Sensitive ELISA detection of amyloid-beta protofibrils in biological samples

Amyloid-beta (Abeta) protofibrils are known intermediates of the in vitro Abeta aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Abeta protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Abeta protofibril immunoassay, Abeta conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Abeta protofibrils without interference from Abeta monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Abeta protofibrils in both cell and animal models, proving that Abeta protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Abeta protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Abeta protofibrils in AD and has the potential of becoming an important diagnostic assay.     

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.     

Abeta protofibrils possess a stable core structure resistant to hydrogen exchange

Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer's disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Abeta(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Abeta protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Abeta is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease.     

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.     

Rapid assembly of amyloid-beta peptide at a liquid/liquid interface produces unstable beta-sheet fibers

Accumulation of aggregated amyloid-beta peptide (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Abeta peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Abeta peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Abeta aggregation, but detailed characterization of these aggregates has not been presented. In this study Abeta(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Abeta in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Abeta aggregates had a beta-sheet secondary structure. However, these aggregates were far less stable than Abeta(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Abeta protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Abeta with beta-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic beta-sheet structure of an in vitro Abeta aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.     

Amyloid-beta protofibrils differ from amyloid-beta aggregates induced in dilute hexafluoroisopropanol in stability and morphology

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.     

The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation

4-Hydroxynonenal (4-HNE), formed as a consequence of oxidative stress, exists at increased concentrations in Alzheimer's disease (AD) patients and is found in amyloid beta peptide (Abeta) plaques associated with AD. Although it remains an open question as to whether oxidative stress is a causative factor or a consequence of AD, we show here that 4-HNE, putatively resulting from the peroxidation of lipids, covalently modifies Abeta, triggering its aggregation. These Abeta modifications result from 1,4 conjugate addition and/or Schiff base formation, they occur at multiple locations on a single Abeta peptide, and they result in covalent cross-linking of Abeta peptides. The consequence of these reactions is that 4-HNE accelerates the formation of Abeta protofibrils while inhibiting the production of straight, mature fibrils. Recent studies implicating Abeta oligomers and protofibrils in the neurotoxic process that ultimately leads to AD suggest that the Abeta aggregates induced by 4-HNE may be important in the pathogenesis of AD. These results provide further incentive to understand the role of oxidative stress and small-molecule Abeta modifications in sporadic AD.     

Identification of a mutant amyloid peptide that predominantly forms neurotoxic protofibrillar aggregates

The amyloid peptide (Abeta), derived from the proteolytic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases, undergoes multistage assemblies to fibrillar depositions in the Alzheimer's brains. Abeta protofibrils were previously identified as an intermediate preceding insoluble fibrils. While characterizing a synthetic Abeta variant named EV40 that has mutations in the first two amino acids (D1E/A2V), we discerned unusual aggregation profiles of this variant. In comparison of the fibrillogenesis and cellular toxicity of EV40 to the wild-type Abeta peptide (Abeta40), we found that Abeta40 formed long fibrillar aggregates while EV40 formed only protofibrillar aggregates under the same in vitro incubation conditions. Cellular toxicity assays indicated that EV40 was slightly more toxic than Abeta40 to human neuroblastoma SHEP cells, rat primary cortical, and hippocampal neurons. Like Abeta40, the neurotoxicity of the protofibrillar EV40 could be partially attributed to apoptosis since multiple caspases such as caspase-9 were activated after SHEP cells were challenged with toxic concentrations of EV40. This suggested that apoptosis-induced neuronal loss might occur before extensive depositions of long amyloid fibrils in AD brains. This study has been the first to show that a mutated Abeta peptide formed only protofibrillar species and mutations of the amyloid peptide at the N-terminal side affect the dynamic amyloid fibrillogenesis. Thus, the identification of EV40 may lead to further understanding of the structural perturbation of Abeta to its fibrillation.     

Secondary structure and interfacial aggregation of amyloid-beta(1-40) on sodium dodecyl sulfate micelles

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Abeta aggregates generated from monomeric Abeta in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily beta-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Abeta aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Abeta aggregation have found that amphiphilic interactions and micellar-like Abeta structures may play a role. We previously described the formation of Abeta(1-40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Abeta(1-40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Abeta(1-40) was introduced. With HFIP, the change involved an increase in predominant beta-structure content and in fluorescence with thioflavin T, while with SDS, a partial alpha-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in beta-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Abeta(1-40) monomers effectively.     

Transglutaminase induces protofibril-like amyloid beta-protein assemblies that are protease-resistant and inhibit long-term potentiation

An increasing body of evidence suggests that soluble assemblies of amyloid beta-protein (Abeta) play an important role in the initiation of Alzheimer disease (AD). In vitro studies have found that synthetic Abeta can form soluble aggregates through self-assembly, but this process requires Abeta concentrations 100- to 1000-fold greater than physiological levels. Tissue transglutaminase (TGase) has been implicated in neurodegeneration and can cross-link Abeta. Here we show that TGase induces rapid aggregation of Abeta within 0.5-30 min, which was not observed with chemical cross-linkers. Both Abeta40 and Abeta42 are good substrates for TGase but show different aggregation patterns. Guinea pig and human TGase induced similar Abeta aggregation patterns, and oligomerization was observed with Abeta40 concentrations as low as 50 nm. The formed Abeta40 species range from 5 to 6 nm spheres to curvilinear structures of the same width, but up to 100 nm in length, that resemble the previously described self-assembled Abeta protofibrils. TGase-induced Abeta40 assemblies are resistant to a 1-h incubation with either neprilysin or insulin degrading enzyme, whereas the monomer is rapidly degraded by both proteases. In support of these species being pathological, TGase-induced Abeta40 assemblies (100 nm) inhibited long term potentiation recorded in the CA1 region of mouse hippocampus slices. Our data suggest that TGase can contribute to AD by initiating Abeta oligomerization and aggregation at physiological levels, by reducing the clearance of Abeta due to the generation of protease-resistant Abeta species, and by forming Abeta assemblies that inhibit processes involved in memory and learning. Our data suggest that TGase might constitute a specific therapeutic target for slowing or blocking the progression of AD.     

FAD mutants unable to increase neurotoxic Abeta 42 suggest that mutation effects on neurodegeneration may be independent of effects on Abeta

Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.     

Abeta42 mutants with different aggregation profiles induce distinct pathologies in Drosophila

Aggregation of the amyloid-beta-42 (Abeta42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer's disease (AD), and the prevention of Abeta aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Abeta can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Abeta42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Abeta42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Abeta42Arc) and an artificial mutation (Abeta42art) that is known to suppress aggregation and toxicity of Abeta42 in vitro. In the Drosophila brain, Abeta42Arc formed more oligomers and deposits than did wild type Abeta42, while Abeta42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Abeta peptides. Surprisingly, however, Abeta42art caused earlier onset of memory defects than Abeta42. More remarkably, each Abeta induced qualitatively different pathologies. Abeta42Arc caused greater neuron loss than did Abeta42, while Abeta42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Abeta aggregates: Abeta42Arc formed large deposits in the cell body, Abeta42art accumulated preferentially in the neurites, while Abeta42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Abeta42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.     

Physiochemical characterization of the Alzheimer's disease-related peptides A beta 1-42Arctic and A beta 1-42wt

The amyloid beta peptide (A beta) is crucial for the pathogenesis of Alzheimer's disease. Aggregation of monomeric A beta into insoluble amyloid fibrils proceeds through several soluble A beta intermediates, including protofibrils, which are believed to be central in the disease process. The main reason for this is their implication in familial Alzheimer's disease with the Arctic amyloid precursor protein mutation (E693G). This mutation gives rise to early onset Alzheimer's disease, and synthetic A beta 1-40Arctic displays an enhanced rate of protofibril formation in vitro[Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J & Lannfelt L. (2001) Nat Neurosci4, 887-893]. To increase our understanding of the mechanisms involved in A beta aggregation, especially A beta monomer oligomerization into protofibrils and protofibril fibrillization into fibrils, the kinetics of A beta 1-42wt and A beta 1-42Arctic aggregation were examined under different physiochemical conditions, such as concentration, temperature, ionic strength and pH. We used size exclusion chromatography for this purpose, where monomers are separated from protofibrils, and fibrils are separated from protofibrils in a centrifugation step. The Arctic mutation significantly accelerated both A beta 1-42wt protofibril formation and protofibril fibrillization. In addition, we demonstrated that two distinct chemical processes - monomer oligomerization and protofibril fibrillization - were affected differently by changes in the micro-environment and that the Arctic mutation alters the peptide response to such changes.     

Aggressive amyloidosis in mice expressing human amyloid peptides with the Arctic mutation

The Arctic mutation within the amyloid-beta (Abeta) peptide causes Alzheimer disease. In vitro, Arctic-mutant Abeta forms (proto)fibrils more effectively than wild-type Abeta. We generated transgenic mouse lines expressing Arctic-mutant human amyloid precursor proteins (hAPP). Amyloid plaques formed faster and were more extensive in Arctic mice than in hAPP mice expressing wild-type Abeta, even though Arctic mice had lower Abeta(1-42/1-40) ratios. Thus, the Arctic mutation is highly amyloidogenic in vivo.     

Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange--mass spectrometry with on-line proteolytic fragmentation

We report here structural differences between Abeta(1-40) protofibrils and mature amyloid fibrils associated with Alzheimer's disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Abeta(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 degrees C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the beta-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal approximately ten residues appear to be unstructured in both protofibrils and fibrils. The 20-30 segment of Abeta(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Abeta peptide.     

Molecular mechanisms initiating amyloid beta-fibril formation in Alzheimer's disease

The deposition of aggregated amyloid beta-protein (Abeta) in the human brain is a major lesion in Alzheimer' disease (AD). The process of Abeta fibril formation is associated with a cascade of neuropathogenic events that induces brain neurodegeneration leading to the cognitive and behavioral decline characteristic of AD. Although a detailed knowledge of Abeta assembly is crucial for the development of new therapeutic approaches, our understanding of the molecular mechanisms underlying the initiation of Abeta fibril formation remains very incomplete. The genetic defects responsible for familial AD influence fibrillogenesis. In a majority of familial cases determined by amyloid precursor protein (APP) and presenilin (PS) mutations, a significant overproduction of Abeta and an increase in the Abeta42/Abeta40 ratio are observed. Recently, it was shown that the two main alloforms of Abeta have distinct biological activity and behaviour at the earliest stage of assembly. In vitro studies demonstrated that Abeta42 monomers, but not Abeta40, form initial and minimal structures (pentamer/hexamer units called paranuclei) that can oligomerize to larger forms. It is now apparent that Abeta oligomers and protofibrils are more neurotoxic than mature Abeta fibrils or amyloid plaques. The neurotoxicity of the prefibrillar aggregates appears to result from their ability to impair fundamental cellular processes by interacting with the cellular membrane, causing oxidative stress and increasing free Ca(2+) that eventually lead to apoptotic cell death.     

Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice

Although soluble oligomeric and protofibrillar assemblies of Abeta-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Abeta-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds 'forward' in a near-irreversible manner from the monomeric Abeta peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Abeta amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Abeta but rather toward soluble amyloid protofibrils. We characterized these 'backward' Abeta protofibrils generated from mature Abeta fibers and compared them with previously identified 'forward' Abeta protofibrils obtained from the aggregation of fresh Abeta monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Abeta-aggregates that could readily be activated by exposure to biological lipids.     

Studies on the in vitro assembly of a beta 1-40: implications for the search for a beta fibril formation inhibitors

The progressive deposition of the amyloid beta peptide (Abeta) in fibrillar form is a key feature in the development of the pathology in Alzheimer's disease (AD). We have characterized the time course of Abeta fibril formation using a variety of assays and under different experimental conditions. We describe in detail the morphological development of the Abeta polymerization process from pseudo-spherical structures and protofibrils to mature thioflavin-T-positive/Congo red-positive amyloid fibrils. Moreover, we structurally characterize the various polymorphic fibrillar assemblies using transmission electron microscopy and determine their mass using scanning transmission electron microscopy. These results provide the framework for future investigations into how target compounds may interfere with the polymerization process. Such substances might have a therapeutic potential in AD.     

Accelerating amyloid-beta fibrillization reduces oligomer levels and functional deficits in Alzheimer disease mouse models

Many proteins suspected of causing neurodegenerative diseases exist in diverse assembly states. For most, it is unclear whether shifts from one state to another would be helpful or harmful. We used mutagenesis to change the assembly state of Alzheimer disease (AD)-associated amyloid-beta (Abeta) peptides. In vitro, the "Arctic" mutation (AbetaE22G) accelerated Abeta fibrillization but decreased the abundance of nonfibrillar Abeta assemblies, compared with wild-type Abeta. In human amyloid precursor protein (hAPP) transgenic mice carrying mutations adjacent to Abeta that increase Abeta production, addition of the Arctic mutation markedly enhanced the formation of neuritic amyloid plaques but reduced the relative abundance of a specific nonfibrillar Abeta assembly (Abeta*56). Mice overexpressing Arctic mutant or wild-type Abeta had similar behavioral and neuronal deficits when they were matched for Abeta*56 levels but had vastly different plaque loads. Thus, Abeta*56 is a likelier determinant of functional deficits in hAPP mice than fibrillar Abeta deposits. Therapeutic interventions that reduce Abeta fibrils at the cost of augmenting nonfibrillar Abeta assemblies could be harmful.     

Cross-seeding of WT amyloid-β with Arctic but not Italian familial mutants accelerates fibril formation in Alzheimer's disease

Alzheimer’s disease (AD) involves the neurotoxic self-assembly of a 40 and 42 residue peptide, Amyloid-β (Aβ). Inherited early-onset AD can be caused by single point mutations within the Aβ sequence, including Arctic (E22G) and Italian (E22K) familial mutants. These mutations are heterozygous, resulting in an equal proportion of the WT and mutant Aβ isoform expression. It is therefore important to understand how these mixtures of Aβ isoforms interact with each other and influence the kinetics and morphology of their assembly into oligomers and fibrils. Using small amounts of nucleating fibril seeds, here, we systematically monitored the kinetics of fibril formation, comparing self-seeding with cross-seeding behavior of a range of isoform mixtures of Aβ42 and Aβ40. We confirm that Aβ40(WT) does not readily cross-seed Aβ42(WT) fibril formation. In contrast, fibril formation of Aβ40(Arctic) is hugely accelerated by Aβ42(WT) fibrils, causing an eight-fold reduction in the lag-time to fibrillization. We propose that cross-seeding between the more abundant Aβ40(Arctic) and Aβ42(WT) may be important for driving early-onset AD and will propagate fibril morphology as indicated by fibril twist periodicity. This kinetic behavior is not emulated by the Italian mutant, where minimal cross-seeding is observed. In addition, we studied the cross-seeding behavior of a C-terminal-amidated Aβ42 analog to probe the coulombic charge interplay between Glu22/Asp23/Lys28 and the C-terminal carboxylate. Overall, these studies highlight the role of cross-seeding between WT and mutant Aβ40/42 isoforms, which can impact the rate and structure of fibril assembly.