Alzheimer β-Amyloid Homodimers Facilitate Aβ Fibrillization and the Generation of Conformational Antibodies

Abstract

We reported previously that stabilized ␤-amyloid peptide dimers were derived from mutant amyloid precursor protein with a single cysteine in the ectodomain juxtamembrane position. In vivo studies revealed that two forms of SDS-stable A␤ homodimers exist, species ending at A␤40 and A␤42. The phenomenon of the transformation of the initially deposited 42-residue ␤-amyloid peptide into the amyloid fibrils of Alzheimer's disease plaques remains to be explained in physical terms, i.e. energetically and structurally. We therefore performed spectroscopic analyses revealing that engineered dimeric peptides ending at residue 42 displayed a much more pronounced ␤-structural transition than corresponding monomers. Specifically, the single chemically induced dimerization of A␤ peptides significantly increased the ␤-sheet content by a factor of 2. The Cterminal residues Ile-41 and Ala-42 of dimeric forms further increased the ␤-sheet content by roughly one-third. In contrast to A␤42, the ␤-sheet content of the ␣-and ␥-secretase-generated p3 fragments did not necessarily correlate with the tendency to form fibrils, although p3/17-42 had a pronounced thread forming character with fibril lengths of up to 2.5 m. Electron microscopic images show that forms of p3/17-42 generated smaller granular particles than forms ending at residue 40. We discuss these findings in terms of A␤1-42 dimers representing paranuclei, which self-aggregate into ribbonlike ordered fibrils by elongation. Based on A␤42 dimerspecific titers of a polyclonal antiserum we propose that the A␤ homodimer represents a nidus for plaque formation and a well defined novel therapeutic target.In the amyloidogenic processing of APP, 1 the A␤ peptide is produced, circulates extracellularly, and usually does not deposit as plaques (1, 2). In the secretory pathway, APP is cleaved C-terminal from A␤ residue 16, thus precluding the formation of full-length A␤1-40/42 but generating 3-kDa fragments, termed A␤17-40/42 or p3/40/42 (3, 4). Familial AD-linked mutations in presenilins 1 and 2 and APP can cause Alzheimer's disease (AD) by increasing the cellular production of A␤42 (5), thereby accelerating the polymerization of A␤42 and promoting cerebral accumulation of A␤ as an essential early event in AD pathogenesis (6, 7). Furthermore, peptides ending at A␤42, i.e. A␤1-42 and A␤17-42, appear to be a major constituent of the diffuse plaques seen initially in AD and Down syndrome, and they have been proposed to serve as a nidus for the aggregation of the more abundant A␤1-40 peptides (8, 9). The presence of mixtures of A␤ assemblies, ranging from monomers to insoluble amyloid fibrils, has made it difficult to ascribe amyloidogenicity or toxicity principally to one or another A␤ species. Naturally secreted oligomers of A␤ are believed to have a direct role in AD pathology, both because of their short and long term neurotoxic effects and their ability to give rise to fibrillar assemblies characterized by the adopted ␤-sheet structure (10 -12). Fibrillar A␤ has been observed to bind...

Effects of Peptides Derived from Terminal Modifications of the Aβ Central Hydrophobic Core on Aβ Fibrillization

“…We have previously reported similar results where large sizes of particles formed with N-terminal modification of RRAA-KLVFF compared to C-terminal modification using six oligolysine chains (25). The particle size difference is closely Article associated with the disruption of the hydrophobic C-terminal group (50), which has more impact on aggregation than the hydrophilic N-terminal (51). The different morphologies of particles formed with AAMP-19 (fibrils) and AAMP-16 (spherical aggregates) are attributable of the N-and C-terminal modifications, respectively.…”

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

“…21 and 29. Aβ42 was monomerized as described before (30,31). Briefly, Aβ42 was dissolved in 98% formic acid.…”

“…In brief, PBS was used as running buffer at 25°C. Monomerized Aβ42 peptide was dissolved in 0.12% ammonia to 1 mg∕mL, diluted 1∶10 with 10 mM sodium acetate (pH 3.4), and subsequently immobilized to the sensor chip surface by amine coupling (30,31). Compounds were diluted from DMSO stock solutions in running buffer and injected for at least 1 min at a flow rate of 30 μL∕ min using the KINJECT command.…”

“…SPR experiments were performed using a BIACORE 3000 instrument and CM5 sensor chips (GE Healthcare) as described previously (31). In brief, PBS was used as running buffer at 25°C.…”

Kinetic control of dimer structure formation in amyloid fibrillogenesis

“…However, it is possible that kinetics control the early intermediates, which form metastable structures that are stabilized when larger aggregates are formed. Experimentally, it has recently been suggested that engineered dimeric Alzheimer's ␤ (A␤) peptides significantly promote the growth of ␤-sheet fibrils (22). Furthermore, Walsh et al (23) have shown a potential synaptotoxicity of the A␤ dimer in vivo.…”