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.

Prior quasielastic light scattering (QLS) studies of fibrillogenesis of synthetic amyloid ␤-protein (A␤)-(1-40) at low pH have suggested a kinetic model in which: (i) fibrillogenesis requires a nucleation step; (ii) nuclei are produced by A␤ micelles in addition to seeds initially present; and (iii) fibril elongation occurs by irreversible binding of A␤ monomers to the fibril ends. Here we present the full mathematical formulation of this model. We describe the temporal evolution of the concentrations of A␤ monomers and micelles as well as the concentration and size distribution of fibrils. This formulation enables deduction of the fundamental parameters of the model-e.g., the nucleation and elongation rate constants k n and k e -from the time dependency of the apparent diffusion coefficient measured by QLS. The theory accurately represents the experimental observations for A␤ concentrations both below and above c*, the critical concentration for A␤ micelle formation. We suggest that the method of QLS in combination with this theory can serve as a powerful tool for understanding the molecular factors that control A␤ plaque formation.A seminal pathogenetic event in Alzheimer disease (AD) is the formation of fibrous amyloid plaques in the brain parenchyma and vasculature (1). The primary protein component of plaques is the amyloid ␤-protein (A␤) (2). In the plasma and cerebrospinal fluid, amyloid ␤-protein exists primarily as a soluble peptide 40 or 42 residues long (3). However, in the senile plaque, A␤ exists in the form of amyloid fibers (4). The conversion of soluble A␤ into insoluble fibers produces structures with neurotoxic activity (5). This observation, coupled with accumulating genetic evidence which links increased production of fibrillogenic forms of A␤ with familial AD (6), makes inhibiting fibrillogenesis an attractive therapeutic strategy.To properly target inhibitors, the structural stages and the kinetics of A␤ fibrillogenesis must be determined. In the past, turbidity and thioflavin T binding have been used to quantify levels of A␤ aggregation and amyloidogenesis, respectively (7-9). However, both methods detect only large polymeric structures and provide no information on either nucleation or elongation rate, the two key parameters controlling the kinetics of A␤ fibrillogenesis. Recently, however, static and dynamic light scattering, sophisticated optical methods capable of monitoring fibril length and structure, have been applied to the A␤ fibrillogenesis problem (10-12).In a previous communication (12) we reported an experimental study of the temporal evolution of A␤ fibrils using quasielastic light scattering (QLS). That study introduced an in vitro model system which permits quantitative monitoring of the kinetics of A␤ fibrillogenesis and enables the determination of the numerical values of the nucleation rate and elongation rate of the fibrils. Knowledge of these rate constants is vital in the effort to understand the molecular factors that control fibril creation and growth. This ...

Fibrillogenesis of the amyloid ␤-protein (A␤) is a seminal pathogenetic event in Alzheimer's disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of A␤ fibrillogenesis. We report results of studies designed to examine the initial stages of A␤ oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 M Tris-HCl, pH 7.4, and removal of preexistent seeds, A␤ chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 ؎ 0.2 nm, consistent with the predicted size of dimeric A␤. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6 -8 nm in diameter and <200 nm in length, which we have termed "protofibrils." The kinetics of protofibril formation and disappearance are consistent with protofibrils being intermediates in the evolution of amyloid fibers. Protofibrils appeared during the polymerization of A␤-(1-40), A␤-(1-42), and A␤-(1-40)-Gln 22 , peptides associated with both sporadic and inherited forms of Alzheimer's disease, suggesting that protofibril formation may be a general phenomenon in A␤ fibrillogenesis. If so, protofibrils could be attractive targets for fibrillogenesis inhibitors.Fibrillar amyloid plaques in the cerebral parenchyma and vasculature are a cardinal neuropathologic feature of Alzheimer's disease (AD) 1 (1). Plaques are composed predominantly of insoluble fibers of the amyloid ␤-protein (A␤) (2). A␤ is a normal component of the plasma and cerebrospinal fluid, occurring as a soluble 40-or 42-residue peptide (3, 4). Thus, a central question in the etiology of AD is the mechanism(s) by which these soluble A␤ molecules are converted into plaqueassociated fibers (5). This question is particularly relevant because A␤ fibers, unlike nonfibrillar A␤, are neurotoxic in vitro and are associated with damaged neuropil in vivo (6). These observations suggest that inhibiting fiber formation would be an effective approach toward AD therapy. However, if these efforts are to succeed, fiber formation must be understood at the molecular level.A␤ fibrillogenesis is a nucleation-dependent polymerization process (7,8). The kinetics of this type of process is controlled by two key parameters, nucleation rate and elongation rate. Past studies of the kinetics of A␤ fibrillogenesis, utilizing techniques including turbidity, sedimentation, and thioflavine T binding, could only provide information on the appearance of high molecular weight aggregates (7, 9) or the disappearance of soluble peptide (10 -13). Neither rate constants nor structures of fibrillogenesis intermediates could be determined by these approaches. In contrast, the technique of quasielastic light scattering spectroscopy (QLS) is particularly well suited for resolv...

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly therefore is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass-spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were non-toxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of non-toxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

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.

The phase diagram of globular colloids is studied using a combined analytic and computational representation of the relevant chemical potentials. It is shown how the relative positions of the phase boundaries are related to the range of interaction and the number of contacts made per particle in the solid phase. The theory presented successfully describes the features of the phase diagrams observed in a wide variety of colloidal systems. [S0031-9007(96)