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.

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.

Characteristic fragment ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine.

Glutamine residues susceptible to transglutaminase-catalyzed crosslinking can be identified by incorporation of dansyl cadaverine or biotin cadaverine. Bacterial transglutaminase and human transglutaminase 2 were used to modify residues in beta-casein with dansyl cadaverine. Bacterial transglutaminase was used to modify residues in human butyrylcholinesterase with biotin cadaverine. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector searches of mass spectrometry data. The MS/MS spectra from modified casein included intense peaks at 336.2, 402.2, and 447.2 for fragments of dansyl cadaverine adducts on glutamine. The MS/MS spectra from modified butyrylcholinesterase included intense peaks at 329.2, 395.2, and 440.2 for fragments of biotin cadaverine adducts on glutamine. No evidence for transglutaminase-catalyzed adducts on glutamic acid, aspartic acid, or asparagine was found. Consistent with expectation, it was concluded that bacterial transglutaminase and human transglutaminase 2 specifically modify glutamine. The characteristic ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine are useful markers for modified peptides.

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

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.

Azure B affects amyloid precursor protein metabolism in PS70 cells.

Alzheimer's disease (AD), the most common form of dementia, is characterized by abundant deposition of amyloid-ß (Aß) peptide that is the result of sequential cleavage of amyloid precursor protein (APP) by ß-secretase and γ-secretase. Several studies have documented that inhibition of Aß peptide synthesis or facilitating its degradation is one of the attractive therapeutic strategies in AD. Methylene blue (MethB), which has recently been investigated in Phase II clinical trials, is a prominent inhibitor in reducing Aß oligomers. Herein, we wonder whether the mitigating effects of MethB on amyloid metabolism are related to the activity of its major metabolite, azure B. The goal of this study was to investigate the effects of azure B, which is also a cholinesterase inhibitor, on APP processing by using Chinese hamster ovary cells stably expressing human wild-type APP and presenilin 1 (PS70). Azure B significantly decreased the levels of secreted APPα (sAPPα) and Aß40/42 in culture medium with a dose-dependent manner. A significant decrease was also observed in the levels of intracellular APP without affecting the cell viability. In parallel with the decrease of APP and APP metabolites, the activity of ß-secretase 1 (BACE1) was significantly attenuated compared to control. Overall, our results show that azure B has a large contribution for the pharmacological profile of MethB in APP metabolism.

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.

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.

Toluidine blue O is a potent inhibitor of human cholinesterases.

In this study, the inhibitory effects of three phenothiazines [toluidine blue O (TBO), thionine (TH) and methylene violet (MV)] were tested on human plasma butyrylcholinesterase (BChE) and their inhibitory mechanisms were studied in detail. MV acted as a linear mixed type inhibitor of human BChE with Ki = 0.66 ± 0.06 µM and α = 13.6 ± 3.5. TBO and TH caused nonlinear inhibition of human BChE, compatible to double occupancy. Ki values estimated by nonlinear regression analysis for TBO and TH were 0.008 ± 0.003 µM and 2.1 ± 0.42 µM, respectively. The inhibitory potential of TBO was also tested on human erythrocyte AChE. TBO acted as a linear mixed type inhibitor of human AChE with Ki = 0.041 ± 0.005 µM and α = 1.6 ± 0.007. Using four site-directed BChE mutants, the role of peripheral anionic site residues of human BChE was also investigated in the binding of TBO to BChE. The peripheral anionic site mutants of BChE caused 16-69-fold increase in Ki value of TBO, compared to recombinant wild-type, suggesting that peripheral anionic site residues are involved in the binding of TBO to human BChE. In conclusion, TBO which is a potent inhibitor of human cholinesterases, may be a potential drug candidate for the treatment of Alzheimer's disease.

Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase.

Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.

Determination of binding points of methylene blue and cationic phenoxazine dyes on human butyrylcholinesterase.

In this study, the binding points of MethB and two structurally-related cationic phenoxazine dyes [meldola blue (MB) and nile blue (NB)] to human butyrylcholinesterase (BChE) were investigated by molecular docking and site directed mutagenesis. The comparative inhibitory effects of MethB, MB and NB on recombinant wild type BChE and six human BChE mutants were spectrophotometrically studied. Kinetic analyses yielded the following information: MethB and MB were found to cause nonlinear inhibition of all recombinant BChEs except Y332A, compatible with a multi-site binding model. On the other hand, MethB and MB caused linear mixed inhibition of Y332A mutant, compatible with a single binding mode. Comparing the inhibitory effects in aspect of Ki values with recombinant wild type BChE (Ki=0.042 µM), MethB was found to be ∼30, 80 and 270-fold less effective as an inhibitor of Y332A, F329A and T120F, respectively. NB caused nonlinear inhibition of all recombinant BChEs. The inhibitory effect of NB on Y332A mutant was ∼370-fold lower, compared to recombinant wild type BChE (Ki=0.006 µM). Considering both kinetic and molecular docking results together, it was concluded that threonine 120, phenylalanine 329 and tyrosine 332 are critical amino acids in binding of cationic phenoxazine/phenothiazine structured ligands to human BChE.

The proline-rich tetramerization peptides in equine serum butyrylcholinesterase.

Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline-rich region of human lamellipodin. Our goal was to examine the tetramer-organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in MALDI-TOF-TOF and linear ion trap quadrupole Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to nine proteins, although not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse butyrylcholinesterase. The function of these polyproline peptides is to serve as tetramer-organizing peptides.

The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase.

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25°C in 50mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K(i) values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 µM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K(i) values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.