Cu（Ⅱ） Potentiation of Alzheimer Aβ1-40 Cytotoxicity and Transition on its Secondary Structure

Mounting evidence has shown that dyshomeostasis of the redox-active biometals such as Cuand Fe can lead to oxidative stress,which plays a key role in the neuropathology of Alzheimer's disease(AD).Here we demonstrate that with the formation of Cu(Ⅱ)·Aβ1-40 complexes,copper markedly potentiatesthe neurotoxicity exhibited by β-amyloid peptide (Aβ).A greater amount of hydrogen peroxide was releasedwhen Cu(Ⅱ)·Aβ1-40 complexes was added to the xanthine oxidase/xanthine system detected by potassiumiodide spectrophotometry.Copper bound to Aβ1-40 was observed by electron paramagnetic resonance(EPR) spectroscopy.Circular dichroism (CD) studies indicated that copper chelation could cause a structuraltransition of Aβ.The addition of copper to Aβ introduced an increase on β-sheet as well as α-helix,whichmay be responsible for the aggregation of Aβ.We hypothesized that Aβ aggregation induced by copper maybe responsible for local injury in AD.The interaction between Cu~(2 ) and Aβ also provides a possible mechanismfor the enrichment of metal ions in amyloid plaques in the AD brain.     

Structure of the Alzheimer's disease amyloid precursor protein copper binding domain. A regulator of neuronal copper homeostasis

A major source of free radical production in the brain derives from copper. To prevent metal-mediated oxidative stress, cells have evolved complex metal transport systems. The Alzheimer's disease amyloid precursor protein (APP) is a major regulator of neuronal copper homeostasis. APP knockout mice have elevated copper levels in the cerebral cortex, whereas APP-overexpressing transgenic mice have reduced brain copper levels. Importantly, copper binding to APP can greatly reduce amyloid beta production in vitro. To understand this interaction at the molecular level we solved the structure of the APP copper binding domain (CuBD) and found that it contains a novel copper binding site that favors Cu(I) coordination. The surface location of this site, structural homology of CuBD to copper chaperones, and the role of APP in neuronal copper homeostasis are consistent with the CuBD acting as a neuronal metallotransporter.     

Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits

Amyloid beta peptide (Abeta) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimer's disease. Cu(2+) and Zn(2+) bind Abeta, inducing aggregation and giving rise to reactive oxygen species. These reactions may play a deleterious role in the disease state, because high concentrations of iron, copper, and zinc have been located in amyloid in diseased brains. Here we show that coordination of metal ions to Abeta is the same in both aqueous solution and lipid environments, with His(6), His(13), and His(14) all involved. At Cu(2+)/peptide molar ratios >0.3, Abeta coordinated a second Cu(2+) atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at N(epsilon)2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu(2+) or Zn(2+) to Abeta in a negatively charged lipid environment caused a conformational change from beta-sheet to alpha-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Abeta generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.     

Intrinsic origin of amyloid aggregation: Behavior of histidine (εεε) and (δδδ) tautomer homodimers of Aβ (1–40)

Amyloid-beta protein (Aβ) accumulation in the brain, which is influenced by several factors, is a hallmark of Alzheimer's disease (AD). Despite the important role of histidine in stabilizing the fibrillar structure of the Aβ peptide at neutral pH, the effect of histidine tautomerism on Aβ peptide aggregation is still largely unknown. Histidine is in equilibrium between δ and ε tautomers and there are three histidine residues (H6, H13, and H14) in the Aβ(1–40) peptide. We performed molecular dynamics simulation on (δδδ) and (εεε) histidine tautomers with different initial homodimeric configurations to elucidate structural and aggregation features. Results indicate that (εεε) homodimers have very low propensity or almost no tendency to form β-sheets, whereas (δδδ) dimers predominantly form β-sheets due to interactions between central hydrophobic core (CHC) residues and C-terminal residues. β-sheet formation occurred in the same regions of each dimer chain at the CHC and C-/N- terminals for different configurations of (δδδ). These results suggest that (δδδ) has an important role in AD progression. Our study provides deeper insight into the effect of tautomerism of histidine residues in Aβ(1–40) on amyloid aggregation.     

Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence.

A distinguishing feature of Alzheimer's disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4-amyloid peptide and the intracellular accumulation of potential amyloidogenic and non-amyloidogenic fragments. The relative amount of amyloid-containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. 'Humanizing' the rodent APP sequence by introducing three mutations in the beta A4-region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4-peptide to non-amyloidogenic p3-peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed-back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)     

The redox chemistry of the Alzheimer's disease amyloid beta peptide

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid beta peptide (Abeta), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Abeta from the brain. Abeta can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Abeta will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Abeta oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Abeta levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Abeta species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.     

Mechanisms of A beta mediated neurodegeneration in Alzheimer's disease

Development of a comprehensive therapeutic treatment for the neurodegenerative Alzheimer's disease (AD) is limited by our understanding of the underlying biochemical mechanisms that drive neuronal failure. Numerous dysfunctional mechanisms have been described in AD, ranging from protein aggregation and oxidative stress to biometal dyshomeostasis and mitochondrial failure. In this review we discuss the critical role of amyloid-beta (A beta) in some of these potential mechanisms of neurodegeneration. The 39-43 amino acid A beta peptide has attracted intense research focus since it was identified as a major constituent of the amyloid deposits that characterise the AD brain, and it is now widely recognised as central to the development of AD. Familial forms of AD involve mutations that lead directly to altered A beta production from the amyloid-beta A4 precursor protein, and the degree of AD severity correlates with specific pools of A beta within the brain. A beta contributes directly to oxidative stress, mitochondrial dysfunction, impaired synaptic transmission, the disruption of membrane integrity, and impaired axonal transport. Further study of the mechanisms of A beta mediated neurodegeneration will considerably improve our understanding of AD, and may provide fundamental insights needed for the development of more effective therapeutic strategies.     

Amyloid beta-Cu2+ complexes in both monomeric and fibrillar forms do not generate H2O2 catalytically but quench hydroxyl radicals

Oxidative stress plays a key role in Alzheimer's disease (AD). In addition, the abnormally high Cu(2+) ion concentrations present in senile plaques has provoked a substantial interest in the relationship between the amyloid beta peptide (Abeta) found within plaques and redox-active copper ions. There have been a number of studies monitoring reactive oxygen species (ROS) generation by copper and ascorbate that suggest that Abeta acts as a prooxidant producing H2O2. However, others have indicated Abeta acts as an antioxidant, but to date most cell-free studies directly monitoring ROS have not supported this hypothesis. We therefore chose to look again at ROS generation by both monomeric and fibrillar forms of Abeta under aerobic conditions in the presence of Cu(2+) with/without the biological reductant ascorbate in a cell-free system. We used a variety of fluorescence and absorption based assays to monitor the production of ROS, as well as Cu(2+) reduction. In contrast to previous studies, we show here that Abeta does not generate any more ROS than controls of Cu(2+) and ascorbate. Abeta does not silence the redox activity of Cu(2+/+) via chelation, but rather hydroxyl radicals produced as a result of Fenton-Haber Weiss reactions of ascorbate and Cu(2+) rapidly react with Abeta; thus the potentially harmful radicals are quenched. In support of this, chemical modification of the Abeta peptide was examined using (1)H NMR, and specific oxidation sites within the peptide were identified at the histidine and methionine residues. Our studies add significant weight to a modified amyloid cascade hypothesis in which sporadic AD is the result of Abeta being upregulated as a response to oxidative stress. However, our results do not preclude the possibility that Abeta in an oligomeric form may concentrate the redox-active copper at neuronal membranes and so cause lipid peroxidation.     

Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

Aggregation of Aβ peptides into amyloid plaques is considered to trigger the Alzheimer’s disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aβ aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H₂O₂. The fibrillization of Aβ with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aβ with oxidized Met35 in the presence of H₂O₂, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ–Cu(II) complex.     

Neurotoxicity and oxidative stress in D1M-substituted Alzheimer's A beta(1-42): relevance to N-terminal methionine chemistry in small model peptides

Small model peptides containing N-terminal methionine are reported to form sulfur-centered-free radicals that are stabilized by the terminal N atom. To test whether a similar chemistry would apply to a disease-relevant longer peptide, Alzheimer's disease (AD)-associated amyloid beta-peptide 1-42 was employed. Methionine at residue 35 of this 42-mer has been shown to be a key amino acid residue involved in amyloid beta-peptide 1-42 [A beta1-42]-mediated toxicity and therefore, the pathogenesis of AD. Previous studies have shown that mutation of the methionine residue to norleucine abrogates the oxidative stress and neurotoxic properties of A beta(1-42). In the current study, we examined if the position of methionine at residue 35 is a criterion for toxicity. In doing so, we tested the effects of moving methionine to the N-terminus of the peptide in a synthetic peptide, A beta(1-42)D1M, in which methionine was substituted for aspartic acid at the N-terminus of the peptide and all subsequent residues from D1 to L34 were shifted one position towards the carboxy-terminus. A beta(1-42)D1M exhibited oxidative stress and neurotoxicity properties similar to those of the native peptide, A beta(1-42), all of which are inhibited by the free radical scavenger Vitamin E, suggesting that reactive oxygen species may play a role in the A beta-mediated toxicity. Additionally, substitution of methionine at the N-terminus by norleucine, A beta(1-42)D1Nle, completely abrogated the oxidative stress and neurotoxicity associated with the A beta(1-42)D1M peptide. The results of this study validate the chemistry reported for short peptides with N-terminal methionines in a disease-relevant peptide.     

Promotion of oxidative lipid membrane damage by amyloid beta proteins

Senile plaques in the cerebral parenchyma are a pathognomonic feature of Alzheimer's disease (AD) and are mainly composed of aggregated fibrillar amyloid beta (Abeta) proteins. The plaques are associated with neuronal degeneration, lipid membrane abnormalities, and chemical evidence of oxidative stress. The view that Abeta proteins cause these pathological changes has been challenged by suggestions that they have a protective function or that they are merely byproducts of the pathological process. This investigation was conducted to determine whether Abeta proteins promote or inhibit oxidative damage to lipid membranes. Using a mass spectrometric assay of oxidative lipid damage, the 42-residue form of Abeta (Abeta42) was found to accelerate the oxidative lipid damage caused by physiological concentrations of ascorbate and submicromolar concentrations of copper(II) ion. Under these conditions, Abeta42 was aggregated, but nonfibrillar. Ascorbate and copper produced H(2)O(2), but Abeta42 reduced H(2)O(2) concentrations, and its ability to accelerate oxidative damage was not affected by catalase. Lipids could be oxidized by H(2)O(2) and copper(II) in the absence of ascorbate, but only at significantly higher concentrations, and Abeta42 inhibited this reaction. These results indicate that the ability of Abeta42 to promote oxidative damage is more potent and more likely to be manifest in vivo than its ability to inhibit oxidative damage. In conjunction with prior results demonstrating that oxidatively damaged membranes cause Abeta42 to misfold and form fibrils, these results suggest a specific chemical mechanism linking Abeta42-promoted oxidative lipid damage to amyloid fibril formation.     

Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer's disease

Increased levels of a 40-42 amino-acid peptide called the amyloid beta protein (A beta) and evidence of oxidative damage are early neuropathological markers of Alzheimer's disease (AD). Previous investigations have demonstrated that melatonin is decreased during the aging process and that patients with AD have more profound reductions of this hormone. It has also been recently shown that melatonin protects neuronal cells from A beta-mediated oxidative damage and inhibits the formation of amyloid fibrils in vitro. However, a direct relationship between melatonin and the biochemical pathology of AD had not been demonstrated. We used a transgenic mouse model of Alzheimer's amyloidosis and monitored over time the effects of administering melatonin on brain levels of A beta, abnormal protein nitration, and survival of the mice. We report here that administration of melatonin partially inhibited the expected time-dependent elevation of beta-amyloid, reduced abnormal nitration of proteins, and increased survival in the treated transgenic mice. These findings may bear relevance to the pathogenesis and therapy of AD.     

Human apolipoprotein A–I binds amyloid-β and prevents Aβ-induced neurotoxicity

Aggregates of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer's disease (AD). Identification of proteins that physiologically bind Aβ and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Aβ_1–42, we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A–I (apoA-I). We show that purified human apoA-I and Aβ form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Aβ. Significantly, Aβ/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Aβ-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Aβ aggregation and Aβ-induced neuronal damage and that the Aβ-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Aβ toxicity.     

Aggregation State-Dependent Activation of the Classical Complement Pathway by the Amyloid β Peptide

Abstract: Activation of the classical complement pathway has been widely investigated in recent years as a potential mechanism for the neuronal loss and neuritic dystrophy characteristic of Alzheimer's disease (AD) pathogenesis. We have previously shown that amyloid β peptide (Aβ) is a potent activator of complement, and recent evidence suggesting that the assembly state of Aβ is crucial to the progress of the disease prompted efforts to determine whether the ability of Aβ to activate the classical complement pathway is a function of the aggregation state of the peptide. In this report, we show that the fibrillar aggregation state of Aβ, as determined by thioflavin T fluorometry, electron microscopy, and staining with Congo red and thioflavine S, is precisely correlated with the ability of the peptide to induce the formation of activated fragments of the complement proteins C4 and C3. These results suggest that the classical complement pathway provides a mechanism whereby complement-dependent processes may contribute to neuronal injury in the proximity of fibrillar but not diffuse Aβ deposits in the AD brain.     

Substitution of isoleucine-31 by helical-breaking proline abolishes oxidative stress and neurotoxic properties of Alzheimer's amyloid beta-peptide

Alzheimer's disease (AD) brain is characterized by excess deposition of the 42-amino acid amyloid beta-peptide [A(beta)(1-42)]. AD brain is under intense oxidative stress, and we have previously suggested that A(beta)(1-42) was associated with this increased oxidative stress. In addition, we previously demonstrated that the single methionine residue of A(beta)(1-42), residue 35, was critical for the oxidative stress and neurotoxic properties of this peptide. Others have shown that the C-terminal region of A(beta)(1-42) is helical in aqueous micellar solutions, including that part of the protein containing Met35. Importantly, Cu(II)-binding induces alpha-helicity in A(beta) in aqueous solution. Invoking the i + 4 rule of helices, we hypothesized that the carbonyl oxygen of Ile31 would interact with the S atom of Met35 to change the electronic environment of the sulfur such that molecular oxygen could lead to the production of a sulfuramyl free radical on Met35. If this hypothesis is correct, a prediction would be that breaking the helical interaction of Ile31 and Met35 would abrogate the oxidative stress and neurotoxic properties of A(beta)(1-42). Accordingly, we investigated A(beta)(1-42) in which the Ile31 residue was replaced with the helix-breaking amino acid, proline. The alpha-helical environment around Met35 was completely abolished as indicated by circular dichroism (CD)-spectroscopy. As a consequence, the aggregation, oxidative stress, Cu(II) reduction, and neurotoxic properties of A(beta)(1-42)I31P were completely altered compared to native A(beta)(1-42). The results presented here are consistent with the notion that interaction of Ile31 with Met35 may play an important role in the oxidative processes of Met35 contributing to the toxicity of the peptide.     

Beta amyloid peptide: from different aggregation forms to the activation of different biochemical pathways

Amyloid beta peptide (Aβ) is the major component of amyloid plaques in the brain of individuals affected by Alzheimer’s disease (AD). The formation of the plaques is due to an overproduction of Aβ by APP processing, its precursor, and to its ability to convert under specific conditions from its soluble form into highly ordered fibrillar aggregates. Although neuronal degeneration occurs near the amyloid plaques, some studies have suggested that intermediates such as protofibrils or simple oligomers are also involved in AD pathogenesis and even appear to be the more dangerous species in the onset of the pathology. Further, toxic properties of aggregates of different size have been investigated and the obtained results support the hypothesis that different aggregate sizes can induce different degeneration pathways. In the present review some of the knowledge about the biochemical routes of Aβ processing and production and the relationship among Aβ and oxidative stress, metal homeostasis, inflammatory process, and cell death are summarized. Moreover, current strategies addressing both fibrillogenesis process and different Aβ altered biochemical pathways utilized for therapies are described.     

Alzheimer's disease amyloid β-protein mutations and deletions that define neuronal binding/internalization as early stage nonfibrillar/fibrillar aggregates and late stage fibrils

Accumulation of amyloid β-protein (Aβ) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aβ accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aβ might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aβ40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aβ40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aβ lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aβ in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aβ40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aβ seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aβ39E22Δ, Aβ41E22Δ, and Aβ42 but not Aβ40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aβ39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aβ, therefore, results from aggregate binding to neurons, which requires H13 for Aβ40 but not for E22Δ or Aβ42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aβ40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aβ40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aβ proteins and their aggregation states or fibrillar structure.     

A synthetic amino acid substitution of Tyr10 in Aβ peptide sequence yields a dominant negative variant in amyloidogenesis

Alzheimer's disease (AD) is the most common cause of dementia in elderly people, and age is the major nongenetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly of the amyloid beta-peptide (Aβ) in the form of oligomers and fibrils. However, how aging induces Aβ aggregation is not yet fully determined. Some residues in the Aβ sequence seem to promote Aβ-induced toxicity in association with age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Aβ sequence in promoting aggregation following interaction with the plasma membrane is not yet demonstrated. As Tyr10 is implicated in the induction of oxidative stress and stabilization of Aβ aggregation, we substituted Tyr 10 with a synthetic amino acid that abolishes Aβ-induced oxidative stress and shows an accelerated interaction with GM1. This variant peptide shows impaired aggregation properties and increased affinity for GM1. It has a dominant negative effect on amyloidogenesis in vitro, in cellulo, and in isolated synaptosomes. The present study shed new light in the understanding of Aβ-membrane interactions in Aβ-induced neurotoxicity. It demonstrates the relevance of Aβ sequence in (i) Aβ-membrane interaction, underlining the role of age-dependent enhanced GM1 content in promoting Aβ aggregation, (ii) Aβ aggregation, and (iii) Aβ-induced oxidative stress. Our results open the way for the design of peptides aimed to inhibit Aβ aggregation and neurotoxicity.     

Differential effects of apolipoprotein E isoforms on metal-induced aggregation of A beta using physiological concentrations

The epsilon 4 allele of apolipoprotein E (APOE) has been found to be a risk factor for late-onset Alzheimer's disease (AD). While the pathogenic mechanism of APOE in AD is not yet clear, APOE isoforms appear to differentially influence the aggregation of A beta, the principal component of Alzheimer-associated beta-amyloid deposits. To date, no data are available for the propensity of A beta to aggregate in the presence of APOE under conditions where these components are at physiological concentrations (in cerebrospinal fluid, APOE and A beta are approximately 100 nM and approximately 5 nM, respectively). We employed a novel in vitro filtration assay for detecting zinc(II)- and copper(II)-induced aggregation of A beta in solutions containing concentrations of the peptide that are similar to those reported for human cerebrospinal fluid. The potential for resolubilization with EDTA and the relative densities of zinc- and copper-induced A beta aggregates were also compared. Zinc-induced A beta aggregates were found to be denser and less easily resolubilized than copper-induced precipitates. Metal-induced aggregation of A beta was studied in the presence of purified apolipoprotein E2, apolipoprotein E3, and apolipoprotein E4 under conditions that approximate the physiological concentrations and ratios of these proteins. In the presence of all three APOE isoforms, zinc-induced aggregation of A beta was attenuated, while precipitation with copper was enhanced. Consistent with the increased risk for AD associated with the epsilon 4 allele of APOE, metal-induced aggregation of A beta was highest for both zinc and copper in the presence of apolipoprotein E4. Our data are consistent with a role for APOE as an in vivo molecular chaperone for A beta.     

Silibinin: a novel inhibitor of Aβ aggregation

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid β peptide (Aβ) into extracellular fibrillar deposits known as amyloid plaque. Inhibition of Aβ aggregation is therefore viewed as a potential method to halt or slow the progression of AD. It is reported that silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), attenuates cognitive deficits induced by Aβ25-35 peptide and methamphetamine. However, it remains unclear whether silibinin interacts with Aβ peptide directly and decreases Aβ peptide-induced neurotoxicity. In the present study, we identified, through employing a ThT assay and electron microscopic imaging that silibinin also appears to act as a novel inhibitor of Aβ aggregation and this effect showed dose-dependency. We also show that silibinin prevented SH-SY5Y cells from injuries caused by Aβ(1-42)-induced oxidative stress by decreasing H(2)O(2) production in Aβ(1-42)-stressed neurons. Taken together, these results indicate that silibinin may be a novel therapeutic agent for the treatment of AD.