Abeta-polyacrolein aggregates: novel mechanism of plastic formation in senile plaques

High levels of acrolein (H2C=HC-CH=O) occur in Alzheimer's brain. Amyloid-beta (Abeta) peptide co-localizes with acrolein presumably due to Abeta-induced lipid peroxidation. Focal production of acrolein may yield a transient elevation in the concentration of acrolein that may be susceptible to polymerization via basic latex polymer chemistry. Following incubation of Abeta with acrolein (16-750 mM), we observed the formation of thin plastic fragments that were extensively punctuated. Planar aggregates stained for protein and for cross-beta structures suggesting an Abeta-polyacrolein colloidal mixture. Depending on acrolein concentration and incubation time, we observed uniformly sized planar aggregates (approximately 10 microm2) or monolayers (>100 mm2) of thin polyacrolein films embedded with Abeta oligomers. The ability of Abeta to catalyze the polymerization of acrolein is likely due to Abeta's surfactant and redox properties. These observations suggest that plastic in the form of Abeta-polyacrolein latexes may exist in neural tissue contributing to the pathogenesis of Alzheimer's disease.     

Specific compositions of amyloid-beta peptides as the determinant of toxic beta-aggregation

Alzheimer's disease (AD) may be caused by toxic aggregates formed from amyloid-beta (Abeta) peptides. By using Thioflavin T, a dye that specifically binds to beta-sheet structures, we found that highly toxic forms of Abeta-aggregates were formed at the initial stage of fibrillogenesis, which is consistent with recent reports on Abeta oligomers. Formation of such aggregates depends on factors that affect both nucleation and elongation. As reported previously, addition of Abeta42 systematically accelerated the nucleation of Abeta40, most likely because of the extra hydrophobic residues at the C terminus of Abeta42. At Abeta42-increased specific ratio (Abeta40: Abeta42 = 10: 1), on the other hand, not only accelerated nucleation but also induced elongation were observed, suggesting pathogenesis of early-onset AD. Because a larger proportion of Abeta40 than Abeta42 was still required for this phenomenon, we assumed that elongation does not depend only on hydrophobic interactions. Without any change in the C-terminal hydrophobic nature, elongation was effectively induced by mixing wild type Abeta40 with Italian variant Abeta40 (E22K) or Dutch variant (E22Q). We suggest that Abeta peptides in specific compositions that balance hydrophilic and hydrophobic interactions promote the formation of toxic beta-aggregates. These results may introduce a new therapeutic approach through the disruption of this balance.     

Disassembly of amyloid beta-protein fibril by basement membrane components

Amyloid beta-protein (A3) fibril in senile plaque may be related to the pathogenesis of Alzheimer's disease (AD). Basement membrane (BM) components are associated with the plaques in AD brain. It suggests that the BM components may play an important role in the deposition of the plaque. We investigated the potential of BM components, such as type IV collagen (collagen IV) and entactin, to induce disassembly of preformed Abeta1-42 (Abeta42) fibrils in direct comparison to laminin. Thioflavin T assays revealed that these BM components disrupted preformed Abeta42 fibrils in a dose-dependent manner. The high concentration of BM components, 100 microg/mL laminin, 50 microg/mL collagen IV and 50 microg/mL entactin, had most effect on disassembly of preformed Abeta42 fibrils (Molar ratio; Abeta42:laminin = 90:1, Abeta42:collagen IV = 34:1, Abeta42:entactin = 20:1). Circular dichroism spectroscopy data indicated that the high concentration of BM components induced structural transition in Abeta42 from beta-sheet to random structures. These results suggest that collagen IV and entactin, as well as laminin, are effective inducers of disassembly of Abeta42 fibrils. The ability of these BM components to induce random structures may be linked to the disassembly of preformed Abeta42 fibrils.     

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.     

Metal ions differentially influence the aggregation and deposition of Alzheimer's beta-amyloid on a solid template

The abnormal deposition and aggregation of beta-amyloid (Abeta) on brain tissues are considered to be one of the characteristic neuropathological features of Alzheimer's disease (AD). Environmental conditions such as metal ions, pH, and cell membranes are associated with Abeta deposition and plaque formation. According to the amyloid cascade hypothesis of AD, the deposition of Abeta42 oligomers as diffuse plaques in vivo is an important earliest event, leading to the formation of fibrillar amyloid plaques by the further accumulation of soluble Abeta under certain environmental conditions. In order to characterize the effect of metal ions on amyloid deposition and plaque growth on a solid surface, we prepared a synthetic template by immobilizing Abeta oligomers onto a N-hydroxysuccinimide ester-activated solid surface. According to our study using ex situ atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and thioflavin T (ThT) fluorescence spectroscopy, Cu2+ and Zn2+ ions accelerated both Abeta40 and Abeta42 deposition but resulted only in the formation of "amorphous" aggregates. In contrast, Fe3+ induced the deposition of "fibrillar" amyloid plaques at neutral pH. Under mildly acidic environments, the formation of fibrillar amyloid plaques was not induced by any metal ion tested in this work. Using secondary ion mass spectroscopy (SIMS) analysis, we found that binding Cu ions to Abeta deposits on a solid template occurred by the possible reduction of Cu ions during the interaction of Abeta with Cu2+. Our results may provide insights into the role of metal ions on the formation of fibrillar or amorphous amyloid plaques in AD.     

Alternate aggregation pathways of the Alzheimer beta-amyloid peptide. An in vitro model of preamyloid

Deposition of amyloid-beta (Abeta) aggregates in the brain is a defining characteristic of Alzheimer's disease (AD). Fibrillar amyloid, found in the cores of senile plaques, is surrounded by dystrophic neurites. In contrast, the amorphous Abeta (also called preamyloid) in diffuse plaques is not associated with neurodegeneration. Depending on the conditions, Abeta will also form fibrillar or amorphous aggregates in vitro. In this present study, we sought to characterize the properties of the amorphous aggregate and determine whether we could establish an in vitro model for amorphous Abeta. CD data indicated that Abeta40 assembled to form either a beta-structured aggregate or an unfolded aggregate with the structured aggregate forming at high peptide concentrations and the unstructured aggregate forming at low Abeta40 levels. The critical concentration separating these two pathways was 10 microm. Fluorescence emission and polarization showed the structured aggregate was tightly packed containing peptides that were not accessible to water. Peptides in the unstructured aggregate were loosely packed, mobile, and accessible to water. When examined by electron microscopy, the structured aggregate appeared as protofibrillar structures and formed classic amyloid fibrils over a period of several weeks. The unstructured aggregate was not visible by electron microscopy and did not generate fibrils. These findings suggest that the unstructured aggregate shares many properties with the amorphous Abeta of AD and that conditions can be established to form amorphous Abeta in vitro. This would allow for investigations to better understand the relationship between fibrillar and amorphous Abeta and could have significant impact upon efforts to find therapies for AD.     

Amyloid-beta(1-42) rapidly forms protofibrils and oligomers by distinct pathways in low concentrations of sodium dodecylsulfate

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.     

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.     

Characterization of chemical exchange between soluble and aggregated states of beta-amyloid by solution-state NMR upon variation of salt conditions

Alzheimer's disease (AD) is characterized by the accumulation of insoluble fibrillar aggregates of beta-amyloid peptides (Abeta), a 39-42 residue peptide, in the brain of AD patients. It is hypothesized that the disease causing form is not the fibrillar species but an oligomeric Abeta molecule, which is often referred to as the "critical oligomer" of Abeta. We show in this paper that Abeta(1-40) undergoes chemical exchange between a monomeric, soluble state and an oligomeric, aggregated state under physiological conditions. In circular dichroism spectroscopy, we observe for this intermediate an alpha-helical structure. The oligomer is assigned a molecular weight of >100 kDa by diffusion-ordered spectroscopy-solution-state NMR spectroscopy (NMR). We can show by saturation transfer difference NMR experiments that the oligomer is related to monomeric Abeta. This experiment also allows us to identify the chemical groups that are involved in interactions between mono- and oligomeric Abeta molecules. Variation of the anionic strength in the buffer induces a shift of equilibrium between mono- and oligomeric states and possibly allows for the stabilization of these intermediate structures.     

Dimerisation of N-acetyl-L-tyrosine ethyl ester and Abeta peptides via formation of dityrosine

Alzheimer's disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid beta-peptide (Abeta). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H(2)O(2)). The generation of H(2)O(2) has been linked with Abeta neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of Abeta. This residue has also been shown to be important to Abeta aggregation and amyloid formation. It is possible that the formation of an Abeta tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in Abeta aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of Abeta dimers containing dityrosine. Here we report the use of horseradish peroxidase and H(2)O(2) to dimerise N-acetyl-L-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected Abeta peptides. We also report a simple fluorescent plate reader method for monitoring Abeta dimerisation via dityrosine formation.     

BACE1 suppression by RNA interference in primary cortical neurons

Extracellular deposition of amyloid-beta (Abeta) aggregates in the brain represents one of the histopathological hallmarks of Alzheimer's disease (AD). Abeta peptides are generated from proteolysis of the amyloid precursor proteins (APPs) by beta- and gamma-secretases. Beta-secretase (BACE1) is a type I integral membrane glycoprotein that can cleave APP first to generate C-terminal 99- or 89-amino acid membrane-bound fragments containing the N terminus of Abeta peptides (betaCTF). As BACE1 cleavage is an essential step for Abeta generation, it is proposed as a key therapeutic target for treating AD. In this study, we show that small interfering RNA (siRNA) specifically targeted to BACE1 can suppress BACE1 (but not BACE2) protein expression in different cell systems. Furthermore, BACE1 siRNA reduced APP betaCTF and Abeta production in primary cortical neurons derived from both wild-type and transgenic mice harboring the Swedish APP mutant. The subcellular distribution of APP and presenilin-1 did not appear to differ in BACE1 suppressed cells. Importantly, pretreating neurons with BACE1 siRNA reduced the neurotoxicity induced by H2O2 oxidative stress. Our results indicate that BACE1 siRNA specifically impacts on beta-cleavage of APP and may be a potential therapeutic approach for treating AD.     

Abeta40 protects non-toxic Abeta42 monomer from aggregation

Abeta40 and Abeta42 are the predominant Abeta species in the human body. Toxic Abeta42 oligomers and fibrils are believed to play a key role in causing Alzheimer's disease (AD). However, the role of Abeta40 in AD pathogenesis is not well established. Emerging evidence indicates a protective role for Abeta40 in AD pathogenesis. Although Abeta40 is known to inhibit Abeta42 fibril formation, it is not clear whether the inhibition acts on the non-toxic monomer or acts on the toxic Abeta42 oligomers. In contrast to conventional methods that detect the appearance of fibrils, in our study Abeta42 aggregation was monitored by the decreasing NMR signals from Abeta42 monomers. In addition, differential NMR isotope labelling enabled the selective observation of Abeta42 aggregation in a mixture of Abeta42 and Abeta40. We found Abeta40 monomers inhibit the aggregation of non-toxic Abeta42 monomers, in an Abeta42/Abeta40 ratio-dependent manner. NMR titration revealed that Abeta40 monomers bind to Abeta42 aggregates with higher affinity than Abeta42 monomers. Abeta40 can also release Abeta42 monomers from Abeta42 aggregates. Thus, Abeta40 likely protects Abeta42 monomers by competing for the binding sites on pre-existing Abeta42 aggregates. Combining our data with growing evidence from transgenic mice and human genetics, we propose that Abeta40 plays a critical, protective role in Alzheimer's by inhibiting the aggregation of Abeta42 monomer. Abeta40 itself, a peptide already present in the human body, may therefore be useful for AD prevention and therapy.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Analysis of single Alzheimer solid plaque cores by laser capture microscopy and nanoelectrospray/tandem mass spectrometry

Aggregation of the 40-42 residue amyloid beta-peptide (Abeta) into amyloid plaques is a central event in Alzheimer's disease (AD) pathogenesis. Many proteins have by immunohistochemical techniques been shown to codeposit with Abeta in AD plaques. It is possible that some of these could seed Abeta aggregation and therefore be found in the actual core of the plaque. Here, we present a highly sensitive method for unbiased biochemical analysis of plaque cores. A mild purification protocol based on centrifugation and filtration was used to purify intact plaque cores from human AD brain. The purified plaques were dispensed on a glass slide and viewed in a laser capture microscope, and plaque cores were catapulted into a tube cap by a laser beam. After dissolution in formic acid, plaques were digested and analyzed by liquid chromatography coupled online to electrospray/tandem mass spectrometry. One single plaque was found to be sufficient for positive identification of the main amyloid component. Remarkably, Abeta was the only protein identified when 200 plaques were isolated and analyzed with the present method. Thus, it is possible that no proteins copolymerize with Abeta in the plaque cores and that Abeta alone is sufficient for formation of plaque cores. In support of this notion, core-like structures were observed after incubation of synthetic Abeta for 2 weeks. We suggest that the method described here could be used for the general analysis of amyloid aggregates and inclusion bodies found in other neurodegenerative disorders and that plaque cores in AD brain are molecularly homogeneous structures.     

Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been assumed to be as a result of the extensive accumulation of beta-amyloid (Abeta). In addition to Abeta fibrillar assemblies, there are pre-fibrillar forms that have been shown to be neurotoxic, although their role in cholinergic degeneration is still not known. Using the cholinergic cell line SN56.B5.G4, we investigated the effect of different Abeta(1-42) aggregates on cell viability. In our model, only soluble oligomeric but not fibrillar Abeta(1-42) forms induced toxicity in cholinergic cells. To determine whether the neurotoxicity of oligomeric Abeta(1-42) was caused by its oxidative potential, we performed microarray analysis of SN56.B5.G4 cells treated either with oligomeric Abeta(1-42) or H(2)O(2). We showed that genes affected by Abeta(1-42) differed from those affected by non-specific oxidative stress. Many of the genes affected by Abeta(1-42) were present in the endoplasmic reticulum (ER), Golgi apparatus and/or otherwise involved in protein modification and degradation (chaperones, ATF6), indicating a possible role for ER-mediated stress in Abeta-mediated toxicity. Moreover, a number of genes, which are known to be involved in AD (clusterin, Slc18a3), were identified. This study provides important leads for the understanding of oligomeric Abeta(1-42) toxicity in cholinergic cells, which may account in part for cholinergic degeneration in AD.     

Inhibition of beta-amyloid peptide aggregation and neurotoxicity by alpha-d-mannosylglycerate, a natural extremolyte

The aggregation of soluble beta-amyloid (Abeta) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The use of naturally occurring small molecules for inhibiting protein aggregation has recently attracted many interests due to their effectiveness for treating protein folding diseases such as AD, Parkinson's, Huntington's disease, and other amyloidosis diseases. alpha-d-Mannosylglycerate (MG), a natural extremolyte identified in microorganisms growing under extremely high temperatures up to 100 degrees C, had been shown to protect proteins against various stress conditions such as heat, freezing, thawing, and drying. Here, we report the effectiveness of MG on the suppression of Alzheimer's Abeta aggregation and neurotoxicity to human neuroblastoma cells. According to our study--carried out by using thioflavin-T induced fluorescence, atomic force microscopy, and cell viability assay--MG had significant inhibitory effect against Abeta amyloid formation and could reduce the toxicity of amyloid aggregates to human neuroblastoma cells while MG itself was innocuous to cells. On the other hand, the structural analogs of MG such as alpha-d-mannosylglyceramide, mannose, methylmannoside, glycerol, showed negligible effect on Abeta aggregate formation. The results suggest that MG could be a potential drug candidate for treating Alzheimer's disease.     

Oxidative metabolites accelerate Alzheimer's amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.     

Abeta immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome

Amyloid-beta (Abeta) plaques and neurofibrillary tangles are the hallmark neuropathological lesions of Alzheimer's disease (AD). Using a triple transgenic model (3xTg-AD) that develops both lesions in AD-relevant brain regions, we determined the consequence of Abeta clearance on the development of tau pathology. Here we show that Abeta immunotherapy reduces not only extracellular Abeta plaques but also intracellular Abeta accumulation and most notably leads to the clearance of early tau pathology. We find that Abeta deposits are cleared first and subsequently reemerge prior to the tau pathology, indicative of a hierarchical and direct relationship between Abeta and tau. The clearance of the tau pathology is mediated by the proteasome and is dependent on the phosphorylation state of tau, as hyperphosphorylated tau aggregates are unaffected by the Abeta antibody treatment. These findings indicate that Abeta immunization may be useful for clearing both hallmark lesions of AD, provided that intervention occurs early in the disease course.     

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.     

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.