Soluble Amyloid-beta Aggregates from Human Alzheimer's Disease Brains.

Thomas J Esparza,David L Brody,Randall J Bateman,Norelle C Wildburger,Hao Jiang,Mihika Gangolli,Nigel J Cairns

doi:10.1038/srep38187

Abstract

Soluble amyloid-beta (Aβ) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Aβ aggregates in the human Alzheimer’s disease brain. Here we present a new method for extracting soluble Aβ aggregates from human brains, separating them from insoluble aggregates and Aβ monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. The method resulted in <40% loss of starting material, no detectible ex vivo aggregation of monomeric Aβ, and no apparent ex vivo alterations in soluble aggregate sizes. By immunoelectron microscopy, soluble Aβ aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2-3 amino-terminal Aβ antibody binding sites, distinct from previously characterized structures. This approach may facilitate investigation into the characteristics of native soluble Aβ aggregates, and deepen our understanding of Alzheimer’s dementia.

Highlights

Soluble amyloid-beta (Aβ) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease
We tested multiple different methods to address each of these tasks, and used four criteria to quantitatively assess the overall results: 1) Quantitative completeness of separation of the soluble Aβaggregates from soluble monomers and insoluble aggregates; 2) Fold enrichment of Aβcompared to total protein; 3) Quantitative completeness of the recovery of the soluble Aβaggregates present in the starting material, i.e. minimization of loss during purification; and 4) Minimization of ex vivo aggregation or disaggregation of Aβduring the extraction and purification process
In summary we have developed a method for reliably enriching soluble Aβaggregates from human brain tissue, separating them from other forms of Aβ,and purifying them more than 6000 fold with less than 40% loss of starting material

Summary

Introduction

Soluble amyloid-beta (Aβ) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease. Despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Aβ aggregates in the human Alzheimer’s disease brain. Soluble Aβ aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2-3 amino-terminal Aβ antibody binding sites, distinct from previously characterized structures This approach may facilitate investigation into the characteristics of native soluble Aβ aggregates, and deepen our understanding of Alzheimer’s dementia. The assay uses the monoclonal antibody HJ3.4 which is specific for the canonical N-terminus of Aβ40; it does not recognize amyloid precursor protein, unlike other commonly used antibodies such as 6E10 or 4G8 Using this assay, we were able to fully distinguish between DAT patients and high pathology non-demented controls with no overlap between groups based on the ratio of soluble Aβaggregates to plaque area[40]. We report a method to purify soluble Aβaggregates directly from frozen human AD brain tissue, reasoning this would be the most relevant source for the species directly underlying dementia in humans

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Results

Conclusion