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...

We have studied the fibrillogenesis of synthetic amyloid 13-protein-(1-40) fragment (Aj3) in 0.1 M HCI.At low pH, Af3 formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii ofA,8 aggregates during the entire process of fibril nucleation and growth. Above an Aj3 concentration of :0.1 mM, the initial rate of elongation and the final size of fibrils were independent of Aj3 concentration. Below an A,j concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n- (21,22) and to "inhibit" or destabilize (23-25) amyloid fibril formation.Fibrillization of many proteins [for example, of actin (26-28)], is controlled by two kinetic parameters: the nucleation rate and the growth rate. Consequently, simple terms such as "inhibition" or "promotion" are inadequate and even misleading descriptors of the effect of external agents on fibrillogenesis. For example, conditions inhibiting nucleation could be interpreted both as "inhibiting" fibrillogenesis, since the total number of fibers will be small, and as "promoting" it, since longer fibrils will be formed. Similarly, conditions promoting nucleation could be interpreted as "promoting" fibrillogenesis because fibers will be more numerous and as "inhibiting" it because shorter fibers will be formed. It has been suggested that these considerations also apply to A,B polymerization (29).Therefore, a complete characterization of AP3 fibrillogenesis must include quantitation of both fibril concentration and fibril dimensions throughout the polymerization process.Previous efforts (12)(13)(14)23) to investigate the kinetics of AP3 fibrillogenesis have had methodological limitations. CD and Fourier-transform IR spectroscopies, turbidity, or thioflavin T binding could not provide direct information on fibril size, while EM, which could elucidate fibril dimensions, was not appropriate for real-time kinetic studies. In contrast, quasielastic light-scattering spectroscopy (QLS) was long recognized as a powerful tool for the study of aggregation kinetics (30). However, since AP fibrillogenesis occurs very rapidly at neutral pH, previous applications of QLS to the AP3 problem Abbreviations: AP3, amyloid ,B-protein-(1-40) fragment; C12E6, ndodecylhexaoxyethylene glycol monoether; QLS, quasi-elastic lightscattering spectroscopy.