Abeta-induced Cytotoxicity Research Articles

Maslinic Acid Protected PC12 Cells Differentiated by Nerve Growth Factor against β-Amyloid-Induced Apoptosis.

β-Amyloid peptide (Abeta) was used to induce apoptosis in PC12 cells differentiated by nerve growth factor, and the protective activities of maslinic acid (MA) at 2-16 μM were examined. Abeta treatment lowered Bcl-2 expression, raised Bax expression, and decreased cell viability. MA pretreatments decreased Bax expression, raised the Bcl-2/Bax ratio, and increased cell viability. MA pretreatments retained glutathione content and decreased subsequent Abeta-induced release of reactive oxygen species, tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Abeta treatment up-regulated protein expression of p47(phox), gp91(phox), mitogen-activated protein kinase, advanced glycation end product receptor (RAGE), and nuclear factor-κ B (NF-κB). MA pretreatments at 2-16 μM suppressed the expression of proteins including gp91(phox), p47(phox), p-p38, and NF-κB p65, at 4-16 μM down-regulated RAGE and NF-κB p50 expression, and at 8 and 16 μM reduced p-ERK1/2 expression. These novel findings suggest that maslinic acid is a potent compound against Abeta-induced cytotoxicity.

Chemical Nanotherapy: Nitroxyl radical-containing Nanoparticle Protects Neuroblastoma SH-SY5Y Cells From Aβ-induced Oxidative Stress

Excessive accumulation of beta-amyloid (Abeta) has been proposed as a pivotal event in the pathogenesis of Alzheimer's disease. Possible mechanisms underlying Abeta-induced neuronal cytotoxicity include excess production of reactive oxygen species (ROS) and apoptosis. We have designed novel nanoparticles, nitroxyl radical-containing nanoparticles (RNPs), which possess nitroxyl radical in the core and chemically scavenges ROS. This study aimed to determine the potential neuroprotective role of RNPs on Abeta-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were preincubated with 0.1-1 mM RNP for 24 h and then incubated with 20 microM Abeta1-42, for 48 h. In every group, cell viability, apoptotic rate, ROS levels including superoxide anion radicals and hydroxyl radicals, ROS production including lipid peroxidation, protein oxidation and DNA oxidation were measured. SH-SY5Y cells preincubated with 0.1-2 mM RNP for 24 h were protected from Abeta-induced damage. SH-SY5Y cells preincubated with more than 2 mM RNPfor 24 h showed cytotoxicity. From the quantitative analyses, it was observed that RNPs reduced intracellular oxidative stress. RNP treatment significantly reduced the amount of oxidized lipids, proteins and DNA. It also reduced DNA fragmentations, which caused lower apoptosis levels. RNPs are promising intracellular ROS scavengers.

Engineered Proteolytic Nanobodies Reduce Aβ Burden and Ameliorate Aβ-Induced Cytotoxicity

Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD), and reduction of Abeta levels in the brain could be a viable therapeutic approach. A potentially noninflammatory route to facilitate clearance and reduce toxicity of Abeta is to degrade the peptide using proteolytic nanobodies. Here we show that a proteolytic nanobody engineered to cleave Abeta at its alpha-secretase site has potential therapeutic value. The Asec-1A proteolytic nanobody, derived from a parent catalytic light chain antibody, prevents aggregation of monomeric Abeta, inhibits further aggregation of preformed Abeta aggregates, and reduces Abeta-induced cytotoxicity toward a human neuroblastoma cell line. The nanobody also reduces toxicity induced by overexpression of the human amyloid precursor protein (APP) in a Chinese hamster ovary (CHO) cell line by cleaving APP at the alpha-secretase site which precludes formation of Abeta. Targeted proteolysis of APP and Abeta with catalytic nanobodies represents a novel therapeutic approach for treating AD where potentially harmful side effects can be minimized.

Aβ20–29 peptide blocking apoE/Aβ interaction reduces full-length Aβ42/40 fibril formation and cytotoxicity in vitro

A key event in the pathogenesis of Alzheimer's disease (AD) is the conversion of the peptide beta-amyloid (Abeta) from its soluble monomeric form into various aggregated morphologies in the brain. Apolipoprotein E (apoE) is known to act as a pathological chaperone of Abeta in this process, promoting its fibril formation from soluble Abeta by binding interaction between carboxy-terminal domain of apoE and residues 12-28 of full-length Abeta. Therefore, blocking apoE/Abeta interaction is being actively pursued as a primary therapeutic strategy for AD. Abeta20-29, a short peptide, contains the residues to competitively bind to apoE and may potentially block the interaction between apoE and full-length Abeta. However, little is known whether Abeta20-29 could block apoE/Abeta interaction to play an effective role in reducing full-length Abeta fibrillization and cytotoxicity. Utilizing fluorescence spectroscopic analysis with thioflavin T and electron microscopic study, we show here that Abeta20-29 alone was non-fibrillogenic, and had no direct effects on Abeta1-42 or Abeta1-40 aggregation. Moreover, apoE can directly promote both Abeta1-42 and Abeta1-40 aggregation and fibril formation, while this promoting effect was inhibited when adding Abeta20-29, with a dose-dependent manner. In the series of cell culture experiments, Abeta20-29 alone shows no cytotoxicity to PC12 cells as demonstrated by MTT assay, while co-incubation apoE isoforms and Abeta1-42 or Abeta1-40 shows stronger cytotoxicity as compared to Abeta1-42 or Abeta1-40 alone. When incubated with Abeta20-29, whereas such strong cytotoxic effect was concentration-dependently reduced. Taken together, we demonstrate for the first time that Abeta20-29 has no direct effect on full-length Abeta aggregation, and may competitively block the binding of full-length Abeta to apoE, resulting in an inhibitory effect on apoE's promoting full-length Abeta fibrillogenesis and Abeta-induced cytotoxicity. Our results raise the possibility that Abeta20-29 peptide blocking the interaction between full-length Abeta and apoE isoforms may be effective as a therapeutic agent for AD.

Colostrinin™ Alleviates Amyloid-β Induced Toxicity in Rat Primary Hippocampal Cultures

Colostrinin (CLN), a complex mixture of proline-rich polypeptides derived from colostrums, can alleviate cognitive decline in early Alzheimer's disease patients. The molecular basis of the action of CLN has been studied in vitro using human neuroblastoma cell lines. The aim of the present study was to use quantitative immunocytochemistry and immunoblotting to investigate the ability of CLN to relieve amyloid-beta (Abeta)-induced cytotoxicity in rat primary hippocampal neuronal cells. Our data confirm that CLN alleviates the effect of Abeta-induced cytotoxicity and causes a significant reduction in the elevated levels of the antioxidant enzyme SOD1.

Neuroprotective effect of epigallocatechin-3-gallate against β-amyloid-induced oxidative and nitrosative cell death via augmentation of antioxidant defense capacity

beta-Amyloid (Abeta) peptide, a major component of senile plaques has been regarded to play a crucial role in the development and neuropathogenesis of Alzheimer's disease (AD). Increasing data from in vitro and in vivo studies indicate that Abeta-induced damages in neurons and glia are mediated via nitrosative as well as oxidative stress. Therefore, recent researches have been focused on searching for dietary and herbal manipulations to protect against the Abeta-induced oxidative and/or nitrosative cell death. Epigallocatechin-3-gallate (EGCG), one of these candidates is a major polyphenolic compound present in green tea and has been reported to exhibit potent antioxidant and anti-inflammatory properties. In the present study, we have investigated the effect of EGCG against Abeta-induced oxidative and/or nitrosative cell death in BV2 microglia. Abeta treatment led to apoptosis in BV2 cells as revealed by DNA fragmentation, perturbation of mitochondrial transmembrane potential, and alterations in the expression of apoptosis-regulator Bcl-2 family proteins. EGCG pretreatment effectively ameliorated Abeta-induced cytotoxicity and manifestation of proapoptotic signals. Furthermore, BV2 cells exposed to Abeta underwent nitrosative stress as shown by the increased expression of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) and peroxynitrite, which were effectively suppressed by EGCG pretreatment. To elucidate a molecular mechanism underlying the neuroprotective effect of EGCG, we have examined the cellular metabolism of reduced glutathione (GSH) with antioxidant properties. EGCG treatment fortified cellular GSH pool through elevated mRNA expression of gamma-glutamylcysteine ligase (GCL), the rate limiting enzyme in the glutathione biosynthesis. These results suggest that EGCG may have preventive and/or therapeutic potential in AD patients by augmenting cellular antioxidant defense capacity and attenuating Abeta-mediated oxidative and/or nitrosative cell death.

Effects of Grape Seed-derived Polyphenols on Amyloid β-Protein Self-assembly and Cytotoxicity*♦

Epidemiological evidence suggests that moderate consumption of red wine reduces the incidence of Alzheimer disease (AD). To study the protective effects of red wine, experiments recently were executed in the Tg2576 mouse model of AD. These studies showed that a commercially available grape seed polyphenolic extract, MegaNatural-AZ (MN), significantly attenuated AD-type cognitive deterioration and reduced cerebral amyloid deposition (Wang, J., Ho, L., Zhao, W., Ono, K., Rosensweig, C., Chen, L., Humala, N., Teplow, D. B., and Pasinetti, G. M. (2008) J. Neurosci. 28, 6388-6392). To elucidate the mechanistic bases for these observations, here we used CD spectroscopy, photo-induced cross-linking of unmodified proteins, thioflavin T fluorescence, size exclusion chromatography, and electron microscopy to examine the effects of MN on the assembly of the two predominant disease-related amyloid beta-protein alloforms, Abeta40 and Abeta42. We also examined the effects of MN on Abeta-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism and lactate dehydrogenase activity in Abeta-treated, differentiated pheochromocytoma (PC12) cells. Initial studies revealed that MN blocked Abeta fibril formation. Subsequent evaluation of the assembly stage specificity of the effect showed that MN was able to inhibit protofibril formation, pre-protofibrillar oligomerization, and initial coil --> alpha-helix/beta-sheet secondary structure transitions. Importantly, MN had protective effects in assays of cytotoxicity in which MN was mixed with Abeta prior to peptide assembly or following assembly and just prior to peptide addition to cells. These data suggest that MN is worthy of consideration as a therapeutic agent for AD.

Lack of oestrogen protection in amyloid-mediated endothelial damage due to protein nitrotyrosination

Amyloid beta-peptide (Abeta) cytotoxicity, the hallmark of Alzheimer's disease, implicates oxidative stress in both neurons and vascular cells, particularly endothelial cells. Consequently, antioxidants have shown neuroprotective activities against Abeta-induced cytotoxicity. Among the different antioxidants used in both in vitro and in vivo studies, 17beta-oestradiol (E2) has garnered the most attention. Oestrogen attenuated Abeta(E22Q)-induced toxicity in neurons but failed to protect endothelial cells. Here we show that E2-mediated activation of endothelial nitric oxide synthase (eNOS) increases the production of nitric oxide (NO), which, under Abeta(E22Q)-induced oxidative damage, results in the formation of peroxynitrite and increased nitration of tyrosine residues. Inhibition of eNOS prevents nitrotyrosination and permits E2-mediated protection against Abeta(E22Q) on endothelial cells. The main nitrotyrosinated proteins in the presence of E2 and Abeta(E22Q) were identified by MALDI-TOF mass spectrometry. These proteins are key players in the regulation of energy production, cytoskeletal integrity, protein metabolism and protection against oxidative stress. Our data highlight the potential damaging consequences of E2 in vascular disorders dealing with oxidative stress conditions, such as cerebral amyloid angiopathy, stroke and ischaemia-reperfusion conditions.

Specific interaction of VEGF165 with β‐amyloid, and its protective effect on β‐amyloid‐induced neurotoxicity

beta-amyloid (Abeta) is a major component of senile plaques that is commonly found in the brain of Alzheimer's disease (AD) patient. In the previous report, we showed that an important angiogenic factor, vascular endothelial growth factor (VEGF) interacts with Abeta and is accumulated in the senile plaques of AD patients' brains. Here we show that Abeta interacts with VEGF(165) isoform, but not with VEGF(121). Abeta binds to the heparin-binding domain (HBD) of VEGF(165) with similar affinity as that of intact VEGF(165). Abeta binds mostly to the C-terminal subdomain of HBD, but with greatly reduced affinity than HBD. Therefore, the full length of HBD appears to be required for maximal binding of Abeta. Although Abeta binds to heparin-binding sequence of VEGF, it does not bind to other heparin-binding growth factors except midkine. Thus it seems that Abeta recognizes unique structural features of VEGF HBD. VEGF(165) prevents aggregation of Abeta through its HBD. We localized the core VEGF binding site of Abeta at around 26-35 region of the peptide. VEGF(165) and HBD protect PC12 cells from the Abeta-induced cytotoxicity. The mechanism of protection appears to be inhibition of both Abeta-induced formation of reactive oxygen species and Abeta aggregation.

Epicatechin and Catechin in Cocoa Inhibit Amyloid β Protein Induced Apoptosis

To elucidate additional health benefits of cocoa phytochemicals on the neurotoxicity induced by amyloid beta protein (Abeta), PC12 cells were treated with toxic peptide (Abeta(25)(-)(35)) and the effects of epicatechin, catechin, and cocoa were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase (LDH) release, and trypan blue exclusion methods. Significant increase in neuronal cell death was observed on PC12 cells treated with Abeta(25)(-)(35) (25 microM), while epicatechin and catechin and their mixture prevented the Abeta-induced neuronal cell death. Abeta treatment also led to the increased membrane instability of PC12 cells. The membrane protective effects of the phenolics determined by LDH release and trypan blue exclusion assays demonstrated that epicatechin, catechin, and their mixture protect cellular membrane from Abeta-induced cytotoxicity. In these three different cell viability assays, the mixture of epicatechin and catechin showed the highest protective effect and synergistic activity. The present results showed that the major flavonoids of cocoa, epicatechin and catechin, protect PC12 cells from Abeta-induced neurotoxicity, and suggest that cocoa may have anti-neurodegenerative effect in addition to other known chemopreventive effects.

Protective Effects of Oligomers of Grape Seed Polyphenols Against β‐Amyloid‐Induced Oxidative Cell Death

beta-Amyloid (Abeta) is considered to be responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimer's disease (AD). There is compelling evidence supporting the notion that Abeta-induced cytotoxicity is mediated through the generation of reactive oxygen species (ROS). Recently, considerable attention has been focused on a wide array of non-vitamin antioxidants present in edible plants that are able to scavenge ROS, thereby protecting against oxidative damage. In this study, we have investigated the possible protective effects of formulated polyphenol oligomers (Oligonol) derived from grape seed extracts on Abeta-induced oxidative cell death. Rat pheochromocytoma (PC12) cells treated with Abeta exhibited increased accumulation of intracellular ROS and underwent apoptosis, as determined by positive in situ terminal end labeling, decreased mitochondrial membrane potential, and the cleavage of poly(ADP-ribose)polymerase. Oligonal attenuated Abeta-induced cytotoxicity, apoptotic features, intracellular ROS accumulation, and lipid peroxidation and increased the cellular glutathione pool. Moreover, Abeta transiently induced the activation of nuclear factor kappaB in PC12 cells, which was suppressed by pretreatment with Oligonol.

The relationship between the aggregational state of the amyloid-beta peptides and free radical generation by the peptides.

In the present study, we investigated whether or not the amyloid-beta protein (Abeta) peptide itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of Abeta and Abeta-induced cytotoxicity. The present results demonstrated a four-line spectrum in the presence of both Abeta40 and Abeta42 with Ntert-butyl-alpha-phenylnitrone (PBN), but not in the presence of PBN alone in phosphate-buffered saline (PBS). The fact that the four-line spectrum obtained for the Abeta/PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observed four-line spectrum to be iron-dependent. The present study also revealed that either Abeta40 or Abeta42 with PBN in phosphate buffer (PB) did not produce any definite four-line spectrum. Both a thioflavine-T (Th-T) fluorometric assay and circular dichroism (CD) spectroscopy showed the amyloid fibril formation of Abeta in PBS to be much higher than that of Abeta in PB. Moreover, Abeta-induced cytotoxicity assays showed Abeta incubated in PBS to be more cytotoxic than that incubated in PB. These results thus suggest that Abeta-associated free radical generation is strongly influenced by the aggregational state of the peptides.

Apolipoprotein A-I directly interacts with amyloid precursor protein and inhibits A beta aggregation and toxicity.

Amyloid precursor protein (APP) is the source of the neurotoxic amyloid beta (Abeta) peptide associated with Alzheimer's disease. Apolipoprotein A-I (apoA-I), a constituent of high-density lipoprotein complexes, was identified by a yeast two-hybrid system as a strong and specific binding partner of full-length APP (APPfl). This association between apoA-I and APPfl was localized to the extracellular domain of APP (APPextra). Furthermore, the interaction between apoA-I and APPfl was confirmed by coprecipitation using recombinant epitope-tagged APPextra and purified apoA-I. Several functional domains have been identified in APPextra, and we focused on a possible interaction between apoA-1 and the pathologically important Abeta peptide, because APPextra contains the nontransmembrane domain of Abeta. The binding between apoA-I and Abeta was saturable (K(d) = 6 nM), specific, and reversible. APPextra also competed with apoA-I for binding to Abeta. Direct evidence for this interaction was obtained by the formation of an SDS-resistant Abeta-apoA-I complex in polyacrylamide gels. Competitive experiments with apolipoprotein E (isoforms E2 and E4) showed that apoA-I had a higher binding affinity for Abeta. We also found that apoA-I inhibited the beta-sheet formation of Abeta with a mean inhibitory concentration close to that of alpha2-macroglobulin. Finally, we demonstrated that apoA-I attenuated Abeta-induced cytotoxicity. These results suggest apoA-I binds to at least one extracellular domain of APP and has a functional role in controlling Abeta aggregation and toxicity.

Role of ERAB/l-3-Hydroxyacyl-coenzyme A Dehydrogenase Type II Activity in Aβ-induced Cytotoxicity

Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.