Structure and orientation of peptide inhibitors bound to beta-amyloid fibrils

Polymerization of the soluble beta-amyloid peptide into highly ordered fibrils is hypothesized to be a causative event in the development of Alzheimer's disease. Understanding the interactions of Abeta with inhibitors on an atomic level is fundamental for the development of diagnostics and therapeutic approaches, and can provide, in addition, important indirect information of the amyloid fibril structure. We have shown recently that trRDCs can be measured in solution state NMR for peptide ligands binding weakly to amyloid fibrils. We present here the structures for two inhibitor peptides, LPFFD and DPFFL, and their structural models bound to fibrillar Abeta(14-23) and Abeta(1-40) based on transferred nuclear Overhauser effect (trNOE) and transferred residual dipolar coupling (trRDC) data. In a first step, the inhibitor peptide structure is calculated on the basis of trNOE data; the trRDC data are then validated on the basis of the trNOE-derived structure using the program PALES. The orientation of the peptide inhibitors with respect to Abeta fibrils is obtained from trRDC data, assuming that Abeta fibrils orient such that the fibril axis is aligned in parallel with the magnetic field. The trRDC-derived alignment tensor of the peptide ligand is then used as a restraint for molecular dynamics docking studies. We find that the structure with the lowest rmsd value is in agreement with a model in which the inhibitor peptide binds to the long side of an amyloid fibril. Especially, we detect interactions involving the hydrophobic core, residues K16 and E22/D23 of the Abeta sequence. Structural differences are observed for binding of the inhibitor peptide to Abeta14-23 and Abeta1-40 fibrils, respectively, indicating different fibril structure. We expect this approach to be useful in the rational design of amyloid ligands with improved binding characteristics.     

Influence of fluorinated and hydrogenated nanoparticles on the structure and fibrillogenesis of amyloid beta-peptide

Peptide aggregation in amyloid fibrils is implicated in the pathogenesis of several diseases such as Alzheimer's disease. There is a strong correlation between amyloid fibril formation and a decrease in conformational stability of the native state. Amyloid-beta peptide (Abeta), the aggregating peptide in Alzheimer's disease, is natively unfolded. The deposits found in Alzheimer's disease are composed of Abeta fibrillar aggregates rich in beta-sheet structure. The influence of fluorinated complexes on the secondary structure and fibrillogenesis of Abeta peptide was studied by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). CD spectra show that complexes of polyampholyte and fluorinated dodecanoic acid induce alpha-helix structure in Abeta, but their hydrogenated analogous lead to beta-sheet formation and aggregation. The fluorinated nanoparticles with highly negative zeta potential and hydrophobic fluorinated core have the fundamental characteristics to prevent Abeta fibrillogenesis.     

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.     

Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.     

Humoral immune response to fibrillar beta-amyloid peptide

The beta-amyloid peptide (Abeta) is a normal product of the proteolytic processing of its precursor (beta-APP). Normally, it elicits a very low humoral immune response; however, the aggregation of monomeric Abeta to form fibrillar Abeta amyloid creates a neo-epitope, to which antibodies are generated. Rabbits were injected with fibrillar human Abeta(1-42), and the resultant antibodies were purified and their binding properties characterized. The antibodies bound to an epitope in the first eight residues of Abeta and required a free amino terminus. Additional residues did not affect the affinity of the epitope as long as the peptide was unaggregated; the antibody bound Abeta residues 1-8, 1-11, 1-16, 1-28, 1-40, and 1-42 with similar affinities. In contrast, the antibodies bound approximately 1000-fold more tightly to fibrillar Abeta(1-42). Their enhanced affinity did not result from their bivalent nature: monovalent Fab fragments exhibited a similar affinity for the fibrils. Nor did it result from the particulate nature of the epitope: monomeric Abeta(1-16) immobilized on agarose and soluble Abeta(1-16) exhibited similar affinities for the antifibrillar antibodies. In addition, antibodies raised to four nonfibrillar peptides corresponding to internal Abeta sequences did not exhibit enhanced affinity for fibrillar Abeta(1-42). Antibodies directed to the C-terminus of Abeta bound poorly to fibrillar Abeta(1-42), which is consistent with models where the carboxyl terminus is buried in the interior of the fibril and the amino terminus is on the surface. When used as an immunohistochemical probe, the antifibrillar Abeta(1-42) IgG exhibited enhanced affinity for amyloid deposits in the cerebrovasculature. We hypothesize either that the antibodies recognize a specific conformation of the eight amino-terminal residues of Abeta, which is at least 1000-fold more favored in the fibril than in monomeric peptides, or that affinity maturation of the antibodies produces an additional binding site for the amino-terminal residues of an adjacent Abeta monomer. In vivo this specificity would direct the antibody primarily to fibrillar vascular amyloid deposits even in the presence of a large excess of monomeric Abeta or its precursor. This observation may explain the vascular meningeal inflammation that developed in Alzheimer's disease patients immunized with fibrillar Abeta. Passive immunization with an antibody directed to an epitope hidden in fibrillar Abeta and in the transmembrane region of APP might be a better choice in the search for an intervention to remove Abeta monomers without provoking an inflammatory response.     

Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease.

The extracellular accumulation of amyloid-beta (Abeta) in neuritic plaques is one of the characteristic hallmarks of Alzheimer's disease (AD), a progressive dementing neurodegenerative disorder of the elderly. By virtue of its structure, Abeta is able to bind to a variety of biomolecules, including lipids, proteins and proteoglycans. The binding of the various forms of Abeta (soluble or fibrillar) to plasma membranes has been studied with regard to the direct toxicity of Abeta to neurons, and the activation of a local inflammation phase involving microglia. The binding of Abeta to membrane lipids facilitates Abeta fibrillation, which in turn disturbs the structure and function of the membranes, such as membrane fluidity or the formation of ion channels. A subset of membrane proteins binds Abeta. The serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind the monomeric form of Abeta. The alpha7nicotinic acetylcholine receptor (alpha7nAChR), integrins, RAGE (receptor for advanced glycosylation end-products) and FPRL1 (formyl peptide receptor-like 1) are able to bind the monomeric and fibrillar forms of Abeta. In addition, APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), the P75 neurotrophin receptor (P75NTR), the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, alpha6beta1-integrin and CD47 have been reported to bind the fibrillar form of Abeta. Heparan sulfate proteoglycans have also been described as cell-surface binding sites for Abeta. The various effects of Abeta binding to these membrane molecules are discussed.     

High density lipoproteins bind Abeta and apolipoprotein C-II amyloid fibrils

Disease-associated amyloid deposits contain both fibrillar and nonfibrillar components. The majority of these amyloid components originate or coexist in the bloodstream. To understand the nature of the interaction between the nonfibrillar and fibrillar components, we have developed a centrifugation method to isolate fibril binding proteins from human serum. Amyloid fibrils composed of either Abeta peptide or apolipoprotein C-II (apoC-II) cosedimented with specific serum proteins. Gel electrophoresis, mass spectrometry peptide fingerprinting, and Western analysis identified the major binding species as proteins found in HDL particles, including apoA-I, apoA-II, apoE, clusterin, and serum amyloid A. Sedimentation analysis showed that purified human HDL and recombinant apoA-I lipid particles bound directly to Abeta and apoC-II amyloid fibrils. These studies reveal a novel function of HDL that may contribute to the well-established protective effect of this lipoprotein class in heart disease.     

Amyloid-beta peptide assembly: a critical step in fibrillogenesis and membrane disruption

Identifying the mechanisms responsible for the assembly of proteins into higher-order structures is fundamental to structural biology and understanding specific disease pathways. The amyloid-beta (Abeta) peptide is illustrative in this regard as fibrillar deposits of Abeta are characteristic of Alzheimer's disease. Because Abeta includes portions of the extracellular and transmembrane domains of the amyloid precursor protein, it is crucial to understand how this peptide interacts with cell membranes and specifically the role of membrane structure and composition on Abeta assembly and cytotoxicity. We describe the results of a combined circular dichroism spectroscopy, electron microscopy, and in situ tapping mode atomic force microscopy (TMAFM) study of the interaction of soluble monomeric Abeta with planar bilayers of total brain lipid extract. In situ extended-duration TMAFM provided evidence of membrane disruption via fibril growth of initially monomeric Abeta1-40 peptide within the total brain lipid bilayers. In contrast, the truncated Abeta1-28 peptide, which lacks the anchoring transmembrane domain found in Abeta1-40, self-associates within the lipid headgroups but does not undergo fibrillogenesis. These observations suggest that the fibrillogenic properties of Abeta peptide are in part a consequence of membrane composition, peptide sequence, and mode of assembly within the membrane.     

Mechanism of accelerated assembly of beta-amyloid filaments into fibrils by KLVFFK(6)

Extracellular senile plaques are a central pathological feature of Alzheimer's disease. At the core of these plaques are fibrillar deposits of beta-amyloid peptide (Abeta). In vitro, Abeta spontaneously assembles into amyloid fibrils of cross-beta sheet structure. Although it was once believed that the fibrils themselves were toxic, more recent data supports the hypothesis that aggregation intermediates, rather than fully formed fibrils, are the most damaging to neuronal tissue. In previously published work, we identified several small peptides that interact with Abeta and increase its aggregation rate while decreasing its toxicity. In this work, we examined in detail the interaction between Abeta and one of these peptides. Using a mathematical model of Abeta aggregation kinetics, we show that the dominant effect of the peptide is to accelerate lateral association of Abeta filaments into fibrils.     

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

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

Polymorphic fibril formation by residues 10-40 of the Alzheimer's beta-amyloid peptide

We report investigations of the morphology and molecular structure of amyloid fibrils comprised of residues 10-40 of the Alzheimer's beta-amyloid peptide (Abeta(10-40)), prepared under various solution conditions and degrees of agitation. Omission of residues 1-9 from the full-length Alzheimer's beta-amyloid peptide (Abeta(1-40)) did not prevent the peptide from forming amyloid fibrils or eliminate fibril polymorphism. These results are consistent with residues 1-9 being disordered in Abeta(1-40) fibrils, and show that fibril polymorphism is not a consequence of disorder in residues 1-9. Fibril morphology was analyzed by atomic force and electron microscopy, and secondary structure and inter-side-chain proximity were probed using solid-state NMR. Abeta(1-40) fibrils were found to be structurally compatible with Abeta(10-40): Abeta(1-40) fibril fragments were used to seed the growth of Abeta(10-40) fibrils, with propagation of fibril morphology and molecular structure. In addition, comparison of lyophilized and hydrated fibril samples revealed no effect of hydration on molecular structure, indicating that Abeta(10-40) fibrils are unlikely to contain bulk water.     

Domains 16 and 17 of tropoelastin in elastic fibre formation

AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39-43 residue long Abeta (amyloid-beta)-peptide. The most abundant species, Abeta(1-40) and Abeta(1-42), are both present within senile plaques, but Abeta(1-42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Abeta-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Abeta(1-40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Abeta(1-42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Abeta(1-40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two beta-strands connected with a bend. This protection pattern partly resembles the pattern found in Abeta(1-42) fibrils, but the Abeta(1-40) fibrils display a significantly increased protection for the N-terminal residues Phe4-His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37-Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.     

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.     

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.     

Surface structure of amyloid-beta fibrils contributes to cytotoxicity

Amyloid beta (Abeta) toxicity has been hypothesized to initiate the pathogenesis of Alzheimer's disease (AD). The characteristic fibrillar morphology of Abeta-aggregates, that constitute the main components of senile plaque, has long been considered to account for the neurotoxicity. But recent reports argue against a primary role for mature fibrils in AD pathogenesis because of the lack of a robust correlation between the severity of neurological impairment and the extent of amyloid deposition. Toxicity from the soluble prefibrillar intermediate entity of aggregates often called oligomer has recently proposed a plausible explanation for this inconsistency. An alternative explanation is based on the observation that certain amyloid fibril morphologies are more toxic than others, indicating that not all amyloid fibrils are equally toxic. Here, we report that it is not only the beta-sheeted fibrillar structure but also the surface physicochemical composition that affects the toxicity of Abeta fibrils. For the first time, colloidal gold was used to visualize by electron microscopy positive-charge clusters on Abeta fibrils. Chemical modifications as well as point-mutated Abeta synthesis techniques were applied to change the surface structures of Abeta and to show how local structure affects surface properties that are responsible for electrostatic and hydrophobic interactions with cells. We also report that covering the surface of Abeta fibers with myelin basic protein, which has surface properties contrary to those of Abeta, suppresses Abeta toxicity. On the basis of these results, we propose that the surface structure of Abeta fibrils plays an important role in Abeta toxicity.     

Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR

Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled peptide are necessary. Bacterial expression of recombinant amyloid proteins and peptides allows uniform isotopic labeling, as well as other patterns of isotope incorporation. However, large-scale production of recombinant amyloidogenic peptides has proven particularly difficult, due to their inherent propensity for aggregation and the associated toxicity of fibrillar material. Yields of recombinant protein are further reduced by the small molecular weights of short amyloidogenic fragments. Here, we report high-yield expression and purification of a peptide comprising residues 11-26 of the Alzheimer's beta-amyloid protein (Abeta(11-26)), with homoserine lactone replacing serine at residue 26. Expression in inclusion bodies as a ketosteroid isomerase fusion protein and subsequent purification under denaturing conditions allows production of milligram quantities of uniformly labeled (13)C- and (15)N-labeled peptide, which forms amyloid fibrils suitable for solid-state NMR spectroscopy. Initial structural data obtained by atomic force microscopy, electron microscopy, and solid-state NMR measurements of Abeta(11-26) fibrils are also presented.     

Fibrillar amyloid beta-protein binds protease nexin-2/amyloid beta-protein precursor: stimulation of its inhibition of coagulation factor XIa

Cerebrovascular deposition of fibrillar 39-42 amino acid amyloid beta-protein (Abeta), a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and related disorders including hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Severe cases of CAA, particularly in HCHWA-D, lead to recurrent and often fatal hemorrhagic strokes. Although the reasons for this pathological consequence remain unclear, alterations in proteolytic hemostasis mechanisms have been implicated. For example, the Abeta parent molecule protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP), which is elevated in HCHWA-D cerebral vessels with Abeta deposits, is a potent inhibitor of coagulation factor XIa (FXIa). Here we show that fibrillar HCHWA-D Abeta binds PN-2/AbetaPP, but not its isolated Kunitz-type proteinase inhibitor (KPI) domain, in a saturable, dose-dependent manner with a K(d) of approximately 28 nM. Neither PN-2/AbetaPP nor its KPI domain bound to nonfibrillar HCHWA-D Abeta. The fibrillar Abeta binding domain on PN-2/AbetaPP was localized to residues 18-119. PN-2/AbetaPP that bound to fibrillar HCHWA-D Abeta immobilized either in plastic wells or on the surface of cultured cerebrovascular smooth muscle cells was active in inhibiting FXIa. Quantitative kinetic measurements revealed that fibrillar HCHWA-D Abeta caused a >5-fold enhancement of FXIa inhibition by PN-2/AbetaPP. Similar stimulatory effects on FXIa inhibition by PN-2/AbetaPP were also observed with fibrillar wild-type Abeta. However, fibrillar Abeta had no effect on the inhibition of trypsin by PN-2/AbetaPP. These findings suggest that fibrillar Abeta deposits in cerebral vessels can effectively localize and enhance the anticoagulant functions of PN-2/AbetaPP, thereby contributing to a microenvironment conducive to hemorrhaging.     

Conformational dynamics of amyloid beta-protein assembly probed using intrinsic fluorescence

Formation of toxic oligomeric and fibrillar structures by the amyloid beta-protein (Abeta) is linked to Alzheimer's disease (AD). To facilitate the targeting and design of assembly inhibitors, intrinsic fluorescence was used to probe assembly-dependent changes in Abeta conformation. To do so, Tyr was substituted in Abeta40 or Abeta42 at position 1, 10 (wild type), 20, 30, 40, or 42. Fluorescence then was monitored periodically during peptide monomer folding and assembly. Electron microscopy revealed that all peptides assembled readily into amyloid fibrils. Conformational differences between Abeta40 and Abeta42 were observed in the central hydrophobic cluster (CHC) region, Leu17-Ala21. Tyr20 was partially quenched in unassembled Abeta40 but displayed a significant and rapid increase in intensity coincident with the maturation of an oligomeric, alpha-helix-containing intermediate into amyloid fibrils. This process was not observed during Abeta42 assembly, during which small decreases in fluorescence intensity were observed in the CHC. These data suggest that the structure of the CHC in Abeta42 is relatively constant within unassembled peptide and during the self-association process. Solvent accessibility of the Tyr ring was studied using a mixed solvent (dimethyl sulfoxide/water) system. [Tyr40]Abeta40, [Tyr30]Abeta42, and [Tyr42]Abeta42 all were relatively shielded from solvent. Analysis of the assembly dependence of the site-specific intrinsic fluorescence data suggests that the CHC is particularly important in controlling Abeta40 assembly, whereas the C-terminus plays the more significant role in Abeta42 assembly.     

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.     

Cytochemical study of the involvement of cell organelles in formation and accumulation of fibrillar amyloid in the pancreas of NORbeta transgenic mice

Phosphatase ultrastructural cytochemistry was used to evaluate the participation of cytoplasmic organelles in the accumulation of fibrillar amyloid beta (Abeta) in exocrine acinar cells and in macrophages of the pancreas of transgenic mice overexpressing a carboxy-terminal fragment of Abeta protein precursor (ABPP). Nucleoside diphosphatase (NDPase) and glucose-6-phosphatase (G6Pase) were used as cytochemical markers of the endoplasmic reticulum (ER), thiamine pyrophosphatase (TPPase) as a marker of the Golgi apparatus (GA), and acid phosphatase (AcPase) as a marker of lysosomes. Monoclonal antibody 4G8 raised against the 17-24 aa sequence of human Abeta protein was used for immunogold localization of fibrillar Abeta. The results of this study indicate that the formation of Abeta in acinar cells occurs directly in the vacuolar areas of the rough ER (RER) without evident participation of the elements of the GA, whereas an intimate structural relation with primary lysosomes suggests their role in modification or digestion of the deposited amyloid. In macrophages, fibrillar amyloid was present in numerous cytoplasmic vacuoles located frequently in close proximity to flattened saccules of the ER. This structural pattern revealed similarity to that observed previously in microglial cells producing fibrillar PrP amyloid in scrapie-infected mice and Abeta in brains of human elderly patients and in Alzheimer's type brain pathology.