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.

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.

Deposition of amyloid fibrils is a pathological hallmark of Alzheimer's disease. Aβ42 is the major protein whose aggregation leads to the formation of these fibrils. Understanding the detailed structure of Aβ42 fibrils is of particular importance for delineating the mechanism of Aβ42 aggregation and developing specific amyloid-targeting drugs. Here, we use site-directed spin labelling and electron paramagnetic resonance spectroscopy to study the site-specific structural order at each and every residue position in Aβ42 fibrils. Strong interactions between spin labels indicate highly ordered protein backbone at the labelling site, while weak interactions suggest disordered local structure. Our results show that Aβ42 consists of five β-strands (residues 2-7, 10-13, 17-20, 31-36, 39-41), three turns (residues 7-8, 14-16, 37-38) and one ordered loop (residues 21-30). Spin labels introduced at β-strand sites show strong spin-spin interactions, while spin labels at turn or loop sites show weak interactions. However, residues 24, 25 and 28 also show strong interactions between spin labels, suggesting that the loop 21-30 is partly ordered. In the context of recent structural work using solid-state NMR and cryoEM, the site-specific structural order revealed in this study provides a different perspective on backbone and side chain dynamics of Aβ42 fibrils.

Amyloid-β (Aβ) oligomers play a central role in the pathogenesis of Alzheimer's disease. Oligomers of different sizes, morphology and structures have been reported in both in vivo and in vitro studies, but there is a general lack of understanding about where to place these oligomers in the overall process of Aβ aggregation and fibrillization. Here, we show that Aβ42 spontaneously forms oligomers with a wide range of sizes in the same sample. These Aβ42 samples contain predominantly oligomers, and they quickly form fibrils upon incubation at 37°C. When fractionated using ultrafiltration filters, the samples enriched with smaller oligomers form fibrils at a faster rate than the samples enriched with larger oligomers, with both a shorter lag time and faster fibril growth rate. This observation is independent of Aβ42 batches and hexafluoroisopropanol treatment. Furthermore, the fibrils formed by the samples enriched with larger oligomers are more readily solubilized by epigallocatechin gallate, a main catechin component of green tea. These results suggest that the fibrils formed by larger oligomers may adopt a different structure from fibrils formed by smaller oligomers, pointing to a link between oligomer heterogeneity and fibril polymorphism.

Formation of polymorphic amyloid fibrils is a common feature in neurodegenerative diseases involving protein aggregation. In Alzheimer's disease, different fibril structures may be associated with different clinical sub-types. Structural basis of fibril polymorphism is thus important for understanding the role of amyloid fibrils in the pathogenesis and progression of these diseases. Here we studied two types of Aβ42 fibrils prepared under quiescent and agitated conditions. Quiescent Aβ42 fibrils adopt a long and twisted morphology, while agitated fibrils are short and straight, forming large bundles via lateral association. EPR studies of these two types of Aβ42 fibrils show that the secondary structure is similar in both fibril polymorphs. At the same time, agitated Aβ42 fibrils show stronger interactions between spin labels across the full range of the Aβ42 sequence, suggesting a more tightly packed structure. Our data suggest that cross-strand side chain packing interactions within the same β-sheet may play a critical role in the formation of polymorphic fibrils.