Alzheimer's Disease Senile Plaques Research Articles

Characterization of AβpN3 deposition in the brains of dogs of various ages and other animal species

Senile plaques (SP) are characteristic histopathological manifestations of Alzheimer's disease (AD), but are also found in normal aging (NA). Recent studies have demonstrated that beta amyloid (Aβ) proteins that have been truncated at the N-terminal position 3 (AβpN3) are the predominant component of SP in AD, but not in NA. The present study revealed that AβpN3 was deposited in an age-dependent manner in canine brains. Moreover, AβpN3 was the main component of the SP that developed in very old dogs. The deposition of AβpN3 increased in accordance with the number of SP, but that of N-terminally intact Aβ (AβN1) did not. In addition, AβpN3 was also deposited in the SP of a Japanese macaque and an American black bear, but not in a feline brain. Focal microvascular cerebral amyloid angiopathy was also observed in the deep cortices and the white matter of the dogs and a woodpecker. Those were always composed of both AβpN3 and AβN1. In conclusion, though non-human animals do not develop full pathology of AD of the human type, AβpN3 is widely deposited in the brains of senescent vertebrates.

Distinct Effects of Zn2+, Cu2+, Fe3+, and Al3+ on Amyloid-β Stability, Oligomerization, and Aggregation: AMYLOID-β DESTABILIZATION PROMOTES ANNULAR PROTOFIBRIL FORMATION

Abnormally high concentrations of Zn(2+), Cu(2+), and Fe(3+) are present along with amyloid-β (Aβ) in the senile plaques in Alzheimer disease, where Al(3+) is also detected. Aβ aggregation is the key pathogenic event in Alzheimer disease, where Aβ oligomers are the major culprits. The fundamental mechanism of these metal ions on Aβ remains elusive. Here, we employ 4,4'-Bis(1-anilinonaphthalene 8-sulfonate) and tyrosine fluorescence, CD, stopped flow fluorescence, guanidine hydrochloride denaturation, and photo-induced cross-linking to elucidate the effect of Zn(2+), Cu(2+), Fe(3+), and Al(3+) on Aβ at the early stage of the aggregation. Furthermore, thioflavin T assay, dot blotting, and transmission electron microscopy are utilized to examine Aβ aggregation. Our results show that Al(3+) and Zn(2+), but not Cu(2+) and Fe(3+), induce larger hydrophobic exposures of Aβ conformation, resulting in its significant destabilization at the early stage. The metal ion binding induces Aβ conformational changes with micromolar binding affinities and millisecond binding kinetics. Cu(2+) and Zn(2+) induce similar assembly of transiently appearing Aβ oligomers at the early state. During the aggregation, we found that Zn(2+) exclusively promotes the annular protofibril formation without undergoing a nucleation process, whereas Cu(2+) and Fe(3+) inhibit fibril formation by prolonging the nucleation phases. Al(3+) also inhibits fibril formation; however, the annular oligomers co-exist in the aggregation pathway. In conclusion, Zn(2+), Cu(2+), Fe(3+), and Al(3+) adopt distinct folding and aggregation mechanisms to affect Aβ, where Aβ destabilization promotes annular protofibril formation. Our study facilitates the understanding of annular Aβ oligomer formation upon metal ion binding.

Mechanism of neuroprotection of melatonin against beta-amyloid neurotoxicity

The main component of senile plaques in Alzheimer's disease (AD), aggregated amyloid beta peptide (βA), is neurotoxic and implicated in AD pathology. Melatonin is a hormone secreted from the pineal gland, levels of which are decreased in aging, particularly in AD subjects. This hormone is known to possess neuroprotective properties against βA toxicity in vivo, but the mechanism of protection remains controversial. In cultures of mixed neurones and astrocytes, we find that melatonin is protective against neuronal and astrocytic death induced by aggregated full length βA 1-40 and the fragments βA 25–40 and βA 1-28. Melatonin had no effect on the process of fibrillation of βA and did not alter βA-induced calcium signalling in astrocytes, but did significantly reduce the rate of βA-induced reactive oxygen species production and also protected astrocytes against the mitochondrial depolarisation. Thus, scavenging of reactive oxygen species by melatonin appears to be the primary effect of melatonin in protecting neurones and astrocytes against βA toxicity.

Molecular-level examination of Cu2+ binding structure for amyloid fibrils of 40-residue Alzheimer's β by solid-state NMR spectroscopy.

Cu(2+) binding to Alzheimer's β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer's senile plaque and such association of Aβ with Cu(2+) triggers the production of neurotoxic reactive oxygen species (ROS) such as H(2)O(2). However, detailed binding sites and binding structures of Cu(2+) to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu(2+) binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution (13)C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1-40). Selective quenching observed in (13)C SSNMR of Cu(2+)-bound Aβ(1-40) suggested that primary Cu(2+) binding sites in Aβ(1-40) fibrils include N(ε) in His-13 and His-14 and carboxyl groups in Val-40 as well as in Glu sidechains (Glu-3, Glu-11, and/or Glu-22). (13)C chemical shift analysis demonstrated no major structural changes upon Cu(2+) binding in the hydrophobic core regions (residues 18-25 and 30-36). Although the ROS production via oxidization of Met-35 in the presence of Cu(2+) has been long suspected, our SSNMR analysis of (13)C(ε)H(3)-S- in M35 showed little changes after Cu(2+) binding, excluding the possibility of Met-35 oxidization by Cu(2+) alone. Preliminary molecular dynamics (MD) simulations on Cu(2+)-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu(2+)-binding modes unique in Aβ fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.

Lipoprotein Lipase Is a Novel Amyloid β (Aβ)-binding Protein That Promotes Glycosaminoglycan-dependent Cellular Uptake of Aβ in Astrocytes

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.

Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide

Phospholipids are known to influence fibril formation of amyloid beta (Aβ) peptide. Here, we show that lysophosphatidylcholine (LPC), a polar phospholipid, enhances Aβ(1-42) fibril formation, by decreasing the lag time and the critical peptide concentration required for fibril formation, and increasing the fibril elongation rate. Conversely, LPC did not have an enhancing effect on Aβ(1-40) fibril formation, and appeared to be inhibitory. Tyrosine fluorescence spectroscopy showed that LPC altered the fluorescence spectra of Aβ(1-40) and Aβ(1-42) in opposite ways. Further, 8-anilino-1-naphthalene sulfonic acid fluorescence spectroscopy showed that LPC significantly increased the hydrophobicity of Aβ(1-42), but not of Aβ(1-40). Tris-tricine gradient SDS/PAGE revealed that LPC increased the formation of higher-molecular-weight species of Aβ(1-42), including trimers and tetramers. LPC had no such effect on Aβ(1-40), and thus may specifically influence the oligomerization and nucleation processes of Aβ(1-42) in a manner dependent on its native structure. Dot-blot assays confirmed that LPC induced Aβ(1-42) oligomer formation at an early time point. Thus our results indicate that LPC specifically enhances the formation of Aβ(1-42) fibrils, the main component of senile plaques in Alzheimer's disease patients, and may be involved in Alzheimer's disease pathology.

Monitoring the aggregation processes of amyloid-β using a spin-labeled, fluorescent nitroxyl radical

Amyloid nitroxyl radical (nitroxide) ligands were used to detect amyloid-β fibrils, the main constituents of senile plaques in Alzheimer's disease, using anisotropic ESR spectra, and were found to affect the aggregation process due to the radical functionality. These compounds have great potential as novel and multifunctional probes, combining spin labels, spin probes, and fluorescence probes.

Impacts of Membrane Biophysics in Alzheimer′s Disease: From Amyloid Precursor Protein Processing to Aβ Peptide‐Induced Membrane Changes

An increasing amount of evidence supports the notion that cytotoxic effects of amyloid-β peptide (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD), are strongly associated with its ability to interact with membranes of neurons and other cerebral cells. Aβ is derived from amyloidogenic cleavage of amyloid precursor protein (AβPP) by β- and γ-secretase. In the nonamyloidogenic pathway, AβPP is cleaved by α-secretases. These two pathways compete with each other, and enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of AD. Since AβPP, α-, β-, and γ-secretases are membrane-associated proteins, AβPP processing and Aβ production can be affected by the membrane composition and properties. There is evidence that membrane composition and properties, in turn, play a critical role in Aβ cytotoxicity associated with its conformational changes and aggregation into oligomers and fibrils. Understanding the mechanisms leading to changes in a membrane's biophysical properties and how they affect AβPP processing and Aβ toxicity should prove to provide new therapeutic strategies for prevention and treatment of AD.

Detection of Aβ Plaques by a Novel Specific MRI Probe Precursor CR‐BSA‐(Gd‐DTPA)n in APP/PS1 Transgenic Mice

Amyloid plaques are one of the hallmarks of Alzheimer's disease (AD). The lack of specific probes that can detect individual senile plaques in AD has prompted the development of magnetic resonance imaging (MRI) probes. In this study, based on DTPA-gadolinium (III) and congo red (CR), a novel specific MRI probe precursor CR-BSA-(Gd-DTPA)n was successfully synthesized. Its ability to bind to amyloid plaques was evaluated by brain sections from APP/PS1 transgenic mice. Its specificity for Aβ plaques was further demonstrated by immunohistochemistry (IHC) staining with the monoclonal antibody to the Aβ protein. Meanwhile, the amyloid deposits detected by the CR-BSA-(Gd-DTPA)n were matched to the amyloid deposits detected by Aβ specific antibody. We also found that a few amyloid-like deposits which was not detected by IHC. The findings indicated that the probe perhaps could detect the neurofibrillary tangles (NFT) similar to the effect of CR itself, and this will be verified in future experiments. The works suggested that the Aβ protein-specific magnetic resonance contrast agent precursor CR-BSA-(Gd-DTPA)n can be used as a potential fluorescence and MR multi-modal imaging probe precursor to display individual senile plaques in AD.

Hypothesis: Soluble Aβ Oligomers in Association with Redox‐Active Metal Ions Are the Optimal Generators of Reactive Oxygen Species in Alzheimer′s Disease

Considerable evidence points to oxidative stress in the brain as an important event in the early stages of Alzheimer's disease (AD). The transition metal ions of Cu, Fe, and Zn are all enriched in the amyloid cores of senile plaques in AD. Those of Cu and Fe are redox active and bind to Aβ in vitro. When bound, they can facilitate the reduction of oxygen to hydrogen peroxide, and of the latter to the hydroxyl radical. This radical is very aggressive and can cause considerable oxidative damage. Recent research favours the involvement of small, soluble oligomers as the aggregating species responsible for Aβ neurotoxicity. We propose that the generation of reactive oxygen species (i.e., hydrogen peroxide and hydroxyl radicals) by these oligomers, in association with redox-active metal ions, is a key molecular mechanism underlying the pathogenesis of AD and some other neurodegenerative disorders.

Novel synthetic small‐molecule activators of AMPK as enhancers of autophagy and amyloid‐β peptide degradation

AMP-activated protein kinase (AMPK) is a metabolic sensor involved in intracellular energy metabolism through the control of several homeostatic mechanisms, which include autophagy and protein degradation. Recently, we reported that AMPK activation by resveratrol promotes autophagy-dependent degradation of the amyloid-β (Aβ) peptides, the core components of the cerebral senile plaques in Alzheimer's disease. To identify more potent enhancers of Aβ degradation, we screened a library of synthetic small molecules selected for their structural similarities with resveratrol. Here, we report the identification of a series of structurally related molecules, the RSVA series, which inhibited Aβ accumulation in cell lines nearly 40 times more potently than did resveratrol. Two of these molecules, RSVA314 and RSVA405, were further characterized and were found to facilitate CaMKKβ-dependent activation of AMPK, to inhibit mTOR (mammalian target of rapamycin), and to promote autophagy to increase Aβ degradation by the lysosomal system (apparent EC(50) ∼ 1 μM). This work identifies the RSVA compounds as promising lead molecules for the development of a new class of AMPK activating drugs controlling mTOR signaling, autophagy, and Aβ clearance.

Heavy-Chain Complementarity-Determining Regions Determine Conformation Selectivity of Anti-Aβ Antibodies

Background/Aims: Amyloid-β (Aβ) protofibrils are neurotoxic soluble intermediates in the Aβ aggregation process eventually forming senile plaques in Alzheimer’s disease. This Aβ species is a potential biomarker for Alzheimer’s disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent Aβ antibodies specific for Aβ protofibrils. Methods: Mice were immunized with Aβ protofibrils to generate hybridomas producing Aβ-specific monoclonal antibodies. Binding of antibodies to different Aβ conformations was investigated with inhibition ELISA. The antibodies’ complementarity-determining region (CDR) sequences were determined and compared. Results: A majority of the antibodies were of the IgM class, all selectively binding to aggregated Aβ. Two IgG antibodies were generated: one with selective affinity for Aβ protofibrils and the other bound Aβ in all conformations. A high degree of similarity between the heavy-chain CDRs of the conformation-dependent antibodies was found, and all high-affinity Aβ antibodies displayed a high degree of sequence similarity in the light-chain CDRs. Conclusion: Sequence similarity in the heavy-chain CDRs is associated with conformation selectivity of the antibodies, while sequence similarity in the light-chain CDRs correlates with the affinity for Aβ.

Cellular Source of Apolipoprotein E4 Determines Neuronal Susceptibility to Excitotoxic Injury in Transgenic Mice

The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimer's disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.

Using single-point pharmacokinetic/pharmacodynamic (PK/PD) analyses to support early discovery of inhibitors of β-site APP cleaving enzyme 1 (BACE1)

BACE1 is responsible for the first step of amyloidogenic cleavage of the amyloid precursor protein (APP), leading to production of amyloid-β (Aβ), the principal component of senile plaques in Alzheimer's disease. Several in vitro assays have been employed to measure compound potency in inhibiting BACE1, such as enzyme activity assay (EAA) and whole-cell assays (WCA) with cells overexpressing wild-type (WT) or mutant APP. To expedite early discovery of BACE1 inhibitors, it is critical to address two issues: which in vitro potency measure best predicts in vivo efficacy and how the relevant in vitro potency translates to in vivo efficacy. A series of compounds were profiled for IC50's for BACE1 inhibition in the EAA, WT-WCA, and mutant-WCA using ELISAs. A subset of the series was tested in WT and Pgp knockout mice for the effects on brain Aβ40 and Aβ42 and the associated drug exposures at 3 hrs after a single dose. We then (1) examined the correlation between any two in vitro potency measures of the series, (2) inspected the relationships of the brain Aβ lowering and the respective exposures normalized by various potency measures (in vitro-in vivo correlation, IVIVC) and identified the best IVIVC, and (3) analyzed the best IVIVC quantitatively to understand in vitro-in vivo translation. Within the potency range of up to 2500 nM, the WT-WCA and mutant-WCA IC50's are modestly correlated (R2 = 0.8); both of them are only weakly correlated with EAA IC50 (R2 < 0.45). The IVIVC inspection found that free brain drug exposure (Cb,u) is more relevant than total brain exposure and that the EAA IC50 best predicts in vivo effect of brain Aβ reduction. Further quantitative analyses of the brain Aβ-Cb,u/EAA IC50 correlation suggested that the maximum brain Aβ reduction under those experimental settings was about 70% and that 50% of reduction from baseline was achieved at Cb,u equivalent to EAA IC50. An IVIVC has been established among brain Aβ reduction, free brain drug exposure, and enzyme activity assay IC50. This correlation is instrumental in expediting early discovery of BACE1 inhibitors.

β-secretase inhibitors for treatment of Alzheimer's disease

The Amyloid Precursor Protein (APP) undergoes sequential enzymatic cleavage by β-secretase (BACE1) and subsequently by γ-secretase to form the amyloid-β peptide (Aβ), the principal component of senile plaques in Alzheimer's disease (AD). BACE is a membrane bound aspartic acid protease found in the CNS. BACE is characterized by a large binding pocket designed to recognize 6-8 amino acid residues of APP. Small molecule BACE inhibitors would be expected to prevent the generation of the Aβ peptides and consequently reduce the detrimental effects of Aβ toxicity and the formation of amyloid plaques in the brain. While inhibition of BACE1 represents one of the promising therapeutic strategies for AD, development of centrally active, small molecule inhibitors have been challenging. We targeted selective inhibitors of human BACE with the appropriate balance of potency and CNS penetration using rational drug design. Inhibitor design was established from the analysis of BACE-ligand X-ray crystallographic data using a diverse array of inhibitors combined with measurement of the energetics of binding interactions. In vitro screening provided guidance on key issues of permeability and efflux due to P-glycoprotein (Pgp). This set of design features was also coupled with our contemporary understanding of the desirable physical property profile of CNS drugs. A strategy to align a set of physicochemical properties into a single molecule led to the discovery of a novel class of BACE inhibitors. Optimization of the lead compounds using SBDD has resulted in a series of BACE inhibitors with a profile that was shown to inhibit Aβ production in WT and mutant APP whole cell assays. These BACE inhibitors were shown to penetrate the CNS and acutely lower brain Aβ levels in wild type and APP transgenic mice. Our results suggest that it is possible to design BACE inhibitors with the desired physical properties to overcome brain penetration issues and achieve efficacy in the CNS. These results support the utility of BACE inhibitors as a viable approach to therapeutic intervention in AD.

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Aβ), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for β-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPβ, but not sAPPα. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that – in addition to its role in APP trafficking – LRP1 affects APP processing by competing for cleavage by BACE1.

SDS-PAGE/Immunoblot Detection of A&amp;beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the beta-amyloid (Abeta) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis(1). Abeta is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into beta-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Abeta multimers are more toxic than fibrillar Abeta amyloid(2). The identification and quantification of low- and high-molecular weight multimeric Abeta species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies(3). Naturally occurring Abeta multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Abeta-specific antibodies. However, the separation and detection of multimeric Abeta requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Abeta peptide. Using this protocol, we consistently detect Abeta monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

Brain Endothelial Cells Synthesize Neurotoxic Thrombin in Alzheimer’s Disease

Alzheimer's disease (AD) is characterized by neuronal death; thus, identifying neurotoxic proteins and their source is central to understanding and treating AD. The multifunctional protease thrombin is neurotoxic and found in AD senile plaques. The objective of this study was to determine whether brain endothelial cells can synthesize thrombin and thus be a source of this neurotoxin in AD brains. Microvessels were isolated from AD patient brains and from age-matched controls. Reverse transcription-PCR demonstrated that thrombin message was highly expressed in microvessels from AD brains but was not detectable in control vessels. Similarly, Western blot analysis of microvessels showed that the thrombin protein was highly expressed in AD- but not control-derived microvessels. In addition, high levels of thrombin were detected in cerebrospinal fluid obtained from AD but not control patients, and sections from AD brains showed reactivity to thrombin antibody in blood vessel walls but not in vessels from controls. Finally, we examined the ability of brain endothelial cells in culture to synthesize thrombin and showed that oxidative stress or cell signaling perturbations led to increased expression of thrombin mRNA in these cells. The results demonstrate, for the first time, that brain endothelial cells can synthesize thrombin, and suggest that novel therapeutics targeting vascular stabilization that prevent or decrease release of thrombin could prove useful in treating this neurodegenerative disease.

NMR Studies of Zinc, Copper, and Iron Binding to Histidine, the Principal Metal Ion Complexing Site of Amyloid-β Peptide

Amyloid-beta (Abeta), the major component of senile plaques in Alzheimer's disease, is known to complex transition metal ions mainly through histidine residues. In this study, using 1H NMR titration experiments, we show that histidine binds strongly to Zn(II), Cu(II), and Fe(III) ions at a biologically relevant pH (pH 7.4), with a stoichiometry of Zn(II): histidine binding of 1:2. The observed deshielding of the chemical shifts and relative line broadening indicate that Fenton-active Cu(II) and Fe(III) bind to histidine relatively more efficiently as compared to Zn(II). Parallel studies showed that glutamic acid and aspartic acid are relatively inefficient in metal ion binding. From these studies, we suggest that Abeta-chelated Zn(II) is readily displaced by Cu(II) and Fe(III) ions and leads to a propagation of oxidative stress.

Oxidation of methionine 35 reduces toxicity of the amyloid beta-peptide(1–42) in neuroblastoma cells (IMR-32) via enzyme methionine sulfoxide reductase A expression and function

The beta amyloid peptide (Aβ), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals that may lead to neurodegeneration. Our recent evidence suggest that the redox state of methionine residue in position 35 (Met-35) of Aβ has the ability to deeply modify peptide's neurotoxic actions. Reversible oxidation of methionine in proteins involving the enzyme methionine sulfoxide reductase type A (MsrA) is postulated to serve a general antioxidant role and a decrease in MsrA has been implicated in Alzheimer's disease. In rat neuroblastoma cells (IMR-32), we used Aβ(1–42), in which the Met-35 is present in the reduced state, with a modified peptide with oxidized Met-35 (Aβ(1–42)Met35 OX), as well as an Aβ-derivative in which Met-35 is substituted with norleucine (Aβ(1–42)Nle35) to investigate the relationship between Met-35 redox state, expression and function of MsrA and reactive oxygen species (ROS) generation. The obtained results shown that MsrA activity, as well as mRNA levels, increase in IMR-32 cells treated with Aβ(1–42)Met35 OX, differently to that shown by the reduced derivative. The increase in MsrA function and expression was associated with a decline of ROS levels. None of these effects were observed when cells were exposed to Aβ containing oxidized Met35 (Aβ1–42)Met35 OX. Taken together, the results of the present study indicate that the differential toxicity of Aβ peptides containing reduced or oxidised Met-35 depends on the ability of the latter form to reduce ROS generation by enhancing MsrA gene expression and function and suggests the therapeutic potential of MsrA in Alzheimer's disease.