Membrane-mediated amyloidogenesis and the promotion of oxidative lipid damage by amyloid beta proteins

Evidence of oxidative stress and the accumulation of fibrillar amyloid beta proteins (Abeta) in senile plaques throughout the cerebral cortex are consistent features in the pathology of Alzheimer disease. To define a mechanistic link between these two processes, various aspects of the relationship between oxidative lipid membrane damage and amyloidogenesis were characterized by chemical and physical techniques. Earlier studies of this relationship demonstrated that oxidatively damaged synthetic lipid membranes promoted amyloidogenesis. The studies reported herein specify that 4-hydroxy-2-nonenal (HNE) is produced in both synthetic lipids and human brain lipid extracts by oxidative lipid damage and that it can account for accelerated amyloidogenesis. Abeta promotes the copper-mediated generation of HNE from polyunsaturated lipids, and in turn, HNE covalently modifies the histidine side chains of Abeta. HNE-modified Abeta have an increased affinity for lipid membranes and an increased tendency to aggregate into amyloid fibrils. Thus, the prooxidant activity of Abeta leads to its own covalent modification and to accelerated amyloidogenesis. These results illustrate how lipid membranes may be involved in templating the pathological misfolding of Abeta, and they suggest a possible chemical mechanism linking oxidative stress with amyloid formation.     

Promotion of oxidative lipid membrane damage by amyloid beta proteins

Senile plaques in the cerebral parenchyma are a pathognomonic feature of Alzheimer's disease (AD) and are mainly composed of aggregated fibrillar amyloid beta (Abeta) proteins. The plaques are associated with neuronal degeneration, lipid membrane abnormalities, and chemical evidence of oxidative stress. The view that Abeta proteins cause these pathological changes has been challenged by suggestions that they have a protective function or that they are merely byproducts of the pathological process. This investigation was conducted to determine whether Abeta proteins promote or inhibit oxidative damage to lipid membranes. Using a mass spectrometric assay of oxidative lipid damage, the 42-residue form of Abeta (Abeta42) was found to accelerate the oxidative lipid damage caused by physiological concentrations of ascorbate and submicromolar concentrations of copper(II) ion. Under these conditions, Abeta42 was aggregated, but nonfibrillar. Ascorbate and copper produced H(2)O(2), but Abeta42 reduced H(2)O(2) concentrations, and its ability to accelerate oxidative damage was not affected by catalase. Lipids could be oxidized by H(2)O(2) and copper(II) in the absence of ascorbate, but only at significantly higher concentrations, and Abeta42 inhibited this reaction. These results indicate that the ability of Abeta42 to promote oxidative damage is more potent and more likely to be manifest in vivo than its ability to inhibit oxidative damage. In conjunction with prior results demonstrating that oxidatively damaged membranes cause Abeta42 to misfold and form fibrils, these results suggest a specific chemical mechanism linking Abeta42-promoted oxidative lipid damage to amyloid fibril formation.     

Distinct early folding and aggregation properties of Alzheimer amyloid-beta peptides Abeta40 and Abeta42: stable trimer or tetramer formation by Abeta42

The amyloid beta peptide (Abeta), composed of 40 or 42 amino acids, is a critical component in the etiology of the neurodegenerative Alzheimer disease. Abeta is prone to aggregate and forms amyloid fibrils progressively both in vitro and in vivo. To understand the process of amyloidogenesis, it is pivotal to examine the initial stages of the folding process. We examined the equilibrium folding properties, assembly states, and stabilities of the early folding stages of Abeta40 and Abeta42 prior to fibril formation. We found that Abeta40 and Abeta42 have different conformations and assembly states upon refolding from their unfolded ensembles. Abeta40 is predominantly an unstable and collapsed monomeric species, whereas Abeta42 populates a stable structured trimeric or tetrameric species at concentrations above approximately 12.5 microm. Thermodynamic analysis showed that the free energies of Abeta40 monomer and Abeta42 trimer/tetramer are approximately 1.1 and approximately 15/ approximately 22 kcal/mol, respectively. The early aggregation stages of Abeta40 and Abeta42 contain different solvent-exposed hydrophobic surfaces that are located at the sequences flanking its protease-resistant segment. The amyloidogenic folded structure of Abeta is important for the formation of spherical beta oligomeric species. However, beta oligomers are not an obligatory intermediate in the process of fibril formation because oligomerization is inhibited at concentrations of urea that have no effect on fibril formation. The distinct initial folding properties of Abeta40 and Abeta42 may play an important role in the higher aggregation potential and pathological significance of Abeta42.     

Promotion of amyloid beta protein misfolding and fibrillogenesis by a lipid oxidation product

Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid β proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain ω-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Aβ proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid β proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease.     

Accelerated accumulation of amyloid beta proteins on oxidatively damaged lipid membranes

The fully developed lesion of Alzheimer's Disease is a dense plaque composed of fibrillar amyloid beta-proteins with a characteristic and well-ordered beta-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form beta-sheet secondary structure, it is important to understand factors that induce amyloid beta-proteins to adopt this conformation. In this investigation we used a novel form of infrared spectroscopy that can characterize the conformation, orientation, and rate of accumulation of the protein on various lipid membranes to determine whether oxidatively damaged phospholipid membranes induce the formation of beta-sheet secondary structure in a 42-residue amyloid beta-protein. We found that membranes containing oxidatively damaged phospholipids accumulated amyloid beta-protein significantly faster than membranes containing only unoxidized or saturated phospholipids. Accelerated accumulation was also seen when 3 mol % G(M1) ganglioside was incorporated into a saturated phosphatidylcholine membrane. The accumulated protein more completely adopted a beta-sheet conformation on oxidized membranes, and the plane of the beta-sheet was oriented parallel to the plane of the membrane. These results indicate that oxidatively damaged phospholipid membranes promote beta-sheet formation by amyloid beta-proteins, and they suggest a possible role for lipid peroxidation in the pathogenesis of Alzheimer's Disease.     

Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo

Alzheimer's disease (AD) involves amyloid beta (Abeta) accumulation, oxidative damage, and inflammation, and risk is reduced with increased antioxidant and anti-inflammatory consumption. The phenolic yellow curry pigment curcumin has potent anti-inflammatory and antioxidant activities and can suppress oxidative damage, inflammation, cognitive deficits, and amyloid accumulation. Since the molecular structure of curcumin suggested potential Abeta binding, we investigated whether its efficacy in AD models could be explained by effects on Abeta aggregation. Under aggregating conditions in vitro, curcumin inhibited aggregation (IC(50) = 0.8 microM) as well as disaggregated fibrillar Abeta40 (IC(50) = 1 microM), indicating favorable stoichiometry for inhibition. Curcumin was a better Abeta40 aggregation inhibitor than ibuprofen and naproxen, and prevented Abeta42 oligomer formation and toxicity between 0.1 and 1.0 microM. Under EM, curcumin decreased dose dependently Abeta fibril formation beginning with 0.125 microM. The effects of curcumin did not depend on Abeta sequence but on fibril-related conformation. AD and Tg2576 mice brain sections incubated with curcumin revealed preferential labeling of amyloid plaques. In vivo studies showed that curcumin injected peripherally into aged Tg mice crossed the blood-brain barrier and bound plaques. When fed to aged Tg2576 mice with advanced amyloid accumulation, curcumin labeled plaques and reduced amyloid levels and plaque burden. Hence, curcumin directly binds small beta-amyloid species to block aggregation and fibril formation in vitro and in vivo. These data suggest that low dose curcumin effectively disaggregates Abeta as well as prevents fibril and oligomer formation, supporting the rationale for curcumin use in clinical trials preventing or treating AD.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

Amyloid beta-protein (Abeta)1-40 protects neurons from damage induced by Abeta1-42 in culture and in rat brain

Previously, we found that amyloid beta-protein (Abeta)1-42 exhibits neurotoxicity, while Abeta1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Abeta1-40 against Abeta1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Abeta1-42 at concentrations higher than 2 microm, which was prevented by concurrent treatment with Abeta1-40 in a dose-dependent manner. However, metal chelators did not prevent Abeta1-42-induced neuronal death. Circular dichroism spectroscopy showed that Abeta1-40 inhibited the beta-sheet transformation of Abeta1-42. Thioflavin-T assay and electron microscopy analysis revealed that Abeta1-40 inhibited the fibril formation of Abeta1-42. In contrast, Abeta1-16, Abeta25-35, and Abeta40-1 did not inhibit the fibril formation of Abeta1-42 nor prevent Abeta1-42-induced neuronal death. Abeta1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Abeta1-40. These results indicate that Abeta1-40 protects neurons from Abeta1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the beta-sheet transformation and fibril formation of Abeta1-42. Our data suggest a mechanism by which elevated Abeta1-42/Abeta1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.     

Structural and dynamic features of Alzheimer's Abeta peptide in amyloid fibrils studied by site-directed spin labeling

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimer's amyloid beta (Abeta) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23-26). Despite their different aggregation properties and roles in disease, the two peptides, Abeta40 and Abeta42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.     

The effect of cholesterol and monosialoganglioside (GM1) on the release and aggregation of amyloid beta-peptide from liposomes prepared from brain membrane-like lipids

In order to investigate the influence of cholesterol (Ch) and monosialoganglioside (GM1) on the release and subsequent deposition/aggregation of amyloid beta peptide (Abeta)-(1-40) and Abeta-(1-42), we have examined Abeta peptide model membrane interactions by circular dichroism, turbidity measurements, and transmission electron microscopy (TEM). Model liposomes containing Abeta peptide and a lipid mixture composition similar to that found in the cerebral cortex membranes (CCM-lipid) have been prepared. In all, four Abeta-containing liposomes were investigated: CCM-lipid; liposomes with no GM1 (GM1-free lipid); those with no cholesterol (Ch-free lipid); liposomes with neither cholesterol nor GM1 (Ch-GM1-free lipid). In CCM liposomes, Abeta was rapidly released from membranes to form a well defined fibril structure. However, for the GM1-free lipid, Abeta was first released to yield a fibril structure about the membrane surface, then the membrane became disrupted resulting in the formation of small vesicles. In Ch-free lipid, a fibril structure with a phospholipid membrane-like shadow formed, but this differed from the well defined fibril structure seen for CCM-lipid. In Ch-GM1-free lipid, no fibril structure formed, possibly because of membrane solubilization by Abeta. The absence of fibril structure was noted at physiological extracellular pH (7.4) and also at liposomal/endosomal pH (5.5). Our results suggest a possible role for both Ch and GM1 in the membrane release of Abeta from brain lipid bilayers.     

Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.     

Abeta42-peptide assembly on lipid bilayers

One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are composed predominantly of the amyloid-beta peptide (Abeta). Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta42-membrane interactions using in situ atomic force microscopy and fluorescence spectroscopy. Our studies demonstrate the association of Abeta42 with planar bilayers composed of total brain lipids, which results initially in peptide aggregation and then fibre formation. Modulation of the cholesterol content is correlated with the extent of Abeta42-assembly on the bilayer surface. Although Abeta42 was not visualized directly on cholesterol-depleted bilayers, fluorescence anisotropy and fluorimetry demonstrate Abeta42-induced membrane changes. Our results demonstrate that the composition of the lipid bilayer governs the outcome of Abeta interactions.     

Abeta46 is processed to Abeta40 and Abeta43, but not to Abeta42, in the low density membrane domains

Gamma-secretase cleaves the transmembrane domain of beta-amyloid precursor protein at multiple sites referred to as gamma-, epsilon-, and zeta-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide gamma-secretase inhibitor, causes differential accumulation of longer amyloid beta-proteins (Abetas) within Chinese hamster ovary cells co-expressing beta C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Abeta46 localized exclusively to low density membrane (LDM) domains. Incubating the Abeta46-accumulating LDM domains at 37 degrees C produced Abeta40, Abeta42, Abeta43, and beta-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented beta-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Abeta46 and the concomitant appearance of Abeta40 and Abeta43 but not Abeta42. Further addition of DAPT suppressed the production of Abeta40/43 and abolished the decrease in the amount of Abeta46. These data indicate that preaccumulated Abeta46 is processed by gamma-secretase to Abeta40/43 but not to Abeta42 in the LDM domains. The amount of newly produced Abeta40 and Abeta43 was roughly equivalent to the decrease in the amount of Abeta46. Temporal profiles did not show a maximal concentration for Abeta43, suggesting that Abeta46 is processed to Abeta40 and Abeta43 through a nonsuccessive process.     

The ratio of monomeric to aggregated forms of Abeta40 and Abeta42 is an important determinant of amyloid-beta aggregation, fibrillogenesis, and toxicity

Aggregation and fibril formation of amyloid-beta (Abeta) peptides Abeta40 and Abeta42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of Abeta40 to Abeta42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of Abeta. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Abeta40 to Abeta42, we probed the concentration- and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric Abeta40 alters the kinetic stability, solubility, and morphological properties of Abeta42 aggregates and prevents their conversion into mature fibrils. Abeta40, at approximately equimolar ratios (Abeta40/Abeta42 approximately 0.5-1), inhibits (> 50%) fibril formation by monomeric Abeta42, whereas inhibition of protofibrillar Abeta42 fibrillogenesis is achieved at lower, substoichiometric ratios (Abeta40/Abeta42 approximately 0.1). The inhibitory effect of Abeta40 on Abeta42 fibrillogenesis is reversed by the introduction of excess Abeta42 monomer. Additionally, monomeric Abeta42 and Abeta40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar Abeta aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, Abeta42 protofibrils selectively seed the fibrillogenesis of monomeric Abeta42 but not monomeric Abeta40. Finally, we also show that the amyloidogenic propensities of different individual and mixed Abeta species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of Abeta40 to Abeta42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease.     

Electrophysiologic properties of channels induced by Abeta25-35 in planar lipid bilayers

Abeta25-35, a fragment of the neurotoxic amyloid beta protein Abeta1-42 found in the brain of Alzheimer patients, possesses amyloidogenic, neurotoxins and channel forming abilities similar to that of Abeta1-42. We have previously reported that Abeta25-35 formed voltage-dependent, relatively nonselective, ion-permeable channels in planar lipid bilayers. Here, we show that Abeta25-35 formed channels in both solvent-containing and solvent-free bilayers. We also report that for Abeta25-35, channel forming activity was dependent on ionic strength, membrane lipid composition, and peptide concentration, but not on pH. Lower ionic strength and negatively charged lipids increased channel formation activity, while cholesterol decreased activity. The nonlinear function relating [Abeta25-35] and membrane activity suggests that aggregation of at least three monomers is required for channel formation.     

Solid-state NMR as a method to reveal structure and membrane-interaction of amyloidogenic proteins and peptides

It is important to understand the Amyloid fibril formation in view of numerous medical and biochemical aspects. Structural determination of amyloid fibril has been extensively studied using electron microscopy. Subsequently, solid state NMR spectroscopy has been realized to be the most important means to determine not only microscopic molecular structure but also macroscopic molecular packing. Molecular structure of amyloid fibril was first predicted to be parallel beta-sheet structure, and subsequently, was further refined for Abeta(1-40) to be cross beta-sheet with double layered in register parallel beta-sheet structure by using solid state NMR spectroscopy. On the other hand, anti-parallel beta-sheet structure has been reported to short fragments of Abeta-amyloid and other amyloid forming peptides. Kinetic study of amyloid fibril formation has been studied using a variety of methods, and two-step autocatalytic reaction mechanism used to explain fibril formation. Recently, stable intermediates or proto-fibrils have been observed by electron microscope (EM) images. Some of the intermediates have the same microscopic structure as the matured fibril and subsequently change to matured fibrils. Another important study on amyloid fibril formation is determination of the interaction with lipid membranes, since amyloid peptide are cleaved from amyloid precursor proteins in the membrane interface, and it is reported that amyloid lipid interaction is related to the cytotoxicity. Finally it is discussed how amyloid fibril formation can be inhibited. Firstly, properly designed compounds are reported to have inhibition ability of amyloid fibril formation by interacting with amyloid peptide. Secondly, it is revealed that site directed mutation can inhibit amyloid fibril formation. These inhibitors were developed by knowing the fibril structure determined by solid state NMR.     

Cellular membrane composition defines A beta-lipid interactions.

Alzheimer's disease pathology has demonstrated amyloid plaque formation associated with plasma membranes and the presence of intracellular amyloid-beta (A beta) accumulation in specific vesicular compartments. This suggests that lipid composition in different compartments may play a role in A beta aggregation. To test this hypothesis, we have isolated cellular membranes from human brain to evaluate A beta 40/42-lipid interactions. Plasma, endosomal, lysosomal, and Golgi membranes were isolated using sucrose gradients. Electron microscopy demonstrated that A beta fibrillogenesis is accelerated in the presence of plasma and endosomal and lysosomal membranes with plasma membranes inducing an enhanced surface organization. Alternatively, interaction of A beta with Golgi membranes fails to progress to fibril formation, suggesting that A beta-Golgi head group interaction stabilizes A beta. Fluorescence spectroscopy using the environment-sensitive probes 1,6-diphenyl-1,3,5-hexatriene, laurdan, N-epsilon-dansyl-L-lysine, and merocyanine 540 demonstrated variations in the inherent lipid properties at the level of the fatty acyl chains, glycerol backbone, and head groups, respectively. Addition of A beta 40/42 to the plasma and endosomal and lysosomal membranes decreases the fluidity not only of the fatty acyl chains but also the head group space, consistent with A beta insertion into the bilayer. In contrast, the Golgi bilayer fluidity is increased by A beta 40/42 binding which appears to result from lipid head group interactions and the production of interfacial packing defects.     

Amyloid-beta peptide disruption of lipid membranes and the effect of metal ions

Beta-amyloid peptide (Abeta), which is cleaved from the larger trans-membrane amyloid precursor protein, is found deposited in the brain of patients suffering from Alzheimer's disease and is linked with neurotoxicity. We report the results of studies of Abeta1-42 and the effect of metal ions (Cu2+ and Zn2+) on model membranes using 31P and 2H solid-state NMR, fluorescence and Langmuir Blodgett monolayer methods. Both the peptide and metal ions interact with the phospholipid headgroups and the effects on the lipid bilayer and the peptide structure were different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induced formation of smaller vesicles but when Abeta1-42 was associated with the bilayer membrane copper did not have this effect. Circular dichroism spectroscopy indicated that Abeta1-42 adopted more beta-sheet structure when incorporated in a lipid bilayer in comparison to the associated peptide, which was largely unstructured. Incorporated peptides appear to disrupt the membrane more severely than associated peptides, which may have implications for the role of Abeta in disease states.     

Apoptosis induced in neuronal cells by C-terminal amyloid beta-fragments is correlated with their aggregation properties in phospholipid membranes

A number of findings suggest that lipophilic monomeric Abeta peptides can interact with the cellular lipid membranes. These interactions can affect the membrane integrity and result in the initiation of apoptotic cell death. The secondary structure of C-terminal Abeta peptides (29-40) and the longer (29-42) variant have been investigated in solution by circular dichroism measurements. The secondary structure of lipid bound Abeta (29-40) and (29-42) peptides prepared at different lipid/peptide ratio's, was investigated by ATR-FTIR spectroscopy. Finally, the changes in secondary structure (i.e. the transition of alpha-helix to beta-sheet) of the lipid bound peptides were correlated with the induction of neurotoxic and apoptotic effects in neuronal cells. The data suggest that the C-terminal fragments of the Abeta peptide induce a significant apoptotic cell death, as demonstrated by caspase-3 measurements and DNA laddering, with consistently a stronger effect of the longer Abeta (29-42) variant. Moreover, the induction of apoptotic death induced by these peptides can be correlated with the secondary structure of the lipid bound amyloid beta peptides. Based on these observations, it is proposed that membrane bound aggregated Abeta peptides (produced locally as the result of gamma-secretase cleavage) can accumulate and aggregate in the membrane. These membrane bound beta-sheet aggregated amyloid peptides induce neuronal apoptotic cell death.     

The Tottori (D7N) and English (H6R) familial Alzheimer disease mutations accelerate Abeta fibril formation without increasing protofibril formation

A subset of Alzheimer disease cases is caused by autosomal dominant mutations in genes encoding the amyloid beta-protein precursor or presenilins. Whereas some amyloid beta-protein precursor mutations alter its metabolism through effects on Abeta production, the pathogenic effects of those that alter amino acid residues within the Abeta sequence are not fully understood. Here we examined the biophysical effects of two recently described intra-Abeta mutations linked to early-onset familial Alzheimer disease, the D7N Tottori-Japanese and H6R English mutations. Although these mutations do not affect Abeta production, synthetic Abeta(1-42) peptides carrying D7N or H6R substitutions show enhanced fibril formation. In vitro analysis using Abeta(1-40)-based mutant peptides reveal that D7N or H6R mutations do not accelerate the nucleation phase but selectively promote the elongation phase of amyloid fibril formation. Notably, the levels of protofibrils generated from D7N or H6R Abeta were markedly inhibited despite enhanced fibril formation. These N-terminal Abeta mutations may accelerate amyloid fibril formation by a unique mechanism causing structural changes of Abeta peptides, specifically promoting the elongation process of amyloid fibrils without increasing metastable intermediates.