Preparation of fluorescently-labeled amyloid-beta peptide assemblies: the effect of fluorophore conjugation on structure and function

Recent research has focused on soluble oligomeric assemblies of the 42 amino acid isoform of the amyloid‐beta peptide (Aβ42) as the proximal cause of neuronal injury, synaptic loss, and the eventual dementia associated with Alzheimer's disease (AD). While neurotoxicity, neuroinflammation, and deficits in behavior and memory have all been attributed to oligomeric Aβ42, the specific roles for this assembly in the cellular neuropathology of AD remain poorly understood. In particular, lack of reliable and well‐characterized forms of easily detectable Aβ42 oligomers has hindered study of the cellular trafficking of exogenous Aβ42 by neurons in vitro and in vivo. Therefore, the objective of this study is to fluorescently label soluble oligomeric Aβ42 without altering the structure or function of this assembly. Previous studies have demonstrated the advantages of using tapping mode atomic force microscopy (AFM) to characterize the structural assemblies formed by synthetic Aβ42 under specific solution conditions (e.g., oligomers, protofibrils, and fibrils). Here, we extend these methods to establish a strategy for fluorescent labeling of oligomeric Aβ42 assemblies that are structurally comparable to unlabeled oligomeric Aβ42. To compare function, we demonstrate that the uptake of labeled and unlabeled oligomeric Aβ42 by neurons in vitro is similar. AFM‐characterized fluorophore‐Aβ42 oligomers are an exciting new reagent for use in a variety of studies designed to elucidate critical cellular and molecular mechanisms underlying the functions of this Aβ42 assembly form in AD. Copyright © 2009 John Wiley & Sons, Ltd.