Genetic evidence predicts a causative role for amyloid-␤ (A␤) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A␤ are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A␤-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and ϳ40-fold more than unaggregated peptide, with oligomeric A␤-(1-42)-induced inhibition significant at 10 nM. Under A␤-(1-42) oligomer-and fibril-forming conditions, A␤-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A␤-(1-42). We applied the aggregation protocols developed for wild type A␤-(1-42) to A␤-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A␤-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A␤-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A␤-(1-42) are useful in distinguishing the structural and functional differences between A␤-(1-42) and A␤-(1-40) and genetic mutations of A␤-(1-42). Amyloid-␤ (A␤)1 is derived by the proteolytic processing of amyloid precursor protein (APP), resulting in a peptide predominantly 40 or 42 amino acids in length. Mutations in APP and the presenilins that increase the amount of A␤-(1-42) cause AD (for review, see Ref. 1). Historically, the "amyloid cascade" hypothesis has defined the fibrillization of A␤ into amyloid deposits, a pathologic hallmark of AD, as a toxic gain of function (2). That A␤-(1-42) is more fibrillogenic than A␤-(1-40) fits well with this hypothesis. However, amyloid plaques do not always correlate in number, tempo, or distribution with neurodegeneration or clinical dementia. Thus, recent debate within the AD community has focused on whether fibrillar (amyloid) or soluble oligomers of A␤ are the active species of the peptide that ultimately cause the synaptic loss and dementia associated with AD (3-7). In vivo, small, stable oligomers of A␤-(1-42) have been isolated from brain, plasma, and cerebrospinal fluid (8 -10) and correlate with the severity of neurodegeneration in AD (11, 12). Thus, although genetic evidence predicts that A␤ is a causative factor in AD, the role of fibrillar and oligomeric A␤ in the pathogenesis of AD remains unclear.Initial in vitro studies suggested that A␤-induced neurotoxicity required the peptide to adopt a fibrillar aggregation state, with unaggregated peptide at low doses actually exhibiting neurotrophic effects (13)(14)(15)(16)(17)(18)). Recent studies demonstrate that non-fibrillar structures, including oligomers and ...

Extensive research causally links amyloid-␤ peptide (A␤) to Alzheimer's disease, although the pathologically relevant A␤ conformation remains unclear. A␤ spontaneously aggregates into the fibrils that deposit in senile plaques. However, recent in vivo and in vitro reports describe a potent biological activity for oligomeric assemblies of A␤. To consistently prepare in vitro oligomeric and fibrillar forms of A␤1-42, a detailed knowledge of how solution parameters influence structure is required. This manuscript represents the first study using a single chemically and structurally homogeneous unaggregated starting material to demonstrate that the formation of oligomers, fibrils, and fibrillar aggregates is determined by time, concentration, temperature, pH, ionic strength, and A␤ species. We recently reported that oligomers inhibit neuronal viability 10-fold more than fibrils and ϳ40-fold more than unaggregated peptide, with oligomeric A␤1-42-induced neurotoxicity significant at 10 nM. In addition, we were able to differentiate by structure and neurotoxic activity wild-type A␤1-42 from isoforms containing familial mutations Amyloid plaques are one of the pathological hallmarks of Alzheimer's disease (AD).1 Genetic findings over the past decade have further supported the pivotal and likely causal role that amyloid-␤ peptide (A␤) plays in the etiology of AD (1-4). This research demonstrates that autosomal dominant mutations affecting the total amount or relative amount of the 42-versus 40-residue form of A␤ are sufficient to cause the disease (for review, see Ref. 5). However, a direct causal relationship between amyloid plaques and cognitive impairment remains unclear (6, 7). This apparent disconnect between plaque burden and neuronal dysfunction and loss has also been described in transgenic mouse models of AD (8, 9). Recent theories that reconcile these findings point to small soluble oligomeric or protofibrillar assemblies of A␤ that would likely escape the immunostaining and histopathological staining used to detect both diffuse amyloid deposits and senile plaques (10 -13).Soluble oligomers of A␤ have been isolated from brain, plasma, cerebrospinal fluid (14, 15), transfected cells (16), and cells derived from human brain (11,17). In vitro, oligomeric and protofibrillar forms of A␤ have been shown to be directly neurotoxic and inhibit electrophysiologic activity that may be necessary for the formation and maintenance of memory (15,(17)(18)(19)(20)(21). Experimental in vivo and in vitro evidence linking oligomeric assemblies to neurodegeneration is reflected in a recent revision to the amyloid hypothesis of AD (22) and other diseases involving amyloidogenic proteins (for review, see Ref. 23). A complete study of A␤, either in vivo or in vitro, requires an understanding of the conditions that drive peptide assembly toward one conformational state or another. Any change that affects the conformation of A␤ likely affects its biological activity.In light of genetic and in vivo evidence, we have chosen to specifical...

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In the course of analyzing the chemical composition of Alzheimer's disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of A␤ peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 M guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic A␤-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8 -10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. Although evidence implicates ␤-amyloid peptide (A␤) in the pathogenesis of Alzheimer's disease (AD) 1 (reviewed in Ref. 1), little is known about the nature of the A␤ mediating the pathology. Toxicity initially was attributed to aggregated A␤ in amyloid plaques (1), the morphological hallmarks of AD brains. A␤-(1-42) is the major peptide constituent of amyloid plaques (2), and increased production of the 42-amino acid peptide correlates with an earlier onset of AD (1). However, recent studies show that small quantities of A␤-(1-42) also exists as soluble peptide in the plasma, cerebrospinal fluid, and cerebral cortex of AD and normal individuals and are also secreted by cells in tissue culture (3-13). Utilizing ultracentrifugation, graded membrane filtration, and ELISA, we have recently isolated and quantitated the oligomeric water-soluble A␤ present in the brains of AD and control individuals (13). The levels of insoluble A␤ in AD brains are at least 100 times higher than those found in control brains. The amounts of water-soluble A␤ in AD brains are approximately six times higher than those detected in control brains. Interestingly, we isolated an A␤ fraction, from the A␤ water-soluble oligomeric pool, with a molecular mass of Ͻ10 kDa containing monomeric and/or dimeric forms of A␤ peptide (13). In all probability these peptides represent the initial building blocks that may ultimately aggregate into insoluble A␤ filaments. In the course of analyzing the chemical composition of AD neuritic plaque and vascular amyloid, we have purified stable dimeric and trimeric components of A␤-(1-40/42) (2, 14 -15). In the present study we report the chemical and morphological characteristics of the dimeric A␤ as elucidated by atomic force microscopy and transmission electron microscopy techniques. In addition, the potential for toxicity of the AD brain-derived A␤-(1-40/42) dimer was assessed on glial-neuronal cell cultures. MATERIALS AND METHODSPurification of A␤-(1-42) from AD Brain-Brains were obtained from eight patients who died of AD (postmortem delay 3-6 h). After separation of the leptomeninges, the right hemispheres were frozen at Ϫ70°C. Examination of the left hemispheres revealed numerous neuritic plaq...