Thioflavin-positive tau aggregates complicating quantification of amyloid plaques in the brain of 5XFAD transgenic mouse model

Abstract 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

Bicine promotes rapid formation of β-sheet-rich amyloid-β fibrils.

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, Aβ40 is a valuable target in cerebral amyloid angiopathy research. However, the slow process of Aβ40 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 Aβ40 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 Aβ40 with high speed and reproducibility, yielding a mixture of aggregates with significant β-sheet-rich fibril formation and toxicity

Solid-phase synthesis and pathological evaluation of pyroglutamate amyloid-β3-42 peptide

Abstract Pyroglutamate amyloid-β3-42 (AβpE3-42) is an N-terminally truncated and pyroglutamate-modified Aβ peptide retaining highly hydrophobic, amyloidogenic, and neurotoxic properties. In Alzheimer’s disease (AD) patients, AβpE3-42 peptides accumulate into oligomers and induce cellular toxicity and synaptic dysfunction. AβpE3-42 aggregates further seed the formation of amyloid plaques, which are the pathological hallmarks of AD. Given that AβpE3-42 peptides play critical roles in the development of neurodegeneration, a reliable and reproducible synthetic access to these peptides may support pathological and medicinal studies of AD. Here, we synthesized AβpE3-42 peptides through the microwave-assisted solid-phase peptide synthesis (SPPS). Utilizing thioflavin T fluorescence assay and dot blotting analysis with anti-amyloid oligomer antibody, the amyloidogenic activity of synthesized AβpE3-42 peptides was confirmed. We further observed the cytotoxicity of AβpE3-42 aggregates in cell viability test. To examine the cognitive deficits induced by synthetic AβpE3-42 peptides, AβpE3-42 oligomers were intracerebroventricularly injected into imprinting control region mice and Y-maze and Morris water maze tests were performed. We found that AβpE3-42 aggregates altered the expression level of postsynaptic density protein 95 in cortical lysates. Collectively, we produced AβpE3-42 peptides in the microwave-assisted SPPS and evaluated the amyloidogenic and pathological function of the synthesized peptides