Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of β-amyloid peptide

Abstract

The beta-amyloid (Abeta) peptide, a major component of senile plaques in Alzheimer's disease brain, has been shown previously to undergo a process of polymerization to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Abeta responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Abeta peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Abeta were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions was determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillar structures, but not to monomeric peptide or sheet-like structures of polymerized Abeta. Biological activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillar Abeta; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Abeta on cell viability is not confined to a single conformational form but that both fibrillar and protofibrillar species have the potential to be active in this assay.

The Role of Prion Peptide Structure and Aggregation in Toxicity and Membrane Binding

“…Analogous to our prion peptide results, relationships between structure and toxicity have been well documented for ␤-amyloid of Alzheimer's disease. In aggregated solutions (containing fibrils, protofibrils, and lowmolecular-weight intermediates), ␤-amyloid has been consistently shown to be toxic to neurons in culture (Pike et al, 1991(Pike et al, , 1993Howlett et al, 1995;Seilheimer et al, 1997;Hartley et al, 1999;Ward et al, 2000). Although there is some disagreement as to the exact structure of the aggregated species associated with toxicity, whether it be a protofibril (Hartley et al, 1999;Ward et al, 2000), a diffusible, nonfibrillar ligand (Lambert et al, 1998), or some other low-molecular-weight intermediate (Hartley et al, 1999), toxicity is associated with peptide structures that are part of an aggregation pathway associated with amyloid formation.…”

Development of photocrosslinked sialic acid containing polymers for use in Aβ toxicity attenuation

“…Given the propensity of Aβ to aggregate as a function of concentration [5,55], the shape of the Aβ binding isotherms may indicate non-specific binding to DSLNT polymers or may be reflective of different aggregation states of Aβ interacting with the DSLNT polymer. The method used to evaluate Aβ binding the DSLNT polymers can not be used to discriminate between these possibilities.…”

“…The main protein component of the plaques is beta amyloid peptide (Aβ) [4][5][6]. It has been hypothesized by many that Aβ plays a causative role in the neurodegeneration associated with AD [3,4,[7][8][9][10].…”

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

“…Treatment of lyophilized Aβ with strong acids and bases to disrupt preformed aggregates and enhance solubility 52 , filtration through LMW cutoff filters 53 , photo-induced crosslinking of unmodified proteins 17 , density gradient centrifugation 54 and size exclusion chromatography (SEC) 13,14,17 have all been used to prepare soluble aggregates of Aβ and yield preparations that vary in size and morphology distribution. SEC, in particular, offers several advantages: (i) a variety of column matrices with different separation capacities are readily available and can be used in isolation or in combination with other columns to obtain high-resolution separation and fractions containing Aβ aggregates of defined size distribution; (ii) generally, the columns are equipped with filters at the top that allow for the removal of fibrillar (or insoluble) material from the injected sample, thus ensuring that the Aβ fractions are free of fibrillar seeds; (iii) if SEC is coupled to a light-scattering detector, accurate determination of Aβ aggregates' size distribution becomes possible 20 ; (iv) in analytical mode, SEC is a valuable tool to monitor early events in amyloid formation and quantification of monomer and/or protofibril loss during the time course of fibril formation 21 ; and (v) by choosing proper running conditions (i.e., buffer pH and contents), fractions are obtained in solution conditions suitable for biological systems, free of harmful or undesired substances (e.g., organic solvents and so on).…”