New Evidence on a Distinction between Aβ40 and Aβ42 Amyloids: Thioflavin T Binding Modes, Clustering Tendency, Degradation Resistance, and Cross-Seeding

Abstract

The relative abundance of two main Abeta-peptide types with different lengths, Aβ40 and Aβ42, determines the severity of the Alzheimer’s disease progression. However, the factors responsible for different behavior patterns of these peptides in the amyloidogenesis process remain unknown. In this comprehensive study, new evidence on Aβ40 and Aβ42 amyloid polymorphism was obtained using a wide range of experimental approaches, including custom-designed approaches. We have for the first time determined the number of modes of thioflavin T (ThT) binding to Aβ40 and Aβ42 fibrils and their binding parameters using a specially developed approach based on the use of equilibrium microdialysis, which makes it possible to distinguish between the concentration of the injected dye and the concentration of dye bound to fibrils. The binding sites of one of these modes located at the junction of adjacent fibrillar filaments were predicted by molecular modeling techniques. We assumed that the sites of the additional mode of ThT-Aβ42 amyloid binding observed experimentally (which are not found in the case of Aβ40 fibrils) are localized in amyloid clots, and the number of these sites could be used for estimation of the level of fiber clustering. We have shown the high tendency of Aβ42 fibers to form large clots compared to Aβ40 fibrils. It is probable that this largely determines the high resistance of Aβ42 amyloids to destabilizing effects (denaturants, ionic detergents, ultrasonication) and their explicit cytotoxic effect, which we have shown. Remarkably, cross-seeding of Aβ40 fibrillogenesis using the preformed Aβ42 fibrils changes the morphology and increases the stability and cytotoxicity of Aβ40 fibrils. The differences in the tendency to cluster and resistance to external factors of Aβ40 and Aβ42 fibrils revealed here may be related to the distinct role they play in the deposition of amyloids and, therefore, differences in pathogenicity in Alzheimer’s disease.

Dissecting the Molecular Mechanisms of the Co-Aggregation of Aβ40 and Aβ42 Peptides: A REMD Simulation Study

“…13,21 Both fibrils and oligomeric aggregates of Aβ42 were reported to have the ability to crossseed Aβ40 monomers in a concentration-dependent manner, 13,29 which changes the morphology of Aβ40 fibrils and increases its stability and cytotoxicity. 30 As a hetero-seed, Aβ40 fibrils can also cross-seed Aβ42 monomers to rapidly aggregate. 21 Based on the important linkage between the crossinteraction of Aβ40−Aβ42 peptides and the AD progression, many research groups were devoted to reveal the molecular mechanisms of the cross-talk between the two peptides and attempted to resolve the structures of their co-aggregates.…”

“…The Chang’s group applied a single-molecule fluorescence resonance energy transfer technique and found that larger and substantially higher neurotoxic hetero-oligomers of Aβ40–Aβ42 were formed in cells compared to those formed from either peptide alone and suggested the synergistic effects between Aβ40 and Aβ42 peptides on altering both the morphology of the oligomers and their cytotoxicity. A large number of research studies reported the cross-seeding behavior between Aβ40 and Aβ42. , Both fibrils and oligomeric aggregates of Aβ42 were reported to have the ability to cross-seed Aβ40 monomers in a concentration-dependent manner, , which changes the morphology of Aβ40 fibrils and increases its stability and cytotoxicity . As a hetero-seed, Aβ40 fibrils can also cross-seed Aβ42 monomers to rapidly aggregate …”

Prediction of the Feasibility of Using the ≪Gold Standard≫ Thioflavin T to Detect Amyloid Fibril in Acidic Media

“…In particular, as already noted, the authors of ref suggested that α-BC fibrils do not stain with ThT due to their structural features. Indeed, it was previously shown that the fluorescence quantum yield of ThT bound to amyloid fibrils with different structures can vary markedly (sometimes by orders of magnitude). In addition, refs , show the possibility of a decrease in ThT absorption and fluorescence values in extremely acidic and alkaline pH.…”

“…To transfer mature amyloids to other conditions, aggregates were precipitated by centrifugation (for 1–2 h at 18,000 rpm) and pellets were dissolved in deionized Milli-Q water or phosphate buffer (20 mM NaH 2 PO 4 /Na 2 HPO 4 ). Mature amyloids formed from Aβ40, Aβ42, and β2m using special protocols ,− were precipitated by centrifugation (for 1–2 h at 18,000 rpm) and pellets were dissolved in 20% acetic acid/100 mM NaCl (pH 2).…”

“…According to a recent review, the most widely used method for studying the formation and degradation of amyloid fibrils in vitro is to analyze the interaction with them of the benzothiazole dye thioflavin T (ThT). − This dye has a significant benefit due to its capacity to bind specifically to amyloid fibrils while not interacting with protein monomers (in native, unfolded, and intermediate partially folded states), oligomers, and amorphous aggregates. ,− Being a fluorescent molecular rotor, ThT exhibits a significantly increased fluorescence quantum yield (by orders of magnitude) when interacting with amyloid fibrils versus free dye in an aqueous solution (about 0.0001) . Recent studies have demonstrated the applicability of ThT not only for the detection of amyloids in vitro but also for the analysis of their structural polymorphism. − …”