Anti-amyloidogenic activity of tannic acid and its activity to destabilize Alzheimer's beta-amyloid fibrils in vitro

Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the destabilization of preformed fAbeta in the CNS would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAbeta formation from Abeta(1-40) and Abeta(1-42) as well as destabilizing preformed fAbeta(1-40) and fAbeta(1-42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of polymeric polyphenol, tannic acid (TA) on the formation, extension, and destabilization of fAbeta(1-40) and fAbeta(1-42) at pH 7.5 at 37 degrees C in vitro. We next compared the anti-amyloidogenic activities of TA with myricetin, rifampicin, tetracycline, and NDGA. TA dose-dependently inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42), as well as their extension. Moreover, it dose-dependently destabilized preformed fAbetas. The effective concentrations (EC50) of TA for the formation, extension and destabilization of fAbetas were in the order of 0-0.1 microM. Although the mechanism by which TA inhibits fAbeta formation from Abeta as well as destabilizes preformed fAbeta in vitro is still unclear, it could be a key molecule for the development of therapeutics for AD.     

Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer's disease

Cerebral deposition of amyloid beta-peptide (Abeta) in the brain is an invariant feature of Alzheimer's disease (AD). A consistent protective effect of wine consumption on AD has been documented by epidemiological studies. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of wine-related polyphenols (myricetin, morin, quercetin, kaempferol (+)-catechin and (-)-epicatechin) on the formation, extension, and destabilization of beta-amyloid fibrils (fAbeta) at pH 7.5 at 37 degrees C in vitro. All examined polyphenols dose-dependently inhibited formation of fAbeta from fresh Abeta(1-40) and Abeta(1-42), as well as their extension. Moreover, these polyphenols dose-dependently destabilized preformed fAbetas. The overall activity of the molecules examined was in the order of: myricetin = morin = quercetin > kaempferol > (+)-catechin = (-)-epicatechin. The effective concentrations (EC50) of myricetin, morin and quercetin for the formation, extension and destabilization of fAbetas were in the order of 0.1-1 micro m. In cell culture experiments, myricetin-treated fAbeta were suggested to be less toxic than intact fAbeta, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which these polyphenols inhibit fAbeta formation from Abeta, and destabilize pre-formed fAbetain vitro are still unclear, polyphenols could be a key molecule for the development of preventives and therapeutics for AD.     

Anti-Parkinsonian agents have anti-amyloidogenic activity for Alzheimer's beta-amyloid fibrils in vitro

Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the destabilization of preformed fAbeta in the central nervous system would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). Many studies have demonstrated that oxidative damage plays a central role in AD pathogenesis, as well as Parkinson disease (PD). Among the antioxidant strategies proposed, increasing evidence points to the possibility of achieving neuroprotection by dopamine agonists, as well as monoamine oxidase B (MAO-B) inhibitors. Actually, the beneficial effect of selegiline, a MAO-B inhibitor, in AD has been noted in several clinical studies. On the reverse, antimuscarinic agents have been reported to accelerate beta-amyloidosis and senile plaque formation in PD. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of anti-Parkinsonian agents, dopamine, levodopa, pergolide, bromocriptine, selegiline, and trihexyphenidyl on the formation, extension, and destabilization of fAbeta(1-40) and fAbeta(1-42) at pH 7.5 at 37 degrees C in vitro. The anti-Parkinsonian agents other than trihexyphenidyl dose-dependently inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42), as well as their extension. Moreover, these agents dose-dependently destabilized preformed fAbetas. The overall activity of the molecules examined was in the order of: dopamine>selegiline>levodopa=pergolide>bromocriptine. Although the exact mechanism of the anti-amyloidogenic activity of these agents is unclear, these and other structurally related compounds could be key molecules for the development of therapeutics for AD and other conformational diseases.     

Ferulic acid destabilizes preformed beta-amyloid fibrils in vitro

Inhibition of the formation of beta-amyloid fibrils (fAbeta), as well as the destabilization of preformed fAbeta in the CNS, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that curcumin (Cur) inhibits fAbeta formation from Abeta and destabilizes preformed fAbeta in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of ferulic acid (FA) on the formation, extension, and destabilization of fAbeta at pH 7.5 at 37 degrees C in vitro. We next compared the anti-amyloidogenic activities of FA with Cur, rifampicin, and tetracycline. Ferulic acid dose-dependently inhibited fAbeta formation from amyloid beta-peptide, as well as their extension. Moreover, it destabilized preformed fAbetas. The overall activity of the molecules examined was in the order of: Cur > FA > rifampicin = tetracycline. FA could be a key molecule for the development of therapeutics for AD.     

The anti-amyloidogenic effect is exerted against Alzheimer's beta-amyloid fibrils in vitro by preferential and reversible binding of flavonoids to the amyloid fibril structure

How various anti-amyloidogenic compounds inhibit the formation of Alzheimer's beta-amyloid fibrils (fAbeta) from amyloid beta-peptide (Abeta) and destabilize fAbeta remains poorly understood. Using spectrophotometry, spectrofluorometry, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and surface plasmon resonance (SPR), we investigated the anti-amyloidogenic effects of five flavonoids on fAbeta in vitro. Oxidized flavonoids generally inhibited fAbeta(1-40) formation significantly more potently than fresh compounds. Characterization of the novel fluorescence of myricetin (Myr) emitted at 575 nm with an excitation maximum at 430 nm in the presence of fAbeta(1-40) revealed the specific binding of Myr to fAbeta(1-40). By SPR analysis, distinct association and dissociation reactions of Myr with fAbeta(1-40) were observed, in contrast to the very weak binding to the Abeta monomer. A significant decrease in the rate of fibril extension was observed when >0.5 microM Myr was injected into the SPR experimental system. These findings suggest that flavonoids, especially Myr, exert an anti-amyloidogenic effect in vitro by preferentially and reversibly binding to the amyloid fibril structure of fAbeta, rather than to Abeta monomers.     

Spontaneous in vitro formation of supramolecular beta-amyloid structures, "betaamy balls", by beta-amyloid 1-40 peptide.

The concentration of beta-amyloid peptide (Abeta), x-42 or x-40 amino acids long, increases in brain with the progression Alzheimer's disease (AD). These peptides are deposited extracellularly as highly insoluble fibrils that form densities of amyloid plaques. Abeta fibrillization is a complex polymerization process preceded by the formation of oligomeric and prefibrillar Abeta intermediates. In some of our in vitro studies, in which the kinetics of intermediate steps of fibril formation were examined, we used concentrations of synthetic Abeta that exceed what is normally employed in fibrillization studies, 300-600 microM. At these concentrations, in a cell free system and under physiological conditions, Abeta 1-40 peptide (Abeta40) forms fibrils that spontaneously assemble into clearly defined spheres, "betaamy balls", with diameters of approximately 20-200 microm. These supramolecular structures show weak birefringence with Congo red staining and high stability with prolonged incubation times (at least 2 weeks) at 30 degrees C, freezing, and dilution in H(2)O. At 600 microM, they are detected after incubation for approximately 20 h. Abeta peptide 1-42 (Abeta42) lacks the ability to form betaamy balls but accelerates Abeta40 betaamy ball formation at low stoichiometric levels (1:20 Abeta42:Abeta40 ratio). Abeta42 levels above this (=10-50% w/w) impede Abeta40 betaamy ball formation. Using light (LM) and electron microscopy (EM), this study examines the gross morphology and ultrastructure of Abeta40 betaamy balls and their time course of formation, in the absence and presence of Abeta42, along with some stability measures. As spheres of a misfolded protein, betaamy balls resemble both AD Abeta senile plaques and neuronal inclusion bodies associated with other neurodegenerative diseases.     

Alpha-lipoic acid exhibits anti-amyloidogenicity for beta-amyloid fibrils in vitro

Inhibition of the formation of beta-amyloid fibrils (fAbeta), as well as the destabilization of preformed fAbeta in the CNS would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of alpha-lipoic acid (LA) and the metabolic product of LA, dihydrolipoic acid (DHLA), on the formation, extension, and destabilization of fAbeta at pH 7.5 at 37 degrees C in vitro. LA and DHLA dose-dependently inhibited fAbeta formation from amyloid beta-protein, as well as their extension. Moreover, they destabilized preformed fAbetas. LA and DHLA could be key molecules for the development of therapeutics for AD.     

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.     

Anti-amyloidogenic effects of antioxidants: implications for the prevention and therapeutics of Alzheimer's disease

Alzheimer's disease (AD) is one of the most common dementing disorders and has profound medical and social consequences. The initiating molecular event is unknown, and its pathophysiology is highly complex. However, free radical injury appears to be a fundamental process contributing to the neuronal death seen in this disorder, and many studies using surrogate markers of oxidative damage have provided evidence supporting this hypothesis. Various compounds with antioxidant ability attenuated the oxidative stress induced by amyloid beta-protein (Abeta) in studies done in vitro and in vivo. Moreover, various antioxidants have been reported to inhibit the formation and extension of beta-amyloid fibrils (fAbeta), as well as to destabilize preformed fAbeta in vitro. In cell culture experiments, destabilized fAbeta were suggested to be less toxic than intact fAbeta. In transgenic mice model studies, some antioxidant compounds reduced plaque burden in vivo. In this article, we review the recent advances in the research on the antioxidants that inhibit the formation of fAbeta, as well as destabilize preformed fAbeta. Although the mechanisms by which these compounds inhibit fAbeta formation from Abeta, and destabilize preformed fAbeta are still unclear, they could be key molecules for the development of preventives and therapeutics for AD.     

The molecular chaperone alphaB-crystallin enhances amyloid beta neurotoxicity.

Amyloid beta (Abeta) is a 40- to 42-residue peptide that is implicated in the pathogenesis of Alzheimer's Disease (AD). As a result of conformational changes, Abeta assembles into neurotoxic fibrils deposited as 'plaques' in the diseased brain. In AD brains, the small heat shock proteins (sHsps) alphaB-crystallin and Hsp27 occur at increased levels and colocalize with these plaques. In vitro, sHsps act as molecular chaperones that recognize unfolding peptides and prevent their aggregation. The presence of sHsps in AD brains may thus reflect an attempt to prevent amyloid fibril formation and toxicity. Here we report that alphaB-crystallin does indeed prevent in vitro fibril formation of Abeta(1-40). However, rather than protecting cultured neurons against Abeta(1-40) toxicity, alphaB-crystallin actually increases the toxic effect. This indicates that the interaction of alphaB-crystallin with conformationally altering Abeta(1-40) may keep the latter in a nonfibrillar, yet highly toxic form.     

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.     

Effect of different anti-Abeta antibodies on Abeta fibrillogenesis as assessed by atomic force microscopy

Extensive data suggest that the conversion of the amyloid-beta (Abeta) peptide from soluble to insoluble forms is a key factor in the pathogenesis of Alzheimer's disease (AD). In recent years, atomic force microscopy (AFM) has provided useful insights into the physicochemical processes involving Abeta morphology, and it can now be used to explore factors that either inhibit or promote fibrillogenesis. We used ex situ AFM to explore the impact of anti-Abeta antibodies directed against different domains of Abeta on fibril formation. For the AFM studies, two monoclonal antibodies (m3D6 and m266.2) were incubated in solution with Abeta(1-42) with a molar ratio of 1:10 (antibody to Abeta) over several days. Fibril formation was analyzed quantitatively by determining the number of fibrils per microm(2) and by aggregate size analysis. m3D6, which is directed against an N-terminal domain of Abeta (amino acid residues 1-5) slowed down fibril formation. However, m266.2, which is directed against the central domain of Abeta (amino acid residues 13-28) appeared to completely prevent the formation of fibrils over the course of the experiment. Inhibition of fibril formation by both antibodies was also confirmed by thioflavin-T (ThT) fluorescence experiments carried out with Abeta(1-40) incubated for five days. However, unlike AFM results, ThT did not differentiate between the samples incubated with m3D6 versus m266.2. These results indicate that AFM can be not only reliably used to study the effect of different molecules on Abeta aggregation, but that it can provide additional information such as the role of epitope specificity of antibodies as potential inhibitors of fibril formation.     

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

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

Globular amyloid beta-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease

Amyloid beta-peptide (Abeta)(1-42) oligomers have recently been discussed as intermediate toxic species in Alzheimer's disease (AD) pathology. Here we describe a new and highly stable Abeta(1-42) oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Abeta(1-42)-overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water-soluble globular 60-kDa oligomer which we named 'Abeta(1-42) globulomer'. Our data indicate that Abeta(1-42) globulomer is a persistent structural entity formed independently of the fibrillar aggregation pathway. It is a potent antigen in mice and rabbits eliciting generation of Abeta(1-42) globulomer-specific antibodies that do not cross-react with amyloid precursor protein, Abeta(1-40) and Abeta(1-42) monomers and Abeta fibrils. Abeta(1-42) globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long-term potentiation in rat hippocampal slices. Our data suggest that Abeta(1-42) globulomer represents a basic pathogenic structural principle also present to a minor extent in previously described oligomer preparations and that its formation is an early pathological event in AD. Selective neutralization of the Abeta globulomer structure epitope is expected to have a high potential for treatment of AD.     

Salvianolic acid B inhibits Abeta fibril formation and disaggregates preformed fibrils and protects against Abeta-induced cytotoxicty

One of the major pathological features of Alzheimer's disease (AD) is the appearance of senile plaques characterized by extracellular aggregation of amyloid beta-peptide (Abeta) fibrils. Inhibition of Abeta fibril aggregation is therefore viewed as one possible method to halt the progression of AD. Salvianolic acid B (Sal B) is an active ingredient isolated from Salvia miltiorrhiza, a Chinese herbal medicine commonly used for the treatment of cardiovascular and cerebrovascular disorders. Recent findings show that Sal B prevents Abeta-induced cytotoxicity in a rat neural cell line. To understand the mechanism of Sal B-mediated neuroprotection, its effects on the inhibition of Abeta1-40 fibril formation and destabilization of the preformed Abeta1-40 fibrils were studied. The results were obtained using Thioflavin T fluorescence assay and Abeta aggregating immunoassay. We found that Sal B can inhibit fibril aggregation (IC(50): 1.54-5.37 microM) as well as destabilize preformed Abeta fibril (IC(50): 5.00-5.19 microM) in a dose- and time-dependent manner. Sal B is a better aggregation inhibitor than ferulic acid but less active than curcumin in the inhibition of Abeta1-40 aggregation. In electron microscope study, Sal B-treated Abeta1-40 fibrils are seen in various stages of shortening or wrinkling with numerous deformed aggregates of amorphous structure. Circular dichroism data indicate that Sal B dose dependently prevents the formation of beta-structured aggregates of Abeta1-40. Addition of preincubated Sal B with Abeta1-42 significantly reduces its cytotoxic effects on human neuroblastoma SH-SY5Y cells. These results suggest that Sal B has therapeutic potential in the treatment of AD, and warrant its study in animal models.     

Inhibitors of amyloid beta-protein aggregation mediated by GM1-containing raft-like membranes

The aggregation (fibril formation) of amyloid beta-protein (Abeta) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Abeta aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Abeta aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Abeta aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Abeta to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Abeta in solution, thus at least partly preventing fibrils from forming. Coincubation of Abeta with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Abeta-induced toxicity in PC12 cells by inhibiting Abeta accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Abeta suggested that the two aggregation processes are not identical.     

Matrix metalloproteinase-9 degrades amyloid-beta fibrils in vitro and compact plaques in situ

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-beta fibrils (fAbeta), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Abeta (sAbeta) could degrade fAbeta as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAbeta and that this ability is not shared by other sAbeta-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAbeta was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAbeta breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAbeta were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Abeta that are cleaved during its degradation. Only MMP-9 digests contained fragments (Abeta(1-20) and Abeta(1-30)) from fAbeta(1-42) substrate; the corresponding cleavage sites are thought to be important for beta-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAbeta and may contribute to ongoing clearance of plaques from amyloid-laden 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.     

A traditional medicinal herb Paeonia suffruticosa and its active constituent 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose have potent anti-aggregation effects on Alzheimer's amyloid β proteins in vitro and in vivo

The deposition of amyloid β (Aβ) protein is a consistent pathological hallmark of Alzheimer's disease (AD) brains; therefore, inhibition of Aβ fibril formation and destabilization of pre-formed Aβ fibrils is an attractive therapeutic and preventive strategy in the development of disease-modifying drugs for AD. This study demonstrated that Paeonia suffruticosa , a traditional medicinal herb, not only inhibited fibril formation of both Aβ_1–40 and Aβ_1–42 but it also destabilized pre-formed Aβ fibrils in a concentration-dependent manner. Memory function was examined using the passive-avoidance task followed by measurement of Aβ burden in the brains of Tg2576 transgenic mice. The herb improved long-term memory impairment in the transgenic mice and inhibited the accumulation of Aβ in the brain. Three-dimensional HPLC analysis revealed that a water extract of the herb contained several different chemical compounds including 1,2,3,4,6-penta- O -galloyl-β- d -glucopyranose (PGG). No obvious adverse/toxic were found following treatment with PGG. As was observed with Paeonia suffruticosa , PGG alone inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils in vitro and in vivo . Our results suggest that both Paeonia suffruticosa and its active constituent PGG have strong inhibitory effects on formation of Aβ fibrils in vitro and in vivo . PGG is likely to be a safe and promising lead compound in the development of disease-modifying drugs to prevent and/or cure AD.     

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.