Hrabovska A


Full name : Paul Hrabovska Anna

First name : Anna

Mail : Department of Pharmacology and Toxicology\; Faculty of Pharmacy\; Comenius University\; Odbojarov 10\; 83232 Bratislava. +421250117377

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Country : Slovakia

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Phone : +421250117153

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References (26)

Title : Serum butyrylcholinesterase as a marker of COVID-19 mortality: Results of the monocentric prospective observational study - Markuskova_2023_Chem.Biol.Interact_14ChEPon_110557
Author(s) : Markuskova L , Javorova Rihova Z , Fazekas T , Martinkovicova A , Havrisko M , Dingova D , Solavova M , Rabarova D , Hrabovska A
Ref : Chemico-Biological Interactions , :110557 , 2023
Abstract : The COVID-19 pandemic represents an excessive burden on health care systems worldwide and the number of patients who require special care in the clinical setting is often hard to predict. Consequently, there is an unmet need for a reliable biomarker that could predict clinical outcomes of high-risk patients. Lower serum butyrylcholinesterase (BChE) activity was recently linked with poor outcomes of COVID-19 patients. In line with this, our monocentric observational study on hospitalized COVID-19 patients focused on changes in serum BChE activity in relation to disease progression. Blood samples from 148 adult patients of both sexes were collected during their hospital stay at the Clinics of Infectiology and Clinics of Anesthesiology and Intensive Care, Trnava University Hospital in alignment with routine blood tests. Sera were analyzed using modified Ellman's method. Patient data with information about the health status, comorbidities and other blood parameters were collected in pseudonymized form. Our results show a lower serum BChE activity together with progressive decline of BChE activity in non-survivors, while higher stable values were present in discharged or transferred patients requiring further care. Lower BChE activity was associated with higher age and lower BMI. Moreover, we observed a negative correlation of serum BChE activity with the routinely used inflammatory markers, C-reactive protein and interleukin-6. Serum BChE activity mirrored clinical outcomes of COVID-19 patients and thus serves as a novel prognostic marker in high-risk patients.
ESTHER : Markuskova_2023_Chem.Biol.Interact_14ChEPon_110557
PubMedSearch : Markuskova_2023_Chem.Biol.Interact_14ChEPon_110557
PubMedID: 37209860

Title : alpha7 nicotinic receptors play a role in regulation of cardiac hemodynamics - Targosova_2023_J.Neurochem_17thISCM__
Author(s) : Targosova K , Kucera M , Fazekas T , Kilianova Z , Stankovicova T , Hrabovska A
Ref : Journal of Neurochemistry , : , 2023
Abstract : The alpha7 nicotinic receptors (NR) have been confirmed in the heart but their role in cardiac functions has been contradictory. To address these contradictory findings, we analyzed cardiac functions in alpha7 NR knockout mice (alpha7(-/-) ) in vivo and ex vivo in isolated hearts. A standard limb leads electrocardiogram was used, and the pressure curves were recorded in vivo, in Arteria carotis and in the left ventricle, or ex vivo, in the left ventricle of the spontaneously beating isolated hearts perfused following Langedorff's method. Experiments were performed under basic conditions, hypercholinergic conditions, and adrenergic stress. The relative expression levels of alpha and beta NR subunits, muscarinic receptors, beta1 adrenergic receptors, and acetylcholine life cycle markers were determined using RT-qPCR. Our results revealed a prolonged QT interval in alpha7(-/-) mice. All in vivo hemodynamic parameters were preserved under all studied conditions. The only difference in ex vivo heart rate between genotypes was the loss of bradycardia in prolonged incubation of isoproterenol-pretreated hearts with high doses of acetylcholine. In contrast, left ventricular systolic pressure was lower under basal conditions and showed a significantly higher increase during adrenergic stimulation. No changes in mRNA expression were observed. In conclusion, alpha7 NR has no major effect on heart rate, except when stressed hearts are exposed to a prolonged hypercholinergic state, suggesting a role in acetylcholine spillover control. In the absence of extracardiac regulatory mechanisms, left ventricular systolic impairment is revealed.
ESTHER : Targosova_2023_J.Neurochem_17thISCM__
PubMedSearch : Targosova_2023_J.Neurochem_17thISCM__
PubMedID: 37017608

Title : Aortic butyrylcholinesterase is reduced in spontaneously hypertensive rats - Szmicsekova_2021_Physiol.Res__
Author(s) : Szmicsekova K , Pivackova LB , Kilianova Z , Slobodova , Krenek P , Hrabovska A
Ref : Physiol Res , : , 2021
Abstract : Despite the fact that vessels have sparse cholinergic innervation, acetylcholine (ACh), the primary neurotransmitter of parasympathetic nervous system, has been commonly used in physiological experiments to assess vascular function. ACh is hydrolyzed by two cholinesterases (ChE), namely acetylcholin-esterase and butyrylcholinesterase (BChE). However, little is known about these enzymes in blood vessels. The aim of the project was to characterize the expression and activity of ChE in rat aorta. As the effect of ACh on vascular tone depends on the presence of endothelium, Wistar rats were used as a model with intact endothelium and spontaneously hypertensive rats as a model of impaired endothelial function. Relative expression of both ChE in different parts of the aorta were determined using RT-qPCR. Enzyme activities were assessed in tissue homogenates by Ellman's assay. Here we showed that both ChE are present in each part of rat aorta, while mRNA is more abundant for BChE than for AChE, irrespective of aortic compartment or genotype. Normotensive Wistar rats possess higher aortic mRNA expression and activity of BChE compared to SHR. We concluded that BChE is the dominant type of ChE in rat aorta and it might play an important role in the regulation of vascular tone.
ESTHER : Szmicsekova_2021_Physiol.Res__
PubMedSearch : Szmicsekova_2021_Physiol.Res__
PubMedID: 34505519

Title : Expression of cholinesterases and their anchoring proteins in rat heart - Kilianova_2020_Can.J.Physiol.Pharmacol__
Author(s) : Kilianova Z , Ciznarova N , Szmicsekova K , Slobodova L , Hrabovska A
Ref : Canadian Journal of Physiology & Pharmacology , : , 2020
Abstract : Acetylcholine-mediated vagal transmission as well as non-neuronal acetylcholine (ACh) release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemic-reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q anchoring protein (ColQ) and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. AChE-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. BChE is found in all heart compartments and is accompanied with both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the non-neuronal cholinergic system (NNCS). Additionally, two PRiMA variants are detected throughout the rat heart.
ESTHER : Kilianova_2020_Can.J.Physiol.Pharmacol__
PubMedSearch : Kilianova_2020_Can.J.Physiol.Pharmacol__
PubMedID: 32017610

Title : [Mice lacking individual molecular forms of cholinesterases] - Kucera_2016_Ceska.Slov.Farm_65_52
Author(s) : Kucera M , Hrabovska A
Ref : Ceska a Slovenska Farmacie , 65 :52 , 2016
Abstract : Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) represent a small family of enzymes called cholinesterases. These enzymes are in the organisms either soluble or anchored through anchoring proteins collagen Q (ColQ) and proline-rich membrane anchor (PRiMA). Knowledge of molecular biology and genetics of cholinesterase and their anchoring proteins resulted in the preparation of mutant mice with the absence of different molecular forms of cholinesterases. So far a number of mutant mice were prepared with a genetic modification on the genes encoding cholinesterases or anchoring proteins. The mice with mutation in the genes encoding the cholinesterases are: the mice with the absence of AChE, mice with the absence of BChE, mice with a deletion of exon 5 and 6 in the AChE gene and mice with the absence of AChE in muscles. The mice with a mutation in the genes encoding anchoring proteins include the mice with the absence of AChE and BChE anchored by ColQ and mice with the absence of AChE and BChE anchored by PRiMA. The study of adaptation changes results from the absence of cholinesterases led to the enrichment of existing knowledge about cholinesterases and the cholinergic nervous system. KEY WORDS: absence of cholinesterases mutant mice acetylcholinesterase butyrylcholinesterase.
ESTHER : Kucera_2016_Ceska.Slov.Farm_65_52
PubMedSearch : Kucera_2016_Ceska.Slov.Farm_65_52
PubMedID: 27356594

Title : Low Plasma Cholinesterase Activities are Associated with Deficits in Spatial Orientation, Reduced Ability to Perform Basic Activities of Daily Living, and Low Body Mass Index in Patients with Progressed Alzheimer's Disease - Dingova_2016_J.Alzheimers.Dis_51_801
Author(s) : Dingova D , Fazekas T , Okuliarova P , Strbova J , Kucera M , Hrabovska A
Ref : J Alzheimers Dis , 51 :801 , 2016
Abstract : Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder characterized by a central cholinergic deficit. Non-neuronal cholinergic changes are, however, described as well. Here we focused on possible changes in the activity of the plasma cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), in hospitalized AD patients. We analyzed plasma AChE and BChE activities with regards to age, gender, body mass index (BMI), cognitive functions, and ability to perform activities of daily living in AD patients in comparison to healthy subjects. We observed lower AChE activity and trend toward lower BChE activity in AD patients, which both correlated with low BMI. AD patients unable to perform basic activities of daily living (feeding, bathing, dressing, and grooming) showed reduced plasma AChE activities, while worse spatial orientation was linked to lower BChE activities. Three out of four AD patients with the lowest BChE activities died within one year. In conclusion, progressed AD was accompanied by lower plasma AChE activity and trend toward lower BChE activity, which correlated with BMI and deficits in different components of the AD.
ESTHER : Dingova_2016_J.Alzheimers.Dis_51_801
PubMedSearch : Dingova_2016_J.Alzheimers.Dis_51_801
PubMedID: 26890780

Title : Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma - Peng_2016_Chem.Biol.Interact_243_82
Author(s) : Peng H , Brimijoin S , Hrabovska A , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 243 :82 , 2016
Abstract : Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.
ESTHER : Peng_2016_Chem.Biol.Interact_243_82
PubMedSearch : Peng_2016_Chem.Biol.Interact_243_82
PubMedID: 26585590

Title : Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: K values, binding pairs, and amino acid sequences - Peng_2015_Chem.Biol.Interact_240_336
Author(s) : Peng H , Brimijoin S , Hrabovska A , Targosova K , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 240 :336 , 2015
Abstract : Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 degrees C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10-9 M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.
ESTHER : Peng_2015_Chem.Biol.Interact_240_336
PubMedSearch : Peng_2015_Chem.Biol.Interact_240_336
PubMedID: 26343001

Title : [Methods for determination of cholinesterase activity] - Dingova_2015_Cesk.Fysiol_64_79
Author(s) : Dingova D , Hrabovska A
Ref : Cesk Fysiol , 64 :79 , 2015
Abstract : Cholinesterases hydrolyze acetylcholine and thus they play a key role in a process of cholinergic neurotransmission. Changes in their activities are linked to many diseases (e.g Alzheimer disease, Parkinson disease, lipid disorders). Thus, it is important to determine their activity in a fast, simply and precise way. In this review, different approaches of studying cholinesterase activities (e.g pH-dependent, spectrophotometric, radiometric, histochemical methods or biosensors) are discussed. Comparisons, advantages or disadvantages of selected methods (e.g most widely used Ellman's assay, extremely sensitive Johnson Russell method or modern technique with golden nanoparticles) are presented. This review enables one to choose a suitable method for determination of cholinesterase activities with respect to laboratory equipment, type of analysis, pH, temperature scale or special conditions.
ESTHER : Dingova_2015_Cesk.Fysiol_64_79
PubMedSearch : Dingova_2015_Cesk.Fysiol_64_79
PubMedID: 26852525

Title : [Cholinergic system of the heart] - Kucera_2015_Ceska.Slov.Farm_64_254
Author(s) : Kucera M , Hrabovska A
Ref : Ceska a Slovenska Farmacie , 64 :254 , 2015
Abstract : The cholinergic system of the heart can be either of neuronal or non-neuronal origin. The neuronal cholinergic system in the heart is represented by preganglionic parasympathetic pathways, intracardiac parasympathetic ganglia and postganglionic parasympathetic neurons projecting to the atria, SA node and AV node. The non-neuronal cholinergic system consists of cardiomyocytes that have complete equipment for synthesis and secretion of acetylcholine. Current knowledge suggests that the non-neuronal cholinergic system in the heart affects the regulation of the heart during sympathetic activation. The non-neuronal cholinergic system of the heart plays also a role in the energy metabolism of cardimyocites. Acetylcholine of both neuronal and non-neuronal origin acts in the heart through muscarinic and nicotinic receptors. The effect of acetylcholine in the heart is terminated by cholinesterases acetylcholinesterase and butyrylcholinesterase. Recently, papers suggest that the increased cholinergic tone in the heart by cholinesterase inhibitors has a positive effect on some cardiovascular disorders such as heart failure. For this reason, the cholinesterase inhibitors might be used in the treatment of certain cardiovascular disorders in the future. KEY WORDS: cholinergic system heart innervation non-neuronal cholinergic system of the heart receptors cholinesterases in the heart.
ESTHER : Kucera_2015_Ceska.Slov.Farm_64_254
PubMedSearch : Kucera_2015_Ceska.Slov.Farm_64_254
PubMedID: 26841700

Title : Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman's assay - Dingova_2014_Anal.Biochem_462C_67
Author(s) : Dingova D , Leroy J , Check A , Garaj V , Krejci E , Hrabovska A
Ref : Analytical Biochemistry , 462C :67 , 2014
Abstract : Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09M Hepes with 0.05M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.
ESTHER : Dingova_2014_Anal.Biochem_462C_67
PubMedSearch : Dingova_2014_Anal.Biochem_462C_67
PubMedID: 24929086

Title : Reassessment of the role of the central cholinergic system - Hrabovska_2014_J.Mol.Neurosci_53_352
Author(s) : Hrabovska A , Krejci E
Ref : Journal of Molecular Neuroscience , 53 :352 , 2014
Abstract : The central cholinergic system is believed to be involved in the control of many physiological functions and is an important pharmacological target for numerous neurological pathologies. Here, we summarize our recent observations regarding this topic that we obtained by studying genetically modified mice devoid of particular cholinesterase molecular forms. Our results, collected from mice with deficits of functional cholinesterases in the brain, suggest that the increase in the level of acetylcholine (ACh) has an impact on cognition only in the situation when extracellular ACh is low. Furthermore, we confirmed the central control of movement coordination, which could be of importance for the management of motor problems in patients with Parkinson's disease. At last, we provide clear evidence that while the hypothermic effect of the muscarinic agonist oxotremorine is based on a central mechanism, in contrast, the acetylcholinesterase inhibitor donepezil decreases body temperature by its action in the periphery.
ESTHER : Hrabovska_2014_J.Mol.Neurosci_53_352
PubMedSearch : Hrabovska_2014_J.Mol.Neurosci_53_352
PubMedID: 24214089

Title : Developmental adaptation of central nervous system to extremely high acetylcholine levels - Farar_2013_PLoS.One_8_e68265
Author(s) : Farar V , Hrabovska A , Krejci E , Myslivecek J
Ref : PLoS ONE , 8 :e68265 , 2013
Abstract : Acetylcholinesterase (AChE) is a key enzyme in termination of fast cholinergic transmission. In brain, acetylcholine (ACh) is produced by cholinergic neurons and released in extracellular space where it is cleaved by AChE anchored by protein PRiMA. Recently, we showed that the lack of AChE in brain of PRiMA knock-out (KO) mouse increased ACh levels 200-300 times. The PRiMA KO mice adapt nearly completely by the reduction of muscarinic receptor (MR) density. Here we investigated changes in MR density, AChE, butyrylcholinesterase (BChE) activity in brain in order to determine developmental period responsible for such adaptation. Brains were studied at embryonal day 18.5 and postnatal days (pd) 0, 9, 30, 120, and 425. We found that the AChE activity in PRiMA KO mice remained very low at all studied ages while in wild type (WT) mice it gradually increased till pd120. BChE activity in WT mice gradually decreased until pd9 and then increased by pd120, it continually decreased in KO mice till pd30 and remained unchanged thereafter. MR number increased in WT mice till pd120 and then became stable. Similarly, MR increased in PRiMA KO mice till pd30 and then remained stable, but the maximal level reached is approximately 50% of WT mice. Therefore, we provide the evidence that adaptive changes in MR happen up to pd30. This is new phenomenon that could contribute to the explanation of survival and nearly unchanged phenotype of PRiMA KO mice.
ESTHER : Farar_2013_PLoS.One_8_e68265
PubMedSearch : Farar_2013_PLoS.One_8_e68265
PubMedID: 23861875

Title : Monoclonal antibodies to mouse butyrylcholinesterase - Mrvova_2013_Chem.Biol.Interact_203_348
Author(s) : Mrvova K , Obzerova L , Girard E , Krejci E , Hrabovska A
Ref : Chemico-Biological Interactions , 203 :348 , 2013
Abstract : Our immunization strategy introduced recombinant mouse butyrylcholinesterase (BChE) to naive BChE knockout mice. An extraordinarily strong immune reaction gave rise to a whole spectrum of antibodies with different properties. Two selective and highly efficient monoclonal anti-mouse BChE antibodies 4H1 (IgG1) and 4C9 (IgG2a), with Kd values in the nanomolar range were generated. ELISA detected BChE in as little as 20-50nl of mouse plasma using 2mug (4H1) or 4mug (4C9). Both antibodies cross-reacted with BChE in dog plasma but only 4H1 reacted with rat BChE, suggesting that the antibodies are targeted towards different epitopes. Surprisingly, neither recognized human BChE. The anti-mouse BChE antibodies were used in immunohistochemistry analysis of mouse muscle where they specifically stained the neuromuscular junction. The antibodies enable visualization of the BChE protein in the mouse tissue, thus complementing activity assays. They can be used to study a long-lasting question about the existence of mixed acetylcholinesterase/BChE oligomers in mouse tissues. Moreover, monoclonal anti-mouse BChE antibodies can provide a simple, fast and efficient way to purify mouse BChE from small amounts of starting material by using a single-step immunomagnetic bead-based protocol.
ESTHER : Mrvova_2013_Chem.Biol.Interact_203_348
PubMedSearch : Mrvova_2013_Chem.Biol.Interact_203_348
PubMedID: 23099085

Title : Near-complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase - Farar_2012_J.Neurochem_122_1065
Author(s) : Farar V , Mohr F , Legrand M , Lamotte d'Incamps B , Cendelin J , Leroy J , Abitbol M , Bernard V , Baud F , Fournet V , Houze P , Klein J , Plaud B , Tuma J , Zimmermann M , Ascher P , Hrabovska A , Myslivecek J , Krejci E
Ref : Journal of Neurochemistry , 122 :1065 , 2012
Abstract : Acetylcholinesterase AChE rapidly hydrolyzes acetylcholine. At the neuromuscular junction AChE is mainly anchored in the extracellular matrix by the collagen Q whereas in the brain AChE is tethered by the proline-rich membrane anchor PRiMA The AChE-deficient mice in which AChE has been deleted from all tissues have severe handicaps Surprisingly PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors are also observed in the brain of PRiMA KO. However the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors and that its absence in AChE deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain the adaptation to the absence of AChE is nearly complete.
ESTHER : Farar_2012_J.Neurochem_122_1065
PubMedSearch : Farar_2012_J.Neurochem_122_1065
PubMedID: 22747514

Title : Distinct localization of collagen Q and PRiMA forms of acetylcholinesterase at the neuromuscular junction - Bernard_2011_Mol.Cell.Neurosci_46_272
Author(s) : Bernard V , Girard E , Hrabovska A , Camp S , Taylor P , Plaud B , Krejci E
Ref : Molecular & Cellular Neurosciences , 46 :272 , 2011
Abstract : Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic postsynaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the postsynaptic surface, but is instead clustered along the presynaptic membrane and deep in the postsynaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extrajunctional regions.
ESTHER : Bernard_2011_Mol.Cell.Neurosci_46_272
PubMedSearch : Bernard_2011_Mol.Cell.Neurosci_46_272
PubMedID: 20883790

Title : Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse - Hrabovska_2010_Chem.Biol.Interact_183_194
Author(s) : Hrabovska A , Farar V , Bernard V , Duysen EG , Brabec J , Lockridge O , Myslivecek J
Ref : Chemico-Biological Interactions , 183 :194 , 2010
Abstract : BACKGROUND: The acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels. HYPOTHESIS: The hypothesis was tested that the dopaminergic neuronal system had also adapted. METHODS: Radioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity. RESULTS: Dopamine receptor levels were decreased 28-fold for the D(1)-like, and more than 37-fold for the D(2)-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity. CONCLUSION: Survival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.
ESTHER : Hrabovska_2010_Chem.Biol.Interact_183_194
PubMedSearch : Hrabovska_2010_Chem.Biol.Interact_183_194
PubMedID: 19818744

Title : A novel system for the efficient generation of antibodies following immunization of unique knockout mouse strains - Hrabovska_2010_PLoS.One_5_e12892
Author(s) : Hrabovska A , Bernard V , Krejci E
Ref : PLoS ONE , 5 :e12892 , 2010
Abstract : BACKGROUND: We wished to develop alternate production strategies to generate antibodies against traditionally problematic antigens. As a model we chose butyrylcholinesterase (BChE), involved in termination of cholinergic signaling, and widely considered as a poor immunogen. METHODOLOGY/PRINCIPAL FINDINGS: Jettisoning traditional laborious in silico searching methods to define putative epitopes, we simply immunized available BChE knock-out mice with full-length recombinant BChE protein (having been produced for crystallographic analysis). Immunization with BChE, in practically any form (recombinant human or mouse BChE, BChE purified from human serum, native or denatured), resulted in strong immune responses. Native BChE produced antibodies that favored ELISA and immunostaining detection. Denatured and reduced BChE were more selective for antibodies specific in Western blots. Two especially sensitive monoclonal antibodies were found capable of detecting 0.25 ng of BChE within one min by ELISA. One is specific for human BChE; the other cross-reacts with mouse and rat BChE. Immunization of wild-type mice served as negative controls. CONCLUSIONS/SIGNIFICANCE: We examined a simple, fast, and highly efficient strategy to produce antibodies by mining two expanding databases: namely those of knock-out mice and 3D crystallographic protein-structure analysis. We conclude that the immunization of knock-out mice should be a strategy of choice for antibody production.
ESTHER : Hrabovska_2010_PLoS.One_5_e12892
PubMedSearch : Hrabovska_2010_PLoS.One_5_e12892
PubMedID: 20886120

Title : Rat butyrylcholinesterase-catalysed hydrolysis of N-alkyl homologues of benzoylcholine - Hrabovska_2006_FEBS.J_273_1185
Author(s) : Hrabovska A , Debouzy JC , Froment MT , Devinsky F , Paulikova I , Masson P
Ref : Febs J , 273 :1185 , 2006
Abstract : The purpose of this work was to study the catalytic properties of rat butyrylcholinesterase with benzoylcholine (BzCh) and N-alkyl derivatives of BzCh (BCHn) as substrates. Complex hysteretic behaviour was observed in the approach to steady-state kinetics for each ester. Hysteresis consisted of a long lag phase with damped oscillation. The presence of a long lag phase, with no oscillations, in substrate hydrolysis by rat butyrylcholinesterase was also observed with N-methylindoxyl acetate as substrate. Hysteretic behaviour was explained by the existence of two interconvertible butyrylcholinesterase forms in slow equilibrium, while just one of them is catalytically active. The damped oscillations were explained by the existence of different substrate conformational states and/or aggregates (micelles) in slow equilibrium. Different substrate conformational states were confirmed by 1H-NMR. The K(m) values for substrates decreased as the length of the alkyl chain increased. High affinity of the enzyme for the longest alkyl chain length substrates was explained by multiple hydrophobic interactions of the alkyl chain with amino acid residues lining the active site gorge. Molecular modelling studies supported this interpretation; docking energy decreased as the length of the alkyl chain increased. The long-chain substrates had reduced k(cat) values. Docking studies showed that long-chain substrates were not optimally oriented in the active site for catalysis, thus explaining the slow rate of hydrolysis. The hydrolytic rate of BCH12 and longer alkyl chain esters vs. substrate concentration showed a premature plateau far below V(max). This was due to the loss of substrate availability. The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh - BCH4.
ESTHER : Hrabovska_2006_FEBS.J_273_1185
PubMedSearch : Hrabovska_2006_FEBS.J_273_1185
PubMedID: 16519684

Title : Delivery of human acetylcholinesterase by adeno-associated virus to the acetylcholinesterase knockout mouse - Hrabovska_2005_Chem.Biol.Interact_157-158_71
Author(s) : Hrabovska A , Duysen EG , Sanders JD , Murrin LC , Lockridge O
Ref : Chemico-Biological Interactions , 157-158 :71 , 2005
Abstract : The purpose of this work was to develop a gene delivery system that expressed acetylcholinesterase (AChE) for prolonged periods. An adeno-associated virus (AAV) expressing human AChE was constructed by co-transfecting three plasmids into HEK 293T cells. The purified vector expressed 0.17 microg AChE per 1 million viral particles in culture medium in 23 h, or 0.8 U/ml. The AAV/hAChE was injected into muscle of adult AChE knockout mice and into the brains of 3-6 week old AChE knockout mice. Intramuscular injection yielded plasma AChE levels approaching 50% of the AChE activity of wild-type mouse plasma. The highest AChE activity was found on day 3 post-injection. AChE activity declined thereafter to a constant 7% of normal. The decreased level was accompanied by the appearance of anti-human AChE antibodies, suggesting partial clearance of AChE from plasma by antibodies. Intrastriatal injection resulted in AChE expression in the striatum. No antibodies were detected in animals treated intrastriatally. Motor coordination was improved and the lifespan of intrastriatally-treated AChE knockout mice was prolonged. Human AChE was expressed in mouse brain for up to 7 months after intrastriatal injection of an AAV/hAChE construct. Gene-therapy to supply AChE to the striatum improved motor coordination and prolonged the life of mice genetically deficient in AChE, probably by reducing their susceptibility to spontaneous seizures. This supports the hypothesis that their seizures are induced by excess acetylcholine.
ESTHER : Hrabovska_2005_Chem.Biol.Interact_157-158_71
PubMedSearch : Hrabovska_2005_Chem.Biol.Interact_157-158_71
PubMedID: 16243306

Title : Hysteresis of butyrylcholinesterase in the approach to steady-state kinetics - Masson_2005_Chem.Biol.Interact_157-158_143
Author(s) : Masson P , Schopfer LM , Froment MT , Debouzy JC , Nachon F , Gillon E , Lockridge O , Hrabovska A , Goldstein BN
Ref : Chemico-Biological Interactions , 157-158 :143 , 2005
Abstract : Butyrylcholinesterase (BChE) displays hysteretic behavior with certain neutral and charged substrates in the approach to steady state. Previous studies led us to interpret this phenomenon in terms of slow transitions between two enzyme conformers E and E'. This kinetic peculiarity is observed in human, horse and rat BChE. Oscillations that superimpose on the hysteretic lag are observed when benzoylcholine and N-alkyl derivatives of benzoylcholine are used as substrate. Hysteresis of BChE can be modulated by medium parameters (pH, salts, temperature, and pressure). Though mutant enzymes show different hysteretic behavior, so far attempts to provide a molecular mechanism of BChE hysteresis from mutagenesis studies have been unproductive. However, the substrate dependence of the hysteretic induction times, using wild-type BChE and several mutants, allowed us to build a general, mechanistic model for the hysteresis. In this model, substrate can bind to E, E', or both conformers, and ES and/or E'S can be catalytically active. The exact pathway followed depends on both the nature of the substrate and the structure of the BChE mutant under study. We propose that oscillations develop when substrate exists in different, slowly interconvertible, conformational and/or aggregation forms, of which only the minor form is capable of reacting with BChE. In support of this proposal, NMR studies have provided direct evidence for slow equilibria between monomeric and micellar forms of long-chain, alkyl derivatives of benzoyl-(N-substituted) choline. There is no direct evidence that hysteresis plays a role in BChE function(s). However, the "new view" of protein dynamics proposes that proteins are normally in equilibrium between pre-existing, functional and non-functional conformers; and that binding a ligand to the functional form shifts that equilibrium towards the functional conformation. Therefore, a physiological or toxicological relevance for the hysteresis in BChE cannot be ruled out.
ESTHER : Masson_2005_Chem.Biol.Interact_157-158_143
PubMedSearch : Masson_2005_Chem.Biol.Interact_157-158_143
PubMedID: 16256969

Title : Poster (82) Acetylcholinesterase wild-type and knock-out mice show different locomotor activity after scopolamine injection -
Author(s) : Hrabovska A , Lockridge O
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :364 , 2004

Title : Acetylcholinesterase knockout mice have increased sensitivity to scopolamine and atropine. -
Author(s) : Hrabovska A , Duysen EG , Lockridge O
Ref : Cholinergic Mechanisms, CRC Press :593 , 2004

Title : Acetylcholinesterase wild-type and knockout mice show different locomotor activity after scopolamine injection -
Author(s) : Hrabovska A , Lockridge O
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :315 , 2004

Title : Poster (55) Life without acetylcholinesterase -
Author(s) : Lockridge O , Duysen EG , Li B , Hrabovska A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :350 , 2004

Title : Altered striatal function and muscarinic cholinergic receptors in acetylcholinesterase knockout mice - Volpicelli-Daley_2003_Mol.Pharmacol_64_1309
Author(s) : Volpicelli-Daley LA , Hrabovska A , Duysen EG , Ferguson SM , Blakely RD , Lockridge O , Levey AI
Ref : Molecular Pharmacology , 64 :1309 , 2003
Abstract : Cholinesterase inhibitors are commonly used to improve cognition and treat psychosis and other behavioral symptoms in Alzheimer's disease, Parkinson's disease, and other neuropsychiatric conditions. However, mechanisms may exist that down-regulate the synaptic response to altered cholinergic transmission, thus limiting the efficacy of cholinomimetics in treating disease. Acetylcholinesterase knockout (AChE-/-) mice were used to investigate the neuronal adaptations to diminished synaptic acetylcholine (ACh) metabolism. The striatum of AChE-/- mice showed no changes in choline acetyltransferase activity or levels of the vesicular ACh transporter but showed striking 60% increases in the levels of the highaffinity choline transporter. This transporter takes choline from the synapse into the neuron for resynthesis of ACh. In addition, the striata of AChE-/- mice showed dramatic reductions in levels of the M1, M2, and M4 muscarinic ACh receptors (mAChRs), but no alterations in dopamine receptors or the beta2 subunit of nicotinic receptors. M1, M2, and M4 also showed decreased dendritic and cell surface distributions and enhanced intracellular localizations in striatal neurons of AChE-/- mice. mAChR antagonist treatment reversed the shifts in mAChR distribution, indicating that internalized receptors in AChE-/- mice can recover to basal distributions. Finally, AChE-/- mice showed increased sensitivity to mAChR antagonist-induced increases in locomotor activity, demonstrating functional mAChR down-regulation. mAChR downregulation in AChE-/- mice has important implications for the long-term use of cholinesterase inhibitors and other cholinomimetics in treating disorders characterized by perturbed cholinergic function.
ESTHER : Volpicelli-Daley_2003_Mol.Pharmacol_64_1309
PubMedSearch : Volpicelli-Daley_2003_Mol.Pharmacol_64_1309
PubMedID: 14645660