Amyloidosis is a condition in which amyloid fibrils, as well as β-sheet structures, accumulate and are deposited extracellularly. Approximately 50 different peptides or proteins associated with amyloid diseases in humans or domestic animals have been reported worldwide [1
]. Alzheimer’s disease (AD), Parkinson’s disease (PD), and other amyloidosis-related diseases are induced when misfolded Amyloid β (Aβ), tau, and α-synuclein proteins aggregate in the brain or in other organs and when hyperphosphorylated tau aggregates in neurofibrillary tangles [5
]. The process of aggregation of these proteins is basically the same as that which forms amyloid fibrils rich in β-sheets. Furthermore, as these proteins aggregate, they can become generic toxins in higher organisms [10
]. The aggregation of these proteins or peptides are a key step in the pathogenesis of amyloid diseases. Although cryo-electron microscopy has allowed the structure of these proteins to be elucidated and made available, the mechanism by which these proteins cause amyloid diseases is currently mostly unknown [14
]. The diversity of amyloid formation may signal why amyloidosis is so difficult to treat with different clinical presentations, even when the same protein or peptide aggregates [16
In amyloid disease, misfolded aggregation and accumulation of amyloid fibrils, when deposited in organs, constitutes a significant step towards the development of amyloidosis [2
]. Various proteins or peptides serve as precursors for the formation of amyloid fibrils. Although effective therapy still currently remains out of reach [16
], target therapy and plasma biomarkers are used to prevent and diagnose amyloidosis [17
]. Therefore, it is important to find an effective compound or therapy to inhibit amyloid aggregation. Among recent research, none has been certified as medicine for the treatment of amyloidosis. Previously, we developed an Aβ-based real-time imaging method with quantum-dot (QD) nanoprobes, which have evolved as highly useful fluorescence probes in biological staining and diagnostics based on confocal and fluorescence microscopy over the past decade [21
]. QDs are useful for long-term, single-molecule imaging in vitro. For these reasons, QDs could be an excellent tool for real-time monitoring of the aggregation of various amyloid proteins with nonspecific-binding-labeled fibrils. Utilizing this method, we developed a Microliter-Scale High-throughput Screening (MSHTS) system to screen Aβ aggregation inhibitors with QDs [21
]. In this method (Supplementary Figure S1
), only 5-μL of a sample can be analyzed in a 1536-well plate, and the half-maximal effective concentration (EC50
) is estimated as an inhibitory activity [25
]. Furthermore, we successfully automated the MSHTS system using an auto-work station and demonstrated that this method could be applied to another protein, tau [21
]. However, since the conventional MSHTS method requires labeling of the target protein by QDs, the issue of versatility remains unresolved. In the first report, we found that unlabeled QDs were able to bind to Aβ fibrils [25
]. Therefore, in this study, to simplify the conventional MSHTS method, we attempted to visualize the 2D and 3D aggregation of various amyloid proteins using nonspecific binding. Consequently, we succeed in evaluating the aggregation inhibitory activity in each amyloid protein with the novel MSHTS method using nonspecific QD binding.
First, we compared the aggregation processes of Aβ, tau, and α-synuclein using unlabeled QDs and noted that the detailed aggregate structures of their proteins were not exactly the same. The results also showed that α-synuclein protein aggregation took a longer time to achieve than Aβ and tau. Using confocal microscopy and transmission electron microscopy (TEM), we detected details of their protein structure and analyzed amyloid fibrils after incubation with QD nanoprobes. As previously described [24
], an inhibitory compound, rosmarinic acid (RA), was extracted from Satureja hortensis
(summer savory), a spice belonging to the Lamiaceae
family. RA has high Aβ aggregation inhibition activity, antioxidant properties, and can also inhibit xanthine oxidase [26
]. An aggregation inhibitory test of these proteins using RA was performed, noting that RA inhibited the aggregation of Aβ but not tau. These results indicate that the modified MSHTS method using unlabeled QDs has the potential to be an easy and useful tool to search for aggregation inhibitors of various amyloids.
The global amyloidosis epidemic is a major complex combination of many diseases involving chronic inflammatory and misfolding of proteins that are characterized by the accumulation of amyloid-plaques and neurofibrillary tangles in tissues and organs [27
]. Although there have been breakthroughs related to the structure and molecular mechanisms of amyloid proteins [30
], the pathogenic mechanism of amyloidosis is still largely unknown. Over the years, therapy that directly targets amyloid aggregation and deposition in organs or tissues in order to clear it has so far been unsuccessful, and no approved treatment can revert or arrest the progression of this disease [7
]. The procedure of amyloidosis in vivo is very sophisticated, and approximately 50 precursors (protein or peptides) have been reported, with some articles suggesting that pre-amyloid aggregates are the main cause of the induction of amyloidosis [35
In this study, we attempted to elucidate the process of amyloid protein aggregation and assess the effects of RA, an aggregation inhibitor, by using a QD-based imaging and MSHTS system that employs fluorescence and confocal microscopy. In this procedure, we used commercially available QDs without any protein-labeling. The MSHTS system can be analyzed with a 5-µL sample volume when a 1536-well plate is used, and inhibitory activity can be estimated as EC50
. QDs bound to amyloid fibrils, and the intervening space became dark, allowing images of the aggregates to be caught by fluorescence microscopy. The micrographs showed real-time aggregation, and the SD values also increased in a real-time manner. The real-time graphs (Figure 1
B) showed a typical kinetic curve for amyloid aggregation that consisted of time lag, growth, and steady state phases, similar to recent 3D volume data that was obtained by confocal microscopy [21
]. The lag time of Tau aggregation was probably too short to be detected under the MSHTS system. Since the time-dependent data revealed that the aggregation reached a plateau around a certain time, which was from 0 h until the amount of aggregation reaches saturation, the incubation period was fixed to that plateau time in the following screening steps.
Herein, we successful elucidated the images of real-time aggregation of three amyloid proteins (Aβ, tau, and α-synuclein) using the MSHTS system coupled to QD nanoprobes [21
]. After these amyloid proteins were incubated in a 1536-well plate at 37 °C, their aggregation over time could be observed by confocal and fluorescence microscopy (Figure 1
and Figure 2
). The accompanying SD values also increased concomitantly over the same period of time. The real-time 2D and 3D images showed that Aβ, tau, and α-synuclein aggregation occurred by 24 h, 24 h, and 168 h, respectively, while SD values reached saturation. In particular, the tau protein began to aggregate earlier than Aβ and α-synuclein. Moreover, 2D slice images from confocal microscopy (Figure 2
) showed that the shapes of aggregates of these proteins were different, but the difference was not significant when observed by TEM (Figure 3
We also analyzed the structural details of these amyloid protein aggregates by confocal microscopy. An important property of these proteins is that their aggregation structures are very similar, gathering in a spiral-like manner and exhibiting a mesh-like structure without the need for any incubation period. These amyloid fibril aggregations that form in vivo and that are deposited in organs or tissues can induce amyloidosis [39
]. Based on previous data, we demonstrated that the aggregation and formation of amyloid proteins such as Aβ, tau, and α-synuclein are almost similar. Three-dimensional aspects related to the detailed structure of amyloid protein aggregation are highly nuanced, and TEM results reveal that amyloid fibrils had a linear morphology with cross structures and irregular length in vitro. The same fibrils can be extracted from diseased tissues or organs, although it cannot be proved whether these amyloid protein aggregation mechanisms are the same. Differences between each protein fiber are difficult to see with the naked eye [21
]. Analysis of 3D images of aggregates using QDs may provide new information about the aggregation of different amyloid proteins.
In our previous researches [21
], we found that the EC50
were different between the MSHTS system and thioflavin-T (ThT) assay in the Aβ protein, in which the EC50
of the MSHTS system was higher than ThT, because ThT has the potent of false positive effects in inner filter effects [42
]. By contrast, the inner filter effects of the MSHTS system were smaller than ThT, which it adopts as a longer excitation spectrum and quantification from variability data of fluorescence intensity. In this study, we used RA to inhibit these amyloid protein aggregations based on the MSHTS system and calculated their EC50
values. Our data demonstrates that RA has high inhibitory activity of the aggregation in vitro of Aβ and α-synuclein. The aggregation inhibitory activity of RA on Aβ and tau was similar to the results using QD-labeled with these peptides [21
]. This result demonstrated that the aggregation inhibitory activity could be evaluated using a commercially available QD that was not labeled with any peptides.
Although enormous efforts have been made by many researchers, currently there are no effective disease-modifying therapies available. Mutations of the amyloid precursor and some external factors, such as heating, create large challenges for the treatment of amyloidosis [43
]. At present, some proposals and research involve treatments for amyloidosis based on antibodies, metal ions, and RNA interference, but none have been certified as medicine for amyloidosis therapy and applied clinically [17
]. In this study, we elucidated 2D and 3D aggregation of three amyloid proteins (Aβ, tau, and α-synuclein) using QD, which has ability to directly observe the processes of aggregation and inhibition of these amyloid proteins in vitro. These aggregation processes, which involve proteins misfolding in human and animal diseases, are basically the same for these three amyloid proteins. Although these amyloid fibrils are polymorphic and consist of similarly structured proto-filaments in different species or in vivo and in vitro, amyloid proteins adopt highly similar β-arch conformations [15
]. We used RA to inhibit amyloid protein (Aβ and α-synuclein) aggregation, showing a high inhibitory activity, but RA had no inhibitory effect on the tau protein. Our findings will be helpful to better understand the pathogenesis of amyloidosis, especially the progression of this disease. With more testing, over time, RA could be used for amyloidosis treatment as an effective medicine. It is our hope that real-time imaging using QDs may serve as an easy and useful tool in the future for the analysis of protein aggregation and to better understand amyloid proteins.