Amyloid ␤-protein (A␤) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of A␤ in AD is new evidence that oligomeric forms of A␤ are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of A␤, A␤42, with the disease. Detailed knowledge of the structure and assembly dynamics of A␤ thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that A␤ oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, sizeexclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of A␤40 and A␤42. Carefully prepared aggregate-free A␤40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, A␤42 preferentially formed pentamer͞hexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to A␤40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that A␤42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of A␤42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.A myloid ␤-protein (A␤) fibril formation and deposition long have been linked to the neuropathogenesis of Alzheimer's disease (AD) (1-5). However, recent data have shown that oligomeric A␤ assembly intermediates are potent neurotoxins, and that these intermediates may be the key effectors of neurotoxicity in AD (6). In transgenic mice expressing the human amyloid ␤-protein precursor (A␤PP) and A␤, neurologic deficits develop before and independently of the appearance of amyloid deposits (6, 7). Importantly, soluble oligomeric forms of A␤ are neurotoxic in vitro (8-15) and in vivo (15). The main alloforms of A␤ found in amyloid deposits are 40 and 42 amino acids long (designated A␤40 and A␤42, respectively). Despite the small structural difference between these two peptides, they display distinct clinical, biological, and biophysical behavior. The concentration of secreted A␤42 is Ϸ10% that of A␤40, yet the longer form is the predominant component in parenchymal plaques (16)(17)(18)). An increase in the A␤42͞A␤40 concentration ratio is associated with familial forms of early onset AD (19,20). Treatments that reduce A␤42 levels have been shown to correlate with decreased risk for AD (21). In addition, A␤42 displays enhanced neurotoxicity relative to A␤40 (22-24). Studies of the kinetics of A␤ fibril formation have shown that A␤42 forms fibrils significantly faster than A␤40 (25), leading to the oftrepeated statement ''A␤42 is mor...

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