Beta-amyloid (Abeta40, Abeta42) binding to modified LDL accelerates macrophage foam cell formation

Apart from its role as a risk factor in arteriosclerosis, plasma cholesterol is increasingly recognized to play a major role in the pathogenesis of Alzheimer's disease (AD). Moreover, alterations of intracellular cholesterol metabolism in neuronal and vascular cells are of considerable importance for the understanding of AD. Cellular cholesterol accumulation enhances the deposition of insoluble beta-amyloid peptides, which is considered a hallmark in the pathogenesis of AD. In order to test the hypothesis, whether exogenous beta-amyloid peptides (Abeta42, Abeta40) might contribute to cellular cholesterol accumulation by opsonization of lipoproteins, we compared the binding and uptake of native LDL, enzymatically modified LDL (E-LDL), copper oxidized LDL (Ox-LDL) and HDL as control, preincubated either in the absence or presence of Abeta42 or Abeta40, by human monocytes or monocyte-derived macrophages. Incubation of monocytes and macrophages with Abeta-lipoprotein-complexes lead to increased cellular free and esterified cholesterol when compared to non-opsonized lipoproteins, except for HDL. Furthermore, the cellular uptake of these complexes regulated Abeta-receptors such as FPRL-1 or LRP/CD91. In summary, our results suggest that Abeta42 and Abeta40 act as potent opsonins for LDL, E-LDL and Ox-LDL and enhance cellular cholesterol accumulation as well as Abeta-deposition in vessel wall macrophages.     

Biogenesis and metabolism of Alzheimer's disease Abeta amyloid peptides

Biochemical and genetic evidence indicates the balance of biogenesis/clearance of Abeta amyloid peptides is altered in Alzheimer's disease. Abeta is derived, by two sequential cleavages, from the receptor-like amyloid precursor protein (APP). The proteases involved are beta-secretase, identified as the novel aspartyl protease BACE, and gamma-secretase, a multimeric complex containing the presenilins (PS). Gamma-secretase can release either Abeta40 or the more aggregating and cytotoxic Abeta42. Secreted Abeta peptides become either degraded by the metalloproteases insulin-degrading enzyme (IDE) and neprilysin or metabolized through receptor uptake mediated by apolipoprotein E. Therapeutic approaches based on secretase inhibition or amyloid clearance are currently under development.     

Abeta42 is more rigid than Abeta40 at the C terminus: implications for Abeta aggregation and toxicity

Abeta40 and Abeta42 are the major forms of amyloid beta peptides (Abeta) in the brain. Although Abeta42 differs from Abeta40 by only two residues, Abeta42 is much more prone to aggregation and more toxic to neurons than Abeta40. To probe whether dynamics contribute to such dramatic difference in function, backbone ps-ns dynamics of native Abeta monomers were characterized by 15N spin relaxation at 273.3 K and 800 MHz. Abeta42 aggregates much faster than Abeta40 in the NMR tube. The effect of Abeta aggregation was removed from the relaxation measurement by interleaved data collection. R1, R2 and nuclear Overhauser enhancement (NOE) values are similar in Abeta40 and Abeta42, except at the C terminus, indicating Abeta42 and Abeta40 monomers have identical global motions. Comparisons of the spectral density function J(0.87omegaH) and order parameters (S2) indicate that the Abeta42 C terminus is more rigid than the Abeta40 C terminus. At 280.4 K and 287.6 K, the Abeta42 C terminus remains more rigid than the Abeta40 C terminus, suggesting such a dynamical difference is likely present at the physiological temperature. The Abeta42 monomer likely has less configurational entropy due to restricted motion in the C terminus and may pay a smaller entropic price to form fibrils than the Abeta40 monomer. We hypothesize that the entropic difference between Abeta40 and Abeta42 monomers might partly account for the fact that Abeta42 is the major Abeta species in parenchymal senile plaques in most Alzheimer's diseased brains in spite of the predominance of Abeta40 in plasma. The increased rigidity of the Abeta42 C terminus is likely due to its pre-ordering for beta-conformation present in soluble oligomers and fibrils. The Abeta42 C terminus may therefore serve as an internal seed for aggregation.     

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.     

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.     

Early synergy between Abeta42 and oxidatively damaged membranes in promoting amyloid fibril formation by Abeta40

Oxidative lipid membrane damage is known to promote the misfolding of Abeta42 into pathological beta structure. In fully developed senile plaques of Alzheimer's disease, however, it is the shorter and more soluble amyloid beta protein, Abeta40, that predominates. To investigate the role of oxidative membrane damage in the misfolding of Abeta40, we have examined its interaction with supported lipid monolayer membranes using internal reflection infrared spectroscopy. Oxidatively damaged lipids modestly increased Abeta40 accumulation, with adsorption kinetics and a conformation that are distinct from that of Abeta42. In stark contrast, pretreatment of oxidatively damaged monolayer membranes with Abeta42 vigorously promoted Abeta40 accumulation and misfolding. Pretreatment of saturated or undamaged membranes with Abeta42 had no such effect. Parallel studies of lipid bilayer vesicles using a dye binding assay to detect fibril formation and electron microscopy to examine morphology demonstrated that Abeta42 pretreatment of oxidatively damaged membranes promoted the formation of mature Abeta40 amyloid fibrils. We conclude that oxidative membrane damage and Abeta42 act synergistically at an early stage to promote fibril formation by Abeta40. This synergy could be detected within minutes using internal reflection spectroscopy, whereas a dye-binding assay required several days and much higher protein concentrations to demonstrate this synergy.     

Urea-based two-dimensional electrophoresis of beta-amyloid peptides in human plasma: evidence for novel Abeta species

The detailed analysis of beta-amyloid (Abeta) peptides in human plasma is still hampered by the limited sensitivity of available mass spectrometric methods and the lack of appropiate ELISAs to measure Abeta peptides other than Abeta(1-38), Abeta(1-40), and Abeta(1-42). By combining high-yield Abeta immuno- precipitation (IP), IEF, and urea-based Abeta-SDS-PAGE-immunoblot, at least 30 Abeta-immuno-reactive spots were detected in human plasma samples as small as 1.6 mL. This approach clearly resolved Abeta peptides Abeta(1-40), Abeta(1-42), Abeta(1-37), Abeta(1-38), Abeta(1-39), the N-truncated Abeta(2-40), Abeta(2-42), and, for the first time, also Abeta(1-41). Relative quantification indicated that Abeta(1-40) and Abeta(1-42) accounted for less than 60% of the total amount of Abeta peptides in plasma. All other Abeta peptides appear to be either C-terminally or N-terminally truncated forms or as yet uncharacterized Abeta species which migrated as trains of spots with distinct pIs. The Abeta pattern found in cerebrospinal fluid (CSF) was substantially less complex. This sensitive method (2-D Abeta-WIB) might help clarifying the origin of distinct Abeta species from different tissues, cell types, or intracellular pools as well as their amyloidogenicity. It might further help identifying plasma Abeta species suitable as biomarkers for the diagnosis of Alzheimer's disease (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.     

Methyl dynamics of the amyloid-beta peptides Abeta40 and Abeta42

To probe the role of side chain dynamics in Abeta aggregation, we studied the methyl dynamics of native Abeta40 and Abeta42 by measuring cross relaxation rates with interleaved data collection. The methyl groups in the C-terminus are in general more rigid in Abeta42 than in Abeta40, consistent with previous results from backbone (15)N dynamics. This lends support to the hypothesis that a rigid C-terminus in Abeta42 may serve as an internal aggregation seed. Interestingly, two methyl groups of V18 located in the central hydrophobic cluster are more mobile in Abeta42 than in Abeta40, most likely due to the paucity of V18 intra-molecular interactions in Abeta42. V18 may then be more available for inter-molecular interactions to form Abeta42 aggregates. Thus, the side chain mobility of the central hydrophobic cluster may play an important role in Abeta aggregation and may contribute to the difference in aggregation propensity between Abeta40 and Abeta42.     

Plasma beta carotene in Alzheimer's disease. Association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), beta-amyloid 1-42 (Abeta42) and total Tau

We studied the plasma beta carotene concentrations in 40 Alzheimer's disease patients and the association with cerebrospinal fluid beta-amyloid 1-40, (Abeta40), cerebrospinal fluid beta-amyloid 1-42 (Abeta42) and cerebrospinal fluid total Tau. We found that patients with plasma beta carotene levels below the 25th percentile had 55% reduced ratios of Abeta40/Tau and 51% reduced ratios of Abeta 40/Abeta 42 compared with patients in the highest quartile. Mean Tau concentrations in the lowest quartile of plasma beta-carotene levels were 74% higher compared with the highest quartile of plasma beta-carotene levels. Thus, we could demonstrate an statistically significant association between beta carotene levels in plasma and neurochemical markers in the cerebrospinal fluid of Alzheimer's disease patients.     

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.     

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.     

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.     

Independent generation of Abeta42 and Abeta38 peptide species by gamma-secretase

Proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase generates the amyloid-beta (Abeta) peptides, which are principal drug targets in Alzheimer disease therapeutics. gamma-Secretase has imprecise cleavage specificity and generates the most abundant Abeta40 and Abeta42 species together with longer and shorter peptides such as Abeta38. Several mechanisms could explain the production of multiple Abeta peptides by gamma-secretase, including sequential processing of longer into shorter Abeta peptides. A novel class of gamma-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Abeta42 levels without a change in Abeta40 levels. A signature of GSMs is the concomitant increase in shorter Abeta peptides, such as Abeta38, leading to the suggestion that generation of Abeta42 and Abeta38 peptide species by gamma-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Abeta42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Abeta38 production would be similarly affected indicating coupled generation of Abeta42 and Abeta38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Abeta38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Abeta peptide species. These data provide evidence that Abeta42 and Abeta38 species can be independently generated by gamma-secretase and argue against a precursor-product relationship between these peptides.     

Cytokine-mediated inhibition of fibrillar amyloid-beta peptide degradation by human mononuclear phagocytes

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-beta peptides (Abeta) in brain. Although Abeta uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Abeta degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Abeta using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer (125)I-Abeta40 and Abeta42. Initial uptake of fibrillar Abeta40 and Abeta42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Abeta less than 0.5% and saturation of degradation within 24 h. IFN-gamma increased the intracellular retention of fibrillar Abeta40 and Abeta42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-beta1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Abeta degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-gamma and TNF-alpha directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Abeta degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Abeta degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.     

{gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE

Mutation of the amyloid precursor protein (APP), presenilin-1, or presenilin-2 results in the development of early onset autosomal dominant forms of Alzheimer disease (AD). These mutations lead to an increased Abeta42/Abeta40 ratio that correlates with the onset of disease. However, it remains unknown how these mutations affect gamma-secretase, a protease that generates the termini of Abeta40 and Abeta42. Here we have determined the reaction mechanism of gamma-secretase with wild type and three mutated APP substrates. Our findings indicate that despite the overall outcome of an increased Abeta42/Abeta40 ratio, these mutations each display rather distinct reactivity to gamma-secretase. Intriguingly, we found that the ratio of Abeta42/Abeta40 is variable with substrate concentration; increased substrate concentrations result in higher ratios of Abeta42/Abeta40. Moreover, we demonstrated that reduction of gamma-secretase substrate concentration by BACE1 inhibition in cells decreased the Abeta42/Abeta40 ratio. This study indicates that biological factors affecting targets such as BACE1 and APP, which ultimately cause an increased concentration of gamma-secretase substrate, can augment the Abeta42/Abeta40 ratio and may play a causative role in sporadic AD. Therefore, strategies lowering the Abeta42/Abeta40 ratio through partial reduction of gamma-secretase substrate production may introduce a practical therapeutic modality for treatment of AD.     

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.     

High levels of circulating Abeta42 are sequestered by plasma proteins in Alzheimer's disease

A previously unrecognized large pool of Abeta was discovered in freshly drawn plasma of patients diagnosed with Alzheimer's disease (AD) and non-demented control subjects. This Abeta pool was revealed after acid denaturation and chromatographic separation of plasma proteins followed by Abeta quantitation in the 4.5 kDa fractions by europium immunoassay. The mean values of Abeta42 in the AD and control individuals amounted to 236 ng/ml and 38 ng/ml, respectively. These Abeta values are on the average far higher than previously measured. Surprisingly, the circulating Abeta42 is about 16 times more abundant than Abeta40 in the AD population. Addition of Abeta to freshly drawn plasma demonstrated the rapid disappearance of Abeta epitopes, as detected by immunochemical techniques, suggesting either proteolytic degradation or Abeta sequestration. Incubation of Abeta with purified plasma proteins and lipoproteins rapidly decreases detectable levels of free Abeta suggesting epitope masking as the likely mechanism. The free and protein-bound Abetab in the circulation may represent a potential source for deposition in the cerebrovasculature and brain parenchyma of AD.     

Secreted Abeta does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein

Antibodies against APP, a precursor of Abeta deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Abeta mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Abeta40 and Abeta42 in the cultured media were undetectable by an assay capable of detecting 100 nM Abeta peptides. However, exogenously treated Abeta1-42 or Abeta1-43 required >3 microM to exert neurotoxicity, and 25 microM Abeta1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Abeta1-42, whereas serum attenuated toxicity by Abeta1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His(657)-Lys(676), but not the Abeta1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Abeta.     

The effect of Abeta conformation on the metal affinity and aggregation mechanism studied by circular dichroism spectroscopy

The conformational change and associated aggregation of beta amyloid (Abeta) with or without metals is the main cause of Alzheimer's disease (AD). In order to further understand the effects of Abeta and its associated metals on the aggregation mechanism, the influence of Abeta conformation on the metal affinity and aggregation was investigated using circular dichroism (CD) spectroscopy. The Abeta conformation is dependent on pH and trifluoroethanol (TFE). The binding of metals to Abeta was found to be dependent on the Abeta conformation. The aggregation induced by Abeta itself or its associated metals is completely diminished for Abeta in 40% TFE. Only in 5% and 25% TFE can Abeta undergo an alpha-helix to beta-sheet aggregation, which involve a three-state mechanism for the metal-free state, and a two-state transition for the metal-bound state, respectively. The aggregation-inducing activity of metals is in the order, Cu2+ > Fe3+ > or = Al3+ > Zn2+.