Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.     

The Arctic mutation alters helix length and type in the 11-28 beta-amyloid peptide monomer-CD, NMR and MD studies in an SDS micelle

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease, and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial Alzheimer's-like diseases with extensive cerebrovascular pathology. It has been demonstrated that such mutations alter the aggregation ability of Abeta and its neurotoxicity. Among the five mutations at positions 21-23 there is one with distinct clinical characteristics and a potentially distinct pathogenic mechanism-the Arctic (E22G) mutation. We have examined the structures of fragment 11-28 of the native peptide and its E22G variant. This fragment was chosen because it has been shown to be a good model for conformational and aggregation studies as it contains the hydrophobic core responsible for aggregation and the residues critical to alpha-secretase cleavage of APP. The detailed structure of the two peptides was determined using CD, 2D NMR and molecular dynamics techniques under water-SDS micelle conditions. Our studies indicated the existence of partially alpha- and 3(10)-helical conformations in the native and mutated peptide, respectively.     

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.     

FTIR spectroscopic studies on aggregation process of the β‐amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimer's disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 3₁₀‐helix/3₁₀‐turn structures. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Neurotoxicity and physicochemical properties of Abeta mutant peptides from cerebral amyloid angiopathy: implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease

Cerebral amyloid angiopathy (CAA) due to beta-amyloid (Abeta) is one of the specific pathological features of familial Alzheimer's disease. Abeta mainly consisting of 40- and 42-mer peptides (Abeta40 and Abeta42) exhibits neurotoxicity and aggregative abilities. All of the variants of Abeta40 and Abeta42 found in CAA were synthesized in a highly pure form and examined for neurotoxicity in PC12 cells and aggregative ability. All of the Abeta40 mutants at positions 22 and 23 showed stronger neurotoxicity than wild-type Abeta40. Similar tendency was observed for Abeta42 mutants at positions 22 and 23 whose neurotoxicity was 50-200 times stronger than that of the corresponding Abeta40 mutants, suggesting that these Abeta42 mutants are mainly involved in the pathogenesis of CAA. Although the aggregation of E22G-Abeta42 and D23N-Abeta42 was similar to that of wild-type Abeta42, E22Q-Abeta42 and E22K-Abeta42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis. In contrast, A21G mutation needs alternative explanation with the exception of physicochemical properties of Abeta mutants. Attenuated total reflection-Fourier transform infrared spectroscopy spectra suggested that beta-sheet content of the Abeta mutants correlates with their aggregation. However, beta-turn is also a critical secondary structure because residues at positions 22 and 23 that preferably form two-residue beta-turn significantly enhanced the aggregative ability.     

Differential degradation of amyloid beta genetic variants associated with hereditary dementia or stroke by insulin-degrading enzyme

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.     

Structure of the 21-30 fragment of amyloid beta-protein

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.     

Secondary structure and interfacial aggregation of amyloid-beta(1-40) on sodium dodecyl sulfate micelles

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Abeta aggregates generated from monomeric Abeta in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily beta-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Abeta aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Abeta aggregation have found that amphiphilic interactions and micellar-like Abeta structures may play a role. We previously described the formation of Abeta(1-40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Abeta(1-40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Abeta(1-40) was introduced. With HFIP, the change involved an increase in predominant beta-structure content and in fluorescence with thioflavin T, while with SDS, a partial alpha-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in beta-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Abeta(1-40) monomers effectively.     

Molecular dynamics studies of hexamers of amyloid-beta peptide (16-35) and its mutants: influence of charge states on amyloid formation

To study the early stage of amyloid-beta peptide (Abeta) aggregation, hexamers of the wild-type (WT) Abeta(16-35) and its mutants with amyloid-like conformations have been studied by molecular dynamics simulations in explicit water for a total time of 1.7 micros. We found that the amyloid-like structures in the WT oligomers are destabilized by the solvation of ionic D23/K28 residues, which are buried in the fibrils. This means that the desolvation of D23/K28 residues may contribute to the kinetic barrier of aggregation in the early stage. In the E22Q/D23N, D23N/K28Q, and E22Q/D23N/K28Q mutants, hydration becomes much less significant because the mutated residues have neutral amide side-chains. These amide side-chains can form linear cross-strand hydrogen bond chains, or "polar zippers", if dehydrated. These "polar zippers" increase the stability of the amyloid-like conformation, reducing the barrier for the early-stage oligomerization. This is in accord with experimental observations that both the D23/K28 lactamization and the E22Q/D23N mutation promote aggregation. We also found that the E22Q/D23N mutant prefers an amyloid-like conformation that differs from the one found for WT Abeta. This suggests that different amyloid structures may be formed under different conditions.     

Pathogenic effects of D23N Iowa mutant amyloid beta -protein.

Cerebral amyloid beta-protein angiopathy (CAA) is a key pathological feature of patients with Alzheimer's disease and certain related disorders. In these conditions the CAA is characterized by the deposition of Abeta within the cerebral vessel wall and, in severe cases, hemorrhagic stroke. Several mutations have been identified within the Abeta region of the Abeta protein precursor (AbetaPP) gene that appear to enhance the severity of CAA. We recently described a new mutation within the Abeta region (D23N) of AbetaPP that is associated with severe CAA in an Iowa kindred (Grabowski, T. J., Cho, H. S., Vonsattel, J. P. G., Rebeck, G. W., and Greenberg, S. M. (2001) Ann. Neurol. 49, 697-705). In the present study, we investigated the effect of this new D23N mutation on the processing of AbetaPP and the pathogenic properties of Abeta. Neither the D23N Iowa mutation nor the E22Q Dutch mutation affected the amyloidogenic processing of AbetaPP expressed in H4 cells. The A21G Flemish mutation, in contrast, resulted in a 2.3-fold increase in secreted Abeta peptide. We also tested synthetic wild-type and mutant Abeta40 peptides for fibrillogenesis and toxicity toward cultured human cerebrovascular smooth muscle (HCSM) cells. The E22Q Dutch, D23N Iowa, and E22Q,D23N Dutch/Iowa double mutant Abeta40 peptides rapidly assembled in solution to form fibrils, whereas wild-type and A21G Flemish Abeta40 peptides exhibited little fibril formation. Similarly, the E22Q Dutch and D23N Iowa Abeta40 peptides were found to induce robust pathologic responses in cultured HCSM cells, including elevated levels of cell-associated AbetaPP, proteolytic breakdown of smooth muscle cell alpha-actin, and cell death. Double mutant E22Q,D23N Dutch/Iowa Abeta40 was more potent than either single mutant form of Abeta in causing pathologic responses in HCSM cells. These data suggest that the different CAA mutations in AbetaPP may exert their pathogenic effects through different mechanisms. Whereas the A21G Flemish mutation appears to enhance Abeta production, the E22Q Dutch and D23N Iowa mutations enhance fibrillogenesis and the pathogenicity of Abeta toward HCSM cells.     

Structures and free-energy landscapes of the wild type and mutants of the Abeta(21-30) peptide are determined by an interplay between intrapeptide electrostatic and hydrophobic interactions

The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Aβ_21-30 peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated pK_a values that compare well with the experimental estimates. The pK_a values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22-K28 are more probable in “dried” salt bridges, whereas D23-K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23N < WT < E22Q < K28A. Free-energy profiles and disconnectivity representation of the energy landscapes show that the monomer structures partition into four distinct basins. The hydrophobic interactions cluster the Aβ_21-30 peptide into two basins, differentiated by the relative position of the DVG(23-25) and GSN(25-27) fragments about the G25 residue. The E22Q mutation increases the population with intact VGSN turn compared to the wild-type (WT) peptide. The increase in the population of the structures in the aggregation-prone Basin I in E22Q, which occurs solely due to the difference in charge states between the Dutch mutant and the WT, gives a structural explanation of the somewhat larger aggregation rate in the mutant. The D23N mutation dramatically reduces the intrapeptide interactions. The K28A mutation increases the intrapeptide hydrophobic interactions that promote population of structures in Basin I and Basin II whose structures are characterized by hydrophobic interaction between V24 and K28 side chains but with well-separated ends of the backbone atoms in the VGSN turn. The intrapeptide electrostatic interactions in the WT and E22Q peptides roughen the free-energy surface compared to the K28A peptide. The D23N mutation has a flat free-energy surface, corresponding to an increased population of random coil-like structures with weak hydrophobic and electrostatic interactions. We propose that mutations or sequences that enhance the probability of occupying Basin I would promote aggregation of Aβ peptides.     

Substitutions at codon 22 of Alzheimer's abeta peptide induce diverse conformational changes and apoptotic effects in human cerebral endothelial cells

Cerebral amyloid angiopathy is commonly associated with normal aging and Alzheimer's disease and it is also the principal feature of hereditary cerebral hemorrhage with amyloidosis Dutch type, a familial condition associated to a point mutation G to C at codon 693 of the amyloid beta (Abeta) precursor protein gene resulting in a Glu to Gln substitution at position 22 of the Abeta (E22Q). The patients carrying the AbetaE22Q variant usually present with lobar cerebral hemorrhages before 50 years of age. A different mutation described in several members of three Italian kindred who presented with recurrent hemorrhagic strokes late in life, between 60 and 70 years of age, also associated with extensive cerebrovascular amyloid deposition has been found at the same position 22, this time resulting in a Glu to Lys substitution (E22K). We have compared the secondary structure, aggregation, and fibrillization properties of the two Abeta40 variants and the wild type peptide. Using flow cytometry analysis after staining with propidium iodide and annexin V, we also evaluated the cytotoxic effects of the peptides on human cerebral endothelial cells in culture. Under the conditions tested, the E22Q peptide exhibited the highest content of beta-sheet conformation and the fastest aggregation/fibrillization properties. The Dutch variant also induced apoptosis of cerebral endothelial cells at a concentration of 25 micrometer, whereas the wild type Abeta and the E22K mutant had no effect. The data suggest that different amino acids at position 22 confer distinct structural properties to the peptides that appear to influence the onset and aggressiveness of the disease rather than the phenotype.     

Insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced activity in cerebral amyloid angiopathy

The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.     

Verification of the turn at positions 22 and 23 of the beta-amyloid fibrils with Italian mutation using solid-state NMR

The aggregation of 42-mer amyloid beta (Abeta42) plays a central role in the pathogenesis of Alzheimer's disease. Our recent research on proline mutagenesis of Abeta42 suggested that the formation of a turn structure at positions 22 and 23 could play a crucial role in its aggregative ability and neurotoxicity. Since E22K-Abeta42 (Italian mutation) aggregated more rapidly and with more potent neurotoxicity than wild-type Abeta42, the tertiary structure at positions 21-24 of E22K-Abeta42 fibrils was analyzed by solid-state NMR using dipolar-assisted rotational resonance (DARR) to identify the 'malignant' conformation of Abeta42. Two sets of chemical shifts for Asp-23 were observed in a ratio of about 2.6:1. The 2D DARR spectra at the mixing time of 500 ms suggested that the side chains of Asp-23 and Val-24 in the major conformer, and those of Lys-22 and Asp-23 in the minor conformer could be located on the same side, respectively. These data support the presence of a turn structure at positions 22 and 23 in E22K-Abeta42 fibrils. The formation of a salt bridge between Lys-22 and Asp-23 in the minor conformer might be a reason why E22K-Abeta42 is more pathogenic than wild-type Abeta42.     

Effects of familial Alzheimer's disease mutations on the folding nucleation of the amyloid beta-protein

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid β-protein (Aβ) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Aβ. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Aβ. Enhanced aggregation in Aβ with familial Alzheimer's disease substitutions may result from the depletion of the E22-K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21-30 segment that can impact the aggregation of Aβ. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Aβ folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Aβ are fundamentally different.     

On the nucleation of amyloid beta-protein monomer folding

Neurotoxic assemblies of the amyloid beta-protein (Abeta) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Abeta assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Abeta(1-40) and Abeta(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Abeta(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a beta-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Abeta nucleates the intramolecular folding of Abeta monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Abeta(1-40) and Abeta(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Abeta(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Abeta(1-42)-specific C-terminal fold may hold promise.     

Inhibition of beta-amyloid-induced neurotoxicity by imidazopyridoindoles derived from a synthetic combinatorial library

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the deposit of amyloid fibrils in the brain that result from the self-aggregative polymerization of the beta-amyloid peptide (Abeta). Evidence of a direct correlation between the ability of Abeta to form stable aggregates in aqueous solution and its neurotoxicity has been reported. The cytotoxic effects of Abeta have been attributed to the aggregation properties of a domain corresponding to the peptide fragment Abeta25-35. In an effort to generate novel inhibitors of Abeta neurotoxicity and/or aggregation, a mixture-based synthetic combinatorial library composed of 23 375 imidazopyridoindoles was generated and screened for inhibition of Abeta25-35 neurotoxicity toward the rat pheochromocytoma PC-12 cell line. The effect of the identified lead compounds on Abeta25-35 aggregation was then evaluated by means of circular dichroism (CD) and thioflavin-T fluorescence spectroscopy. Their activity against Abeta1-42 neurotoxicity toward the PC-12 cell line was also determined. The most active imidazopyridoindoles inhibited both Abeta25-35 and Abeta1-42 neurotoxicity in the low- to mid-micromolar range. Furthermore, inhibition of the random coil to beta-sheet transition and self-aggregation of Abeta25-35 was observed by CD and fluorescence spectroscopy, supporting the relationship between inhibition of the Abeta aggregation process and neurotoxicity.     

Synthesis,aggregation, neurotoxicity,and secondary structure of various A beta 1-42 mutants of familial Alzheimer's disease at positions 21-23

Cerebral amyloid angiopathy (CAA) due to amyloid beta (A beta) deposition is a key pathological feature of Alzheimer's disease (AD), especially in some form of familial Alzheimer's disease (FAD) including hereditary cerebral hemorrhage with amyloidosis-Dutch type. A beta mainly consists of 40- and 42-mer peptides (Abeta 1-40 and A beta 1-42), which accumulate in senile plaques of AD brains and show neurotoxicity for cultured nerve cells. We synthesized all variant forms of A beta 1-42 associated with reported FAD, such as A21G (Flemish), E22Q (Dutch), E22K (Italian), E22G (Arctic), and D23N (Iowa) along with three potential mutants by one point missense mutation (E22A, E22D, and E22V) in a highly pure form, and examined their ability to aggregate and their neurotoxicity in PC12 cells. The mutants at positions 22 and 23 showed potent aggregative ability and neurotoxicity whereas the potential mutants did not, indicating that A beta 1-42 mutants at positions 22 and 23 play a critical role in FAD of Dutch-, Italian-, Arctic-, and Iowa-types. However, Flemish-type FAD needs alternative explanation except the aggregation and neurotoxicity of the corresponding A beta 1-42 mutant.     

Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid β Peptide

Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid β peptide involved in Alzheimer's disease. We focused our study on the Glu²² residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and β-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu²² in wild-type) or hydrophobic (Val²² in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and α-helix structures (with low β-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these Aβ_12-28 variants does not correlate with the amount of β-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.     

NMR and CD studies on the interaction of Alzheimer beta-amyloid peptide (12-28) with beta-cyclodextrin

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has emerged as an approach for developing therapeutics for AD. NMR and CD spectra were used to investigate the interaction between cyclodextrin and Abeta(12-28) peptide, which was reported to be an important region for forming amyloid fibrils. CD spectral analyses show that of the alpha-, beta- and gamma-cyclodextrins only beta-cyclodextrin inhibits the aggregation of Abeta(12-28) at pH 5.0. Analysis of the one-dimensional proton NMR spectra of Abeta(12-28) and the mixture of Abeta(12-28) with beta-cyclodextrin clearly indicates that there are chemical shift changes in the aromatic ring of Phe19 and the methyl groups of Val18 in the peptide. The NOESY spectra show cross-peaks between H-3 and H-5 of beta-cyclodextrin and the aromatic protons of Phe19 and Phe20. These chemical shift differences and NOEs demonstrate that there is an interaction between Abeta(12-28) and beta-cyclodextrin. Analysis of the cross-peak intensity in the NOESY spectra reveals that the aromatic rings of Phe19 and 20 are generally inserted into beta-cyclodextrin at the broad side and are oriented toward the narrow side of the cavity.