Visualizing the growth of Alzheimer's Aβ amyloid-like fibrils

Abstract

Polymerization of normally soluble, β-sheet-forming proteins or peptides yielding fibrillar amyloid deposits is a hallmark of many neurodegenerative diseases[1xAmyloid diseases: abnormal protein aggregation in neurodegeneration. Koo, E.H. et al. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9989–9990Crossref | PubMed | Scopus (455)See all References][1]. Alzheimer's disease (AD) is in part neuropathologically defined by senile plaques constituting extracellular deposits of β-amyloid (Aβ) fibrils. Aβ, a 39–43 amino acid peptide is derived from endoproteolytic cleavage of the transmembrane amyloid precursor protein (APP). Mutations in APP and in presenilin, a protein associated with intramembranous cleavage of APP, are linked to early onset forms of inherited AD. Mutations all give rise to increased levels of Aβ peptides, thus suggesting a central role of Aβ in the disease process[2xAlzheimer's disease: genes, proteins, and therapy. Selkoe, D.J. Physiol. Rev. 2001; 81: 741–766PubMedSee all References][2]. Consequently, reduction of Aβ production by blocking the APP cleavage enzymes, stimulating the clearance of amyloid deposits by immunization with fibrillar Aβ, or inhibiting Aβ fibril formation itself[3xApproaches to discovery and characterization of inhibitors of amyloid β-peptide polymerization. Findeis, M.A. Biochim. Biophys. Acta Mol. Basis Dis. 2000; 1502: 76–84Crossref | PubMed | Scopus (156)See all References][3], are all current strategies for the development of therapeutic interventions.With the aim of developing fibril formation inhibitors, the Aβ fibril assembly process and the structures of its products have been characterized in some detail using in vitro assays[4xStudies on the in vitro assembly of Aβ1–40: implications for the search for Aβ fibril formation inhibitors. Goldsbury, C. et al. J. Struct. Biol. 2000; 130: 217–231Crossref | PubMed | Scopus (224)See all References][4]. We have developed an atomic force microscopy (AFM) assay whereby effects can be measured at the single-fibril level. The distinct advantage of AFM over other techniques is its ability to provide direct time-lapse images of changes in the aggregation state of macromolecules in a physiological buffer environment[5xWatching amyloid fibrils grow by time-lapse atomic force microscopy. Goldsbury, C. et al. J. Mol. Biol. 1999; 285: 33–39Crossref | PubMed | Scopus (247)See all References][5]. The assembly of synthetic Aβ1–40 fibrils is carried out in the fluid cell of an AFM, and the growth of individual fibrils attached to a piece of mica is recorded over periods of several hours. To study the effect of inhibitors of fibril elongation, the process is induced by first seeding the surface with pre-assembled fibrils before injecting fresh Aβ1–40. The added monomeric Aβ1–40 is then consumed by the process of elongating the pre-adsorbed fibril seeds (Fig. 1Fig. 1). As well as from direct elongation of pre-adsorbed fibrils, new fibrils often appear to originate from small aggregates on the surface. Aβ fibrils appear to be polymorphic[4xStudies on the in vitro assembly of Aβ1–40: implications for the search for Aβ fibril formation inhibitors. Goldsbury, C. et al. J. Struct. Biol. 2000; 130: 217–231Crossref | PubMed | Scopus (224)See all References][4]. Generally, fibrils appear to follow the same morphology as the adsorbed fibril seed, so that the growing fibril evolves to have the same axial periodicity as the pre-adsorbed seed (Fig. 1Fig. 1, arrows). We are currently in the process of testing potential fibril elongation inhibitors using this assay system. On a related note, the AFM technique could also be applied to determine the fibril-nucleating ability and structural incorporation of other amyloid components, for example heparan sulfate proteoglycans, phospholipids, lipoproteins or metal ions. Such investigations might ultimately aid our understanding of disease-related amyloid-fibril deposition processes.Fig. 1Seeded growth of Aβ1–40 amyloid-like fibrils on a mica surface imaged by atomic force microscopy (AFM). An aliquot of mature Aβ fibrils (‘seeds’) was adsorbed to the mica surface and the surface was then washed several times with buffer (top left panel). Sequential images of the same surface area are shown at the times indicated after injection of a freshly prepared non-fibrillar Aβsolution.View Large Image | Download PowerPoint Slide