Selective intracellular release of copper and zinc ions from bis(thiosemicarbazonato) complexes reduces levels of Alzheimer disease amyloid-beta peptide

Copper and zinc play important roles in Alzheimer disease pathology with recent reports describing potential therapeutics based on modulation of metal bioavailability. We examined the ability of a range of metal bis(thiosemicarbazonato) complexes (MII(btsc), where M=CuII or ZnII) to increase intracellular metal levels in Chinese hamster ovary cells overexpressing amyloid precursor protein (APP-CHO) and the subsequent effect on extracellular levels of amyloid-beta peptide (Abeta). The CuII(btsc) complexes were engineered to be either stable to both a change in oxidation state and dissociation of metal or susceptible to intracellular reduction and dissociation of metal. Treatment of APP-CHO cells with stable complexes resulted in elevated levels of intracellular copper with no effect on the detected levels of Abeta. Treatment with complexes susceptible to intracellular reduction increased intracellular copper levels but also resulted in a dose-dependent reduction in the levels of monomeric Abeta. Treatment with less stable ZnII(btsc) complexes increased intracellular zinc levels with a subsequent dose-dependent depletion of monomeric Abeta levels. The increased levels of intracellular bioavailable copper and zinc initiated a signaling cascade involving activation of phosphoinositol 3-kinase and c-Jun N-terminal kinase. Inhibition of these enzymes prevented Abeta depletion induced by the MII(btsc) complexes. Inhibition of metalloproteases also partially restored Abeta levels, implicating metal-driven metalloprotease activation in the extracellular monomeric Abeta depletion. However, a role for alternative metal-induced Abeta metabolism has not been ruled out. These studies demonstrate that MII(btsc) complexes have potential for Alzheimer disease therapy.     

Presenilins promote the cellular uptake of copper and zinc and maintain copper chaperone of SOD1-dependent copper/zinc superoxide dismutase activity

Dyshomeostasis of extracellular zinc and copper has been implicated in β-amyloid aggregation, the major pathology associated with Alzheimer disease. Presenilin mediates the proteolytic cleavage of the β-amyloid precursor protein to release β-amyloid, and mutations in presenilin can cause familial Alzheimer disease. We tested whether presenilin expression affects copper and zinc transport. Studying murine embryonic fibroblasts (MEFs) from presenilin knock-out mice or RNA interference of presenilin expression in HEK293T cells, we observed a marked decrease in saturable uptake of radiolabeled copper and zinc. Measurement of basal metal levels in 6-month-old presenilin 1 heterozygous knock-out (PS1(+/-)) mice revealed significant deficiencies of copper and zinc in several tissues, including brain. Copper/zinc superoxide dismutase (SOD1) activity was significantly decreased in both presenilin knock-out MEFs and brain tissue of presenilin 1 heterozygous knock-out mice. In the MEFs and PS1(+/-) brains, copper chaperone of SOD1 (CCS) levels were decreased. Zinc-dependent alkaline phosphatase activity was not decreased in the PS null MEFs. These data indicate that presenilins are important for cellular copper and zinc turnover, influencing SOD1 activity, and having the potential to indirectly impact β-amyloid aggregation through metal ion clearance.     

A new naphthalene derivative with anti-amyloidogenic activity as potential therapeutic agent for Alzheimer's disease

The aggregation of β-amyloid peptides is associated to neurodegeneration in Alzheimer’s disease (AD) patients. Consequently, the inhibition of both oligomerization and fibrillation of β-amyloid peptides is considered a plausible therapeutic approach for AD. Herein, the synthesis of new naphthalene derivatives and their evaluation as anti-β-amyloidogenic agents are presented. Molecular dynamic simulations predicted the formation of thermodynamically stable complexes between the compounds, the Aβ_1-42 peptide and fibrils. In human microglia cells, these compounds inhibited the aggregation of Aβ_1-42 peptide. The lead compound 8 showed a high affinity to amyloid plaques in mice brain ex vivo assays and an adequate log P_oct/PBS value. Compound 8 also improved the cognitive function and decreased hippocampal β-amyloid burden in the brain of 3xTg-AD female mice. Altogether, our results suggest that 8 could be a novel therapeutic agent for AD.     

Inhibition of TDP-43 Accumulation by Bis(thiosemicarbazonato)-Copper Complexes

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable bis(thiosemicarbazonato)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu(II)(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, Cu(II)(atsm) reduced accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu(II)(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.     

ESI-MS identification of the minimal zinc-binding center in natural isoforms of β-amyloid domain 1–16

Alzheimer’s disease is a lethal neurodegenerative pathology accompanied by the formation of water-soluble neurotoxic oligomers of the human β-amyloid peptide, Aβ, which are then accumulated as polymeric extracellular aggregates (the so-called amyloid plaques). The human Aβ isoform isomerized at aspartate 7 (isoAβ) is the major component of amyloid plaques and is regarded as a potential causative agent for Alzheimer’s disease. A mechanism for producing this isoform from a genetically determined variant of D7N β-amyloid (Tottori mutation) has been proposed. However, the rat/mouse Aβ (ratAβ), which carries three amino acid substitutions in metal-binding domain 1–16, is not susceptible to pathogenic aggregation in vivo, unlike the other known genetically determined or chemically modified natural Aβ isoforms. The interactions with zinc ion play a key role in the in vitro and in vivo aggregation of monomeric human Aβ. Here, we have used high-resolution ESI-MS to demonstrate for the first time that domains 1–16 of the isoforms isoAβ and D7N-Aβ bind zinc ion in exactly the same manner as human 1–16 Aβ domain. On the other hand, the structure of the minimal zinc-binding center in ratAβ differs significantly. These results confirm the general mechanism underlying the interaction of zinc ions with human Aβ isoforms and suggest that structural modulations of Aβ region 6–14 can be used as a promising approach to the therapy of Alzheimer’s disease.     

Subcellular localization of a fluorescent derivative of CuII(atsm) offers insight into the neuroprotective action of CuII(atsm)

Copper complexes of bis(thiosemicarbazone) (Cu(II)(btsc)s) have been studied as potential anti-cancer agents and hypoxia imaging agents. More recently, Cu(II)(btsc)s have been identified as possessing potent neuroprotective properties in cell and animal models of neurodegenerative disease. Despite their broad range of pharmacological activity little is known about how cells traffic Cu(II)(btsc)s and how this relates to potential anti-cancer or neuroprotective outcomes. One method of investigating sub-cellular localization of metal complexes is through confocal fluorescence imaging of the compounds in cells. Previously we harnessed the fluorescence of a pyrene group attached to diacetyl-bis(N4-methylthiosemicarbazonato)copper(ii)) (Cu(II)(atsm)), (Cu(II)L(1)). We demonstrated that Cu(II)L(1) was partially localized to lysosomes in HeLa cancer epithelial cells. Here we extend these studies to map the sub-cellular localization of Cu(II)L(1) in M17 neuroblastoma cells. Treatment of M17 or HeLa cells led to rapid association of the Cu-complex into distinct punctate structures that partially co-localized with lysosomes as assessed by co-localization with Lysotracker and acridine orange. No localization to early or late endosomes, the nucleus or mitochondria was observed. We also found evidence for a limited association of Cu(II)L(1) with autophagic structures, however, this did not account for the majority of the punctate localization of Cu(II)L(1). In addition, Cu(II)L(1) revealed partial localization with ER Tracker and was found to inhibit ER stress induced by tunicamycin. This is the first report to comprehensively characterize the sub-cellular localization of a Cu(II)(atsm) derivative in cells of a neuronal origin and the partial association with lysosome/autophagic structures and the ER may have a potential role in neuroprotection.     

Physiological cholesterol concentration is a neuroprotective factor against β-amyloid and β-amyloid-metal complexes toxicity

Alzheimer's disease is one of the most common forms of dementia in the elderly. One of its hallmarks is the abnormal aggregation and deposition of β-amyloid (Aβ). Endogenous and exogenous metal ions seem to influence β-amyloid folding process, aggregation and deposition. Besides these variables other elements appear to affect β-amyloid behavior, such as cholesterol. The physiological concentration of cholesterol in the cerebrospinal fluid (CSF) was used in order to determine the extent in which Aβ and Aβ-metal complexes in vitro aggregation and their toxicity on human neuroblastoma cell cultures is affected. Cholesterol did not appear to influence Aβ and Aβ-metal complexes aggregation, but it was effective in protecting neuroblastoma cells against Aβ complexes' toxicity. The Aβ-Al complex seemed to be the most effective in disrupting and damaging membrane external layer, and simultaneously it appears to increase its toxicity on cell cultures; both of these effects are preventable by cholesterol. The presence in physiological concentrations of cholesterol seemed to compensate membrane damage that occurred to neuroblastoma cells. These findings appear to contradict some data reported in literature. We believe that our results might shed some light on the role played by cholesterol at physiological concentrations in both cellular balance and membrane protection.     

Zinc-induced interactions of the metal-binding domain of beta-amyloid with nucleic acids and glycosaminoglycans

Zinc ions form complexes with β-amyloid peptides and play an important role in Alzheimer’s disease pathogenesis. It has been demonstrated by turbidimetry and correlation spectroscopy that synthetic peptide Aβ16 representing the metal-binding domain of β-amyloid is able to interact with nucleic acids, chondroitin polysulfate, and dextran sulfates in the presence of zinc ions. The amino acid D7H substitution enhanced the peptide binding to polyanions, whereas the H6R and H6A-H13A substitutions abolished this interaction. It is suggested that the metal-binding domain may serve as a zinc-dependent site of β-amyloid interaction with biological polyanions including DNA, RNA, and glycosaminoglycans.     

维生素C保护Aβ1-40Cu(Ⅱ)复合物引起的神经元氧化损伤

老年斑中存在大量β 淀粉样蛋白（β-amyloid, Aβ）是老年痴呆症（Alzheimer′s disease, AD）的重要病理特征.大量数据表明,Aβ上具有与过渡态金属离子共价结合的位点,二者能结合成为寡聚复合物. Aβ_1-40Cu（Ⅱ）复合物通过Cu²⁺的还原催化O₂产生H₂O₂但反应机制不清.本文尝试以天然抗氧化剂维生素C（VC）来对抗Aβ_1-40及Aβ_1-40Cu（Ⅱ）复合物产生的H₂O₂对原代培养的神经细胞的毒性.结果表明,VC能够起到显著的保护作用,其有效浓度为1mmol/L.本文用胞外乳酸脱氢酶泄漏量和H₂O₂生成量的数据证实了细胞存活率（MTT实验）的实验结果.这些结果表明,Aβ_1-40Cu（Ⅱ）复合物能够释放更多的H₂O₂,引发细胞膜破裂并最终引起细胞死亡.加入VC后,神经元受到的损伤较轻,提示VC在保护细胞免受氧化损伤方面发挥了重要作用.     

Design,synthesis and evaluation of flurbiprofen-clioquinol hybrids as multitarget-directed ligands against Alzheimer’s disease

A series of novel flurbiprofen-clioquinol hybrids were designed and synthesized as multifunctional agents for Alzheimer’s disease therapy, and their potential was evaluated through various biological experiments. In vitro studies showed that most target compounds exhibited significant ability to inhibit self- and Cu²⁺-induced β-amyloid aggregation. Furthermore, some target compounds, especially 7i and 7r, also showed biometal chelating abilities, antioxidant activity, anti-neuroinflammatory activity and appropriate BBB permeability. These biological activities indicated that the representative compound 7i and 7r might be promising multifunctional agents for AD treatment.     

The synthesis and structure–activity relationship of substituted N-phenyl anthranilic acid analogs as amyloid aggregation inhibitors

It is believed that β-amyloid aggregation is an important event in the development of Alzheimer’s disease. In the course of our studies to identify β-amyloid aggregation inhibitors, a series of N-phenyl anthranilic acid analogs were synthesized and studied for β-amyloid inhibition activity. The synthesis, structure–activity relationship, and in vivo activity of these analogs are discussed.     

Phenylpropanoids and lignans from Prunus tomentosa seeds as efficient β-amyloid (Aβ) aggregation inhibitors

Alzheimer’s disease (AD) is characterized by the progressive accumulation of extracellular β-amyloid (Aβ) aggregates. Recently, lignans and phenylpropanoids are attracting increasing attention to discovery useful agents of inhibition on Aβ aggregation. In the present study, to develop potential agents for slowing the progression of AD, Prunus tomentosa seeds were selected as a raw material for bioactive compounds, which led to the separation of two pairs of new enantiomeric lignans and phenylpropanoids using chiral HPLC. The planar structures of these compounds were elucidated by spectroscopic data analyses. And their absolute configurations were determined by comparing of experimental and calculated electronic circular dichroism (ECD). The biosynthesis pathway was also discussed. Additionally, the inhibitory activity on Aβ aggregation of all optical pure compounds was tested by thioflavin T (ThT) assay. The isolates (1a, 1b, 2a and 2b) showed more potent inhibitory activity than positive control curcumin with inhibitory rate of 73.89 ± 3.41% 78.69 ± 1.50%, 63.25 ± 2.68%, and 67.13 ± 0.90% at 20 μM, respectively. More importantly, the inhibition profiles were explained by molecular dynamics and docking simulation studies.     

Neuronal Autophagy: Self-eating or Self-cannibalism in Alzheimer’s Disease

Autophagy is a major intracellular degeneration pathway involved in the elimination and recycling of damaged organelles and long-lived proteins by lysosomes. Many of the pathological factors, which trigger neurodegenerative diseases, can perturb the autophagy activity, which is associated with misfolded protein aggregates accumulation in these disorders. Alzheimer’s disease, the first neurodegenerative disorder between dementias, is characterized by two aggregating proteins, β-amyloid peptide (plaques) and τ-protein (tangles). In Alzheimer’s disease autophagosomes dynamically form along neurites within neuronal cells and in synapses but effective clearance of these structures needs retrograde transportation towards the neuronal soma where there is a major concentration of lysosomes. Maturation of autophago-lysosomes and their retrograde trafficking are perturbed in Alzheimer’s disease, which causes a massive concentration of autophagy elements along degenerating neurites. Transportation system is disturbed along defected microtubules in Alzheimer’s disease brains. τ-protein has been found to control the stability of microtubules, however, phosphorylation of τ-protein or an increase in the total level of τ-protein can cause dysfunction of neuronal cells microtubules. Current evidence has shown that autophagy is developing in Alzheimer’s disease brains because of ineffective degradation of autophagosomes, which hold amyloid precursor protein-rich organelles and secretases important for β-amyloid peptides generation from amyloid precursor. The combination of raised autophagy induction and abnormal clearance of β-amyloid peptide-generating autophagic vacuoles creates circumstances helpful for β-amyloid peptide aggregation and accumulation in Alzheimer’s disease. However, the key role of autophagy in Alzheimer’s disease development is still under consideration today. One point of view suggests that abnormal autophagy induction causes a concentration of autophagic vacuoles rich in amyloid precursor protein, β-amyloid peptide and the elements crucial for its formation, whereas other hypothesis points to marred autophagic clearance or even decrease in autophagic effectiveness playing a role in maturation of Alzheimer’s disease. In this review we present the recent evidence linking autophagy to Alzheimer’s disease and the role of autophagic regulation in the development of full-blown Alzheimer’s disease.     

Probing copper/tau protein interactions electrochemically

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid β-peptide (Aβ), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140 ± 5 mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.     

β-Amyloid-aluminum complex alters cytoskeletal stability and increases ROS production in cortical neurons

Several lines of evidence have supported the potential involvement of metal ions in the etiology of Alzheimer’s Disease (AD). However, the molecular mechanisms underlying this interaction are still partially unknown. Previous work from our laboratory has shown that β-amyloid peptide (Aβ) aggregation was strongly influenced by the conjugation of the peptide with few metal ions (aluminum, copper, zinc, and iron) that are found in high concentrations in the senile plaque core. The binding of aluminum (Al) to Aβ specifically stabilized the peptide in an oligomeric conformation. Here, we show that the aggregation of Aβ-Al was boosted by sodium dodecyl sulfate, a detergent that mimics some characteristics of biological membrane, suggesting a potential role for membrane components in the Aβ aggregation process. Notably, we also found that Aβ-Al caused mitochondrial dysfunction and reactive oxygen species production in primary cortical neurons. Aβ-Al strongly promoted also alterations in cytoskeleton network as shown by the increased F-actin expression and the occurrence of neuritic beading. Interestingly, the neurotoxic effect of this metal complex was associated with a decreased mRNA expression of ubiquitin thiolesterase, an ubiquitin-dependent protein involved in catabolic process, and by the increased expression of glutaminyl cyclase, responsible for pathological post-translational modification of Aβ. These results suggest that, in neuronal cells, Aβ-Al can induce relevant detrimental changes that resemble pathological hallmarks of AD.     

Electrochemical synthesis and crystal structures of nickel(II), copper(II), zinc(II) and cadmium(II) complexes with N,N′-bis[(4-methylphenyl)sulfonyl]ethylenediamine

The electrochemical oxidation of anodic metal (nickel, copper, zinc and cadmium) in acetonitrile solutions containing N,N′-bis[(4-methylphenyl)sulfonyl]ethylenediamine H₂L and an additional nitrogen coligand, such as 1,10-phenanthroline, yielded mixed complexes of general formula [ML(phen)₂] (M=Ni, Cu, Zn and Cd). The compounds have been characterized by microanalysis, IR and UV-Vis (Ni, Cu complexes) spectroscopy, FAB mass spectrometry, ¹H NMR spectroscopic studies (Zn, Cd complexes) and EPR spectroscopy (Cu and Ni complexes). All compounds have also been characterized by single crystal X-ray diffraction. The molecular structures of these compounds consist of individual monomeric molecules in which the metal atom is in an [MN₆] distorted octahedral environment.     

The reaction of zinc sulphide with ionic copper—the biological implications. Part 1. Surface area studies of zinc sulphide and its reaction with copper complexes and copper containing proteins

The role of metal sulphides vis-à-vis the availability of dietary copper in ruminant animals has been investigated using zinc sulphide as a model metal sulphide and a selection of copper complexes and copper containing proteins as models for sources of dietary copper. The extent of reactivity of zinc sulphide towards the copper complexes is dependent upon the type of donor atom co-ordinated to copper:

The order to reactivity is found to be CuO > CuN > CuS complexes and is in keeping with the reported values for the instability constant pK_n of the complexes. In contrast, no reaction is observed between zinc sulphide and the copper containing proteins studied (azurin, superoxide dismutase and cerulophasmin) and is attributed to the protection of the copper centres by the protein backbone. The results facilitate an understanding of copper metabolism in ruminants and a mechanism is proposed for the removal of dietary copper sources in such species.Reactions between copper(II) sulphate solutions and samples of zinc sulphide having a range of specific surface areas (prepared by sintering at differing temperatures) have been studied. The fact that the reactivity is found to be highly dependent upon the specific surface area of the metal sulphide may well be of significance when considering the fate of copper in sulphur-rich biological systems.     

Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation

Amyloid-β (Aβ) and tau protein are two crucial hallmarks in Alzheimer’s disease (AD). Their aggregation forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aβ and tau aggregation. In vitro studies showed that compounds 25–30 (20?μM) with N-methylation of the quinolone ring effectively inhibited Aβ_1-42 aggregation by 84.7%–99.5% and tau aggregation by 71.2%–101.8%. Their structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to Aβ_1-42 and tau, inhibit Aβ_1-42 β-sheets formation, and prevent tau aggregation in living cells.     

Methionine does not reduce Cu(II)–β-amyloid!—Rectification of the roles of methionine-35 and reducing agents in metal-centered oxidation chemistry of Cu(II)–β-amyloid

The potential risk of metal-centered oxidative catalysis has been overlooked in the research of the copper complexes of the Alzheimer's disease-related β-amyloid (Aβ) peptides. Cu²⁺ complexes of Aβ_1–40 and its 1–16 and 1–20 fragments have recently been shown to exhibit significant metal-centered oxidative activities toward several catecholamine neurotransmitters with and without H₂O₂ around neutral pH [G.F.Z. da Silva, L.-J. Ming, “Metallo-ROS” in Alzheimer's disease: metal-centered oxidation of neurotransmitters by Cu^II–β-amyloid and neuropathology of Alzheimer's disease, Angew. Chem. Int. Ed. 46 (2007) 3337–3341]. The results further support the metallo-Aβ-associated oxidative stress theory often considered to be connected to the neuropathology of the disease. The metal-centered oxidative catalysis of CuAβ_1–16/20 challenges the long-standing proposed redox role of Met35 in Aβ because Aβ_1–16/20 do not contain a Met. External Met has been determined by kinetic, optical, and electron paramagnetic resonance methods to bind directly to the Cu²⁺ center of CuAβ_1–40 and CuAβ_1–20 with K_d = 2.8 mM and 11.3 μM, respectively, which reflects less accessibility of the metal center in the full-length CuAβ_1–40. However, Met does not serve as a reducing agent for the Cu(II) which thus must amplify the observed oxidative catalysis of CuAβ_1–20 through a non-redox mechanism. Conversely, the CuAβ-catalyzed oxidation reaction of dopamine is inhibited by bio-available reducing agents such as ascorbate (competitive K_ic = 66 μM) and glutathione (non-competitive, K_inc = 53 μM). These data indicate that the oxidation chemistry of metallo-Aβ is not initiated by Met35. The results yield further molecular and mechanistic insights into the roles of metallo-Aβ in this disease.     

The inhibitory effects of Escherichia coli maltose binding protein on β-amyloid aggregation and cytotoxicity

The aggregation of β-amyloid (Aβ) peptide from its monomeric to its fibrillar form importantly contributes to the development of Alzheimer’s disease. Here, we investigated the effects of Escherichia coli maltose binding protein (MBP), which has been previously used as a fusion protein, on Aβ42 fibrillization, in order to improve understanding of the self-assembly process and the cytotoxic mechanism of Aβ42. MBP, at a sub-stoichiometric ratio with respect to Aβ42, was found to have chaperone-like inhibitory effects on β-sheet fibril formation, due to the accumulation of Aβ42 aggregates by sequestration of active Aβ42 species as Aβ42-MBP complexes. Furthermore, MBP increased the lag time of Aβ42 polymerization, decreased the growth rate of fibril extension, and suppressed Aβ42 mediated toxicity in human neuroblastoma SH-SY5Y cells. It appears that MBP decreases the active concentration of Aβ42 by sequestering it as Aβ42-MBP complex, and that this sequestration suppresses ongoing nucleation and retards the growth rate of Aβ42 species required for fibril formation. We speculate that inhibition of the growth rate of potent Aβ42 species by MBP suppresses Aβ42-mediated toxicity in SH-SY5Y cells.