Cerebral deposition of amyloid β peptide (Aβ) is an early and critical feature of Alzheimer's disease. Aβ generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: β-secretase and γ-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of β-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of β-secretase cleavage products, and these were cleaved exactly and only at known β-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of β-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as β-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for β-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.

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...

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...

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.

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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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.

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