivo. Here we address the question of whether Aβ42 and Aβ40 interact with each other in the formation of oligomers using electron paramagnetic resonance (EPR) spectroscopy. When the Aβ42 oligomers were formed using only spin-labeled Aβ42, the dipolar interaction between spin labels that are within 20 Å range broadened the EPR spectrum and reduced its amplitude. Oligomers formed with a mixture of spin-labeled Aβ42 and wild-type Aβ42 gave an EPR spectrum with higher amplitude due to weakened spin-spin interactions, suggesting molecular mixing of labeled and wild-type Aβ42. When spin-labeled Aβ42 and wild-type Aβ40 were mixed to form oligomers, the resulting EPR spectrum also showed reduced amplitude, suggesting that wild-type Aβ40 can also form oligomers with spin-labeled Aβ42. Therefore, our results suggest that Aβ42 and Aβ40 form mixed oligomers with direct molecular interactions. Our results point to the importance of investigating Aβ42-Aβ40 interactions in the brain for a complete understanding of Alzheimer's pathogenesis and therapeutic interventions.

Development of therapeutic interventions for Alzheimers over the past three decades has been guided by the amyloid hypothesis, which puts Aβ deposition as the initiating event of a pathogenic cascade leading to dementia. In the current form, the amyloid hypothesis lacks a comprehensive framework that considers the complex nature of Aβ aggregation. The explanation of how Aβ deposition leads to downstream pathology, and how reducing Aβ plaque load via anti-amyloid therapy can lead to improvement in cognition remains insufficient. In this perspective we integrate the concept of Aβ supersaturation into the amyloid hypothesis, laying out a framework for the mechanistic understanding and therapeutic intervention of Alzheimers disease. We discuss the important distinction between in vitro and in vivo patterns of Aβ aggregation, the impact of different aggregation stages on therapeutic strategies, and how future investigations could integrate this concept in order to produce a more thorough understanding and better treatment for Alzheimers and other amyloid-related disorders.

Aβ42 aggregation plays a central role in the pathogenesis of Alzheimer's disease. In addition to the insoluble fibrils that comprise the amyloid plaques, Aβ42 also forms soluble aggregates collectively called oligomers, which are more toxic and pathogenic than fibrils. Understanding the structure and dynamics of Aβ42 oligomers is critical for developing effective therapeutic interventions against these oligomers. Here we studied the structural dynamics of Aβ42 globulomers, a type of Aβ42 oligomers prepared in the presence of sodium dodecyl sulfate, using site-directed spin labeling. Spin labels were introduced, one at a time, at all 42 residue positions of Aβ42 sequence. Electron paramagnetic resonance spectra of spin-labeled samples reveal four structural segments based on site-dependent spin label mobility pattern. Segment-1 consists of residues 1-6, which have the highest mobility that is consistent with complete disorder. Segment-3 is the most immobilized region, including residues 31-34. Segment-2 and -4 have intermediate mobility and are composed of residues 7-30 and 35-42, respectively. Considering the inverse relationship between protein dynamics and stability, our results suggest that residues 31-34 are the most stable segment in Aβ42 oligomers. At the same time, the EPR spectral lineshape suggests that Aβ42 globulomers lack a well-packed structural core akin to that of globular proteins.

Formation of amyloid fibrils by Aβ42 protein is a pathological hallmark of Alzheimer's disease. Aβ42 fibrillization is a nucleation-dependent polymerization process, in which nucleation is the rate-limiting step. Structural knowledge of the fibril nucleus is important to understand the molecular mechanism of Aβ aggregation and is also critical for successful modulation of the fibrillization process. Here, we used a scanning mutagenesis approach to study the role of each residue position in Aβ42 fibrillization kinetics. The side chain we used to replace the native residue is a nitroxide spin label called R1, which was introduced using site-directed spin labeling. In this systematic study, all residue positions of Aβ42 sequence were studied, and we identified six key residues for the Aβ42 fibril formation: H14, E22, D23, G33, G37, and G38. Our results suggest that charges at positions 22 and 23 and backbone flexibilities at positions 33, 37, and 38 play key roles in Aβ42 fibrillization kinetics. Our results also suggest that the formation of a β-strand at residues 15-21 is an important feature in Aβ42 fibril nucleus. In overall evaluation of all of the mutational effects on fibrillization kinetics, we found that the thioflavin T fluorescence at the aggregation plateau is a poor indicator of aggregation rates.

Aβ42 is the major component of parenchymal plaques in the brain of Alzheimer's patients, while Aβ40 is the major component of cerebrovascular plaques. Since Aβ40 and Aβ42 coexist in the brain, understanding the interaction between Aβ40 and Aβ42 during their aggregation is important to delineate the molecular mechanism underlying Alzheimer's disease. Here, we present a rigorous and systematic study of the cross-seeding effects between Aβ40 and Aβ42. We show that Aβ40 fibril seeds can promote Aβ42 aggregation in a concentration-dependent manner, and vice versa. Our results also suggest that seeded aggregation and spontaneous aggregation may be two separate pathways. These findings may partly resolve conflicting observations in the literature regarding the cross-seeding effects between Aβ40 and Aβ42.

Formation of amyloid fibrils underlies a wide range of human disorders, including Alzheimer's and prion diseases. The amyloid fibrils can be readily detected thanks to thioflavin T (ThT), a small molecule that gives strong fluorescence upon binding to amyloids. Using the amyloid fibrils of Aβ40 and Aβ42 involved in Alzheimer's disease, and of yeast prion protein Ure2, here we study three aspects of ThT binding to amyloids: quantification of amyloid fibrils using ThT, the optimal ThT concentration for monitoring amyloid formation and the effect of ThT on aggregation kinetics. We show that ThT fluorescence correlates linearly with amyloid concentration over ThT concentrations ranging from 0.2 to 500 µM. At a given amyloid concentration, the plot of ThT fluorescence versus ThT concentration exhibits a bell-shaped curve. The maximal fluorescence signal depends mostly on the total ThT concentration, rather than amyloid to ThT ratio. For the three proteins investigated, the maximal fluorescence is observed at ThT concentrations of 20-50 µM. Aggregation kinetics experiments in the presence of different ThT concentrations show that ThT has little effect on aggregation at concentrations of 20 µM or lower. ThT at concentrations of 50 µM or more could affect the shape of the aggregation curves, but this effect is protein-dependent and not universal.

Amyloid formation is involved in a wide range of neurodegenerative diseases including Alzheimer's and prion diseases. Structural understanding of the amyloid is critical to delineate the mechanism of aggregation and its pathological spreading. Site-directed spin labelling has emerged as a powerful structural tool in the studies of amyloid structures and provided structural evidence for the parallel in-register β-sheet structure for a wide range of amyloid proteins. It is generally accepted that spin labelling does not disrupt the structure of the amyloid fibrils, the end product of protein aggregation. The effect on the rate of protein aggregation, however, has not been well characterized. Here, we employed a scanning mutagenesis approach to study the effect of spin labelling on the aggregation rate of 79 spin-labelled variants of the Ure2 prion domain. The aggregation of Ure2 protein is the basis of yeast prion [URE3]. We found that all spin-labelled Ure2 mutants aggregated within the experimental timeframe of 15 to 40 h. Among the 79 spin-labelled positions, only five residue sites (N23, N27, S33, I35 and G42) showed a dramatic delay in the aggregation rate as a result of spin labelling. These positions may be important for fibril nucleation, a rate-limiting step in aggregation. Importantly, spin labelling at most of the sites had a muted effect on Ure2 aggregation kinetics, showing a general tolerance of spin labelling in protein aggregation studies.

Deposition of Aβ42 aggregates in the form of amyloid plaques is a pathological hallmark of Alzheimer's disease. A desired avenue of intervention is the inhibition of Aβ42 aggregation. Epigallocatechin gallate (EGCG), the main polyphenol in green tea, has been generally considered an inhibitor of Aβ aggregation. However, previous experiments focused on the reduction of the amount of Aβ42 aggregates, while the effect of EGCG on the rate of Aβ42 aggregation was not critically analyzed. Here we performed an experimental evaluation of Aβ42 aggregation kinetics in the absence and presence of EGCG at a wide range of concentrations. We found that EGCG reduced thioflavin T fluorescence in an EGCG concentration-dependent manner, suggesting that EGCG reduced the amount of Aβ42 fibrils. The effect of EGCG on the rate of Aβ42 aggregation appears to be bimodal. We found that higher EGCG-to-Aβ42 ratios promoted the rate of Aβ42 aggregation, while lower EGCG-to-Aβ42 ratios inhibited the aggregation rate. To confirm that the reduction of thioflavin T fluorescence is due to the lowered aggregate quantity, but not due to perturbation of thioflavin T binding to Aβ42 fibrils, we probed the effect of EGCG on Aβ42 aggregation using site-directed spin labeling. Electron paramagnetic resonance of spin-labeled Aβ42 aggregates suggests that high EGCG-to-Aβ42 ratios led to a greatly reduced amount of Aβ42 fibrils, and these aggregates adopt similar structures as the fibrils in the no-EGCG sample. Potential implications of this work in designing prevention or therapeutic strategies using EGCG are discussed.