Azole derivatives inhibit wildtype butyrylcholinesterase and its common mutants.

Azoles, which have been used for antifungal chemotherapy for decades, have recently been of interest for their efficacy against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). There is little known about the potential of azoles against BChE, however there is none regarding their inhibitory effects against mutants of BChE. In the current study, an azole library of 1-aryl-2-(1H-imidazol-1-yl)ethanol/ethanone oxime esters were tested against AChE and BChE, which yielded derivates more potent than the positive control, galantamine, against both isoforms. Kinetic analyses were performed for wildtype and mutant (A328F and A328Y) inhibition for the two most potent BChE inhibitors, pivalic and 3-bezoylpropanoic acid esters of 2-(1H-imidazol-1-yl)-1-(2-naphthyl)ethanol, which were found to have great affinity to the wildtype and mutant BChE types with Ki values as low as 0.173 ± 0.012 µM. The compounds were identified to show linear competitive or mixed type inhibition. Molecular modeling confirmed these kinetic data and provided further insights regarding molecular basis of BChE inhibition by the active derivatives. Thus, current study suggests new azole derivatives with promising cholinesterase inhibitory effects and reveals the first set of information to promote our understanding for the inhibitory behavior of this class against the mutant BChE forms.

Organophosphorus Pesticides Promote Protein Cross-Linking.

Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a cross-link between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced cross-linking. Our goal was to explore OP-induced cross-linking in a complex protein sample, MAP-rich tubulin from Sus scrofa and to test 8 OP for their capacity to promote isopeptide cross-linking. We treated 100 µg of MAP-rich tubulin with 100 µM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide cross-links. Sixteen spectra yielded convincing evidence for isopeptide cross-linked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein cross-linking is a general property of organophosphorus pesticides and pesticide metabolites. Data are available via ProteomeXchange with identifier PXD034529.

Toluidine blue O attenuates tau phosphorylation in N2a-APPSwe cells.

Alzheimer's disease (AD) is characterized by extracellular amyloid plaques composed of amyloid-ß peptide (Aß), intracellular neurofibrillary tangles containing hyperphosphorylated tau protein and neuronal loss. Most of the FDA-approved AD drugs currently on the market are cholinesterase inhibitors, which are only effective in relieving the symptoms of AD. However, recent studies in AD drug discovery focus on multi-targeted strategies, including anti-amyloid and anti-tau therapy. In the current study, we have investigated the effects of toluidine blue O (TBO), a cholinesterase inhibitor, on amyloid precursor protein (APP) processing, tau phosphorylation, and tau kinases/phosphatase in N2a mouse neuroblastoma cells stably expressing the Swedish mutation of human APP695 (N2a-APPSwe). The results demonstrated that TBO reduces Aß40/42 levels by decreasing expression levels of ß-secretase 1 (BACE1), presenilin 1 (PS1) and total APP without causing cytotoxic effects in N2a-APPSwe cells. TBO also decreased the levels of both total tau and phosphorylated tau at residues Ser202/Thr205, Thr181, Ser396 and Ser 396/Ser404. Moreover, when the possible mechanisms underlying its effects on tau pathology were explored, TBO was found to decrease tau phosphorylation at those sites by reducing the expression levels of Akt, GSK-3ß, Cdk5, inactive p-PP2A and increasing the expression levels of p-Akt Ser473 and inactive p-GSK-3ß Ser9. Our new data support the idea that TBO may be a promising multi-target drug candidate for the treatment of AD.

Chlorpyrifos oxon crosslinking of amyloid beta 42 peptides is a new route for generation of self-aggregating amyloidogenic oligomers that promote Alzheimer's disease.

Epidemiological evidence suggests that people chronically exposed to organophosphorus pesticides are at increased risk of neurodegenerative disease. Covalently linked amyloid beta dimers have been isolated from the brains of Alzheimer's patients. The toxic forms of amyloid beta are amyloid dimers that spontaneously oligomerize. In the present report we treated recombinant and synthetic amyloid beta (1-42) with 1 mM chlorpyrifos oxon or 1 mM paraoxon. The trypsin-digested samples were analyzed by liquid chromatography tandem mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer. Data were searched with Protein Prospector software. We found two new types of crosslinks in amyloid dimers. An isopeptide Asp-Asp link occurred between the N-terminal amine of Asp 1 in one peptide and the beta carboxyl group of Asp 1 in another peptide. An Asp-Arg link occurred between the guanidino group of Arg 5 in one peptide and the beta carboxyl group of Asp 1 in another peptide. These results show that the active metabolites of the pesticides chlorpyrifos and parathion catalyze the crosslinking of amyloid beta (1-42) into toxic dimers. It was concluded that the increased risk of neurodegenerative disease in people exposed to organophosphorus pesticides could be explained by the crosslinking activity of these chemicals. Data are available via ProteomeXchange with identifier PXD034163.

Butyrylcholinesterase in SH-SY5Y human neuroblastoma cells.

Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.

Effects of toluidine blue O and methylene blue on growth and viability of pancreatic cancer cells.

Amyloid precursor-like protein-2 (APLP2) and its C-terminal fragments (CTFs) are expressed at high levels in pancreatic cancer cells and knockdown of APLP2 expression inhibits tumor growth. CTFs are released from APLP2 by beta-secretase (BACE). In this study, our goal was to determine whether methylene blue (MethB) and toluidine blue O (TBO) could be used to slow down the growth and viability of pancreatic cancer cells (Hs 766T). We found that TBO and MethB decreased the growth and viability of Hs 766T cells in a dose- and time-dependent manner compared to vehicle-treated control, as demonstrated by MTT and trypan blue exclusion assays. Although TBO led to decreased expression of APLP2, MethB did not show any significant effect on APLP2. However, both MethB and TBO reduced BACE activity and the levels of APLP2 CTFs in Hs 766T cells. In conclusion, MethB and TBO may be valuable candidates for the treatment of pancreatic cancer by targeting APLP2 processing.

Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry.

Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (Kd 3.2 × 10-8 M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 µM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.

Evaluation of mass spectrometry MS/MS spectra for the presence of isopeptide crosslinked peptides.

Isopeptide crosslinked proteins can be the product of transglutaminase or of exposure to organophosphorus toxicants (OP). Transglutaminase links glutamine to lysine with loss of ammonia. OP toxicants induce a link between glutamic acid and lysine with loss of water. Our goal was to establish criteria to distinguish real from false isopeptide crosslinks reported by software searches of mass spectrometry data. We used fragmentation spectra of tryptic peptides from MAP-rich tubulin Sus scrofa as a test system for detection of naturally-occurring isopeptide crosslinks. Data were analyzed with Protein Prospector. Criteria for the assignments included the presence of at least 1 crosslink specific product ion, fragment ions from both peptides, Protein Prospector scores ≥20, and best fit of the MS/MS data to the crosslinked peptide as opposed to a linear peptide. Out of 301,364 spectra, 15 potential transglutaminase-type crosslinked peptide candidates were identified. Manual evaluation of these MS/MS spectra reduced the number to 1 valid crosslink between Q112 of NFH and K368 of Tau. Immunopurification with anti-isopeptide 81D1C2 confirmed that MAP-rich tubulin contained only one isopeptide. Support for this isopeptide bond was obtained by showing that transglutaminase was capable of incorporating dansyl-aminohexyl -QQIV into K368. A model of the KIETHK-QLEAHNR isopeptide was synthesized with the aid of transglutaminase. MS/MS spectra of the model validated our interpretation of the native isopeptide. An OP-induced isopeptide bond between K163 of tubulin alpha-1A and E158 of tubulin beta-4B was induced by treating MAP-rich tubulin with 100 µM chlorpyrifos oxon. This crosslink was supported by the criteria described above and by the presence of diethoxyphospho-lysine 163 in the tubulin alpha-1A peptide. The information obtained in this work is valuable for future studies that aim to understand why exposure to OP is associated with increased risk of neurodegenerative disease.

Inhibition of cholinesterases by safranin O: Integration of inhibition kinetics with molecular docking simulations.

In the present study, the inhibitory mechanisms and effects of a synthetic phenazine dye, safranin O (SO) on human plasma butyrylcholinesterase (BChE), human erythrocyte acetylcholinesterase (AChE) and recombinant BChE mutants were investigated. Kinetic studies showed the following information: SO leaded to linear competitive inhibition of human plasma BChE with Ki = 0.44 ± 0.085 µM; α = ∞. It acted as a hyperbolic noncompetitive inhibitor of human erythrocyte AChE with Ki = 0.69 ± 0.13; α = 1; ß = 0.08 ± 0.02. On the other hand, the inhibitory effects of SO on two BChE mutants, where A328 was modified to either F or Y, revealed differences in terms of inhibitory patterns and Ki values, compared to the obtained results with recombinant wild type BChE. SO was found to act as a linear competitive inhibitor of A328F and A328Y BChE mutants. Compared to recombinant wild type BChE, A328Y and A328F BChE mutants caused a 4- and 10-fold decrease in Ki value for SO, respectively. These findings were supported by molecular modelling studies. In conclusion, SO is a potent inhibitor of human cholinesterases and may be useful in the design and development of new drugs for the treatment of AD.

The kinetics of inhibition of human acetylcholinesterase and butyrylcholinesterase by methylene violet 3RAX.

Phenazines, naturally produced by bacteria and archaeal Methanosarcina species are nitrogen-containing tricyclic molecules with antibiotic, antitumoral, and antiparasitic activities. Phenazines are used as electron acceptors-donors in wide range of fields including environmental biosensors. In this study, the inhibitory effects of a synthetic phenazine dye, methylene violet 3RAX (also known as diethyl safranine) on human erythrocyte AChE and human plasma BChE were tested and also its inhibitory mechanisms for both enzymes were studied in detail. Kinetic analyses showed that methylene violet 3RAX acts as a hyperbolic noncompetitive inhibitor of AChE with Ki value of 1.58 ±â€¯0.36 µM; α = 1; ß = 0.12 ±â€¯0.0003. On the other hand, it caused linear competitive inhibition of BChE with Ki value of 0.51 ±â€¯0.006 µM; α = ∞. In conclusion, methylene violet 3RAX which is a highly effective inhibitor of both human AChE and human BChE with Ki values in low micromolar range may be a promising candidate for the treatment of Alzheimer's disease.

Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment.

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 ß, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.

Delipidation of Plasma Has Minimal Effects on Human Butyrylcholinesterase.

Human butyrylcholinesterase (BChE) is purified in large quantities from Cohn fraction IV-4 to use for protection against the toxicity of chemical warfare agents. Small scale preliminary experiments use outdated plasma from the American Red Cross as the starting material for purifying BChE (P06276). Many of the volunteer donor plasma samples are turbid with fat, the donor having eaten fatty food before the blood draw. The turbid fat interferes with enzyme assays performed in the spectrophotometer and with column chromatography. Our goal was to find a method to remove fat from plasma without loss of BChE activity. Satisfactory delipidation was achieved by adding a solution of 10% dextran sulfate and calcium chloride to fatty plasma, followed by centrifugation, and filtration through a 0.8 µm filter. Treatment with Aerosil also delipidated fatty plasma, but was accompanied by loss of 50% of the plasma volume. BChE activity and the BChE isozyme pattern on nondenaturing gel electrophoresis were unaffected by delipidation. BChE in delipidated plasma was efficiently captured by immobilized monoclonal antibodies B2 18-5 and mAb2. The immunopurified BChE was released from antibody binding with acid and visualized as a highly enriched, denatured BChE preparation by SDS gel electrophoresis. In conclusion, delipidation with dextran sulfate/CaCl2 preserves BChE activity and the tetramer structure of BChE.

Toluidine blue O modifies hippocampal amyloid pathology in a transgenic mouse model of Alzheimer's disease.

Recently, we have demonstrated that toluidine blue O (TBO), a phenothiazine dye, shows inhibitory effects on both cholinesterases and amyloid pathology in Alzheimer's disease (AD) cellular model. In the present study, we aimed to determine the effects of TBO (in a purity of 85%) on amyloid and tau pathologies in a triple transgenic mouse model of AD (3xTg-AD). Beginning at 7.5 (mild pathology) or 13 (severe pathology) months of age, 3xTg-AD mice were treated intraperitoneally with 4 mg/kg TBO or vehicle daily for 30 days. TBO treatment significantly reduced the levels of insoluble Aß40 and Aß42 in the hippocampi of mild and severe pathology groups compared to vehicle-treated counterparts. When the levels of full-length amyloid precursor protein (APP) and ß-site APP-cleaving enzyme 1 (BACE1) were assessed in 3xTg-AD mice at late pathological stage, no significant changes were observed after TBO treatment. Similarly, TBO did not recover hyperphosphorylation of tau at residues Thr181 and Ser202/Thr205 significantly in soluble and insoluble hippocampal fractions of 3xTg-AD mice. Taken together, the current study is the first in vivo report, to our knowledge, demonstrating that TBO mitigates amyloid pathology in 3xTg-AD mice with no apparent change on tau phosphorylation. Overall, the preliminary data presented here support the possible use of TBO as a disease-modifying drug for AD treatment.

Use of Hupresin To Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure.

Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.

Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 × 10-9 M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10-8 M for OP-peptides and 1 × 10-12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 µg/mL of depY was 0.025 µg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.

Hupresin Retains Binding Capacity for Butyrylcholinesterase and Acetylcholinesterase after Sanitation with Sodium Hydroxide.

Hupresin is a new affinity resin that binds butyrylcholinesterase (BChE) in human plasma and acetylcholinesterase (AChE) solubilized from red blood cells (RBC). Hupresin is available from the CHEMFORASE company. BChE in human plasma binds to Hupresin and is released with 0.1 M trimethylammonium bromide (TMA) with full activity and 10-15% purity. BChE immunopurified from plasma by binding to immobilized monoclonal beads has fewer contaminating proteins than the one-step Hupresin-purified BChE. However, when affinity chromatography on Hupresin follows ion exchange chromatography at pH 4.5, BChE is 99% pure. The membrane bound AChE, solubilized from human RBC with 0.6% Triton X-100, binds to Hupresin and remains bound during washing with sodium chloride. Human AChE is not released in significant quantities with non-denaturing solvents, but is recovered in 1% trifluoroacetic acid. The denatured, partially purified AChE is useful for detecting exposure to nerve agents by mass spectrometry. Our goal was to determine whether Hupresin retains binding capacity for BChE and AChE after Hupresin is washed with 0.1 M NaOH. A 2 mL column of Hupresin equilibrated in 20 mM TrisCl pH 7.5 was used in seven consecutive trials to measure binding and recovery of BChE from 100 mL human plasma. Between each trial the Hupresin was washed with 10 column volumes of 0.1 M sodium hydroxide. A similar trial was conducted with red blood cell AChE in 0.6% Triton X-100. It was found that the binding capacity for BChE and AChE was unaffected by washing Hupresin with 0.1 M sodium hydroxide. Hupresin could be washed with sodium hydroxide at least seven times without losing binding capacity.

Effects of phenothiazine-structured compounds on APP processing in Alzheimer's disease cellular model.

The excess accumulation of amyloid-ß (Aß) peptides derived from the sequential cleavage of amyloid precursor protein (APP) by secretases, is one of the toxic key events leading to neuronal loss in Alzheimer's disease (AD). Studies have shown that cholinergic activity may also be involved in the regulation of APP metabolism. In the current study, we have investigated the roles of toluidine blue O (TBO) and thionine (TH), newly recognized phenothiazine-derived cholinesterase inhibitors, on the metabolism of APP in Chinese hamster ovary cells stably expressing human APP751 and presenilin 1 (PS70 cells). We assessed the effects of both compounds on the levels of Aß, soluble APP-α (sAPPα), intracellular APP and ß-site APP-cleaving enzyme 1 (BACE1). After treatment of PS70 cells with TBO or TH without any side effect on cell viability, the levels of secreted Aß40, Aß42 and sAPPα were assayed by specific sandwich ELISAs while APP and BACE1 in cell lysates were analyzed using Western blot. The secreted Aß40, Aß42 and sAPPα in TBO- and TH-treated cells were found to be reduced in a dose-dependent manner compared to vehicle-treated cells. Results suggest that TH mitigated the Aß pathology by lowering APP levels whereas reduced Aß caused by TBO treatment seems to be the outcome of both less substrate availability and amyloidogenic APP processing. Taken together, our results represent the first report demonstrating that TBO and TH can affect amyloid metabolism in vitro.