Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

• Published in: Nature protocols Vol. 5; no. 6; pp. 1186 - 1209
• Main Authors: Hartley, Dean M, Lashuel, Hilal A, Jan, Asad
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2010; Nature Publishing Group
• Subjects: 631/1647/2204; 631/1647/2230/1378; 631/45/470/2284; 692/699/375/132/1283; Alzheimer's disease; Amyloid beta-protein; Analytical Chemistry; Biological Techniques; Biomedical and Life Sciences; Chemical properties; Computational Biology/Bioinformatics; Development and progression; Genetic aspects; Life Sciences; Microarrays; Organic Chemistry; Physiological aspects; protocol; Structure; United States; Switzerland
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2010.72

The amyloid cascade hypothesis, supported by strong evidence from genetics, pathology and studies using animal models, implicates amyloid-β (Aβ) oligomerization and fibrillogenesis as central causative events in the pathogenesis of Alzheimer's disease (AD). Today, significant efforts in academia, biotechnology and the pharmaceutical industry are devoted to identifying the mechanisms by which the process of Aβ aggregation contributes to neurodegeneration in AD and to the identity of the toxic Aβ species. In this paper, we describe methods and detailed protocols for reproducibly preparing Aβ aggregates of defined size distribution and morphology, including monomers, protofibrils and fibrils, using size exclusion chromatography. In addition, we describe detailed biophysical procedures for elucidating the structural features, aggregation kinetics and toxic properties of the different Aβ aggregation states, with special emphasis on protofibrillar intermediates. The information provided by this approach allows for consistent correlation between the properties of the aggregates and their toxicity toward primary neurons and/or cell lines. A better understanding of the molecular and structural basis of Aβ aggregation and toxicity is crucial for the development of effective strategies aimed at prevention and/or treatment of AD. Furthermore, the identification of specific aggregation states, which correlate with neurodegeneration in AD, could lead to the development of diagnostic tools to detect and monitor disease progression. The procedures described can be performed in as little as 1 day, or may take longer, depending on the exact toxicity assays used.