amyloid-beta-peptides and thiazolyl-blue

Multiple lines of evidence indicate that regional brain eicosanoid signaling is important in initiation and progression of neurodegenerative conditions that have neuroinflammatory pathologic component, such as AD. We hypothesized that PGE(2) receptor subtype 1 (EP1) signaling (linked to intracellular Ca(2+) release) regulates Aβ peptide neurotoxicity and tested this in two complementary in vitro models: a human neuroblastoma cell line (MC65) producing Aβ(1-40) through conditional expression of the APP C-terminal portion, and murine primary cortical neuron cultures exposed to Aβ(1-42). In MC65 cells, EP1 receptor antagonist SC-51089 reduced Aβ neurotoxicity ~50 % without altering high molecular weight Aβ immunoreactive species formation. Inositol-3-phosphate receptor antagonist 2-aminoethoxy-diphenyl borate offered similar protection. SC-51089 largely protected the neuron cultures from synthetic Aβ(1-42) neurotoxicity. Nimodipine, a Ca(2+) channel blocker, was completely neuroprotective in both models. Based on these data, we conclude that suppressing neuronal EP1 signaling may represent a promising therapeutic approach to ameliorate Aβ peptide neurotoxicity.

Topics: Amyloid beta-Peptides; Animals; Blotting, Western; Calcium Channel Blockers; Cell Line, Tumor; Cells, Cultured; Coloring Agents; Dinoprostone; Humans; Hydrazines; Mice; Mice, Inbred C57BL; Neurons; Neurotoxicity Syndromes; Nimodipine; Oxazepines; Peptide Fragments; Prostaglandin Antagonists; Receptors, Prostaglandin E, EP1 Subtype; Tetrazolium Salts; Thiazoles

Previously we reported that endoplasmic reticulum (ER)-mitochondria crosstalk is involved in amyloid-β (Aβ)-induced apoptosis. Now we show that mitochondrial dysfunction affects the ER stress response triggered by Aβ using cybrids that recreate the defect in mitochondrial cytochrome c oxidase (COX) activity detected in platelets from Alzheimer's disease (AD) patients. AD and control cybrids were treated with Aβ or classical ER stressors and the ER stress-mediated apoptotic cell death pathway was accessed. Upon treatment, we found increased glucose-regulated protein 78 (GRP78) levels and caspase-4 activation (ER stress markers) which were more pronounced in AD cybrids. Treated AD cybrids also exhibited decreased cell survival as well as increased caspase-3-like activity, poli-ADP-ribose-polymerase (PARP) levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells. Finally, we showed that Aβ-induced caspase-3 activation in both cybrid cell lines was prevented by dantrolene, thus implicating ER Ca(2+) release in ER stress-mediated apoptosis. Our results demonstrate that mitochondrial dysfunction occurring in AD patients due to COX inhibition potentiates cell susceptibility to Aβ-induced ER stress. This study further supports the close communication between ER and mitochondria during apoptosis in AD.

Topics: Aged; Alzheimer Disease; Amyloid beta-Peptides; Analysis of Variance; Blood Platelets; Brefeldin A; Caspase 3; Cell Death; Cell Fusion; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Female; Gene Expression Regulation; Heat-Shock Proteins; Humans; Hybrid Cells; In Situ Nick-End Labeling; Male; Middle Aged; Mitochondria; Peptide Fragments; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Tetrazolium Salts; Thapsigargin; Thiazoles

The cellular prion protein (PrP(C)) is a neuronal-anchored glycoprotein that has been associated with several functions in the CNS such as synaptic plasticity, learning and memory and neuroprotection. There is great interest in understanding the role of PrP(C) in the deleterious effects induced by the central accumulation of amyloid-β (Aβ) peptides, a pathological hallmark of Alzheimer's disease, but the existent results are still controversial. Here we compared the effects of a single intracerebroventricular (i.c.v.) injection of aggregated Aβ(1-40) peptide (400pmol/mouse) on the spatial learning and memory performance as well as hippocampal cell death biomarkers in adult wild type (Prnp(+/+)), PrP(C) knockout (Prnp(0/0)) and the PrP(C) overexpressing Tg-20 mice. Tg-20 mice, which present a fivefold increase in PrP(C) expression in comparison to wild type mice, were resistant to the Aβ(1-40)-induced spatial learning and memory impairments as indicated by reduced escape latencies to find the platform and higher percentage of time spent in the correct quadrant during training and probe test sessions of the water maze task. The protection against Aβ(1-40)-induced cognitive impairments observed in Tg-20 mice was accompanied by a significant decrease in the hippocampal expression of the activated caspase-3 protein and Bax/Bcl-2 ratio as well as reduced hippocampal cell damage assessed by MTT and propidium iodide incorporation assays. These findings indicate that the overexpression of PrP(C) prevents Aβ(1-40)-induced spatial learning and memory deficits in mice and that this response is mediated, at least in part, by the modulation of programed cell death pathways.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Survival; Cognition Disorders; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Peptide Fragments; Prion Proteins; Prions; Propidium; Proto-Oncogene Proteins c-bcl-2; Reaction Time; Tetrazolium Salts; Thiazoles

Mitochondrial dysfunction is observed in Alzheimer's disease (AD) brain and peripheral tissues. Amyloid-β (Aβ) peptides are known to interact with several proteins inside the mitochondria, leading to mitochondrial dysfunction. Recent studies have provided substantial evidence that mitochondria serve as direct targets for Aβ-mediated neuronal toxicity. The observations that Aβ progressively accumulates in cortical mitochondria from AD patients and transgenic AD type mouse models suggest the role of mitochondrial Aβ in the pathogenesis or development of AD. Herein, we studied the downstream signaling pathways induced by Aβ-mediated mitochondrial metabolism alterations and its consequences on cellular fate. We found that Aβ peptides induced an increase in NAD+levels and a decrease in ATP levels, which was related with decreases in acetylated tubulin levels and tau hyperphosphorylation. As a result of microtubule disruption, alterations in macroautophagy, like a decrease in autophagossome degradation and altered cellular distribution of LC3B, were found. Taxol, a microtubule stabilizer drug, was able to restore microtubule network and to prevent cell death induced by Aβ peptides. Our data shows for the first time that mitochondrial and cytosolic Aβ oligomers were significantly reduced upon microtubule dynamics re-establishment. These observations point out that an intervention at a microtubule level may be effective as a disease modifying therapy.

Topics: Adenine Nucleotides; Amyloid beta-Peptides; Antineoplastic Agents, Phytogenic; Autophagy; Blotting, Western; Caspases; Cell Line, Tumor; Cell Proliferation; Coloring Agents; Electron Transport Complex IV; Enzyme Activation; Humans; Lysosomes; Microscopy, Confocal; Microtubules; Mitochondrial Diseases; Mitochondrial Membranes; NAD; Paclitaxel; Peptide Fragments; Signal Transduction; Tetrazolium Salts; Thiazoles; Tubulin

β-Amyloid (Aβ) plaques are characteristic hallmarks of Alzheimer's disease. In the present study, we examined the neuroprotective effects of S-aspirin, a hydrogen sulfide (H(2)S)-releasing aspirin, on Aβ-induced cell toxicity. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that S-aspirin, but not aspirin, significantly increased cell viability in BV-2 microglial cells, indicating that S-aspirin may protect cells against injury via releasing H(2)S. S-aspirin at 2.5-10 μM significantly increased cell viability and decreased lactate dehydrogenase release in Aβ-treated BV-2 microglial cells. Western blotting analysis showed that S-aspirin suppressed the protein expression levels of cyclooxygenase-2 and growth arrest DNA damage (GADD). These data suggest that S-aspirin may protect microglial cells by inhibition of Aβ-induced inflammation and cell cycle re-entry. To study whether S-aspirin can protect mitochondria function, mitochondria membrane potential was measured with molecular probe JC-1. It was found that S-aspirin protected mitochondria from Aβ-induced loss of mitochondrial member potential. (ΔΨm). In addition, S-aspirin also prevented Aβ-induced activation of p38-mitogen activated protein kinase (MAPK). In conclusion, our results suggest that S-aspirin may protect microglial injury via inhibition of inflammation, prevention of mitochondria function, and stimulation of cell growth via stimulating p38-MAPK pathway. Our study may suggest that S-aspirin may have potential therapeutic value for the treatment of Alzheimer's disease.

Topics: Amyloid beta-Peptides; Animals; Aspirin; Cell Line, Transformed; Cell Survival; Cyclooxygenase 2; Dose-Response Relationship, Drug; Hydrogen Sulfide; Lactate Dehydrogenases; Membrane Potential, Mitochondrial; Mice; Microglia; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Peptide Fragments; Protective Agents; Tetrazolium Salts; Thiazoles; Transcription Factor CHOP; Tumor Necrosis Factor-alpha; Up-Regulation

The cellular pathology of Alzheimer's disease is progressive and protracted leading eventually to considerable neuronal death. The underlying mechanisms of the pathology are complex but changes in the control of intracellular Ca2+ are believed to contribute to the demise of neurons. In this study, we investigated the functional consequences of an increase in the expression of the type 3 isoform of the ryanodine receptor (RyR3). We found that although cortical neurons from TgCRND8 mice secreted significantly more amyloid beta protein and showed significantly increased RyR3 expression, they were no more sensitive to cell stress than non-transgenic neurons. Furthermore, despite increased intracellular Ca2+ release in response to ryanodine, we found that basal Ca2+, K+-evoked Ca2+ responses, and capacitative Ca2+ entry were no different in TgCRND8 neurons compared with non-transgenic neurons. Therefore, as RyR3 up-regulation did not affect neuronal health or global Ca2+ homeostasis, we investigated the effect of reducing RyR3 expression using small interfering RNA. Surprisingly, a reduction of RyR3 expression in TgCRND8, but not in non-transgenic, neurons increased neuronal death. These data reveal a new role for RyR3 and indicate a novel potential therapeutic target to delay or prevent the progression of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Calcium; Cell Death; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Glutamic Acid; Green Fluorescent Proteins; Humans; Mice; Mice, Transgenic; Mutation; Neurons; Peptide Fragments; Phosphopyruvate Hydratase; Potassium; Presenilin-1; RNA, Messenger; RNA, Small Interfering; Ryanodine Receptor Calcium Release Channel; Tetrazolium Salts; Thiazoles; Up-Regulation

Statins, used as cholesterol-lowering drugs, were reported to reduce the progression of Alzheimer's disease (AD). However, the molecular mechanisms underlying these findings remain to be clarified and it is not well understood whether this beneficial effect is due to simply lowering cholesterol levels. This study was aimed to investigate the neuroprotective effect of simvastatin and lovastatin, lipophilic statins that can transverse the blood brain barrier, against the toxicity triggered by the AD-associated amyloid-beta (Abeta) peptides and to analyze if such protection is cholesterol-independent. Using primary cultures of cortical neurons treated with Abeta1-40 peptide, we have demonstrated that pre-incubation with statins prevents the rise in cytosolic Ca2+ concentration and the accumulation of reactive oxygen species induced by Abeta through mechanisms independent of cholesterol reduction. The neuroprotective actions of statins were rather attributable to their ability to reduce isoprenyl intermediates levels in the cholesterol biosynthetic pathway since their effect was reversed by geranyl pyrophosphate while cholesterol addition was ineffective. Consequently, statins were shown to rescue cortical neurons from Abeta-40-induced caspase-3-dependent apoptosis. Moreover, our results revealed that simvastatin, at neuroprotective concentrations against Abeta-induced toxicity, is not able to activate Akt or ERK2, two signaling kinases with neuroprotective roles against apoptosis.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Calcium; Caspase 3; Cell Survival; Cells, Cultured; Cerebral Cortex; Cholesterol; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Extracellular Signal-Regulated MAP Kinases; In Situ Nick-End Labeling; Lovastatin; Neurons; Neuroprotective Agents; Oncogene Protein v-akt; Peptide Fragments; Rats; Rats, Wistar; Reactive Oxygen Species; Simvastatin; Tetrazolium Salts; Thiazoles; Time Factors

The key pathogenic event in the onset of Alzheimer's disease (AD) is believed to be the aggregation of the beta-amyloid (Abeta) peptide into toxic oligomers. Molecules that interfere with this process may therefore act as therapeutic agents for the treatment of AD. N-Methylated peptides (meptides) are a general class of peptide aggregation inhibitors that act by binding to one face of the aggregating peptide but are unable to hydrogen bond on the other face, because of the N-methyl group replacing a backbone NH group. Here, we optimize the structure of meptide inhibitors of Abeta aggregation, starting with the KLVFF sequence that is known to bind to Abeta. We varied the meptide length, N-methylation sites, acetylation, and amidation of the N and C termini, side-chain identity, and chirality, via five compound libraries. Inhibitor activity was tested by thioflavin T binding, affinity chromatography, electron microscopy, and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide toxicity assay. We found that inhibitors should have all d chirality, have a free N terminus but an amidated C terminus, and have large, branched hydrophobic side chains at positions 1-4, while the side chain at position 5 was less important. A single N-methyl group was necessary and sufficient. The most active compound, d-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH(2), was more active than all previously reported peptide inhibitors. Its related non-N-methylated analogues were insoluble and toxic.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Animals; Chromatography, Affinity; Molecular Sequence Data; Oligopeptides; PC12 Cells; Peptide Fragments; Peptide Library; Peptides; Protein Structure, Quaternary; Rats; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Toxicity Tests

Accumulating evidence suggests that amyloid protein aggregation is pathogenic in many diseases, including Alzheimer's disease. However, the mechanisms by which protein aggregation mediates cellular dysfunction and overt cell death are unknown. Recent reports have focused on the potential role of amyloid oligomers or protofibrils as a neurotoxic form of amyloid-beta (Abeta) and related amyloid aggregates. Here we describe studies indicating that overt neuronal cell death mediated by Abeta(1-40) is critically dependent on ongoing Abeta(1-40) polymerization and is not mediated by a single stable species of neurotoxic aggregate. The extent and rate of neuronal cell death can be controlled by conditions that alter the rate of Abeta polymerization. The results presented here indicate that protofibrils and oligomeric forms of Abeta most likely generate neuronal cell death through a nucleation-dependent process rather than acting as direct neurotoxic ligands. These findings bring into question the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide formazan assay (MTT assay) as a reporter of Abeta-mediated neuronal cell death and suggest that diffusible Abeta protofibrils and oligomers more likely mediate subtle alterations of synaptic function and long-term potentiation rather than overt neuronal cell death. These results have been extended to Abeta(1-42), the non-Abeta component of Alzheimer's disease amyloid plaques, and human amylin, suggesting that nucleation-dependent polymerization is a common mechanism of amyloid-mediated neuronal cell death. Our findings indicate that ongoing amyloid fibrillogenesis may be an essential mechanistic process underlying the pathogenesis associated with protein aggregation in amyloid disorders.

Topics: Amyloid; Amyloid beta-Peptides; Artifacts; Biopolymers; Cell Death; Cell Membrane Permeability; Cells, Cultured; Cerebral Cortex; Coloring Agents; Crystallization; Formazans; Humans; Islet Amyloid Polypeptide; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Models, Chemical; Neurons; Oxidation-Reduction; Peptide Fragments; Solubility; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles

High serum cholesterol level has been shown as one of the risk factors for Alzheimer's disease (AD), and epidemiological studies indicate that treatment with cholesterol-lowering substances, statins, may provide protection against AD. An acute-phase reaction and inflammation, with increased levels of proinflammatory cytokines, are well known in the AD brain. Notably, there is evidence for antiinflammatory activities of statins, such as reduction in proinflammatory cytokines. Consequently, it is of interest to analyze the effects of statins on microglia, the main source of inflammatory factors in the brain, such as in AD. The aims of this study were to determine the effects of statins (atorvastatin and simvastatin) on microglial cells with regard to the secretion of the inflammatory cytokine interleukin-6 (IL-6) and cell viability after activation of the cells with bacterial lipopolysaccharides (LPS) or beta-amyloid1-40 (Abeta1-40) and in unstimulated cells. Cells of the human microglial cell line CHME-3 and primary cultures of rat neonatal cortical microglia were used. Incubation with LPS or Abeta1-40 induced secretion of IL-6, and Abeta1-40, but not LPS, reduced cell viability. Both atorvastatin and simvastatin reduced the basal secretion of IL-6 and the cell viability of the microglia, but only atorvastatin reduced LPS- and Abeta1-40-induced IL-6 secretion. Both statins potentiated the Abeta1-40-induced reduction in cell viability. The data indicate the importance of also considering the microglial responses to statins in evaluation of their effects in AD and other neurodegenerative disorders with an inflammatory component.

Topics: Amyloid beta-Peptides; Animals; Atorvastatin; Cell Death; Cell Line; Cell Survival; Cytokines; Dose-Response Relationship, Drug; Drug Interactions; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; L-Lactate Dehydrogenase; Lipopolysaccharides; Microglia; Peptide Fragments; Pyrroles; Rats; Simvastatin; Statistics, Nonparametric; Tetrazolium Salts; Thiazoles

The present work shows that alpha-glycerylphosphorylethanolamine (alpha-GPE) is effective in recovering astrocytes from mitochondrial membrane integrity and potential derangement and cellular oxidative stress that occur under amyloid beta-peptides-induced reactive gliosis.alpha-Glycerylphosphorylethanolamine (alpha-GPE), a new compound with nootropic properties, known to improve in vivo the learning and memory processes, has been tested for its protective properties on an in vitro model of degeneration. Rat primary astrocytic cultures treated with two amyloid-derived peptides, Abeta((1-40)) and Abeta(3(pE)-42), showed a marked reduction of the mitochondrial redox activity and membrane potential, together with an increase of oxidative species production. Plasma membrane lipid peroxidation (LPO) as well as generation of peroxides is greatly increased under Abeta-peptides toxicity. These features, typical of the reactive gliosis that accompanies neuronal degeneration, were readily recovered by pretreatment with alpha-GPE. alpha-GPE, likely improving the fluidity of cell membrane, has the potential to recover astrocytes from the general redox derangement induced by different amyloid fragments and possibly to protect from inflammation, gliosis and neurodegeneration. This is the first evidence of an antioxidant effect of the ethanolamine derivative on a rat model of chronic gliosis.

Topics: Amyloid beta-Peptides; Animals; Astrocytes; Cell Membrane; Cells, Cultured; Gliosis; Lipid Peroxidation; Membrane Fluidity; Membrane Potentials; Microscopy, Confocal; Mitochondria; Nerve Degeneration; Oxidative Stress; Peptide Fragments; Phosphatidylethanolamines; Rats; Tetrazolium Salts; Thiazoles

In this paper, we investigated interactions of the acidic oligosaccharide sugar chain (AOSC), derived from brown algae Echlonia kurome OKAM, with amyloid beta protein (Abeta). We observed that AOSC inhibited the toxicity induced by Abeta in both primarily cortical cells and the SH-SY5Y cell line. We also observed that AOSC inhibited the apoptosis induced by Abeta in SH-SY5Y by reducing the elevated level of intracellular calcium concentration ([Ca(2+)](i)) and suppressing the generation of reactive oxygen species. Surface plasmon resonance analysis demonstrated that AOSC had affinity for both freshly-dissolved Abeta and 48-h incubated Abeta. Furthermore, AOSC blocked the fibril formation of Abeta, which may be responsible for its anti-cytotoxic effects. Thus, our results indicate that AOSC might be a potentially therapeutic compound for Alzheimer's disease.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Calcium; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cerebral Cortex; Humans; Lipid Peroxidation; Membrane Potentials; Mitochondria; Peptide Fragments; Phaeophyceae; Rats; Tetrazolium Salts; Thiazoles; Thiobarbituric Acid Reactive Substances

Progressive deposition of amyloid beta peptide in the senile plaques is a principal event in the neurodegenerative process of Alzheimer's disease. Several reports have demonstrated that amyloid beta is cytotoxic using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of viability in cells. With the MTT assay, we screened an in-house library to find compounds which suppress amyloid beta-induced inhibition of MTT reduction. We have previously reported that 6-ethyl-N,N'-bis(3-hydroxyphenyl)[1,3,5]triazine-2,4-diamine (named RS-0466), found in an in-house library, was capable of significantly inhibiting amyloid beta-induced cytotoxicity in HeLa cells. From further screening hits, we newly focused on 4-(7-hydroxy-2,2,4-trimethyl-chroman-4-yl)benzene-1,3-diol (named RS-4252), which show comparable potency to RS-0466 to ameliorate amyloid beta-induced cytotoxicity. Furthermore, RS-4252 reversed the decrease in phosphorylated Akt by amyloid beta. These results imply that RS-4252 or one of its derivatives has the potential to be a therapeutic for Alzheimer's disease patients, and that activation of Akt is at least in part involved in the effect.

Topics: Amyloid beta-Peptides; Chromans; HeLa Cells; Humans; Oxidation-Reduction; Peptide Fragments; Phenols; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Tetrazolium Salts; Thiazoles; Triazines

Cerebral deposition of amyloid beta-peptide (Abeta) in the brain is an invariant feature of Alzheimer's disease (AD). A consistent protective effect of wine consumption on AD has been documented by epidemiological studies. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of wine-related polyphenols (myricetin, morin, quercetin, kaempferol (+)-catechin and (-)-epicatechin) on the formation, extension, and destabilization of beta-amyloid fibrils (fAbeta) at pH 7.5 at 37 degrees C in vitro. All examined polyphenols dose-dependently inhibited formation of fAbeta from fresh Abeta(1-40) and Abeta(1-42), as well as their extension. Moreover, these polyphenols dose-dependently destabilized preformed fAbetas. The overall activity of the molecules examined was in the order of: myricetin = morin = quercetin > kaempferol > (+)-catechin = (-)-epicatechin. The effective concentrations (EC50) of myricetin, morin and quercetin for the formation, extension and destabilization of fAbetas were in the order of 0.1-1 micro m. In cell culture experiments, myricetin-treated fAbeta were suggested to be less toxic than intact fAbeta, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which these polyphenols inhibit fAbeta formation from Abeta, and destabilize pre-formed fAbetain vitro are still unclear, polyphenols could be a key molecule for the development of preventives and therapeutics for AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Benzothiazoles; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Flavonoids; Humans; Kidney; Kinetics; Microscopy, Electron; Peptide Fragments; Phenols; Polymers; Quercetin; Spectrometry, Fluorescence; Tetrazolium Salts; Thiazoles

beta-Amyloid peptide (beta-AP) elicits a toxic effect on neurons in vitro and in vivo. Many environmental factors including antioxidants, metal ions and proteoglycans modify beta-AP toxicity. We have investigated on PC12 cells, the protective effect from beta-AP (1-41) of two plant polyphenols, resveratrol and catechin. PC12 cells treated with 10(-6)M beta-AP (1-41) for 16h decrease the cell viability at 24% of the control with an IC(50) value of 1.1+/-0.14 x 10(-8)M. Twenty-five micromolar resveratrol and 50 microM catechin protect PC12 cells from beta-AP (1-41) toxicity with the IC(50) value increased at 2.2+/-0.19 x 10(-7)M and at 8.9+/-0.7 x 10(-8)M, respectively. While the protective effect is concentration dependent for catechin, resveratrol shows a concentration-dependent biphasic effect. Up to 50 microM concentration, resveratrol protects PC12 cells from beta-AP (1-41) toxicity. At concentration higher than 50 microM, an inhibitory activity on cell proliferation appears. This antiproliferative effect is shown also in the absence of beta-AP (1-41). Resveratrol and catechin have a synergistic protective action. In the presence of 50 microM catechin and 10 microM resveratrol or 25 microM resveratrol and 10 microM catechin, the toxicity determined by 10(-7)M beta-AP (1-41) is almost completely abolished. Catechin is more effective than resveratrol in protecting PC12 cells from the toxicity of hydrogen peroxide. The protection from Oxygen Reactive Species (ROS) toxicity is concentration dependent for both resveratrol and catechin. In this case the protection is merely additive and the synergistic effect is not observed. These results demonstrate that resveratrol and catechin protect PC12 cells from beta-AP (1-41) toxicity and that their protective effect is synergistic. Such a protective effect probably is not due only to their antioxidant activity. The different chemical and biological activity shown by these compounds on several cell types and the complexity of the beta-AP (1-41) toxicity may explain the synergistic protective effect and suggest that the utilization of different compounds with synergistic activity may protect more effectively from complex mechanisms of toxicity.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Catechin; Cell Count; Cell Death; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Lipid Peroxidation; PC12 Cells; Peptide Fragments; Rats; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Thiobarbiturates

The aggregation process of beta-amyloid peptide Abeta into amyloid is strongly associated with the pathology of Alzheimer's disease (AD). Aggregation may involve a transition of an alpha helix in Abeta(1-28) into beta sheets and interactions between residues 18-20 of the "Abeta amyloid core." We applied an i, i+4 cyclic conformational constraint to the Abeta amyloid core and devised side chain-to-side chain lactam-bridged cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28). In contrast to Abeta(1-28) and [Lys(17), Asp(21)]Abeta(1-28), cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was not able to form beta sheets and cytotoxic amyloid aggregates. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was able to interact with Abeta(1-28) and to inhibit amyloid formation and cytotoxicity. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) also interacted with Abeta(1-40) and interfered with its amyloidogenesis. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) or similarly constrained Abeta sequences may find therapeutic and diagnostic applications in AD.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Benzothiazoles; Cell Survival; Chromatography, Gel; Circular Dichroism; Congo Red; Cyclization; Drug Design; Electrophoresis, Polyacrylamide Gel; Humans; Microscopy, Electron; Molecular Sequence Data; Peptide Fragments; Protein Binding; Protein Conformation; Protein Structure, Secondary; Spectroscopy, Fourier Transform Infrared; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Amyloid beta-peptide (Abeta) effectively inhibits the cellular reduction activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in a variety of cultured cells. Although the inhibitory activity is widely used for the estimation of the biological activity of Abeta, the cellular mechanism is unclear. In the present study, we examined the effect of Abeta on the morphology of early endosomes, in which MTT is accumulated as MTT formazan after cellular reduction. We found that Abeta1-40 alters the distribution of Rab5- and early endosomal auto-antigen 1-positive early endosomes in the presence of MTT in HeLa cells, which are susceptible to the Abeta1-40-induced inhibition of cellular MTT reduction. To obtain a clue to the molecular mechanism, we determined whether Abeta1-40 affects the signal cascade of the phosphatidylinositol-3-OH kinase (PI-3K) pathway that is involved in early endosomal trafficking. MTT induced phosphorylation of Akt and mitogen-activated protein kinase. Abeta1-40 suppressed the PI-3K-dependent Akt phosphorylation but not the mitogen-activated protein kinase phosphorylation. Thus, Abeta seems to modulate early endosomal trafficking via inhibition of the PI-3K pathway in the presence of MTT. Modulation of early endosomal trafficking appears to affect the cellular metabolism of MTT, causing suppression of cellular MTT reduction by Abeta. These findings may help clarify the mechanism of the cytotoxicity of Abeta.

Topics: Amyloid beta-Peptides; Antibodies; Biomarkers; Chromones; Coloring Agents; Endosomes; Enzyme Inhibitors; HeLa Cells; Humans; Immunohistochemistry; Membrane Proteins; Mitogen-Activated Protein Kinases; Morpholines; Oxidation-Reduction; Peptide Fragments; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; rab5 GTP-Binding Proteins; Tetrazolium Salts; Thiazoles; Vesicular Transport Proteins

beta-Amyloid peptide is the principal protein in the senile plaques of Alzheimer's disease and is considered to be responsible for the pathology of Alzheimer's disease. Several studies have shown that beta-amyloid is cytotoxic, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of viability in cells. Utilizing the MTT assay, we screened an in-house library to find compounds that suppress beta-amyloid-induced inhibition of MTT reduction. From among the screening hits, we focused on 6-ethyl-N,N'-bis(3-hydroxyphenyl)[1,3,5]triazine-2,4-diamine (named RS-0466), which had been newly synthesized in our laboratory. This compound was found to be capable of significantly inhibiting beta-amyloid-induced cytotoxicity in HeLa cells and of reversing the decrease of phosphorylated Akt induced by beta-amyloid. Furthermore, RS-0466 reversed the beta-amyloid-induced impairment of long-term potentiation in rat hippocampal slices. These results raise the possibility that RS-0466 or its derivatives have potential as a therapeutic agent for Alzheimer's disease patients, and its effect is at least in part mediated by activation of Akt.

Topics: Amyloid beta-Peptides; Animals; Cells, Cultured; Coloring Agents; Excitatory Postsynaptic Potentials; Hippocampus; Humans; In Vitro Techniques; Long-Term Potentiation; Neurons; Peptide Fragments; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors; Triazines

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

Topics: Acetylcysteine; Amyloid beta-Peptides; Blotting, Western; Cell Survival; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Glutathione; Glutathione Reductase; Humans; Hydrogen Peroxide; Neuroblastoma; Oxidative Stress; Peptide Fragments; Phosphorylation; tau Proteins; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Ultraviolet Rays

Concentrations of heavy metals, including mercury, have been shown to be altered in the brain and body fluids of Alzheimer's disease (AD) patients. To explore potential pathophysiological mechanisms we used an in vitro model system (SHSY5Y neuroblastoma cells) and investigated the effects of inorganic mercury (HgCl2) on oxidative stress, cell cytotoxicity, beta-amyloid production, and tau phosphorylation. We demonstrated that exposure of cells to 50 microg/L (180 nM) HgCl2 for 30 min induces a 30% reduction in cellular glutathione (GSH) levels (n = 13, p<0.001). Preincubation of cells for 30 min with 1 microM melatonin or premixing melatonin and HgCl2 appeared to protect cells from the mercury-induced GSH loss. Similarly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that 50 microg/L HgCl2 for 24 h produced a 50% inhibition of MTT reduction (n = 9, p<0.001). Again, melatonin preincubation protected cells from the deleterious effects of mercury, resulting in MTT reduction equaling control levels. The release of beta-amyloid peptide (Abeta) 1-40 and 1-42 into cell culture supernatants after exposure to HgCl2 was shown to be different: Abeta 1-40 showed maximal (15.3 ng/ml) release after 4 h, whereas Abeta 1-42 showed maximal (9.3 ng/ml) release after 6 h of exposure to mercury compared with untreated controls (n = 9, p<0.001). Preincubation of cells with melatonin resulted in an attenuation of Abeta 1-40 and Abeta 1-42 release. Tau phosphorylation was significantly increased in the presence of mercury (n = 9, p<0.001), whereas melatonin preincubation reduced the phosphorylation to control values. These results indicate that mercury may play a role in pathophysiological mechanisms of AD.

Topics: Amyloid beta-Peptides; Cell Survival; Drug Combinations; Glutathione; Melatonin; Mercuric Chloride; Neuroblastoma; Oxidative Stress; Peptide Fragments; Phosphorylation; tau Proteins; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Laminin has recently been reported to inhibit both Abeta40 and Abeta42 fibril formation in vitro. Laminin was thus suggested to be an effective therapeutic agent for Alzheimer's disease. However, some recent reports have shown that Abeta fibril formation may not necessarily be linked to the development of Abeta neurotoxicity. In the present study, we thus investigated whether or not laminin affects Abeta40 and Abeta42-induced neurotoxicity. The findings of the present study by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye reduction test showed laminin not to have an inhibitory effect on Abeta40 or Abeta42-induced cytotoxicity in PC12 cells while Abeta fibril formation was inhibited under the conditions used in the present study. The findings of the present study therefore do not support the hypothesis that Abeta fibril formation is absolutely required for the development of Abeta cytotoxicity.

Topics: Amyloid beta-Peptides; Animals; Cell Survival; Coloring Agents; Laminin; PC12 Cells; Peptide Fragments; Rats; Tetrazolium Salts; Thiazoles

We have investigated the effect of amyloid beta-peptide (Abeta) in rat pheochromocytoma PC 12h and murine C 1300 neuroblastoma cells by using MTT ¿3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Exposure of the cells to Abeta peptides, Abeta1-40 and its fragment Abeta25-35, induced a concentration-dependent inhibition of MTT reduction in both cell lines, and MTT-dependent LDH release due to cell lysis in PC12h cells. We also found that sodium nitroprusside (SNP), a spontaneous nitric oxide (NO) generator, significantly prevented the inhibition of MTT reduction and MTT-dependent LDH release caused by Abeta peptides at 10-100 microM, although a high concentration of SNP (> or = 333 microM) was remarkably toxic by itself. Since the inhibition of MTT reduction caused by Abeta is known as one of the first indicators of its toxicity, these findings suggest that Abeta peptides have a toxic effect in these cell lines, and SNP may attenuate the Abeta peptide-induced toxicity. In regard the mechanisms of the actions of SNP, hydroxylamine which also generates NO and 8-Br-cGMP, a membrane-permeable analogue of cyclic GMP (cGMP), failed to prevent the inhibition of MTT reduction caused by Abeta25-35 in PC12h cells, implying that the effect of SNP may be mediated by the NO-independent pathway. Since potassium ferrocyanide showed a significant prevention at 333 microM although it had toxic effect at this concentration, it is considered that the ferrocyanide portion of the SNP metabolite may be partially involved. The cell death induced by other oxidative insults, such as glutamate and hydrogen peroxide (H2O2), could not be attenuated by SNP in both cell lines. Thus, the observed effect of SNP might not be due to its direct antioxidative action.

Topics: Amyloid beta-Peptides; Animals; L-Lactate Dehydrogenase; Mice; Neuroblastoma; Nitric Oxide; Nitroprusside; Oxidation-Reduction; PC12 Cells; Peptide Fragments; Rats; Sodium Glutamate; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Mechanism of amyloid beta-peptide (A beta) toxicity in cultured neurons involves the development of oxidative stress in the affected cells. A significant increase in protein carbonyl formation was detected in cultured hippocampal neurons soon after the addition of preaggregated A beta(1-40), indicating oxidative damage of proteins. We report that neurons, subjected to A beta(1-40), respond to A beta oxidative impact by activation of antioxidant defense mechanisms and alternative ATP-regenerating pathway. The study demonstrates an increase of Mn SOD gene expression and the restoration of Cu, Zn SOD gene expression to a normal level after temporary suppression. Partial loss of creatine kinase (CK) BB activity, which is the key enzyme for functioning of the creatine/phosphocreatine shuttle, was compensated in neurons surviving the A beta oxidative attack by increased production of the enzyme. As soon as the oxidative attack triggered by the addition of preaggregated A beta (1-40) to rat hippocampal cell cultures has been extinguished, CK BB expression and SOD isoenzyme-specific mRNA levels in surviving neurons return to normal. We propose that the maintenance of a constant level of CK function by increased CK BB production together with the induction of antioxidant enzyme gene expression in A beta-treated hippocampal neurons accounts for at least part of their adaptation to A beta toxicity.

Topics: Amyloid beta-Peptides; Animals; Blotting, Western; Cell Survival; Cells, Cultured; Creatine Kinase; Hippocampus; Humans; Isoenzymes; Mitochondria; Neurons; Oxidative Stress; Peptide Fragments; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Trypan Blue

An amyloid-binding dye Congo red has been reported to prevent the neurotoxic effect of Alzheimer's amyloid beta protein (Abeta). In the present study, we investigated the effect of Congo red in cultured rat cortical astrocytes. Abeta (1 nM-10 microM) did not cause cell death, but potently inhibited the cellular redox activity as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Congo red (0.2-20 microM), when added together with or prior to Abeta, significantly blocked Abeta-induced inhibition of redox activity. Furthermore, when Congo red was added after treatment with Abeta, the inhibited redox activity was restored to normal, indicating that Congo red can reverse Abeta-induced cellular stress. The reversing effect of Congo red cannot be explained by the inhibition of Abeta fibril formation and suggests a novel aspect of the interaction of Congo red with Abeta.

Topics: Amyloid beta-Peptides; Animals; Astrocytes; Cells, Cultured; Congo Red; Oxidation-Reduction; Peptide Fragments; Rats; Rats, Wistar; Stress, Physiological; Tetrazolium Salts; Thiazoles; Time Factors

Amyloid beta protein (A beta), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of A beta. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of A beta and reaches a plateau in 12 h. This effect of A beta is not blocked by Ca2+ channel blockers or in Ca(2+)-free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of A beta and are blocked by Ca2+ channel blockers such as Co2+, nicardipine, and diltiazem. When A beta was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long-lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that A beta toxicity consists of two phases-a Ca(2+)-independent early phase and a Ca(2+)-dependent late phase- and that the early phase may be required to induce the late phase.

Topics: Amyloid beta-Peptides; Animals; Calcium; Cell Death; Coloring Agents; Embryo, Mammalian; Glutamic Acid; Hippocampus; Kinetics; L-Lactate Dehydrogenase; Neurons; Neurotoxins; Oxidation-Reduction; Peptide Fragments; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Thiazoles; Time Factors

Glycosaminoglycan (GAG)-containing proteoglycans are associated with the neuritic plaques and cerebrovascular beta-amyloid deposits of Alzheimer's disease as well as with the amyloid deposits of prion and other disorders. GAGs and other sulfate-containing compounds have previously been shown to bind beta-amyloid peptide in vitro, suggesting possible effects of beta-amyloid deposition and/or toxicity in vivo. Using reduction of the redox dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure beta-amyloid neurotoxicity in rat pheochromocytoma PC12 cells, several polysulfated GAGs and synthetic sulfate-containing compounds were found to attenuate the neurotoxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

Topics: Amyloid beta-Peptides; Animals; Cell Survival; Coloring Agents; Glycosaminoglycans; Oxidation-Reduction; PC12 Cells; Peptide Fragments; Rats; Tetrazolium Salts; Thiazoles

Topics: Amyloid beta-Peptides; Animals; Cell Survival; Coloring Agents; Congo Red; Dose-Response Relationship, Drug; Glycosaminoglycans; Neurotoxins; PC12 Cells; Peptide Fragments; Rats; Tetrazolium Salts; Thiazoles

The neurotoxic effects of soluble and aggregated synthetic amyloid beta protein (A beta P) have been investigated in rat primary cultures. Freshly solubilized beta(1-40) was neurotoxic not to immature, but to mature hippocampal neurons. On the other hand, aggregated beta(1-40) was neurotoxic to both. Neurotoxicity induced by aggregated beta(1-40) was 10-fold more potent than soluble beta(1-40) and was not prevented by substance P. The neurotoxicity of aggregated beta(1-40) to cultured neurons depended on the peptide concentration and the duration of exposure to it. Cerebral cortical and hippocampal neurons were significantly susceptible to aggregated beta(1-40) than cerebellar granular cells, and cultured astrocytes were not vulnerable to aggregated beta(1-40) even at high concentrations.

Topics: Amyloid beta-Peptides; Animals; Astrocytes; Cell Death; Cells, Cultured; Cerebral Cortex; Hippocampus; Neurons; Peptide Fragments; Rats; Substance P; Tetrazolium Salts; Thiazoles