Transgenic mouse models recapitulating Alzheimer’s disease (AD) pathology are pivotal in molecular studies and drug evaluation. In transgenic models selectively expressing amyloid-β (Aβ), thioflavin S (ThS), a fluorescent dye with β-sheet binding properties, is widely employed to observe amyloid plaque accumulation. In this study, we investigated the possibility that a commonly used Aβ-expressing AD model mouse, 5XFAD, generates ThS-positive aggregates of β-sheet structures in addition to Aβ fibrils. To test this hypothesis, brain sections of male and female 5XFAD mice were double-stained with ThS and monoclonal antibodies against Aβ, tau, or α-synuclein, all of which aggregates are detected by ThS. Our results revealed that, in addition to amyloid plaques, 5XFAD mice express ThS-positive phospho-tau (p-tau) aggregates. Upon administration of a small molecule that exclusively disaggregates Aβ to 5XFAD mice for six weeks, we found that the reduction level of plaques was smaller in brain sections stained by ThS compared to an anti-Aβ antibody. Our findings implicate that the use of ThS complicates the quantification of amyloid plaques and the assessment of Aβ-targeting drugs in 5XFAD mice.

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Background Aggregated amyloid-β (Aβ) is considered a pathogenic initiator of Alzheimer’s disease (AD), in strong association with tau hyperphosphorylation, neuroinflammation, synaptic dysfunction, and cognitive decline. As the removal of amyloid burden from AD patient brains by antibodies has shown therapeutic potential, the development of small molecule drugs inducing chemical dissociation and clearance of Aβ is compelling as a therapeutic strategy. In this study, we synthesized and screened aryloxypropanolamine derivatives and identified 1-(3-(2,4-di-tert-pentylphenoxy)-2-hydroxypropyl)pyrrolidin-1-ium chloride, YIAD002, as a strong dissociator of Aβ aggregates. Methods The dissociative activity of aryloxypropanolamine derivatives against Aβ aggregates were evaluated through in vitro assays. Immunohistochemical staining, immunoblot assays, and the Morris water maze were used to assess the anti-Alzheimer potential in YIAD002-treated 5XFAD and transgenic APP/PS1 mice. Target-ligand interaction mechanism was characterized via a combination of peptide mapping, fluorescence dissociation assays, and constrained docking simulations. Results Among 11 aryloxypropanolamine derivatives, YIAD002 exerted strongest dissociative activity against β-sheet-rich Aβ aggregates. Upon oral administration, YIAD002 substantially reduced amyloid burden and accordingly, improved cognitive performance in the Morris water maze and attenuated major pathological hallmarks of AD including tauopathy, neuroinflammation, and synaptic protein loss. Mechanism studies suggest that YIAD002 interferes with intermolecular β-sheet fibrillation by directly interacting with KLVFFA and IGLMVG domains of Aβ. In addition, YIAD002 was found to possess dissociative activity against aggregates of pyroglutamate-modified Aβ and tau. Conclusions Collectively, our results evince the potential of chemical-driven dissociation of Aβ aggregates by aryloxypropanolamines as a therapeutic modality of the amyloid clearance approach.

Fibrillar aggregates of amyloid-β (Aβ) are the main component of plaques lining the cerebrovasculature in cerebral amyloid angiopathy. As the predominant Aβ isoform in vascular deposits, Aβ 40 is a valuable target in cerebral amyloid angiopathy research. However, the slow process of Aβ 40 aggregation in vitro is a bottleneck in the search for Aβ-targeting molecules. In this study, we sought a method to accelerate the aggregation of Aβ 40 in vitro, to improve experimental screening procedures. We evaluated the aggregating ability of bicine, a biological buffer, using various in vitro methods. Our data suggest that bicine promotes the aggregation of Aβ 40 with high speed and reproducibility, yielding a mixture of aggregates with significant β-sheet-rich fibril formation and toxicity.