Amyloidosis refers to aggregates of protein that accumulate and are deposited as amyloid fibrils into plaques. When these are detected in organs, they are the main hallmark of Alzheimer’s disease, Parkinson’s disease, and other related diseases. Recent medical advances have shown that many precursors and proteins can induce amyloidosis even though the mechanism of amyloid aggregation and the relationship of these proteins to amyloidosis remains mostly unclear. In this study, we report the real-time 3D-imaging and inhibition analysis of amyloid β (Aβ), tau, and α-synuclein aggregation utilizing the affinity between quantum dots (QD) and amyloid aggregates. We successfully visualized these amyloid aggregations in real-time using fluorescence microscopy and confocal microscopy simply by adding commercially available QD. The observation by transmission electron microscopy (TEM) showed that QD particles bound to all amyloid fibrils. The 3D-imaging with QD revealed differences between amyloid aggregates composed of different amyloid peptides that could not be detected by TEM. We were also able to quantify the inhibition activities of these proteins by rosmarinic acid, which has high activity for Aβ aggregation, from fluorescence micrographs as half-maximal effective concentrations. These imaging techniques with QD serve as quick, easy, and powerful tools to understand amyloidosis and to discover drugs for therapies.

Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis, although the underlying aggregation mechanism has not been elucidated. Since SAA aggregation is a key step in this pathogenesis, inhibitors are useful to prevent and treat AA amyloidosis, serving as tools to investigate the pathogenic mechanism. In this study, we showed that rosmarinic acid (RA), which is a well-known inhibitor of the aggregation of amyloid β (Aβ), displayed inhibitory activity against SAA aggregation in vitro using a microliter-scale high-throughput screening (MSHTS) system with quantum-dot nanoprobes. Therefore, we evaluated the amyloid aggregation inhibitory activity of blood and the deposition of SAA in organs by feeding mice with Melissa officinalis extract (ME) containing RA as an active substance. Interestingly, the inhibitory activity of ME-fed mice sera for SAA and Aβ aggregation, measured with the MSHTS system, was higher than that of the control group. The amount of amyloid deposition in the organs of ME-fed mice was lower than that in the control group, suggesting that the SAA aggregation inhibitory activity of serum is associated with SAA deposition. These results suggest that dietary intake of RA-containing ME enhanced amyloid aggregation inhibitory activity of blood and suppressed SAA deposition in organs. This study also demonstrated that the MSHTS system could be applied to in vitro screening and to monitor comprehensive activity of metabolized foods adsorbed by blood.

The aggregation and accumulation of amyloid β (Aβ) in the brain is a trigger of pathogenesis for Alzheimer’s disease. Previously, we developed a microliter-scale high-throughput screening (MSHTS) system for Aβ₄₂ aggregation inhibitors using quantum-dot nanoprobes. The MSHTS system is seldom influenced by contaminants in samples and is able to directly evaluate Aβ₄₂ aggregation inhibitory activity of samples containing various compounds. In this study, to elucidate whether the MSHTS system could be applied to the evaluation of processed foods, we examined Aβ₄₂ aggregation inhibitory activity of salad dressings, including soy sauces. We estimated the 50% effective concentration (EC₅₀) from serial diluted dressings. Interestingly, all 19 commercial dressings tested showed Aβ₄₂ aggregation inhibitory activity. It was suggested that EC₅₀ differed by as much as 100 times between the dressings with the most (0.065 ± 0.020 v/v%) and least (6.737 ± 5.054 v/v%) inhibitory activity. The highest activity sample is traditional Japanese dressing, soy sauce. It is known that soy sauce is roughly classified into a heat-treated variety and a non-heat-treated variety. We demonstrated that non-heat-treated raw soy sauce exhibited higher Aβ₄₂ aggregation inhibitory activity than heat-treated soy sauce. Herein, we propose that MSHTS system can be applied to processed foods.

Amyloid fibrils are characterized by a linear morphology and a cross-β structure. Polymorphic and multiple fibril morphologies can be found when amyloid fibrils are extracted from amyloid-laden tissue. In this study, we report on the purification and transmission electron microscopic analysis of amyloid fibrils from 5 different animal species (mouse, cow, goat, dog, and camel) with AA amyloidosis. The results show that amyloid fibrils had a linear morphology with a cross-structure and irregular length in vivo. Although the fibrils from these different species showed highly similar conformations, there were significant differences in fibril width and crossover distance. We analyzed the sequences of homologous amyloid proteins and serum amyloid A, an evolutionarily conserved protein and a major amyloid precursor. We found 78.23% homology between the most distant amyloid proteins. The findings suggested similar fibril width and crossover distance in different animal species that displayed high homology of amyloid protein sequences. Dog and camel, as well as goat and cow, showed high genetic homology and similar fibril morphology. These data indicate that the fibrils from different animal species have similar genetic homology and morphology, which may provide a better understanding of the pathogenesis of amyloidosis.

Amyloid A (AA) amyloidosis is a condition in which amyloid fibrils characterized by a linear morphology and a cross-β structure accumulate and are deposited extracellularly in organs, resulting in chronic inflammatory diseases and infections. The incidence of AA amyloidosis is high in humans and several animal species. Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis. Amyloid enhancing factor (AEF) serves as a seed for fibril formation and shortens the onset of AA amyloidosis sharply. In this study, we examined whether AEFs extracted and purified from five animal species (camel, cat, cattle, goat, and mouse) could promote mouse SAA (mSAA) protein aggregation in vitro using quantum-dot (QD) nanoprobes to visualize the aggregation. The results showed that AEFs shortened and promoted mSAA aggregation. In addition, mouse and cat AEFs showed higher mSAA aggregation-promoting activity than the camel, cattle, and goat AEFs. Interestingly, homology analysis of SAA in these five animal species revealed a more similar amino acid sequence homology between mouse and cat than between other animal species. Furthermore, a detailed comparison of amino acid sequences suggested that it was important to mSAA aggregation-promoting activity that the 48th amino acid was a basic residue (Lys) and the 125th amino acid was an acidic residue (Asp or Glu). These data imply that AA amyloidosis exhibits higher transmission activity among animals carrying genetically homologous SAA gene, and may provide a new understanding of the pathogenesis of amyloidosis.