CLAC binds to aggregated Abeta and Abeta fragments, and attenuates fibril elongation

Deposition of amyloid beta-peptide (Abeta) into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Proteins that codeposit with Abeta are potentially important for the pathogenesis, and a recently discovered plaque-associated protein is the collagenous Alzheimer amyloid plaque component (CLAC). In this study, we investigated the molecular interactions between Abeta aggregates and CLAC using surface plasmon resonance spectroscopy and a solid-phase binding immunoassay. We found that CLAC binds to Abeta with high affinity, that the central region of Abeta is necessary and sufficient for CLAC interaction, and that the aggregation state of Abeta as well as the presence of negatively charged residues is important. We also show that this binding results in a reduced rate of fibril elongation. Taken together, we suggest that CLAC becomes involved at an intermediate stage in the pathogenesis by binding to Abeta fibrils, including fibrils formed from peptides with truncated N- or C-termini, and thereby slows their growth.     

CLAC binds to amyloid beta peptides through the positively charged amino acid cluster within the collagenous domain 1 and inhibits formation of amyloid fibrils

CLAC (collagenous Alzheimer amyloid plaque component) is a proteolytic fragment derived from a novel membrane-bound collagen, CLAC-P/collagen type XXV, that deposits in senile plaques associated with amyloid beta peptides (Abeta) in the brains of patients with Alzheimer's disease. We previously showed that CLAC binds to the fibrillized form of Abeta in vitro, although the mechanism and the subdomains that mediate interaction of CLAC with Abeta as well as the effect of binding of CLAC on amyloid fibril formation remain unknown. Here we show that the collagenous domain 1 of CLAC, which is rich in positively charged amino acid residues, mediates its interaction with Abeta and that this binding is mediated by an electrostatic interaction and requires formation of the triple helix structure of CLAC. The soluble form of CLAC purified from the media of cells transfected with CLAC-P inhibited fibrillization of Abeta in vitro, especially in its elongation phase. These results suggest the anti-amyloidogenic roles of CLAC in the pathophysiology of Alzheimer's disease.     

Collagenous Alzheimer amyloid plaque component assembles amyloid fibrils into protease resistant aggregates

Recently, a novel plaque-associated protein, collagenous Alzheimer amyloid plaque component (CLAC), was identified in brains from patients with Alzheimer's disease. CLAC is derived from a type II transmembrane collagen precursor protein, termed CLAC-P (collagen XXV). The biological function and the contribution of CLAC to the pathogenesis of Alzheimer's disease and plaque formation are unknown. In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid beta-peptide (Abeta). Here, we examined the effects of CLAC on Abeta fibrils using assays based on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy. The incubation of CLAC with preformed Abeta fibrils led to increased turbidity, indicating that larger aggregates were formed. In support of this contention, more Abeta was sedimented in the presence of CLAC, as determined by gel electrophoresis. Moreover, electron microscopy revealed an increased amount of Abeta fibril bundles in samples incubated with CLAC. Importantly, the frequently used thioflavin T-binding assay failed to reveal these effects of CLAC. Digestion with proteinase K or trypsin showed that Abeta fibrils, incubated together with CLAC, were more resistant to proteolytic degradation. Therefore, CLAC assembles Abeta fibrils into fibril bundles that have an increased resistance to proteases. We suggest that CLAC may act in a similar way in vivo.     

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.     

Familial Danish dementia: co-existence of Danish and Alzheimer amyloid subunits (ADan AND A{beta}) in the absence of compact plaques

Familial Danish dementia is an early onset autosomal dominant neurodegenerative disorder linked to a genetic defect in the BRI2 gene and clinically characterized by dementia and ataxia. Cerebral amyloid and preamyloid deposits of two unrelated molecules (Danish amyloid (ADan) and beta-amyloid (Abeta)), the absence of compact plaques, and neurofibrillary degeneration indistinguishable from that observed in Alzheimer disease (AD) are the main neuropathological features of the disease. Biochemical analysis of extracted amyloid and preamyloid species indicates that as the solubility of the deposits decreases, the heterogeneity and complexity of the extracted peptides exponentially increase. Nonfibrillar deposits were mainly composed of intact ADan-(1-34) and its N-terminally modified (pyroglutamate) counterpart together with Abeta-(1-42) and Abeta-(4-42) in approximately 1:1 mixture. The post-translational modification, glutamate to pyroglutamate, was not present in soluble circulating ADan. In the amyloid fractions, ADan was heavily oligomerized and highly heterogeneous at the N and C terminus, and, when intact, its N terminus was post-translationally modified (pyroglutamate), whereas Abeta was mainly Abeta-(4-42). In all cases, the presence of Abeta-(X-40) was negligible, a surprising finding in view of the prevalence of Abeta40 in vascular deposits observed in sporadic and familial AD, Down syndrome, and normal aging. Whether the presence of the two amyloid subunits is imperative for the disease phenotype or just reflects a conformational mimicry remains to be elucidated; nonetheless, a specific interaction between ADan oligomers and Abeta molecules was demonstrated in vitro by ligand blot analysis using synthetic peptides. The absence of compact plaques in the presence of extensive neuro fibrillar degeneration strongly suggests that compact plaques, fundamental lesions for the diagnosis of AD, are not essential for the mechanism of dementia.     

Cholesterol-dependent formation of GM1 ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid

GM1 ganglioside-bound amyloid beta-protein (GM1/Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. To elucidate the molecular mechanism underlying GM1/Abeta formation, the effects of lipid composition on the binding of Abeta to GM1-containing lipid bilayers were examined in detail using fluorescent dye-labeled human Abeta-(1-40). Increases in not only GM1 but also cholesterol contents in the lipid bilayers facilitated the binding of Abeta to the membranes by altering the binding capacity but not the binding affinity. An increase in membrane-bound Abeta concentration triggered its conformational transition from helix-rich to beta-sheet-rich structures. Excimer formation of fluorescent dye-labeled GM1 suggested that Abeta recognizes a GM1 "cluster" in membranes, the formation of which is facilitated by cholesterol. The results of the present study strongly suggested that increases in intramembrane cholesterol content, which are likely to occur during aging, appear to be a risk factor for amyloid fibril formation.     

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.     

Binding of amyloid beta-peptide to ganglioside micelles is dependent on histidine-13

Amyloid beta-peptide (Abeta) is a major component of plaques in Alzheimer's disease, and formation of senile plaques has been suggested to originate from regions of neuronal membrane rich in gangliosides. Here we demonstrate using NMR on 15N-labelled Abeta-(1-40) and Abeta-(1-42) that the interaction with ganglioside G(M1) micelles is localized to the N-terminal region of the peptide, particularly residues His13 to Leu17, which become more helical when bound. The key interaction is with His13, which undergoes a G(M1)-specific conformational change. The sialic acid residue of the ganglioside headgroup is important for determining the nature of the conformational change. The isolated pentasaccharide headgroup of G(M1) is not bound, suggesting the need for a polyanionic surface. Binding to heparin confirms this suggestion, since binding is of similar affinity but does not produce the same conformational changes in the peptide. A comparison of Abeta-(1-40) and Abeta-(1-42) indicates that binding to G(M1) micelles is not related to oligomerization, which occurs at the C-terminal end. These results imply that binding to ganglioside micelles causes a transition from random coil to alpha-helix in the N-terminal region, leaving the C-terminal region unstructured.     

Attenuated hippocampus-dependent learning and memory decline in transgenic TgAPPswe Fischer-344 rats

Alzheimer's disease (AD) is characterized by increased beta amyloid (Abeta) levels, extracellular Abeta deposits in senile plaques, neurofibrillary tangles, and neuronal loss. However, the physiological role of normal levels of Abeta and its parent protein, the amyloid precursor protein (APP) are unknown. Here we report that low-level transgenic (Tg) expression of the Swedish APP mutant gene (APPswe) in Fischer-344 rats results in attenuated age-dependent cognitive performance decline in 2 hippocampus-dependent learning and memory tasks compared with age-matched nontransgenic Fischer-344 controls. TgAPPswe rats exhibit mild increases in brain APP mRNA (56.8%), Abeta-42 (21%), and Abeta-40 (6.1%) peptide levels at 12 mo of age, with no extracellular Abeta deposits or senile plaques at 6, 12, and 18 mo of age, whereas 3- to 6-fold increases in Abeta levels are detected in plaque-positive human AD patients and transgenic mouse models. The data support the hypothesis that a threshold paradigm underlies Abeta-related pathology, below which APP expression may play a physiological role in specific hippocampus-dependent tasks, most likely related to its neurotrophic role.     

Seeding specificity in amyloid growth induced by heterologous fibrils

Over residues 15-36, which comprise the H-bonded core of the amyloid fibrils it forms, the Alzheimer's disease plaque peptide amyloid beta (Abeta) possesses a very similar sequence to that of another short, amyloidogenic peptide, islet amyloid polypeptide (IAPP). Using elongation rates to quantify seeding efficiency, we inquired into the relationship between primary sequence similarity and seeding efficiency between Abeta-(1-40) and amyloid fibrils produced from IAPP as well as other proteins. In both a solution phase and a microtiter plate elongation assay, IAPP fibrils are poor seeds for Abeta-(1-40) elongation, exhibiting weight-normalized efficiencies of only 1-2% compared with Abeta-(1-40) fibrils. Amyloid fibrils of peptides with sequences completely unrelated to Abeta also exhibit poor to negligible seeding ability for Abeta elongation. Fibrils from a number of point mutants of Abeta-(1-40) exhibit intermediate seeding abilities for wild-type Abeta elongation, with differing efficiencies depending on whether or not the mutation is in the amyloid core region. The results suggest that amyloid fibrils from different proteins exhibit structural differences that control seeding efficiencies. Preliminary results also suggest that identical sequences can grow into different conformations of amyloid fibrils as detected by seeding efficiencies. The results have a number of implications for amyloid structure and biology.     

Common binding sites for beta-amyloid fibrils and fibroblast growth factor-2 in heparan sulfate from human cerebral cortex.

Heparan sulfate found in the cerebral plaques of Alzheimer's disease binds to beta-amyloid (Abeta) fibrils. This interaction has been proposed to enhance fibril deposition and mediate Abeta-induced glia activation and neurotoxicity. On the other hand, heparan sulfate augments signaling of fibroblast growth factor-2 (FGF-2), a neuroprotective factor that antagonizes the neurotoxic effects of Abeta. We defined structures in heparan sulfate from human cerebral cortex that bind Abeta fibrils. The minimal binding site is found in N-sulfated hexasaccharide domains and contains critical 2-O-sulfated iduronic acid residues. By contrast, binding of Abeta monomers requires, in addition, 6-O-sulfate groups on glucosamine residues. The binding specificity of fibrillar Abeta is shared by FGF-2, and we here show that cerebral heparan sulfate domains selected for binding to Abeta-(1-40) fibrils bind also to FGF-2. These data suggest that neurotoxic and neuroprotective signals may converge by competing for the same binding sites on the heparan sulfate chain.     

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.     

Oxidation of methionine 35 attenuates formation of amyloid beta -peptide 1-40 oligomers

Amyloid plaques formed by aggregation of the amyloid beta-peptide (Abeta) are an intrinsic component of Alzheimer disease pathogenesis. It has been suggested that oxidation of methionine 35 in Abeta has implications for Alzheimer disease, and it has been shown that oxidation of Met-35 significantly inhibits aggregation in vitro. In this study, the aggregational properties of Abeta-(1-40) before and after Met-35 oxidation were investigated using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The results show that Abeta-(1-40)Met-35(O) trimer and tetramer formation is significantly attenuated as compared with Abeta-(1-40). This suggests that oxidation of Met-35 inhibits a conformational switch in Abeta-(1-40) necessary for trimer but not dimer formation. Random incorporation of Abeta-(1-40) and Abeta-(1-40)Met-35(O) in homo- and heterooligomers could also be observed. This is the first report of an early rate-limiting step in Abeta-(1-40) aggregation. Slowing of the fibrillization process at this early step is likely to support prolonged solubility and clearance of Abeta from brain and may reduce disease progression.     

Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease

Alzheimer's disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.     

The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.     

CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV

We raised monoclonal antibodies against senile plaque (SP) amyloid and obtained a clone 9D2, which labeled amyloid fibrils in SPs and reacted with approximately 50/100 kDa polypeptides in Alzheimer's disease (AD) brains. We purified the 9D2 antigens and cloned a cDNA encoding its precursor, which was a novel type II transmembrane protein specifically expressed in neurons. This precursor harbored three collagen-like Gly-X-Y repeat motifs and was partially homologous to collagen type XIII. Thus, we named the 9D2 antigen as CLAC (collagen-like Alzheimer amyloid plaque component), and its precursor as CLAC-P/collagen type XXV. The extracellular domain of CLAC-P/collagen type XXV was secreted by furin convertase, and the N-terminus of CLAC deposited in AD brains was pyroglutamate modified. Both secreted and membrane-tethered forms of CLAC-P/collagen type XXV specifically bound to fibrillized Abeta, implicating these proteins in beta-amyloidogenesis and neuronal degeneration in AD.     

Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity

The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.     

Formation of amyloids by Abeta-(1-42) on NGF-differentiated PC12 cells: roles of gangliosides and cholesterol

The conversion of soluble, non-toxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta could be the key step in the development of Alzheimer's disease. Liposomal studies have proposed that Abeta-(1-40) preferentially recognizes a cholesterol-dependent cluster of gangliosides and a conformationally altered form of Abeta promotes the aggregation of the protein. Cell experiments using fluorescein-labeled Abeta-(1-40) supported this model. Here, the interaction of native Abeta-(1-42) with unfixed rat pheochromocytoma PC12 cells was visualized using the amyloid-specific dye Congo red. Abeta-(1-42) preferentially bound to ganglioside and cholesterol-rich domains of cell membranes and formed amyloids in a time-dependent manner. These observations corroborate the model involving ganglioside-mediated accumulation of Abeta. The NGF-induced differentiation of PC12 cells into neuron-like cells caused a marked increase in both gangliosides and cholesterol, and thereby greatly potentiated the accumulation and cytotoxicity of Abeta-(1-42). NGF-differentiated cells exposed to Abeta-(1-42) had degenerated neurites, in which ganglioside and cholesterol-rich domains were localized, preceding cell death. A reduction in the amount of cholesterol by the cholesterol synthesis inhibitor compactin almost nullified the formation of amyloids by Abeta-(1-42). Our system using NGF-differentiated PC12 cells and Congo red is useful for screening inhibitors of the formation of amyloids by and cytotoxicity of Abeta.     

The neuronal adaptor protein X11alpha reduces Abeta levels in the brains of Alzheimer's APPswe Tg2576 transgenic mice

Increased production and deposition of the 40-42-amino acid beta-amyloid peptide (Abeta) is believed to be central to the pathogenesis of Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP), but the mechanisms that regulate APP processing to produce Abeta are not fully understood. X11alpha (also known as munc-18-interacting protein-1 (Mint1)) is a neuronal adaptor protein that binds APP and modulates APP processing in transfected non-neuronal cells. To investigate the in vivo effect of X11alpha on Abeta production in the brain, we created transgenic mice that overexpress X11alpha and crossed these with transgenics harboring a familial Alzheimer's disease mutant APP that produces increased levels of Abeta (APPswe Tg2576 mice). Analyses of Abeta levels in the offspring generated from two separate X11alpha founder mice revealed a significant, approximate 20% decrease in Abeta(1-40) in double transgenic mice expressing APPswe/X11alpha compared with APPswe mice. At a key time point in Abeta plaque deposition (8 months old), the number of Abeta plaques was also deceased in APPswe/X11alpha mice. Thus, we report here the first demonstration that X11alpha inhibits Abeta production and deposition in vivo in the brain.     

Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer disease and familial British dementia

Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the beta-amyloid peptide (Abeta) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early "soluble oligomers" are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of Abeta, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of Abeta and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of Abeta-(1-40) and ABri and found that in both cases it was generated as a short "burst" early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling "soluble oligomers" or "protofibrils" were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from Abeta-(1-40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases.