Methionine residue 35 is critical for the oxidative stress and neurotoxic properties of Alzheimer's amyloid beta-peptide 1-42

Amyloid beta-peptide 1-42 [Abeta(1-42)] is central to the pathogenesis of Alzheimer's disease (AD), and the AD brain is under intense oxidative stress. Our laboratory combined these two aspects of AD into the Abeta-associated free radical oxidative stress model for neurodegeneration in AD brain. Abeta(1-42) caused protein oxidation, lipid peroxidation, reactive oxygen species formation, and cell death in neuronal and synaptosomal systems, all of which could be inhibited by free radical antioxidants. Recent studies have been directed at discerning molecular mechanisms by which Abeta(1-42)-associated free radical oxidative stress and neurotoxicity arise. The single methionine located in residue 35 of Abeta(1-42) is critical for these properties. This review presents the evidence supporting the role of methionine in Abeta(1-42)-associated free radical oxidative stress and neurotoxicity. This work is of obvious relevance to AD and provides a coupling between the centrality of Abeta(1-42) in the pathogenesis of AD and the oxidative stress under which the AD brain exists.     

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.     

Review: Alzheimer's amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity

Alzheimer's disease, the major dementing disorder of the elderly that affects over 4 million Americans, is related to amyloid beta-peptide, the principal component of senile plaques in Alzheimer's disease brain. Oxidative stress, manifested by protein oxidation and lipid peroxidation, among other alterations, is a characteristic of Alzheimer's disease brain. Our laboratory united these two observations in a model to account for neurodegeneration in Alzheimer's disease brain, the amyloid beta-peptide-associated oxidative stress model for neurotoxicity in Alzheimer's disease. Under this model, the aggregated peptide, perhaps in concert with bound redox metal ions, initiates free radical processes resulting in protein oxidation, lipid peroxidation, reactive oxygen species formation, cellular dysfunction leading to calcium ion accumulation, and subsequent neuronal death. Free radical antioxidants abrogate these findings. This review outlines the substantial evidence from multiidisciplinary approaches for amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity and protection against these oxidative processes and cell death by free radical scavengers. In addition, we review the strong evidence supporting the notion that the single methionine residue of amyloid beta-peptide is vital to the oxidative stress and neurotoxicological properties of this peptide. Further, we discuss studies that support the hypothesis that aggregated soluble amyloid beta-peptide and not fibrils per se are necessary for oxidative stress and neurotoxicity associated with amyloid beta-peptide.     

Protective effect of quercetin in primary neurons against Abeta(1-42): relevance to Alzheimer's disease

Quercetin, a flavonoid found in various foodstuffs, has antioxidant properties and increases glutathione (GSH) levels and antioxidant enzyme function. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimer's disease (AD). Amyloid beta-peptide [Abeta(1-42)], elevated in AD brain, is associated with oxidative stress and neurotoxicity. We aimed to investigate the protective effects of quercetin on Abeta(1-42)-induced oxidative cell toxicity in cultured neurons in the present study. Decreased cell survival in neuronal cultures treated with Abeta(1-42) correlated with increased free radical production measured by dichlorofluorescein fluorescence and an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (protein-bound 4-hydroxy-2-nonenal). Pretreatment of primary hippocampal cultures with quercetin significantly attenuated Abeta(1-42)-induced cytotoxicity, protein oxidation, lipid peroxidation and apoptosis. A dose-response study suggested that quercetin showed protective effects against Abeta(1-42) toxicity by modulating oxidative stress at lower doses, but higher doses were not only non-neuroprotective but also toxic. These findings provide motivation to test the hypothesis that quercetin may provide a promising approach for the treatment of AD and other oxidative-stress-related neurodegenerative diseases.     

The Nonfibrillar Amyloid β-Peptide Induces Apoptotic Neuronal Cell Death

The toxicity of the nonaggregated amyloid beta-peptide (1-40) [A beta(1-40)] on the viability of rat cortical neurons in primary culture was investigated. We demonstrated that low concentrations of A beta peptide, in a nonfibrillar form, induced a time- and dose-dependent apoptotic cell death, including DNA condensation and fragmentation. We compared the neurotoxicity of the A beta(1-40) peptide with those of several A beta-peptide domains, comprising the membrane-destabilizing C-terminal domain of A beta peptide (e.g., amino acids 29-40 and 29-42). These peptides reproduced the effects of the (1-40) peptide, whereas mutant nonfusogenic A beta peptides and the central region of the A beta peptide (e.g., amino acids 13-28) had no effect on cell viability. We further demonstrated that the neurotoxicity of the nonaggregated A beta peptide paralleled a rapid and stable interaction between the A beta peptide and the plasma membrane of neurons, preceding apoptosis and DNA fragmentation. By contrast, the peptide in a fibrillar form induced a rapid and dramatic neuronal death mainly through a necrotic pathway, under our conditions. Taken together, our results suggest that A beta induces neuronal cell death by either apoptosis and necrosis and that an interaction between the nonfibrillar C-terminal domain of the A beta peptide and the plasma membrane of cortical neurons might represent an early event in a cascade leading to neurodegeneration.     

The critical role of methionine 35 in Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity

Amyloid beta-peptide (1-42) [Abeta(1-42)] has been proposed to play a central role in the pathogenesis of Alzheimer's disease, a neurodegenerative disorder associated with cognitive decline and aging. AD brain is under extensive oxidative stress, and Abeta(1-42) has been shown to induce protein oxidation, lipid peroxidation, and reactive oxygen species formation in neurons and synaptosomes, all of which are inhibited by the antioxidant vitamin E. Additional studies have shown that Abeta(1-42) induces oxidative stress when expressed in vivo in Caenorhabditis elegans, but when methionine 35 is replaced by cysteine, the oxidative stress is attenuated. This finding coupled with in vitro studies using mutant peptides have demonstrated a critical role for methionine 35 in the oxidative stress and neurotoxic properties of Abeta(1-42). In this review, we discuss the role of methionine 35 in the oxidative stress and neurotoxicity induced by Abeta(1-42) and the implications of these findings in the pathogenesis of AD.     

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid beta-peptide in human neuroblastoma IMR32 cells

Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.     

Amyloid-beta-peptide reduces copper(II) to copper(I) independent of its aggregation state

Alzheimer's disease (AD) is characterized by the deposition of amyloid beta-peptide (A beta) and neuronal degeneration in brain regions involved in learning and memory. One of the leading etiologic hypotheses regarding AD is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Recent evidence suggests that metals concentrated in amyloid deposits may contribute to the oxidative insults observed in AD-affected brains. We hypothesized that A beta peptide in the presence of copper enhances its neurotoxicity generating free radicals via copper reduction. In the present study, we have examined the effect of the aggregation state of amyloid-beta-peptide on copper reduction. In independent experiments we measured the copper-reducing ability of soluble and fibrillar A beta(1-40) forms by bathocuproine assays. As it was previously observed for the amyloid precursor protein (APP), the A beta peptide showed copper-reducing ability. The capacity of A beta to reduce copper was independent of the aggregation state. Finally, the A beta peptide derived from the human sequence has a greater effect than the A beta peptide derived from the rat sequence, suggesting that histidine 13 may play a role in copper reduction. In agreement with this possibility, the A beta peptide reduces less copper in the presence of exogenous histidine.     

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.     

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.     

The various aggregation states of beta-amyloid 1-42 mediate different effects on oxidative stress, neurodegeneration, and BACE-1 expression

The amyloid cascade hypothesis suggests that the insoluble and fibrillar form of beta-amyloid (A beta) may play a primary pathogenic role in Alzheimer disease at the molecular level. However, neither the rate of dementia nor the extent of neuronal change seems to correlate with the levels of amyloidotic plaques (i.e., aggregated/fibrillar A beta). Recent evidence suggests, however, that neurotoxicity may be exerted also by rather small soluble aggregates of A beta, including oligomers. To characterize the mechanisms underlying toxicity mediated by the various aggregation states of A beta peptides is then a major goal of research. In this work we investigated the effects of fibrillar, prefibrillar, and oligomeric A beta(1-42) on the induction of oxidative stress, cell death, and BACE-1 expression in NT2 neuronal cells. We found that prefibrillar and oligomeric A beta(1-42) resulted in a more dramatic increase in the oxidative stress markers 4-hydroxynonenal and hydrogen peroxide compared to fibrillar A beta(1-42). Moreover, increased oxidative stress levels also resulted in a more rapid and significant induction of both apoptotic and necrotic neuronal cell death. Accordingly, fibrillar A beta(1-42), but not the soluble nonfibrillar forms, was the only condition able to up-regulate BACE-1 expression and activity.     

Toxicity of Pyroglutaminated Amyloid β-Peptides 3(pE)-40 and -42 Is Similar to That of Aβ1-40 and -42

An N-terminal truncated isoform of the amyloid beta-peptide (A beta) that begins with a pyroglutamate (pE) residue at position 3 [A beta3(pE)-42] is the predominant isoform found in senile plaques. Based upon previous in vitro studies regarding A beta N-terminal truncated isoforms, it has been hypothesized that A beta3(pE)-x isoforms may aggregate more rapidly and become more toxic than corresponding Abeta1-x peptides. However, the toxicity and aggregation properties of A beta3(pE)-42 and A beta3(pE)-40 have not previously been examined. After initial solubilization and 1-week preaggregation of each peptide at 37 degrees C and pH 7.4, the toxicity of 5-50 microM A beta3(pE)-42 was similar to that of A beta1-42. Moreover, the toxicity of A beta3(pE)-40 paralleled that induced by A beta1-40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged A beta1-42, A beta3(pE)-42, A beta3(pE)-40, and A beta1-40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N-terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of A beta.     

Role of glycine-33 and methionine-35 in Alzheimer's amyloid beta-peptide 1-42-associated oxidative stress and neurotoxicity

Recent theoretical calculations predicted that Gly33 of one molecule of amyloid beta-peptide (1-42) (Abeta(1-42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Abeta(1-42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Abeta(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Abeta(1-42) was substituted by Val (Abeta(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Abeta(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Abeta(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Abeta(1-42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-alpha-phenylnitrone (PBN). While Abeta(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Abeta(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Abeta(1-42G33V) does not form fibrils nearly as well as Abeta(1-42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Abeta(1-42) and that contribute to the peptide's toxicity in Alzheimer's disease brain.     

In vivo oxidative stress in brain of Alzheimer disease transgenic mice: Requirement for methionine 35 in amyloid β-peptide of APP

Numerous studies have demonstrated oxidative damage in the central nervous system in subjects with Alzheimer disease and in animal models of this dementing disorder. In this study, we show that transgenic mice modeling Alzheimer disease—PDAPP mice with Swedish and Indiana mutations in the human amyloid precursor protein (APP)—develop oxidative damage in brain, including elevated levels of protein oxidation (indexed by protein carbonyls and 3-nitrotyrosine) and lipid peroxidation (indexed by protein-bound 4-hydroxy-2-nonenal). This oxidative damage requires the presence of a single methionine residue at position 35 of the amyloid β-peptide (Aβ), because all indices of oxidative damage in brain were completely prevented in genetically and age-matched PDAPP mice with an M631L mutation in APP. No significant differences in the levels of APP, Aβ(1–42), and Aβ(1–40) or in the ratio Aβ(1–42)/Aβ(1–40) were found, suggesting that the loss of oxidative stress in vivo in the brain of PDAPP(M631L) mice results solely from the mutation of the Met35 residue to Leu in the Aβ peptide. However, a marked reduction in Aβ-immunoreactive plaques was observed in the M631L mice, which instead displayed small punctate areas of nonplaque immunoreactivity and a microglial response. In contrast to the requirement for Met at residue 35 of the Aβ sequence (M631 of APP) for oxidative damage, indices of spatial learning and memory were not significantly improved by the M631L substitution. Furthermore, a genetically matched line with a different mutation—PDAPP(D664A)—showed the reverse: no reduction in oxidative damage but marked improvement in memory. This is the first in vivo study to demonstrate the requirement for Aβ residue Met35 for oxidative stress in the brain of a mammalian model of Alzheimer disease. However, in this specific transgenic mouse model of AD, oxidative stress is neither required nor sufficient for memory abnormalities.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Ferulic acid ethyl ester protects neurons against amyloid beta- peptide(1-42)-induced oxidative stress and neurotoxicity: relationship to antioxidant activity

Alzheimer's disease (AD) is neuropathologically characterized by depositions of extracellular amyloid and intracellular neurofibrillary tangles, associated with loss of neurons in the brain. Amyloid beta-peptide (Abeta) is the major component of senile plaques and is considered to have a causal role in the development and progress of AD. Several lines of evidence suggest that enhanced oxidative stress and inflammation play important roles in the pathogenesis or progression of AD. The present study aimed to investigate the protective effects of ethyl-4-hydroxy-3-methoxycinnamic acid (FAEE), a phenolic compound which shows antioxidant and anti-inflammatory activity, on Abeta(1-42)-induced oxidative stress and neurotoxicity. We hypothesized that the structure of FAEE would facilitate radical scavenging and may induce protective proteins. Abeta(1-42) decreases cell viability, which was correlated with increased free radical formation, protein oxidation (protein carbonyl, 3-nitrotyrosine), lipid peroxidation (4-hydroxy-2-trans-nonenal) and inducible nitric oxide synthase. Pre-treatment of primary hippocampal cultures with FAEE significantly attenuated Abeta(1-42)-induced cytotoxicity, intracellular reactive oxygen species accumulation, protein oxidation, lipid peroxidation and induction of inducible nitric oxide synthase. Treatment of neurons with Abeta(1-42) increases levels of heme oxygenase-1 and heat shock protein 72. Consistent with a cellular stress response to the Abeta(1-42)-induced oxidative stress, FAEE treatment increases the levels of heme oxygenase-1 and heat shock protein 72, which may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells against Abeta(1-42)-induced toxicity. These results suggest that FAEE exerts protective effects against Abeta(1-42) toxicity by modulating oxidative stress directly and by inducing protective genes. These findings suggest that FAEE could potentially be of importance for the treatment of AD and other oxidative stress-related diseases.     

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

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

Attenuated cytotoxicity but enhanced betafibril of a mutant amyloid beta-peptide with a methionine to cysteine substitution

Amyloid-beta peptide (Abeta), the major constituent of senile plaques in the Alzheimer's disease (AD) brain, is the main source of oxidative stress leading to neurodegeneration. The methionine residue in this peptide is reported to be responsible for neurotoxicity. Structurally similar substitution with methionine 35 replaced by cysteine in Abeta(40) was synthesized, and this result in enhanced beta-sheet structures according to both circular dichroism (CD) spectra and beta-fibril specific fluorescence assay but attenuated cytotoxicity whether in the presence of copper or not. These findings may provide further evidence on disclosing the connection between amyloid beta-aggregation and Abeta-induced neurotoxicity.     

Differential accumulation of soluble amyloid β peptides 1–40 and 1–42 in human monocytic and neuroblastoma cell lines

Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid beta protein (A(beta)). While A(beta)1-40 predominates in the vascular system, A(beta)1-42 is the major component of the senile plaques in the neuropil. The concentration of both A(beta) species required to form amyloid fibrils in vitro is micromolar, yet soluble A(betas) found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of A(beta) sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of A(beta), we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125I-A(beta)1-40 and 125I-A(beta)1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of A(beta) in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125I-A(betas) than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125I-A(beta)1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125I-A(beta)1-40. These results support that A(beta)1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar A(beta)1-42, we propose that intracellular amyloidogenesis in microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease.     

Trolox and 17beta-estradiol protect against amyloid beta-peptide neurotoxicity by a mechanism that involves modulation of the Wnt signaling pathway

Oxidative stress is a key mechanism in amyloid beta-peptide (A beta)-mediated neurotoxicity; therefore, the protective roles of 17beta-estradiol (E2) and antioxidants (Trolox and vitamin C) were assayed on hippocampal neurons. Our results show the following: 1) E2 and Trolox attenuated the neurotoxicity mediated by A beta and H2O2 as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays, quantification of apoptotic cells, and morphological studies of the integrity of the neurite network. 2) Vitamin C failed to protect neurons from A beta toxicity. 3) A beta-mediated endoperoxide production, reported to induce cell damage, was decreased in the presence of E2 and Trolox. 4) Two key Wnt signaling components were affected by E2 and Trolox; in fact, the enzyme glycogen synthase kinase 3beta was inhibited by both E2 and Trolox, and both compounds were able to stabilize cytoplasmic beta-catenin. 5) E2 activated the expression of the Wnt-5a and Wnt-7a ligands, and at the same time, E2, through the alpha-estrogen receptor, was able to prevent the excitotoxic A beta-induced rise in bulk-free Ca2+ as an alternative pathway to increase cell viability. 6) Finally, the Wnt-7a ligand protected against cytoplasmic calcium disturbances induced by A beta treatment. Our results suggest that control of oxidative stress, regulation of cytoplasmic calcium, and activation of Wnt signaling may prevent A beta neurotoxicity.