Amyloid-β Peptide Neurotoxicity Research Articles

Peptide–Polyphenol (KLVFF/EGCG) Binary Modulators for Inhibiting Aggregation and Neurotoxicity of Amyloid-β Peptide

Alzheimer’s disease (AD) is known as a typical neurodegenerative disease, and the pathogenic hallmark is the aggregation and deposition of amyloid-β peptide (Aβ) fibrils and plaque on neuronal cells resulting in cell dysfunction and cell death. One effective approach to preventing and curing AD lies in tuning Aβ aggregation and inhibiting the neurotoxicity by using molecular modulators. Peptide breakers and antioxidants are widely used inhibitors to modulate Aβ aggregation and neurotoxicity, although less efficiency of single modulators hinders the practical application of these molecules. An integration of different molecular modulators is expected to make use of multiple interactions and modulating sites and therefore synergistically improve the capacity of modulators in inhibiting Aβ aggregation. In this work, the concept of a binary peptide–polyphenol binary modulator (Aβ-segment KLVFF and (−)-epigallocatechin-3-gallate, KLVFF/EGCG) is proposed, and the synergistic effect of the KLVFF/EGCG modulator i...

Deferoxamine enhances alternative activation of microglia and inhibits amyloid beta deposits in APP/PS1 mice

The neurotoxicity of amyloid-β peptide (Aβ), a predominant histopathological hallmark lesion of Alzheimer’s disease (AD), is enhanced by iron, as found in amyloid plaques of Alzheimer’s disease (AD) patients. We investigated whether deferoxamine (DFX) treatment promotes functional recovery and tissue repair in APP/PS1 double transgenic mice. Twelve-month-old APP/PS1 mice were randomly divided into two groups (APP/PS1 and DFX). Neurological deficits were monitored for 2weeks following DFX treatment. To characterize the activation of the microglia, expression of the M1 and M2 phenotypes was analyzed by immunohistochemistry and immunoblotting. Moreover, deposition of iron and Aβ, as well as apoptosis, were examined, and a behavioral test was performed. DFX significantly ameliorated cognitive function and deposition of Aβ as well as inhibited apoptosis in the brain. Consistent with these observations, DFX induced M2 activation of microglia and inhibited M1 activation of microglia in the hippocampus of APP/PS1 mice. In conclusion, DFX treatment improved functional recovery of AD mice, and the mechanism may involve DFX-induced M2 activation of microglia.

Ceria/POMs hybrid nanoparticles as a mimicking metallopeptidase for treatment of neurotoxicity of amyloid-β peptide

Protein misfolding to amyloid aggregates is the hallmark for neurodegenerative disease. While much attention has been paid to screen natural proteases that can degrade amyloid-β peptides (Aβ), it is difficult to apply them in the clinics with the intractable problem of immunogenicity in living organisms. Herein, we rationally designed an artificial nanozyme, Ceria/Polyoxometalates hybrid (CeONP@POMs) with both proteolytic and superoxide dismutase (SOD) activities. Our results indicated that CeONP@POMs could efficiently degrade Aβ aggregates and reduce intracellular reactive oxygen species (ROS). More importantly, CeONP@POMD could not only promote PC12 cell proliferation and can cross blood−brain barrier (BBB), but also inhibit Aβ-induced BV2 microglial cell activation which was demonstrated by immunoluorescence assay and flow cytometry measurements. In vivo studies further indicated that CeONP@POMD as nanozyme possessed good biocompatibility, evidenced by a detailed study of their biodistribution, body weight change, and in vivo toxicology. Therefore, our results pave the way for design of multifunctional artificial nanozyme for treatment of neurotoxicity of amyloid-β peptide.

Increasing anti‐Aβ‐induced neurotoxicity ability of Antrodia camphorata‐fermented product with deep ocean water supplementary

Antrodia camphorata is proven to probably inhibit the neurotoxicity of amyloid β-peptide (Aβ), known as a risk factor toward the development of Alzheimer's disease. Deep ocean water (DOW), drawn from an ocean depth of more than 200 m, has proven to stimulate the growth and metabolite biosynthesis of fungi owing to its rich minerals and trace elements. Based on these advantages of DOW, this study used statistical response surface methodology (RSM) to investigate the effects of DOW on the growth and anti-Aβ-induced neurocytotoxicity ability of A. camphorata. The results showed that DOW was useful for increasing the biomass of A. camphorata and enhancing its neuroprotective capability. The anti-Aβ40-induced neurocytotoxicity ability of filtrate was increased via raising the mycelium-secreted components. Furthermore, the anti-Aβ40-induced neurocytotoxicity ability of mycelium was also increased by the DOW-stimulated intracellular antioxidants. Using 80% DOW concentration, initial pH 3.3 and 20% inoculum size as the optimal culture conditions of A. camphorata significantly stimulated the biomass and mycelium-mediated Aβ40-induced cell viability from 302 ± 14 mg per 100 mL and 49.2 ± 2.2% to 452 ± 33 mg per 100 mL and 65.0 ± 7.4% respectively. This study indicated that DOW could be used as a promising supplementary for the production of A. camphorata secondary metabolites with strong antioxidant activity to protect neuron cells from damage based on Aβ stimulation cytotoxicity. © 2016 Society of Chemical Industry.

Effect of amyloid β-peptide on the fluidity of phosphatidylcholine membranes: Uses and limitations of diphenylhexatriene fluorescence anisotropy

There is accumulating evidence that peptide-induced perturbations in the order and dynamics of cellular membranes may play a role in the neurotoxicity of amyloid β-peptide (Aβ). Several studies have reported that Aβ decreases fluidity of membranes based on an Aβ-induced increase in the fluorescence anisotropy of diphenylhexatriene (DPH). However, the effect of Aβ on the membrane fluidity is still a subject of controversy, because other studies that employed pyrene as a fluorescent probe have shown that Aβ has the opposite effect. To reveal the reason for this discrepancy, we have examined the effect of Aβ on the fluidity of phosphatidylcholine membranes using spectroscopic methods. The fluorescence anisotropy of DPH is dramatically increased on addition of Aβ to DPH-containing phosphatidylcholine membranes. However, Aβ does not affect the Raman spectrum of the membrane, which is sensitive to the packing order of the hydrocarbon chains of lipids. We have also found that circular dichroism (CD) bands of DPH appear during incubation of DPH-containing membranes with Aβ, whereas DPH is an achiral molecule. The observed CD bands of DPH are induced by a chiral environment of Aβ but not by that of the lipids, because positive CD bands appear regardless of the d/l-chirality of phosphatidylcholine. The findings obtained from CD measurements provide evidence that DPH molecules translocate from the membrane to Aβ. The peptide-mediated extraction of DPH from the membrane may cause changes in the fluorescence anisotropy of DPH, even though Aβ does not affect the fluidity of membranes.

Potentiation of amyloid-β peptide neurotoxicity in human dental-pulp neuron-like cells by the membrane lipid peroxidation product 4-hydroxynonenal

Lipid peroxidation is generally considered as primarily implicated in the pathogenesis of Alzheimer's disease (AD); one of its more reactive end products, 4-hydroxynonenal (HNE), has been shown to cause neuron dysfunction and degeneration. HNE production in the brain is stimulated by the amyloid-β peptide (Aβ), whose excessive accumulation in specific brain areas is a hallmark of AD. Conversely, Aβ production is up-regulated by this multifunctional aldehyde. Findings reported here point to the ability of HNE and Aβ to interact, with consequent potentiation of Aβ’s cytotoxicity as determined in vitro using neuron-like cells derived from human dental-pulp progenitor cells. Preincubation of cells with the aldehyde markedly up-regulated Aβ uptake and intracellular accumulation, by overexpressing two of the three components of the plasma membrane multireceptor complex CD36/CD47/β1-integrin: experimental and clinical data indicate that intraneuronal accumulation of Aβ is an early event possibly playing a primary role in AD pathogenesis. That HNE-mediated overexpression of CD36 and β1-integrin, which plays a key role in HNE's potentiating Aβ neurotoxicity, in terms of necrosis, was confirmed when this effect was prevented by specific antibodies against the two receptors.

Overcoming Cell Death and Tau Phosphorylation Mediated by PI3KInhibition: A Cell Assay to Measure Neuroprotection

Few targets for neuroprotection have been defined in Alzheimer's disease (AD). Recent data from the role of Wnt, insulin-like growth factor-1 and estradiol pathways in AD suggest some therapeutic targets for disease treatment, and have led us to evaluate the "common factors" in these pathways as further candidate targets. These data have led us to propose that glycogen synthase kinase-3 (GSK-3) inhibition appears to be a common feature of these pathways. Besides, considering that GSK-3 activation seems to be correlated with neurodegeneration, its selection as a relevant target appears obvious. The capacity of different GSK-3 inhibitors to prevent amyloid β-peptide neurotoxicity and tau phosphorylation has been evaluated in order to develop novel clinical and therapeutic approaches. Different approaches could be used to search for new neuroprotective compounds. The most classical of these is to first define the target and then design a specific in vitro screening assay for it. Alternatively, a cell model of cell culture could be used as a "primary screen". Following this rationale, we have used a combined approach in which we first used an in vitro system to select compounds able to inhibit recombinant GSK3β. Subsequently, we subjected the candidate compounds to three consecutive cell-based complementary screening assays. First, cell viability was assessed using a neuroblastoma cell line before assaying the capacity of the compounds to reduce tau phosphorylation. Finally, we designed a neuronal cell model of apoptosis using the phosphatidylinositol kinase-3 inhibitor LY294002. Finally, we summarize several new compounds with "neuroprotective" properties.

Copper Enhances Amyloid-β Peptide Neurotoxicity and non β-Aggregation: A Series of Experiments Conducted upon Copper-Bound and Copper-Free Amyloid-β Peptide

Alzheimer's disease is characterized by the abnormal aggregation of amyloid-beta peptide (Abeta) in extracellular deposits known as senile plaques. However, the nature of the toxic Abeta species and its precise mechanism of action remain unclear. Previous reports suggest that the histidine residues are involved in copper-Abeta interaction, by which resulting in the neurotoxicity of Abeta and free radical damage. Here, we employed a mutant Abeta (Abeta H13R) in which a histidine residue was replaced by arginine. Copper facilitated the precipitation of both wild-type and mutant Abeta in the spectrophotometric absorbance assay but suppressed beta-structure aggregates according to Thioflavine-T assay. Wild-type Abeta alone is more cytotoxic but produced less amount of H(2)O(2) than AbetaH13R-copper complexes, suggesting that Abeta-membrane interaction may also implicated in the pathologic progress. Abeta toxicity is in positive correlation to its competence to aggregate despite the aggregation is mainly composed of non-beta fibril substances. In short, these findings may provide further evidence on the role of copper in the pathogenesis of Alzheimer's disease.

RAGE Expression in Rhabdomyosarcoma Cells Results in Myogenic Differentiation and Reduced Proliferation, Migration, Invasiveness, and Tumor Growth

Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEDeltacyto) (TE671/RAGEDeltacyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH(2) terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEDeltacyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential.

System Xc−and Apolipoprotein E Expressed by Microglia Have Opposite Effects on the Neurotoxicity of Amyloid-β Peptide 1–40

Because senile plaques in Alzheimer's disease (AD) contain reactive microglia in addition to potentially neurotoxic aggregates of amyloid-beta (Abeta), we examined the influence of microglia on the viability of rodent neurons in culture exposed to aggregated Abeta 1-40. Microglia enhanced the toxicity of Abeta by releasing glutamate through the cystine-glutamate antiporter system Xc-. This may be relevant to Abeta toxicity in AD, because the system Xc(-)-specific xCT gene is expressed not only in cultured microglia but also in reactive microglia within or surrounding amyloid plaques in transgenic mice expressing mutant human amyloid precursor protein or in wild-type mice injected with Abeta. Inhibition of NMDA receptors or system Xc- prevented the microglia-enhanced neurotoxicity of Abeta but also unmasked a neuroprotective effect of microglia mediated by microglial secretion of apolipoprotein E (apoE) in the culture medium. Immunodepletion of apoE or targeted inactivation of the apoE gene in microglia abrogated neuroprotection by microglial conditioned medium, whereas supplementation by human apoE isoforms restored protection, which was potentiated by the presence of microglia-derived cofactors. These results suggest that inhibition of microglial system Xc- might be of therapeutic value in the treatment of AD. Its inhibition not only prevents glutamate excitotoxicity but also facilitates neuroprotection by apoE.

Trolox and 17β-Estradiol Protect against Amyloid β-Peptide Neurotoxicity by a Mechanism That Involves Modulation of the Wnt Signaling Pathway

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

Protective effects of S-nitrosoglutathione against amyloid β-peptide neurotoxicity

Amyloid β-peptide (Aβ) is a major constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. We have previously demonstrated ceramide production secondary to Aβ-induced activation of neutral sphingomyelinase (nSMase) in cerebral endothelial cells and oligodendrocytes, which may contribute to cellular injury during progression of AD. In this study, we first established the “Aβ → nSMase → ceramide → free radical → cell death” pathway in primary cultures of fetal rat cortical neurons. We also provided experimental evidence showing that S-nitrosoglutathione (GSNO), a potent endogenous antioxidant derived from the interaction between nitric oxide (NO) and glutathione, caused dose-dependent protective effects against Aβ/ceramide neurotoxicity via inhibition of caspase activation and production of reactive oxygen species (ROS). This GSNO-mediated neuroprotection appeared to involve activation of cGMP-dependent protein kinase (PKG), phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinase (ERK). Activation of the cGMP/PKG pathway induced expression of thioredoxin and Bcl-2 that were beneficial to cortical neurons in antagonizing Aβ/ceramide toxicity. Consistently, exogenous application of thioredoxin exerted remarkable neuroprotective efficacy in our experimental paradigm. Results derived from the present study establish a neuroprotective role of GSNO, an endogenous NO carrier, against Aβ toxicity via multiple signaling pathways.

CDNA cloning of a novel secreted isoform of the human receptor for advanced glycation end products and characterization of cells co-expressing cell-surface scavenger receptors and Swedish mutant amyloid precursor protein

The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-β protein (Aβ), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-β peptide neurotoxicity in Alzheimer's disease, Nature 382 (1996) 685–691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Aβ and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human βAPP695-SFAD in a human cell and the level of Aβ in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Aβ from the conditioned medium of hRAGE cells and ∼40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Aβ.

Transgenic murine cortical neurons expressing human Bcl-2 exhibit increased resistance to amyloid β-peptide neurotoxicity

The generation of reactive oxygen species has been implicated in the neurotoxicity of amyloid β-peptide, the main constituent of the senile plaques that accumulates in the brain of Alzheimer's disease victims. In this study, we have compared the toxicity of amyloid β-peptide on cultured cortical neurons from control mice and transgenic mice expressing either human copper–zinc superoxide dismutase or human Bcl-2, two proteins that protect cells against oxidative damage. Copper–zinc superoxide dismutase overexpression failed to protect cortical neurons against the toxicity of amyloid β-peptide(25–35) [the minimal cytotoxic fragment of amyloid β-peptide(1–42)] as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and an enzyme-linked immunoabsorbent assay using an antibody directed against microtubule-associated protein-2 (a specific neuronal protein), ruling out a role for superoxide anion and peroxynitrite in amyloid β-peptide-evoked neurotoxicity. On the contrary, cortical neurons expressing human copper–zinc superoxide dismutase exhibited increased apoptotic nuclei in both untreated and amyloid β-peptide(25–35)-exposed neurons. Transgenic neurons expressing human Bcl-2 were partially protected against amyloid β-peptide-induced neuronal death. This neuroprotection appears to be related to the complete inhibition of apoptosis induced by both amyloid β-peptide(25–35) and amyloid β-peptide(1–42). This study may be relevant for developing neuroprotective gene therapy to inhibit neuronal apoptosis in Alzheimer's disease.

Catecholamines Potentiate Amyloid β-Peptide Neurotoxicity: Involvement of Oxidative Stress, Mitochondrial Dysfunction, and Perturbed Calcium Homeostasis

Oxidative stress and mitochondrial dysfunction are implicated in the neuronal cell death that occurs in physiological settings and in neurodegenerative disorders. In Alzheimer's disease (AD) degenerating neurons are associated with deposits of amyloid β-peptide (Aβ), and there is evidence for increased membrane lipid peroxidation and protein oxidation in the degenerating neurons. Cell culture studies have shown that Aβ can disrupt calcium homeostasis and induce apoptosis in neurons by a mechanism involving oxidative stress. We now report that catecholamines (norepinephrine, epinephrine, and dopamine) increase the vulnerability of cultured hippocampal neurons to Aβ toxicity. The catecholamines were effective in potentiating Aβ toxicity at concentrations of 10–200 μM, with the higher concentrations (100–200 μM) themselves inducing cell death. Serotonin and acetylcholine were not neurotoxic and did not modify Aβ toxicity. Levels of membrane lipid peroxidation, and cytoplasmic and mitochondrial reactive oxygen species, were increased following exposure to neurons to Aβ, and catecholamines exacerbated the oxidative stress. Subtoxic concentrations of catecholamines exacerbated decreases in mitochondrial energy charge and transmembrane potential caused by Aβ, and higher concentrations of catecholamines alone induced mitochondrial dysfunction. Antioxidants (vitamin E, glutathione, and propyl gallate) protected neurons against the damaging effects of Aβ and catecholamines, whereas the β-adrenergic receptor antagonist propanolol and the dopamine (D1) receptor antagonist SCH23390 were ineffective. Measurements of intracellular free Ca2+([Ca2+]i) showed that Aβ induced a slow elevation of [Ca2+]iwhich was greatly enhanced in cultures cotreated with catecholamines. Collectively, these data indicate a role for catecholamines in exacerbating Aβ-mediated neuronal degeneration in AD and, when taken together with previous findings, suggest roles for oxidative stress induced by catecholamines in several different neurodegenerative conditions.

In vitro neurotoxicity of amyloid β-peptide cross-linked by transglutaminase.

Transglutaminase catalyzes the intermolecular cross-linking of peptides between Gln and Lys residues, forming an ε-(γ-glutamyl) lysine bond. Amyloid β-peptide, a major constituent of the deposits in Alzheimer disease, contains Lys16, Lys28, and Gln15 which may act as substrates of transglutaminase. Transglutaminase treatment of amyloid β-peptide (1-28) and amyloid β-peptide (1-40) yielded cross-linked oligomers. Transglutaminase-treated Aβ retarded neurite extension of PC12 cells, and rat cultured neurons of hippocampus and septum, brain areas severely affected by Alzheimer disease, and subsequently caused cell death, whereas the transglutaminase-untreated counterparts did not show harmful effects. The transglutaminase-catalyzed oligomers of amyloid β-peptide and their neurotoxicity may be involved in two characteristics in Alzheimer disease, neuronal degeneration and formation of the insoluble deposits. AD - Alzheimer disease, Aβ - amyloid β-peptide, DMEM - Dulbecco's modified Eagle's medium, DMEM/F-12-1:1 mixture of DMEM and Ham's F-12 medium, FCS - fetal calf serum, HS - horse serum, PAGE - polyacrylamide gel electrophoresis, MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, NGF - nerve growth factor, TGase - transglutaminase.

RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease.

Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.