In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimer's disease.Many diseases share the common trait of peptide-protein misfolding that leads to oligomerization and, eventually, formation of plaques of β-sheet structure. Prominent among these are type 2 diabetes 1 , Parkinson's disease 2 and Alzheimer's disease 3,4 . Of these, Alzheimer's disease is the leading cause of late-life dementia and is the focus of this paper. An increasing body of evidence links oligomerization of a ubiquitous peptide, the amyloid-β [3][4][5][6] . For this reason, elucidation of pathways of oligomer formation may be critical for the identification of therapeutic targets.Many types of oligomeric amyloid-β assemblies have been described (for a review, see Lazo et al. 7 ). Recently, Bitan et al. [8][9][10] used photoinduced cross-linking of unmodified proteins (PICUP) to reveal that the 42-residue form of amyloid-β, Aβ42, formed (Aβ42) 5 and (Aβ42) 6 oligomers ('paranuclei') that could oligomerize to form structures of higher order. Aβ40 did not form paranuclei, but instead existed as a mixture of monomers, dimers, trimers and tetramers. Chen and Glabe 11 , in contrast, used fluorescence and gel electrophoresis to determine oligomer states of amyloid-β refolded from denaturing solutions. They observed only Aβ42 monomer and trimer bands, and no oligomers of Aβ40. Differences such as these may exist because of the diverse experimental systems used to monitor amyloid-β selfassociation. Also, it has been argued that, in addition to the intrinsic potential of amyloid-β to traverse different assembly pathways, flaws in experimental design may have misled researchers in their quest to elucidate fully the amyloid-β oligomerization process 12 . Hence there is significant uncertainty about amyloid-β oligomer states and their position and relevance to amyloid-β aggregation. Results and discussionWe used a different, more direct, method to probe the amyloid-β oligomerization process: ion mobility coupled with mass spectrometry [13][14][15] . Details are given in the Methods section.Here the results for Aβ40 are given as an example. The mass spectrum of Aβ40 is s...

Several pathogenic Alzheimer's disease (AD) mutations have been described, all of which cause increased amyloid beta-protein (Abeta) levels. Here we present studies of a pathogenic amyloid precursor protein (APP) mutation, located within the Abeta sequence at codon 693 (E693G), that causes AD in a Swedish family. Carriers of this 'Arctic' mutation showed decreased Abeta42 and Abeta40 levels in plasma. Additionally, low levels of Abeta42 were detected in conditioned media from cells transfected with APPE693G. Fibrillization studies demonstrated no difference in fibrillization rate, but Abeta with the Arctic mutation formed protofibrils at a much higher rate and in larger quantities than wild-type (wt) Abeta. The finding of increased protofibril formation and decreased Abeta plasma levels in the Arctic AD may reflect an alternative pathogenic mechanism for AD involving rapid Abeta protofibril formation leading to accelerated buildup of insoluble Abeta intra- and/or extracellularly.

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Amyloid ␤-protein (A␤) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). ''Oligomer'' is an ill-defined term because many kinds have been reported and they often exist in rapid equilibrium with monomers and higher-order assemblies. We report here results of studies in which specific oligomers have been stabilized structurally, fractionated in pure form, and then studied by using a combination of CD spectroscopy, Thioflavin T fluorescence, EM, atomic force microscopy (AFM), and neurotoxicity assays. A␤ monomers were largely unstructured, but oligomers exhibited order-dependent increases in ␤-sheet content. EM and AFM data suggest that dimerization and subsequent monomer addition are processes in which significant and asymmetric monomer conformational changes occur. Oligomer secondary structure and order correlated directly with fibril nucleation activity. Neurotoxic activity increased disproportionately (order dependence >1) with oligomer order. The structure-activity correlations reported here significantly extend our understanding of the conformational dynamics, structure, and relative toxicity of pure A␤ oligomers of specific order.Alzheimer's disease ͉ toxicity A lzheimer's disease (AD) is the most common form of late-life dementia. Current estimates of AD incidence are Ͼ24 million worldwide, a number that is expected to double every 20 years, reaching 81 million in 2040 (1). AD is a slowly progressive disorder with insidious onset and progressive impairment of episodic memory and executive function coupled with aphasia, apraxia, and agnosia (1).The amyloid ␤-protein (A␤) appears to play an essential role in the pathogenesis of AD. A␤ is produced throughout life through posttranslational processing of the A␤ precursor (APP). Familial forms of AD increase A␤ production or the propensity of A␤ to aggregate (2). The ''amyloid cascade hypothesis'' proposes that assemblies of A␤ initiate a process leading to neuronal dysfunction and cell death (2). The most potent neurotoxic assemblies appear to be oligomeric, rather than fibrillar, in nature (3, 4). For example, oligomers extracted from AD brain potently impair synapse structure and function (5). The smallest of these oligomers appears to be dimeric (5). However, a systematic correlation of oligomer structure and neurotoxic potency has not been reported. This correlation is critical for the targeting and design of disease-modifying therapeutic agents.Efforts to establish rigorous structure-toxicity correlations have been hindered by the complex, dynamic equilibria displayed by A␤ (for recent reviews, see refs. 6 and 7). To enable determination of the oligomer frequency distribution, we have used in situ chemical cross-linking to prevent oligomer dissociation or growth (8-10). Oligomers thus stabilized can be visualized and quantified by SDS/PAGE. However, in theory, the method also could be used to produce pure populations of oligomers of defined order † , enabling the biophysical and biological studies necessary to establish structure-toxic...