Real-Time Imaging and Quantification of Amyloid-β Peptide Aggregates by Novel Quantum-Dot Nanoprobes

Kiyotaka Tokuraku,Meg Marquardt,Tsuneya Ikezu,Ashley M Buckle

doi:10.1371/journal.pone.0008492

Abstract

BackgroundProtein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease. However, direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. Here we demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-β peptide (QDAβ) and its advanced applications.Methodology/Principal FindingsThe QDAβ construct retained Aβ oligomer-forming ability, and the sizes of these oligomers could be estimated from the relative fluorescence intensities of the imaged spots. Both QDAβ coaggregation with intact Aβ42 and insertion into fibrils were detected by fluorescence microscopy. The coaggregation process was observed by real-time 3D imaging using slit-scanning confocal microscopy, which showed a typical sigmoid curve with 1.5 h in the lag-time and 12 h until saturation. Inhibition of coaggregation using an anti-Aβ antibody can be observed as 3D images on a microscopic scale. Microglia ingested monomeric QDAβ more significantly than oligomeric QDAβ, and the ingested QDAβ was mainly accumulated in the lysosome.Conclusions/SignificanceThese data demonstrate that QDAβ is a novel nanoprobe for studying Aβ oligomerization and fibrillization in multiple modalities and may be applicable for high-throughput drug screening systems.

Highlights

Neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease, and prion diseases are characterized by misfolded protein aggregates, termed amyloids, which are usually high in b-sheet content [1]
We confirmed that Ab42, without SDS, begins to aggregate within minutes of preparation—already forming oligomers or protofibrils during the one hour labeling process (Fig. S1a)
Since formation of the toxic, b-sheet rich, Amyloid-b peptide (Ab) oligomer can be enhanced by certain concentrations of SDS [5,6], we examined whether QDAb forms oligomers in the presence or absence of SDS

Summary

Introduction

Neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease, and prion diseases are characterized by misfolded protein aggregates, termed amyloids, which are usually high in b-sheet content [1]. Amyloid-b peptide (Ab) is the major component of senile plaques and is a hallmark of AD [2]. Ab peptide has been observed in various cellular localities, including lysosomes, aggresomes, mitochondria, dendritic spines, and within neurons, microglia, astrocytes and the extra-cellular space [7,8,9,10,11], but the exact cellular origin of Ab aggregation is not known. To understand the mechanism of Ab misfolding and locate the origin of Ab assemblage, we have developed a real-time imaging tool for monitoring Ab aggregation. Protein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease. Direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. We demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-b peptide (QDAb) and its advanced applications

Methods

Results

Conclusion