SOLUBLE AMYLOID-BETA AGGREGATES FROM HUMAN ALZHEIMER’S DISEASE BRAINS

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. Our approach to isolating soluble Aβ aggregates from human brain involved using 2–3 grams of frontal or parietal cortex from pathologically confirmed CDR3 (severe) AD cases. Tissue was homogenized in 1xPBS containing protease inhibitors and subjected to differential ultracentrifugation, and dual antibody immunoprecipitation. We examined the effect of multiple extractions, the use of detergents during extraction, and chromatographic methods to improve enrichment of the Aβ aggregates prior to immunopurification. The stability of the aggregates was characterized during purification using size-exclusion chromatography, sucrose density gradient centrifugation, immunoelectron microscopy, and quantitative tracking of the Aβ aggregates at each step. We developed a method that results in <40% loss of starting material and purifying them >6000 fold with no detectible ex vivo aggregation of monomeric Aβ, and no apparent ex vivo alterations in soluble aggregate sizes. Loss of Aβ was greatly reduced by universal blocking of materials with albumin. 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. A major challenge has been that the specific forms of soluble Aβ aggregates most relevant to human disease have not been determined. Aβ can potentially aggregate into a vast number of forms, consisting of different numbers of Aβ peptides, various size forms of Aβ, multiple Aβ post-translational modifications and alternative structural configurations of Aβ. Here we present a method to purify soluble Aβ aggregates directly from frozen human AD brain tissue, reasoning this would be the most relevant approach to facilitate investigation into the characteristics of native soluble Aβ aggregates, and deepen our understanding of Alzheimer's dementia.