Mechanical manipulation of Alzheimer's amyloid beta1-42 fibrils

The 39- to 42-residue-long amyloid beta-peptide (Abeta-peptide) forms filamentous structures in the neuritic plaques found in the neuropil of Alzheimer's disease patients. The assembly and deposition of Abeta-fibrils is one of the most important factors in the pathogenesis of this neurodegenerative disease. Although the structural analysis of amyloid fibrils is difficult, single-molecule methods may provide unique insights into their characteristics. In the present work, we explored the nanomechanical properties of amyloid fibrils formed from the full-length, most neurotoxic Abeta1-42 peptide, by manipulating individual fibrils with an atomic force microscope. We show that Abeta-subunit sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition. The fundamental unzipping force (approximately 23 pN) was significantly lower than that observed earlier for fibrils formed from the Abeta1-40 peptide (approximately 33 pN), suggesting that the presence of the two extra residues (Ile and Ala) at the peptide's C-terminus result in a mechanical destabilization of the fibril. Deviations from the constant force transition may arise as a result of geometrical constraints within the fibril caused by its left-handed helical structure. The nanomechanical fingerprint of the Abeta1-42 is further influenced by the structural dynamics of intrafibrillar interactions.     

Alzheimer's amyloid fibrils: structure and assembly

Structural studies of Alzheimer's amyloid fibrils have revealed information about the structure at different levels. The amyloid-beta peptide has been examined in various solvents and conditions and this has led to a model by which a conformational switching occurs from alpha-helix or random coil, to a beta-sheet structure. Amyloid fibril assembly proceeds by a nucleation dependent pathway leading to elongation of the fibrils. Along this pathway small oligomeric intermediates and short fibrillar structures (protofibrils) have been observed. In cross-section the fibril appears to be composed of several subfibrils or protofilaments. Each of these protofilaments is composed of beta-sheet structure in which hydrogen bonding occurs along the length of the fibre and the beta-strands run perpendicular to the fibre axis. This hierarchy of structure is discussed in this review.     

Scanning cysteine mutagenesis analysis of Abeta-(1-40) amyloid fibrils

We describe here the use of cysteine substitution mutants in the Alzheimer disease amyloid plaque peptide Abeta-(1-40) to probe amyloid fibril structure and stabilization. In one approach, amyloid fibrils were grown from Cys mutant peptides under reducing conditions and then challenged with an alkylating agent to probe solvent accessibility of different residues in the fibril. In another approach, monomeric Cys mutants, either in the thiol form or modified with iodoacetic acid or methyl iodide, were grown into amyloid fibrils, and the equilibrium position at the end of the amyloid formation reaction was quantified by determining the concentration of monomeric Abeta. The DeltaG values of fibril elongation obtained were then compared in order to provide information on the environment of each residue side chain in the fibril. In general, Cys residues in the N and C termini of Abeta-(1-40) were not only accessible to alkylation in the fibril state but also, when modified in the monomeric state, did not greatly impact fibril stability; these observations were consistent with previous indications that these portions of the peptide are not part of the amyloid core. In contrast, residues 16-19 and 31-34 were not only uniformly inaccessible to alkylation in the fibril state, but their modification with the negatively charged carboxymethyl group in monomeric Abeta also destabilized fibril elongation, confirming other data showing that these segments are likely packed into a hydrophobic amyloid core. Residues 20, 30, and 35, flanking these implicated beta-sandwich regions, are accessible to alkylation in the fibril indicating a location in solvent exposed structure.     

Sulfated polysaccharides promote the assembly of amyloid beta(1-42) peptide into stable fibrils of reduced cytotoxicity

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Abeta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Abeta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Abeta(1-42) species on Ca(2+) homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca(2+) that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca(2+) increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.     

Formation of toxic fibrils of Alzheimer's amyloid beta-protein-(1-40) by monosialoganglioside GM1, a neuronal membrane component

A pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid beta-protein (Abeta) in fibrillar form on neuronal cells. However, the role of Abeta fibrils in neuronal dysfunction is highly controversial. This study demonstrates that monosialoganglioside GM1 (GM1) released from damaged neurons catalyzes the formation of Abeta fibrils, the toxicity and the cell affinity of which are much stronger than those of Abeta fibrils formed in phosphate-buffered saline. Abeta-(1-40) was incubated with equimolar GM1 at 37 degrees C. After a lag period of 6-12 h, amyloid fibrils were formed, as confirmed by circular dichroism, thioflavin-T fluorescence, size-exclusion chromatography, and transmission electron microscopy. The fibrils showed significant cytotoxicity against PC12 cells differentiated with nerve growth factor. Trisialoganglioside GT1b also facilitated the fibrillization, although the effect was weaker than that of GM1. Our study suggests an exacerbation mechanism of AD and an importance of polymorphisms in Abeta fibrils during the pathogenesis of the disease.     

Effect of different salt ions on the propensity of aggregation and on the structure of Alzheimer's abeta(1-40) amyloid fibrils

The formation of amyloid fibrils and other polypeptide aggregates depends strongly on the physico-chemical environment. One such factor affecting aggregation is the presence and concentration of salt ions. We have examined the effects of salt ions on the aggregation propensity of Alzheimer's Abeta(1-40) peptide and on the structure of the dissolved and of the fibrillar peptide. All salts examined promote aggregation strongly. The most pronounced effect is seen within the cationic series, i.e. for MgCl2. Evaluation of different possible explanations suggests that Abeta(1-40) aggregation depends on direct interaction between ions and Abeta(1-40) peptide, and correlates with ion-induced changes of the surface tension. Salts have profound effects on the fibril structure. In the presence of salts, fibrils are associated with smaller diameters, narrower crossover distances and lower amide I maxima. Since Abeta(1-40) aggregation responds to salts in a manner unlike that for other polypeptides, such as glucagon, beta2-microglobulin or alpha-synuclein; these data argue that there is no fully uniform way in which salts affect aggregation of different polypeptide chains. These observations are important for understanding and predicting aggregation on the basis of simple physico-chemical properties.     

Formation of toxic Abeta(1-40) fibrils on GM1 ganglioside-containing membranes mimicking lipid rafts: polymorphisms in Abeta(1-40) fibrils

The abnormal aggregation and deposition of amyloid β protein (Aβ) on neuronal cells are critical to the onset of Alzheimer's disease. The entity (oligomers or fibrils) of toxic Aβ species responsible for the pathogenesis of the disease has been controversial. We have reported that the Aβ aggregates on ganglioside-rich domains of neuronal PC12 cells as well as in raft-like model membranes. Here, we identified toxic Aβ(1-40) aggregates formed with GM1-ganglioside-containing membranes. Aβ(1-40) was incubated with raft-like liposomes composed of GM1/cholesterol/sphingomyelin at 1:2:2 and 37 °C. After a lag period, toxic amyloid fibrils with a width of 12 nm were formed and subsequently laterally assembled with slight changes in their secondary structure as confirmed by viability assay, thioflavin-T fluorescence, circular dichroism, and transmission electron microscopy. In striking contrast, Aβ fibrils formed without membranes were thinner (6.7 nm) and much less toxic because of weaker binding to cell membranes and a smaller surface hydrophobicity. This study suggests that toxic Aβ(1-40) species formed on membranes are not soluble oligomers but amyloid fibrils and that Aβ(1-40) fibrils exhibit polymorphisms.     

Interaction between A beta(1-42) and A beta(1-40) in Alzheimer's beta-amyloid fibril formation in vitro

We analyzed the interaction of two kinds of amyloid beta-peptides (A beta), i.e., A beta(1-42) and A beta(1-40), in the kinetics of beta-amyloid fibril (fA beta) formation in vitro, based on a nucleation-dependent polymerization model using fluorescence spectroscopy with thioflavin T. When 25 microM A beta(1-42) was incubated with increasing concentrations of amyloidogenic A beta(1-40), the time to proceed to equilibrium was extended dose-dependently. A similar inhibitory effect was observed when 45 microM A beta(1-40) was incubated with increasing concentrations of A beta(1-42). On the other hand, when 50 microM of nonamyloidogenic A beta(1-40) was incubated with A beta(1-42) at a molar ratio of 10:1 or 5:1, A beta(1-42) initiated fA beta formation from A beta(1-40). The lag time of the reaction shortened in a concentration-dependent manner, with A beta(1-42). We next examined the seeding effect of fA beta formed from A beta(1-42) (fA beta(1-42)) on nonamyloidogenic A beta(1-40). When 50 microM of nonamyloidogenic A beta(1-40) was incubated with 10 or 20 microg/mL (2.2 or 4.4 microM) of fA beta(1-42), the fluorescence showed a sigmoidal increase. The lag time of the reaction was shortened by fA beta(1-42) in a concentration-dependent manner. However, the time to proceed to equilibrium was much longer than when an equal concentration of fA beta formed from A beta(1-40) (fA beta(1-40)) was added to A beta(1-40). The fluorescence increased hyperbolically without a lag phase when 25 microM A beta(1-42) was incubated with 10 or 20 microg/mL (2.3 or 4.6 microM) of fA beta(1-40), and proceeded to equilibrium more rapidly than without fA beta(1-40). An electron microscopic study indicated that the morphology of fA beta formed is governed by the major component of fresh A beta peptides in the reaction mixture, not by the morphology of preexisting fibrils. These results may indicate the central role of A beta(1-42) for fA beta deposition in vivo, among the different coexisting A beta species.     

Structural analysis of amyloid beta peptide fragment (25-35) in different microenvironments

Amyloid beta (Abeta) peptides are one of the classes of amphiphilic molecules that on dissolution in aqueous solvents undergo interesting conformational transitions. These conformational changes are known to be associated with their neuronal toxicity. The mechanism of structural transition involved in the monomeric Abeta to toxic assemblage is yet to be understood at the molecular level. Early results indicate that oriented molecular crowding has a profound effect on their assemblage formation. In this work, we have studied how different microenvironments affect the conformational transitions of one of the active amyloid beta-peptide fragments (Abeta(25-35)). Spectroscopic techniques such as CD and Fourier transform infrared spectroscopy were used. It was observed that a stored peptide concentrates on dissolution in methanol adopts a minor alpha-helical conformation along with unordered structures. On changing the methanol concentration in the solvated film form, the conformation switches to the antiparallel beta-sheet structure on the hydrophilic surface, whereas the peptide shows transition from a mixture of helix and unordered structure into predominantly a beta-sheet with minor contribution of helix structure on the hydrophobic surface. Our present investigations indicate that the conformations induced by the different surfaces dictate the gross conformational preference of the peptide concentrate.     

Structural stability of amyloid fibrils of beta(2)-microglobulin in comparison with its native fold

Among various amyloidogenic proteins, beta(2)-microglobulin (beta2-m) responsible for dialysis-related amyloidosis is a target of extensive study because of its clinical importance and suitable size for examining the formation of amyloid fibrils in comparison with protein folding to the native state. The structure and stability of amyloid fibrils have been studied with various physicochemical methods, including H/D exchange of amyloid fibrils combined with dissolution of fibrils by dimethylsulfoxide and NMR analysis, thermodynamic analysis of amyloid fibril formation by isothermal calorimetry, and analysis of the effects of pressure on the structure of amyloid fibrils. The results are consistent with the view that amyloid fibrils are a main-chain-dominated structure with larger numbers of hydrogen bonds and pressure-accessible cavities in the interior, in contrast to the side-chain-dominated native structure with the optimal packing of amino acid residues. We consider that a main-chain dominated structure provides the structural basis for various conformational states even with one protein. When this feature is combined with another unique feature, template-dependent growth, propagation and maturation of the amyloid conformation, which cannot be predicted with Anfinsen's dogma, take place.     

Expression of beta-amyloid precursor protein-CD3gamma chimeras to demonstrate the selective generation of amyloid beta(1-40) and amyloid beta(1-42) peptides within secretory and endocytic compartments.

Amyloid beta-protein (Abeta) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the beta-amyloid precursor protein (APP). We have analyzed the amyloidogenic processing of APP using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3 gamma chain of the T cell antigen receptor (CD3gamma). CD3gamma contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface (Letourneur, F., and Klausner, R.D. (1992) Cell 69, 1143-1157). We used the wild-type sequence of CD3gamma to create an APP chimera predicted to remain in the ER (gammaAPP(ER)). Deletion of the RKK motif at the C terminus directed the protein directly to the lysosomes (gammaAPP(LYS)). A third chimera was created by removing both lysosomal targeting signals in addition to RKK (gammaAPP(DeltaDelta)). This last construct does not contain known targeting signals and consequently accumulates at the cell surface. We show by immunofluorescence and by biochemical methods that all three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. We found that Abeta(1-40) is generated in the early secretory and endocytic pathways, whereas Abeta(1-42) is made mainly in the secretory pathway. More importantly, we provide evidence that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived Abeta forms can enter the secretable pool. Finally, we directly demonstrate that lysosomal processing is not involved in the generation or secretion of either Abeta(1-40) or Abeta(1-42).     

Secondary conformations and temperature effect on structural transformation of amyloid beta (1-28), (1-40) and (1-42) peptides

Secondary structure of three amyloid b-peptides [A beta(1-28), A beta(1-40) and A beta(1-42)] in the solid state was respectively determined by Fourier transform infrared (FT-IR) microspectroscopy. Their thermal-dependent structural transformation were also investigated by FT-IR microspectroscopy equipped with a thermal analyzer. The present result demonstrates that the solid-state A beta(1-28), A beta(1-40) and A beta(1-42) peptides showed a significant IR spectral difference in the amide I and II bands. The secondary conformation of A beta(1-28) peptide was the combination of major beta-sheet and minor alpha-helix with little random coil structures, but A beta(1-40) peptide showed the co-existence of major beta-sheet and minor random coil with little alpha-helix structures. A beta(1-42) peptide mainly consisted of the predominant b-sheet structure. Although the intact A beta(1-28), A beta(1-40) or A beta(1-42) peptide exhibits a different secondary structure, a similar beta-conformation may form after thermal treatment. A thermal-dependent transition was found for solid A beta(1-28) and A beta(1-40) peptides near 40 degrees C and 45 degrees C, respectively. There was no transition temperature for solid A beta(1-42) peptide, however, due to only a very little level of alpha-helix and random coil structure containing in the solid A beta(1-42) peptide. The thermal denaturation plays an important role in the structural transformation from alpha-helix/random coil to beta-sheet.     

Methionine 35 oxidation reduces fibril assembly of the amyloid abeta-(1-42) peptide of Alzheimer's disease

The major component of amyloid plaques in Alzheimer's disease (AD) is Abeta, a small peptide that has high propensity to assemble as aggregated beta-sheet structures. Using three well established techniques for studying amyloid structure, namely circular dichroism, thioflavin-T fluorescence, and atomic force microscopy, we demonstrate that oxidation of the Met-35 side chain to a methionine sulfoxide (Met-35(ox)) significantly hinders the rate of fibril formation for the 42-residue Abeta-(1-42) at physiological pH. Met-35(ox) also alters the characteristic Abeta fibril morphology and prevents formation of the protofibril, which is a key intermediate in beta-amyloidosis and the associated neurotoxicity. The implications of these results for the biological function and role of Abeta with oxidative stress in AD are discussed.     

Expression and purification of uniformly (15)N-labeled amyloid beta peptide 1-40 in Escherichia coli

For biophysical studies using heteronuclear NMR analysis of amyloid beta peptide, construction of an efficient and high yield expression system of uniformly isotopic labeled amyloid beta peptide is desirable. Here we succeeded in obtaining (15)N-labeled amyloid beta 1-40 expressed by attachment to hen egg white lysozyme as a fusion protein.     

Interactions of amyloid beta-peptide (1-40) with ganglioside-containing membranes

Interactions between amyloid beta-peptides (Abeta) and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. To gain insight into the molecular details of this association, we investigated the interactions of Abeta (1-40) with ganglioside-containing membranes by circular dichroism (CD) and Fourier transform infrared-polarized attenuated total reflection (FTIR-PATR) spectroscopy. The CD study revealed that at physiological ionic strength Abeta (1-40) specifically binds to ganglioside-containing membranes inducing a two-state, unordered --> beta-sheet transition above a threshold intramembrane ganglioside concentration, which depends on the host lipid bilayers used. Furthermore, differences in the number and position of sialic acid residues of the carbohydrate backbone significantly affected the conformational transition of the peptide. FTIR-PATR spectroscopy experiments demonstrated that Abeta (1-40) forms an antiparallel beta-sheet, the plane of which lies parallel to the membrane surface, inducing dehydration of lipid interfacial groups and perturbation of acyl chain orientation. These results suggest that Abeta (1-40) imposes negative curvature strain on ganglioside-containing lipid bilayers, disturbing the structure and function of the membranes.     

Y10W beta(1-40) fluorescence reflects epitope exposure in conformers of Alzheimer's beta-peptide

The Alzheimer's beta-peptide in neutral aqueous solution is characterized variously as a random coil or a heterogeneous mixture of conformers. Under conditions of lowered pH characteristic of intracellular compartments such as endosomes or lysosomes, a different conformation is favored, which is reflected in the biophysical and biological properties of the peptide. The reactivity of the epitope of the monoclonal antibody 6F/3D, encompassing residues 9-14, is drastically reduced. The fluorescence of human sequence beta(1-40) with the tyrosine at position 10 substituted with tryptophan (Y10W beta(1-40)) is quenched nearly 50% when the peptide is shifted to pH 4.6. The exposure of the 6F/3D epitope parallels Y10W beta(1-40) fluorescence changes induced by a variety of perturbations. The linkage of the sensitivity of immunological detection with the potential for monitoring rapid changes by fluorescence offers convergence of biology and biophysics in the study of beta-amyloid peptide conformation.     

Diversity of amyloid beta protein fragment [1-40]-formed channels

1. The lipid bilayer technique was used to characterize the biophysical and pharmacological properties of several ion channels formed by incorporating amyloid beta protein fragment (AP) 1–40 into lipid membranes. Based on the conductance, kinetics, selectivity, and pharmacological properties, the following AP[1–40]-formed ion channels have been identified: (i) The AP[1–40]-formed bursting fast cation channel was characterized by (a) a single channel conductance of 63 pS (250/50 mM KCl cis/trans) at +140 mV, 17 pS (250/50 mM KCl cis/trans) at –160 mV, and the nonlinear current–voltage relationship drawn to a third-order polynomial, (b) selectivity sequence P _K > P _Na > P _Li = 1.0:0.60:0.47, (c) P_o of 0.22 at 0 mV and 0.55 at +120 mV, and (d) Zn²⁺-induced reduction in current amplitude, a typical property of a slow block mechanism. (ii) The AP[1–40]-formed spiky fast cation channel was characterized by (a) a similar kinetics to the bursting fast channel with exception for the absence of the long intraburst closures, (b) single channel conductance of 63 pS (250/50 KCl) at +140 mV 17 pS (250/50 KCl) at –160 mV, the current–voltage relationship nonlinear drawn to a third-order polynomial fit, and (c) selectivity sequence P _Rb > P _K > P _Cs > P _Na > P _Li = 1.3:1.0:0.46:0.40:0.27. (iii) The AP[1–40]-formed medium conductance channel was charcterized by (a) 275 pS (250/50 mM KCl cis/trans) at +140 mV and 19 pS (250/50 mM KCl cis/trans) at –160 mV and (b) inactivation at V_ms more negative than –120 and more positive than +120 mV. (iv) The AP[1–40]-formed inactivating large conductance channel was characterized by (a) fast and slow modes of opening to seven multilevel conductances ranging between 0–589 pS (in 250/50 mM KCl) at +140 mV and 0–704 pS (in 250/50 mM KCl) at –160 mV, (b) The fast mode which had a conductance of <250 pS was voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.2 s at +36 mV. The slow mode which had a conductance of >250 pS was also voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.0 s at –76 mV, (c) the value of P _K/P _choline for the fast mode was 3.9 and selectivity sequence P _K > P _Cs > P _Na > P _Li = 1.0:0.94:0.87:0.59. The value of P _K/P _choline for the slow mode was 2.7 and selectivity sequence P _K > P _Na > P _Li > P _Cs = 1.0:0.59:0.49:0.21, and (d) asymmetric blockade with 10 mM Zn²⁺-induced reduction in the large conductance state of the slow mode mediated via slow block mechanism. The fast mode of the large conductance channel was not affected by 10 mM Zn²⁺.2. It has been suggested that, although the bursting fast channel, the spiky fast channel and the inactivating medium conductance channel are distinct, it is possible that they are intermediate configurations of yet another configuration underlying the inactivating large conductance channel. It is proposed that this heterogeneity is one of the most common features of these positively-charged cytotoxic amyloid-formed channels reflecting these channels ability to modify multiple cellular functions.3. Furthermore, the formation of -sheet based oligomers could be an important common step in the formation of cytotoxic amyloid channels.     

Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils

Increasing evidence indicates that oligomeric protein assemblies may represent the molecular species responsible for cytotoxicity in a range of neurological disorders including Alzheimer and Parkinson diseases. We use all-atom computer simulations to reveal that the process of oligomerization can be divided into two steps. The first is characterised by a hydrophobic coalescence resulting in the formation of molten oligomers in which hydrophobic residues are sequestered away from the solvent. In the second step, the oligomers undergo a process of reorganisation driven by interchain hydrogen bonding interactions that induce the formation of β sheet rich assemblies in which hydrophobic groups can become exposed. Our results show that the process of aggregation into either ordered or amorphous species is largely determined by a competition between the hydrophobicity of the amino acid sequence and the tendency of polypeptide chains to form arrays of hydrogen bonds. We discuss how the increase in solvent-exposed hydrophobic surface resulting from such a competition offers an explanation for recent observations concerning the cytotoxicity of oligomeric species formed prior to mature amyloid fibrils.