Amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity: implications for neurodegeneration in Alzheimer's disease brain. A review

Oxidative stress, manifested by protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, among other indices, is observed in Alzheimer's disease (AD) brain. Amyloid beta-peptide (1-42) [Abeta(1-42)] may be central to the pathogenesis of AD. Our laboratory and others have implicated Abeta(1-42)-induced free radical oxidative stress in the neurodegeneration observed in AD brain. This paper reviews some of these studies from our laboratory. Recently, we showed both in-vitro and in-vivo that methionine residue 35 (Met-35) of Abeta(1-42) was critical to its oxidative stress and neurotoxic properties. Because the C-terminal region of Abeta(1-42) is helical, and invoking the i + 4 rule of helices, we hypothesized that the carboxyl oxygen of lle-31, known to be within a van der Waals distance of the S atom of Met-35, would interact with the latter. This interaction could alter the susceptibility for oxidation of Met-35, i.e. free radical formation. Consistent with this hypothesis, substitution of lle-31 by the helix-breaking amino acid, proline, completely abrogated the oxidative stress and neurotoxic properties of Abeta(1-42). Removal of the Met-35 residue from the lipid bilayer by substitution of the negatively charged Asp for Gly-37 abrogated oxidative stress and neurotoxic properties of Abeta(1-42). The free radical scavenger vitamin E prevented A(beta (1-42)-induced ROS formation, protein oxidation, lipid peroxidation, and neurotoxicity in hippocampal neurons, consistent with our model for Abeta-associated free radical oxidative stress induced neurodegeneration in AD. ApoE, allele 4, is a risk factor for AD. Synaptosomes from apoE knock-out mice are more vulnerable to Abeta-induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. We also studied synaptosomes from allele-specific human apoE knock-in mice. Brain membranes from human apoE4 mice have greater vulnerability to Abeta(1-42)-induced oxidative stress than brain membranes from apoE2 or E3, assessed by the same indices, consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Using immunoprecipitation of proteins from AD and control brain obtained no longer than 4h PMI, selective oxidized proteins were identified in the AD brain. Creatine kinase (CK) and beta-actin have increased carbonyl groups, an index of protein oxidation, and Glt-1, the principal glutamate transporter, has increased binding of the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE). Abeta inhibits CK and causes lipid peroxidation, leading to HNE formation. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. Other oxidatively modified proteins have been identified in AD brain by proteomics analysis, and these oxidatively-modified proteins may be related to increased excitotoxicity (glutamine synthetase), aberrant proteasomal degradation of damaged or aggregated proteins (ubiquitin C-terminal hydrolase L-1), altered energy production (alpha-enolase), and diminished growth cone elongation and directionality (dihydropyrimindase-related protein 2). Taken together, these studies outlined above suggest that Met-35 is key to the oxidative stress and neurotoxic properties of Abeta(1-42) and may help explain the apoE allele dependence on risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of Abeta(1-42)-induced oxidative stress and neurodegeneration in AD.     

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.     

Amyloid β-peptide (1-42)-induced Oxidative Stress and Neurotoxicity: Implications for Neurodegeneration in Alzheimer's Disease Brain. A Review

Oxidative stress, manifested by protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, among other indices, is observed in Alzheimer's disease (AD) brain. Amyloid &#103 -peptide (1-42) [A &#103 (1-42)] may be central to the pathogenesis of AD. Our laboratory and others have implicated A &#103 (1-42)-induced free radical oxidative stress in the neurodegeneration observed in AD brain. This paper reviews some of these studies from our laboratory. Recently, we showed both in-vitro and in-vivo that methionine residue 35 (Met-35) of A &#103 (1-42) was critical to its oxidative stress and neurotoxic properties. Because the C-terminal region of A &#103 (1-42) is helical, and invoking the i +4 rule of helices, we hypothesized that the carboxyl oxygen of lle-31, known to be within a van der Waals distance of the S atom of Met-35, would interact with the latter. This interaction could alter the susceptibility for oxidation of Met-35, i.e. free radical formation. Consistent with this hypothesis, substitution of lle-31 by the helix-breaking amino acid, proline, completely abrogated the oxidative stress and neurotoxic properties of A &#103 (1-42). Removal of the Met-35 residue from the lipid bilayer by substitution of the negatively charged Asp for Gly-37 abrogated oxidative stress and neurotoxic properties of A &#103 (1-42). The free radical scavenger vitamin E prevented A &#103 (1-42)-induced ROS formation, protein oxidation, lipid peroxidation, and neurotoxicity in hippocampal neurons, consistent with our model for A &#103 -associated free radical oxidative stress induced neurodegeneration in AD. ApoE, allele 4, is a risk factor for AD. Synaptosomes from apoE knock-out mice are more vulnerable to A &#103 -induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. We also studied synaptosomes from allele-specific human apoE knock-in mice. Brain membranes from human apoE4 mice have greater vulnerability to A &#103 (1-42)-induced oxidative stress than brain membranes from apoE2 or E3, assessed by the same indices, consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Using immunoprecipitation of proteins from AD and control brain obtained no longer than 4 h PMI, selective oxidized proteins were identified in the AD brain. Creatine kinase (CK) and &#103 -actin have increased carbonyl groups, an index of protein oxidation, and Glt-1, the principal glutamate transporter, has increased binding of the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE). A &#103 inhibits CK and causes lipid peroxidation, leading to HNE formation. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. Other oxidatively modified proteins have been identified in AD brain by proteomics analysis, and these oxidatively-modified proteins may be related to increased excitotoxicity (glutamine synthetase), aberrant proteasomal degradation of damaged or aggregated proteins (ubiquitin C-terminal hydrolase L-1), altered energy production ( &#102 -enolase), and diminished growth cone elongation and directionality (dihydropyrimindase-related protein 2). Taken together, these studies outlined above suggest that Met-35 is key to the oxidative stress and neurotoxic properties of A &#103 (1-42) and may help explain the apoE allele dependence on risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of A &#103 (1-42)-induced oxidative stress and neurodegeneration in AD.     

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.     

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.     

N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.     

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.     

Assemblies of Alzheimer's peptides A beta 25-35 and A beta 31-35: reverse-turn conformation and side-chain interactions revealed by X-ray diffraction

Alzheimer's beta amyloid protein (A beta) is a 39 to 43 amino acid peptide that is a major component in the neuritic plaques of Alzheimer's disease (AD). The assemblies constituted from residues 25-35 (A beta 25-35), which is a sequence homologous to the tachykinin or neurokinin class of neuropeptides, are neurotoxic. We used X-ray diffraction and electron microscopy to investigate the structure of the assemblies formed by A beta 25-35 peptides and of various length sequences therein, and of tachykinin-like analogues. Most solubilized peptides after subsequent drying produced diffraction patterns characteristic of beta-sheet structure. Moreover, the peptides A beta 31-35 (Ile-Ile-Gly-Leu-Met) and tachykinin analogue A beta(Phe(31))31-35 (Phe-Ile-Gly-Leu-Met) gave powder diffraction patterns to 2.8A Bragg spacing. The observed reflections were indexed by an orthogonal unit cell having dimensions of a=9.36 A, b=15.83 A, and c=20.10 A for the native A beta 31-35 peptide, and a=9.46 A, b=16.22 A, and c=11.06 A for the peptide having the Ile31Phe substitution. The initial model was a beta strand where the hydrogen bonding, chain, and intersheet directions were placed along the a, b, and c axes. An atomic model was fit to the electron density distribution, and subsequent refinement resulted in R factors of 0.27 and 0.26, respectively. Both peptides showed a reverse turn at Gly33 which results in intramolecular hydrogen bonding between the antiparallel chains. Based on previous reports that antagonists for the tachykinin substance P require a reverse turn, and that A beta is cytotoxic when it is oligomeric or fibrillar, we propose that the tachykinin-like A beta 31-35 domain is a turn exposed at the A beta oligomer surface where it could interact with the ligand-binding site of the tachykinin G-protein-coupled receptor.     

NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque-competence, A beta(1-40)(ox) and A beta(1-42)(ox).

NMR studies of amyloid beta-peptides (A beta) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer's disease-related conformational polymorphism of A beta. In the aqueous medium, neither of the polypeptides A beta(1-40)(ox) or A beta(1-42)(ox) (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of A beta(1-40)(ox) and A beta(1-42)(ox) are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in A beta(1-42)(ox) when compared to A beta(1-40)(ox). The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that A beta(1-42)(ox) in aqueous solution has much higher plaque-competence than A beta(1-40)(ox).     

Cupric-amyloid beta peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical

A growing body of evidence supports an important role for oxidative stress in the pathogenesis of Alzheimer's disease. Recently, a number of papers have shown a synergistic neurotoxicity of amyloid beta peptide and cupric ions. We hypothesized that complexes of cupric ions with neurotoxic amyloid beta peptides (Abeta) can stimulate copper-mediated free radical formation. We found that neurotoxic Abeta (1-42), Abeta (1-40), and Abeta (25-35) stimulated copper-mediated oxidation of ascorbate, whereas nontoxic Abeta (40-1) did not. Formation of ascorbate free radical was significantly increased by Abeta (1-42) in the presence of ceruloplasmin. Once cupric ion is reduced to cuprous ion, it can be oxidized by oxygen to generate superoxide radical or it can react with hydrogen peroxide to form hydroxyl radical. Hydrogen peroxide greatly increased the oxidation of cyclic hydroxylamines and ascorbate by cupric-amyloid beta peptide complexes, implying redox cycling of copper ions. Using the spin-trapping technique, we have shown that toxic amyloid beta peptides led to a 4-fold increase in copper-mediated hydroxyl radical formation. We conclude that toxic Abeta peptides do indeed stimulate copper-mediated oxidation of ascorbate and generation of hydroxyl radicals. Therefore, cupric-amyloid beta peptide-stimulated free radical generation may be involved in the pathogenesis of Alzheimer's disease.     

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.     

Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been assumed to be as a result of the extensive accumulation of beta-amyloid (Abeta). In addition to Abeta fibrillar assemblies, there are pre-fibrillar forms that have been shown to be neurotoxic, although their role in cholinergic degeneration is still not known. Using the cholinergic cell line SN56.B5.G4, we investigated the effect of different Abeta(1-42) aggregates on cell viability. In our model, only soluble oligomeric but not fibrillar Abeta(1-42) forms induced toxicity in cholinergic cells. To determine whether the neurotoxicity of oligomeric Abeta(1-42) was caused by its oxidative potential, we performed microarray analysis of SN56.B5.G4 cells treated either with oligomeric Abeta(1-42) or H(2)O(2). We showed that genes affected by Abeta(1-42) differed from those affected by non-specific oxidative stress. Many of the genes affected by Abeta(1-42) were present in the endoplasmic reticulum (ER), Golgi apparatus and/or otherwise involved in protein modification and degradation (chaperones, ATF6), indicating a possible role for ER-mediated stress in Abeta-mediated toxicity. Moreover, a number of genes, which are known to be involved in AD (clusterin, Slc18a3), were identified. This study provides important leads for the understanding of oligomeric Abeta(1-42) toxicity in cholinergic cells, which may account in part for cholinergic degeneration in AD.     

Glutathione-S-transferase P1 is a critical regulator of Cdk5 kinase activity

Cyclin dependent kinase-5 (Cdk5) activity is deregulated in Alzheimer's disease (AD) and contributes to all three hallmarks: neurotoxic β-amyloid formation, neurofibrillary tangles, and neuronal death. However, the mechanism leading to Cdk5 deregulation remains controversial. Cdk5 deregulation in AD is usually linked to the formation of p25, a proteolysis product of Cdk5 activator p35, which leads to Cdk5 mislocalization and hyperactivation. A few studies have indeed shown increased p25 levels in AD brains; however, others have refuted this observation. These contradictory findings suggest that additional factors contribute to Cdk5 deregulation. This study identified glutathione-S-transferase pi 1 (GSTP1) as a novel Cdk5 regulatory protein. We demonstrate that it is a critical determinant of Cdk5 activity in human AD brains and various cancer and neuronal cells. Increased GSTP1 levels were consistently associated with reduced Cdk5 activity. GSTP1 directly inhibits Cdk5 by dislodging p25/p35, and indirectly by eliminating oxidative stress. Cdk5 promotes and is activated by oxidative stress, thereby engaging a feedback loop which ultimately leads to cell death. Not surprisingly, GSTP1 transduction conferred a high degree of neuroprotection under neurotoxic conditions. Given the critical role of oxidative stress in AD pathogenesis, an increase in GSTP1 level may be an alternative way to modulate Cdk5 signaling, eliminate oxidative stress, and prevent neurodegeneration.     

Fragment 31-35 of beta-amyloid peptide induces neurodegeneration in rat cerebellar granule cells via bax gene expression and caspase-3 activation. A crucial role for the redox state of methionine-35 residue

The amyloid beta-peptide (AbetaP) is the major protein component of brain senile plaques in Alzheimer's disease. The redox state of methionine-35 residue plays a critical role in peptide neurotoxic actions. We used the fragment 31-35 of AbetaP [AbetaP(31-35)], containing a single methionine-35 residue (Met-35), to investigate the relationship between the oxidative state of Met-35 and neurotoxic and pro-apoptotic actions induced by the peptide; in rat cerebellar granule cells (CGC), we compared the effects of AbetaP(31-35), in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 [AbetaP(31-35)Met-35(OX)](,) as well as an AbetaP-derivative with Met-35 substituted by norleucine [AbetaP(31-35)Nle-35]. AbetaP(31-35) induced a time-dependent decrease in cell viability. AbetaP(31-35)Met-35(OX) was significantly less potent, but still induced a significant decrease in cell viability compared to control. No toxic effects were observed after treatment with AbetaP(31-35)Nle-35. AbetaP(31-35) induced a 2-fold increase in bax mRNA levels after 4h, whereas AbetaP(31-35)Met-35(OX) raised bax mRNA levels by 41% and AbetaP(31-35)Nle-35 had no effect. Finally, AbetaP(31-35) caused a 43% increase in caspase-3 activity after 24h; AbetaP(31-35)Met-35(OX) caused only a 18% increase, and AbetaP(31-35)Nle-35 had no effect. These findings suggest that AbetaP(31-35)-induced neurodegeneration in CGC is mediated by a selective early increase in bax mRNA levels followed by delayed caspase-3 activation; the redox state of the single Met-35 residue is crucial in the occurrence and extent of the above phenomena.     

Specific reaction of Met 35 in amyloid beta peptide with hypochlorous acid

Abstract

The reaction of the amyloid beta peptide (Aβ) with hypochlorous acid and hydroxyl radicals was analysed by spectrophotometry and mass spectrometry. N-acetylmethionine, Aβ25-35 and Aβ1-42 reacted rapidly with hypochlorous acid. The relative reaction rates of N-acetylmethionine and Aβ with hypochlorous acid was in the order N-acetylmethionine > Aβ25-35 > Aβ1-42. While the reaction of Aβ25-35 in the presence of a slight excess of hypochlorous acid resulted in complete conversion of Met35 to Met35 sulphoxide, Aβ1-42 required more than a 4-fold excess of hypochlorous acid for complete conversion of Met35. Identical products were obtained when Aβ25-35 and Aβ1-42 were treated with a hypochlorous acid generating system. Conversion of Met35 to Met35 sulphoxide in Aβ abolished the aggregation of Aβ25-35. Reaction of Aβ with hydroxyl radicals resulted in limited conversion of Met35 to Met35 sulphoxide. The specific reaction of Met35 in Aβ with hypochlorous acid to form Met35 sulphoxide has been analysed.     

Amyloid-beta peptide fragments p3 and p4 induce pro-inflammatory cytokine and chemokine production in vitro and in vivo

Alzheimer's disease (AD) pathology is characterized by senile plaques containing amyloid-beta (A beta) peptide, a protein with neurotoxic and glial immune activating potential. In addition to the highly amyloidogenic peptides A beta(1--40/42), plaques contain amino-terminal truncated A beta peptides including the alpha secretase-generated p3 fragments A beta(17--40/42). In the present study, A beta(17--40/42), A beta(1--40/42), A beta(1--16), and A beta(25--35) aged in different solvents exhibited varying capacity to activate the murine microglia cell line MG-7 depending on solvent, peptide 'aging', and peptide sequence that did not strictly correlate with beta-sheet formation. A beta(17--40/42) or A beta(1--42) stimulated production of the pro-inflammatory cytokines interleukin (IL)-1 alpha, IL-1 beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha), and the chemokine MCP-1 from differentiated human monocytes (THP-1) while little or no stimulation was observed with the other A beta fragments. MG7 cells also produced these five pro-inflammatory proteins in response to A beta(1-42) whereas A beta(17--40/42) elicited mainly TNF-alpha and MCP-1. Murine and human astrocyte cell lines (D30 and U373, respectively) were generally less responsive to A beta fragments producing mainly IL-6 and MCP-1 in response to A beta(1--42) or A beta(17--40/42) fragments. In mice, an intracerebroventricular infusion of A beta(1--42) significantly increased IL-1 alpha, IL-1 beta, IL-6 and MCP-1 while A beta(17--40/42) increased MCP-1 and A beta(17--40) increased IL-1 beta. These results demonstrate that p3 and p4 A beta fragments are pro-inflammatory glial modulators and thus may play a role in development of the immunopathology observed in AD.     

Sub-lethal levels of amyloid β-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons

Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aβ) peptide. Current hypotheses regard soluble amyloid beta oligomers (AβOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aβ fibrils and AβOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AβOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AβOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AβOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AβOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AβOs did not potentiate iron-induced cell death while iron chelation did not decrease AβOs-induced cell death. The lack of synergism between iron and AβOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration.     

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.     

Glu11 site cleavage and N-terminally truncated A beta production upon BACE overexpression

Amyloid beta peptides (A beta) are generated by the proteolytic processing of the amyloid beta precursor protein (APP). The newly identified beta-site APP-cleaving enzyme (BACE) cleaves APP at Asp1 as well as between Tyr10 and Glu11 of A beta, producing C-terminal fragments (CTFs) C99 and C89, respectively. Subsequent cleavage by gamma-secretase gives rise to A beta 1-40/42 and A beta 11-40/42. Although both full-length and A beta peptides truncated at residue 11 have been identified in amyloid plaques in the AD brain, the relative proportion of these two cleavage products produced by BACE and secreted into the medium by cultured cells is unknown. Using cell lines stably overexpressing BACE, we found that A beta 11-40 and A beta 11-42 are major A beta cleavage products generated by BACE. We further showed that BACE utilizes both full-length APP as well as C99 as substrates for the production of C89, and that A beta 11-40/42 can be generated by sequential cleavage of single APP molecules by BACE and gamma-secretase. Taken together, the abundance of A beta 11-40/42 produced by BACE suggests that their roles in AD pathogenesis may be underestimated.