Alzheimer's disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid ␤-protein (A␤). Fibrils, both in vitro and in vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control A␤ production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of A␤ fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364 -22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight A␤ (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer's disease and for our understanding of fibril assembly are discussed. Alzheimer's disease (AD)1 is a progressive neurodegenerative disorder defined histologically by the formation in the brain of intracellular neurofibrillary tangles and extracellular amyloid deposits (1). Particular attention has been focused on the role that the amyloid ␤-protein (A␤), the primary protein constituent of amyloid deposits, plays in development of AD. A␤ molecules are fibrillogenic and exist in a number of forms in vivo (2). Among those forms found in amyloid deposits, 40 and 42 residue long species (A␤(1-40) and A␤(1-42), respectively) are particularly important. Genetic studies of AD have shown that mutations in the gene encoding the precursor of A␤ (the amyloid ␤-protein precursor (␤APP) gene) (3-6), or in genes that regulate the proteolytic processing of ␤APP (7-9), cause AD. The phenotypic effects of these mutations show remarkable consistency, they all result in excessive production of A␤ or in an increased A␤(1-42)/A␤(1-40) ratio, facilitating amyloid deposition (10, 11). In addition, specific haplotypes and mutations in genes involved in the extracellular transport or cleavage of A␤ are risk factors for AD (12,13). In vitro and in vivo studies of A␤ toxicity indicate that fibrillar A␤ can directly kill neurons or initiate a cascade of events leading to neuronal cell death (14 -16). For this reason, therapeutic strategies targeting A␤ fibrillogenesis are being pursued actively (17-20). Unfortunately, key areas of A␤ fibrillogenesis are poorly understood. In particular, the three-dimensional structure and organization of fibril subunits are unknown, as are the steps involved in assembly of nascent, monomeric A␤ first into nuclei, then into higher order oligo...

Fibrillogenesis of the amyloid -protein (A ) is believed to play a central role in the pathogenesis of Alzheimer's disease. Previous studies of the kinetics of A fibrillogenesis showed that the rate of fibril elongation is proportional to the concentration of monomers. We report here the study of the temperature dependence of the A fibril elongation rate constant, k e , in 0.1 M HCl. The rate of fibril elongation was measured at A monomer concentrations ranging from 50 to 400 M and at temperatures from 4• • • C to 40 • • • C. Over this temperature range, k e increases by two orders of magnitude. The temperature dependence of k e follows the Arrhenius law, k e ‫؍‬ A exp −E A /kT . The preexponential factor A and the activation energy E A are 8 8 8 6 ؋ 10 18 liter/(mol . sec) and 23 kcal/mol, respectively. Such a high value of E A suggests that significant conformational changes are associated with the binding of A monomers to fibril ends.