Cholinesterase Gene Research Articles

Hermona Soreq: Revolutionizing neuroscience by elucidating the roles of poly(A) tails, mRNA stability, and acetylcholine in brain-body communication throughout the lifespan

Hermona Soreq, PhD, holds the Endowed Slesinger Professorship of Molecular Neuroscience at the Hebrew University of Jerusalem. She is an internationally recognized molecular neuroscientist known for her research into the cholinergic system and the small RNA regulators driving the parasympathetic system in men and women under daily and acute stress responses and neurodegenerative diseases like Alzheimer's and Parkinson's diseases (AD, PD). Her studies have long been focused on the roles of acetylcholine in the mammalian nervous system. In the 1980s, Professor Soreq and coworkers cloned the human cholinesterase genes, identified several disease-related mutations and single nucleotide polymorphisms (SNPs) that may impair their functions, and described the unusual features these mutations conferred on carriers under acute stress, exposure to anticholinesterase poisons and diverse disease conditions, including but not limited to myasthenia gravis, ischemic stroke, schizophrenia, bipolar disorder, AD and PD as well as daily stress responses. Soreq further identified microRNA-132 as a principal controller of the cholinergic pathway and studied its impact as well as of other cholinergic-targeted microRNAs as regulators of parasympathetic brain and body functions and neuroinflammation. More recently, she has shifted her interest to the re-discovered transfer RNA fragments (tRFs), and showed that their rapidly declined control over cholinergic transcripts may lead to the fast cognitive deterioration of women living with AD; that they are prominently altered in PD patients’ biofluids and that their levels are sex-relatedly modified in the blood of newborn babies, dependent on pre-delivery stress. Her multi-leveled interests in the stress and sex-related cholinergic aspects of AD, PD, adult, and pre-delivery trauma further reflect the impact on acute stress responses as the kernel of the neuroscience research in the current Israeli landscape and has further enabled her a wide-angle view of diverse cholinergic-regulated states and diseases. We are delighted that Professor Soreq answered the Genomic Press Interview, generously sharing her life's trajectory with our readers.

Effect of curcumin-donepezil combination on spatial memory, astrocyte activation, and cholinesterase expressions in brain of scopolamine-treated rats.

The study investigated the effect of co-administration of curcumin and donepezil on several markers of cognitive function (such as spatial memory, astrocyte activation, cholinesterase expressions) in the brain cortex and hippocampus of scopolamine-treated rats. For seven consecutive days, a pre-treatment of curcumin (50mg/kg) and/or donepezil (2.5mg/kg) was administered. On the seventh day, scopolamine (1mg/kg) was administered to elicit cognitive impairment, 30min before memory test was conducted. This was followed by evaluating changes in spatial memory, cholinesterase, and adenosine deaminase (ADA) activities, as well as nitric oxide (NO) level were determined. Additionally, RT-qPCR for glial fibrillary acidic protein (GFAP) and cholinesterase gene expressions was performed in the brain cortex and hippocampus. Also, GFAP immunohistochemistry of the brain tissues for neuronal injury were performed in the brain cortex and hippocampus. In comparison to the control group, rats given scopolamine had impaired memory, higher levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and ADA activities, as well as elevated markers of oxidative stress. In addition to enhanced GFAP immunoreactivity, there was also overexpression of the GFAP and BChE genes in the brain tissues. The combination of curcumin and donepezil was, however, observed tobetter ameliorate these impairments in comparison to thedonepezil-administered rat group. Hence, this evidence provides more mechanisms to support the hypothesis that the concurrent administration of curcumin and donepezil mitigates markers of cognitive dysfunction in scopolamine-treated rat model.

Occurrence of butyrylcholinesterase polymorphisms in patients undergoing surgery in Slovakia.

Post-operative residual curarization is a persisting problem, characterized by muscle fatigue, exhaustion or paresis, caused by the use of neuromuscular blocking agents with prolonged postoperative effect. Genetically, determined changes in cholinesterase activity can be a major reason for persistent muscle blockade after administration of muscle relaxants. Regarding the subsistence of polymorphisms in the plasma cholinesterase gene causing change in enzyme activity and metabolism of applied drugs, we investigated the frequency of two polymorphisms known to reduce its activity significantly in patients undergoing surgery. Primary results show a relatively high occurrence of plasma cholinesterase K risk allele (18.75%). Characterization of the lacking information about genetic background of changes in plasma cholinesterase activity within Slovakia may allow for easier decision-making in clinical practice when selecting alternative neuromuscular blocking and also reversal agents.

Preface: Cholinergic Mechanisms

This special issue is a companion to the meeting 'XVth International Symposium on Cholinergic Mechanisms', and is edited by Israel Silman, Marco Prado and Pascale Marchot. In the review articles, renowned researchers in the field capture key mechanisms of cholinergic neurotransmission, from genomic amplification of cholinesterase genes, splicing and post-translational modifications; features of the neuromuscular junction, implications of cholinergic circuitry that are relevant to addiction, anxiety and mood, to preclinical models, protein biomarkers, and clinical findings that are relevant to pathology, for example, developmental neurotoxicity. The broad variety of features reflects the impact of cholinergic mechanisms on many physiological events and emphasizes the importance of research in this area. This is the Preface for thespecial issue XVth International Symposium on Cholinergic Mechanisms.

Natural genomic amplification of cholinesterase genes in animals.

Tight control of the concentration of acetylcholine at cholinergic synapses requires precise regulation of the number and state of the acetylcholine receptors, and of the synthesis and degradation of the neurotransmitter. In particular, the cholinesterase activity has to be controlled exquisitely. In the genome of the first experimental models used (man, mouse, zebrafish and drosophila), there are only one or two genes coding for cholinesterases, whereas there are more genes for their closest relatives the carboxylesterases. Natural amplification of cholinesterase genes was first found to occur in some cancer cells and in insect species subjected to evolutionary pressure by insecticides. Analysis of the complete genome sequences of numerous representatives of the various metazoan phyla show that moderate amplification of cholinesterase genes is not uncommon in molluscs, echinoderms, hemichordates, prochordates or lepidosauria. Amplification of acetylcholinesterase genes is also a feature of parasitic nematodes or ticks. In these parasites, over-production of cholinesterase-like proteins in secreted products and the saliva are presumed to have effector roles related to host infection. These amplification events raise questions about the role of the amplified gene products, and the adaptation processes necessary to preserve efficient cholinergic transmission. This is an article for thespecial issue XVth International Symposium on Cholinergic Mechanisms.

A farmacogenética e a medicina personalizada

Pharmacogenetics aims to identify genetic differences between individuals that may influence the response to drug therapy, improving their effectiveness and safety. Associated with the pharmacogenetics emerges ‘personalized medicine’. In opposition to the existence of a drug that can treat all patients, the individualized treatment seems the most promising as it reduces the risk of side effects for toxicity (safety), reduces losses due to excess or deficit (dose), avoiding the testing methodology in the choice of the correct drug (effectiveness). Pharmacogenetics is relevant to the individual response to the drug in two ways: the pharmacokinetics and pharmacodynamics. The genetic variability can affect the way a drug can be absorbed, metabolized, excreted or activated, and can drive to a difference in the patient response. Among the endless number of possible examples, in this review we present examples related to cytochrome P450 genes, NAT2 gene and the Cholinesterase gene. Genetic differences between individuals can still affect the response to the drug by its pharmacodynamics, drug target-specific response to a particular drug. Among the multitude of existing drug targets, it will be presented examples of the G6PD gene, and the VKORC1 gene. Despite some evidence given for the benefit of the patient, we are still far from Pharmacogenetics to be part of routine clinical practice, perhaps because the cost-benefit have not yet been correctly assessed.

The Progress in Cloning, Expression and Purification of Cholinesterase in <i>Pichia pastoris</i>: One Kind of Biomaterial for the Detection of Residual Insecticide Contamination

Cholinesterase serves as a key enzyme of the cholinergic system, which could be one kind of promising biomaterial to assay OP and CB residues. However, cholinesterase purified from tissues or blood is both time and cost intensive. In this essay, different methods via biotechnology to get this biomaterial in Pichia pastoris is summarized, including cholinesterase gene cloning and expression vector construction, construction of genetic modified yeast, screening high yield transformant, purification from supernatant and activity analysis.

Cholinesterases as Biomarkers for Parasympathetic Dysfunction and Inflammation-Related Disease

Accumulating evidence suggests parasympathetic dysfunction and elevated inflammation as underlying processes in multiple peripheral and neurological diseases. Acetylcholine, the main parasympathetic neurotransmitter and inflammation regulator, is hydrolyzed by the two closely homologous enzymes, acetylcholinesterase and butyrylcholinesterase (AChE and BChE, respectively), which are also expressed in the serum. Here, we consider the potential value of both enzymes as possible biomarkers in diseases associated with parasympathetic malfunctioning. We cover the modulations of cholinesterase activities in inflammation-related events as well as by cholinesterase-targeted microRNAs. We further discuss epigenetic control over cholinesterase gene expression and the impact of single-nucleotide polymorphisms on the corresponding physiological and pathological processes. In particular, we focus on measurements of circulation cholinesterases as a readily quantifiable readout for changes in the sympathetic/parasympathetic balance and the implications of changes in this readout in health and disease. Taken together, this cumulative know-how calls for expanding the use of cholinesterase activity measurements for both basic research and as a clinical assessment tool.

Identification and characterization of three cholinesterases from the common bed bug, Cimex lectularius

We identified and characterized the full-length cDNA sequences encoding two acetylcholinesterases (ClAChE1 and ClAChE2) and a salivary gland-specific cholinesterase-like protein (ClSChE) from the common bed bug, Cimex lectularius. All three cholinesterase genes (Clac1, Clace2 and Clsce) have conserved motifs, including a catalytic triad, a choline-binding site and an acyl pocket. Phylogenetic analysis showed that ClAChE1 belongs to the insect AChE1 clade, whereas ClAChE2 belongs to the insect AChE2 clade. ClSChE was grouped into the clade containing all AChE1s, suggesting a paralogous relationship to ClAChE1. Transcription levels of Clace1 were higher than those of Clace2 in all tissues examined, including the central nervous system (CNS). In contrast, the Clsce transcript was not detected in the CNS but specifically found in the salivary gland at much higher levels (>3000-fold) than those of Clace1 and Clace2. Western blot analysis using anti-ClAChE antibodies, in conjunction with activity staining, revealed that ClAChE1 is more active than ClAChE2, whereas ClSChE has little enzyme activity. Three-dimensional structure modelling suggested that ClAChEs and ClSChE shared structural similarities, but had some differences in the residues forming the acyl pocket and oxyanion hole. The current findings should provide valuable insights into the evolution and functional diversification of insect cholinesterase.

Naturally Occurring Variations in the Human Cholinesterase Genes: Heritability and Association with Cardiovascular and Metabolic Traits

Cholinergic neurotransmission in the central and autonomic nervous systems regulates immediate variations in and longer-term maintenance of cardiovascular function with acetylcholinesterase (AChE) activity that is critical to temporal responsiveness. Butyrylcholinesterase (BChE), largely confined to the liver and plasma, subserves metabolic functions. AChE and BChE are found in hematopoietic cells and plasma, enabling one to correlate enzyme levels in whole blood with hereditary traits in twins. Using both twin and unrelated subjects, we found certain single nucleotide polymorphisms (SNPs) in the ACHE gene correlated with catalytic properties and general cardiovascular functions. SNP discovery from ACHE resequencing identified 19 SNPs: 7 coding SNPs (cSNPs), of which 4 are nonsynonymous, and 12 SNPs in untranslated regions, of which 3 are in a conserved sequence of an upstream intron. Both AChE and BChE activity traits in blood were heritable: AChE at 48.8 ± 6.1% and BChE at 81.4 ± 2.8%. Allelic and haplotype variations in the ACHE and BCHE genes were associated with changes in blood AChE and BChE activities. AChE activity was associated with BP status and SBP, whereas BChE activity was associated with features of the metabolic syndrome (especially body weight and BMI). Gene products from cDNAs with nonsynonymous cSNPs were expressed and purified. Protein expression of ACHE nonsynonymous variant D134H (SNP6) is impaired: this variant shows compromised stability and altered rates of organophosphate inhibition and oxime-assisted reactivation. A substantial fraction of the D134H instability could be reversed in the D134H/R136Q mutant. Hence, common genetic variations at ACHE and BCHE loci were associated with changes in corresponding enzymatic activities in blood.

From Split to Sibenik: The tortuous pathway in the cholinesterase field

The interim between the first and tenth International Cholinesterase Meetings has seen remarkable advances associated with the applications of structural biology and recombinant DNA methodology to our field. The cloning of the cholinesterase genes led to the identification of a new super family of proteins, termed the α,β-hydrolase fold; members of this family possess a four helix bundle capable of linking structural subunits to the functioning globular protein. Sequence comparisons and three-dimensional structural studies revealed unexpected cousins possessing this fold that, in turn, revealed three distinct functions for the α,β-hydrolase proteins. These encompass: (1) a capacity for hydrolytic cleavage of a great variety of substrates, (2) a heterophilic adhesion function that results in trans-synaptic associations in linked neurons, (3) a chaperone function leading to stabilization of nascent protein and its trafficking to an extracellular or secretory storage location. The analysis and modification of structure may go beyond understanding mechanism, since it may be possible to convert the cholinesterases to efficient detoxifying agents of organophosphatases assisted by added oximes. Also, the study of the relationship between the α,β-hydrolase fold proteins and their biosynthesis may yield means by which aberrant trafficking may be corrected, enhancing expression of mutant proteins. Those engaged in cholinesterase research should take great pride in our accomplishments punctuated by the series of ten meetings. The momentum established and initial studies with related proteins all hold great promise for the future.

Amplification and deletion of the ACHE and BCHE cholinesterase genes in sporadic breast cancer

Increasing evidence supports the involvement of acetylcholinesterase and butyrylcholinesterase in cell proliferation control and differentiation, reinforcing the hypothesis that these enzymes might have an influence in tumorigenesis. It has already been shown that the cholinesterase genes are structurally altered or aberrantly expressed in a variety of tumor types. In this study, amplifications and deletions in the ACHE and BCHE genes were investigated in sporadic breast tumors using real-time polymerase chain reaction and the relative quantification method. The majority of the tumor tissues showed a notable number of both deletions and amplifications: 65.7% and 22.9%, respectively, in BCHE and 45.7% and 31.4%, respectively, in ACHE. Deletion of the ACHE gene was significantly correlated with amplification of the protooncogene ERBB2. Tumor size was significantly higher when the ACHE gene was amplified, and the total number of alterations (amplifications plus deletions) of the BCHE gene was positively correlated with tumor malignancy grade.

Contributions of selective knockout studies to understanding cholinesterase disposition and function

The complete knockout of the acetylcholinesterase gene ( AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.

The cholinesterases: Analysis by pharmacogenomics in man

We have undertaken a study on variations in cholinesterase (ChE) genes in relation to cardiovascular (CV) function and the metabolic syndrome. Peripheral and central nervous system control of cardiovascular (CV) function mediated through cholinergic pathways is critical in homeostatic maintenance of blood pressure and responsiveness to stress. For acetylcholinesterase (AChE; EC 3.1.1.7) our focus is to identify single nucleotide polymorphisms (SNPs) in the gene that are linked to cardiovascular function. For butyrylcholinesterase (BChE; EC 3.1.1.8) we examined whether BChE activity correlated with parameters of the metabolic syndrome and cardiovascular function. ChE can be found in whole blood enabling a characterization of biochemical phenotype in addition to correlating genotype with phenotypic physiologic responses. Analysis of enzymatic activity was determined spectrophotometrically in blood samples from twin and other subject registries. Correlation analysis revealed significant relationships between enzyme activity and certain CV endpoints. Linkage analysis with data from a dizygotic (DZ) twin set showed a suggestive linkage at the BChE locus, and statistical analysis revealed a high correlation between BChE activity and variables associated with cardiovascular risk and the metabolic syndrome. Pattern of within-pair twin correlations by zygosity and the ACE model-fitting findings suggest the major source of this variation (65%) is attributable to an additive genetic component. To date 19 SNPs have been identified by the re-sequencing of AChE including four nonsynonymous coding SNPs (cSNPs).

Fresh frozen plasma transfusion for reversal of prolonged post‐anaesthesia apnoea

An 18-year old woman admitted for tonsillectomy developed prolonged post-operative paralysis after anaesthesia with mivacurium. Investigation revealed a decreased cholinesterase activity because of a homozygous atypical and heterozygous K variant of the cholinesterase gene. Transfusion of fresh frozen plasma was associated with reversal of the respiratory paralysis and complete recovery.

Butyrylcholinesterase: Association with the Metabolic Syndrome and Identification of 2 Gene Loci Affecting Activity

Plasma cholinesterase activity is known to be correlated with plasma triglycerides, HDL- and LDL-cholesterol, and other features of the metabolic syndrome. A role in triglyceride metabolism has been proposed. Genetic variants that decrease activity have been studied extensively, but the factors contributing to overall variation in the population are poorly understood. We studied plasma cholinesterase activity in a sample of 2200 adult twins to assess covariation with cardiovascular risk factors and components of the metabolic syndrome, to determine the degree of genetic effects on enzyme activity, and to search for quantitative trait loci affecting activity. Cholinesterase activity was lower in women than in men before the age of 50, but increased to activity values similar to those in males after that age. There were highly significant correlations with variables associated with the metabolic syndrome: plasma triglyceride, HDL- and LDL-cholesterol, apolipoprotein B and E, urate, and insulin concentrations; gamma-glutamyltransferase and aspartate and alanine aminotransferase activities; body mass index; and blood pressure. The heritability of plasma cholinesterase activity was 65%. Linkage analysis with data from the dizygotic twin pairs showed suggestive linkage on chromosome 3 at the location of the cholinesterase (BCHE) gene and also on chromosome 5. Our results confirm and extend the connection between cholinesterase, cardiovascular risk factors, and metabolic syndrome. They establish a substantial heritability for plasma cholinesterase activity that might be attributable to variation near the structural gene and at an independent locus.

Acetyl- and Butyrylcholinesterase Activities Decrease in Human Colon Adenocarcinoma

Apart from the hydrolysis of acetylcholine (ACh), acetyl- (AChE) and butyrylcholinesterase (BChE), through noncatalytic mechanisms, intervene in hematopoiesis, morphogenesis, and neurogenesis (Layer and Willbold, 1995; Soreq and Seidman, 2001). Cholinesterase (ChE) molecules occur as globular (G1, G2, and G4) and asymmetric (A4, A8, and A12) forms (Legay, 2000; Massoulié, 2002). The G species might display amphiphilic (GA) or hydrophilic (GH) properties (Perrier et al., 2002). The involvement of ChEs in tumorigenesis is supported by the measurement of ChE activity in tumors (García-Ayllón et al., 2001; Ruiz-Espejo et al., 2003), the amplification of ChE genes in leukemias and ovarian tumors, and the relationship between the expression of AChE and the aggressiveness of astrocytomas(Perry et al., 2002). This research was undertaken to determine whether ChE activity is altered in gut carcinomas.

Analysis of mutation of the plasma cholinesterase gene in a man who had died following a traffic accident

We analyzed mutation of the butyrylcholinesterase (BCHE) gene in a 69-year-old man on whom a forensic autopsy had been performed after he had died following a traffic accident. Extremely low plasma cholinesterase activity had been pointed out by the emergency doctor at the hospital prior to his death and based on this, organophosphorus poisoning had been suspected. However, no pesticides, which could have reduced the plasma cholinesterase activity, were detected by toxicological analysis using GC/MS. Subsequently, one base insertion was found in exon 2. The frame shift mutation had occurred because a homozygous extra T had been inserted between nucleotides 1343 and 1344, resulting in the appearance of a stop codon in codon 454 (AGA454TAA, Arg454stop). This heterozygous frame shift mutation at this point was identified in the man's son. It is likely that there may be many such latent patients with abnormal plasma cholinesterase activity, and accordingly we should always bear this fact in mind and should carry out molecular genetic testing for an accurate diagnosis of this deficiency.

Frequency of butyrylcholinesterase gene mutations in individuals with abnormal inhibition numbers: an Italian-population study.

More than 30 genetic variants of serum cholinesterase (butyrylcholinesterase, BChE) have been described. Some of them (the atypical and the fluoride-resistant variants) are well known because carriers are prone to develop prolonged apnea following the administration of the muscle relaxant succinylcholine. Genotype characterization is therefore important in order to prevent such episodes. Genetic studies have so far focused on selected individuals or families rather than on the random population. From a large group of healthy blood donors (n = 2609), we selected all the 58 individuals with low serum cholinesterase activity: among them 28 subjects had abnormal dibucaine and fluoride inhibition numbers. Twenty-five mutations in the coding region of the human cholinesterase gene were analyzed. All individuals with abnormal inhibition numbers were homozygotes or double heterozygotes in several mutations. Asp70Gly (Atypical variant) and Ala539Thr (K variant) were the most frequently observed amino acid substitutions. The majority of subjects with low BChE activity but normal dibucaine and fluoride number presented only the K form. We analyzed 106 randomly chosen subjects for K and atypical variants. Carriers of these alleles were at risk of low BChE activity (OR = 9.55, 95%CI, 5.61-16.26 and OR = 30.33, 95%CI, 7.05-130.52 respectively). Data obtained from this study help to better define the etiology of low BChE activity and the role of the rather common K allele. It is the first time that such a large population has been screened for so many mutations. BChE is also implicated in detoxifying cocaine; therefore genetic analysis could be useful in cases of cocaine toxicity in Italian subjects.

Analysis of Mutations in the Plasma Cholinesterase Gene of Patients with a History of Prolonged Neuromuscular Block during Anesthesia

Decreased activity of plasma cholinesterase is responsible for prolonged apnea during anesthesia using neuromuscular blockers such as suxamethonium and mivacurium. More than 20 mutations have been identified so far in the BCHE gene resulting in impaired plasma cholinesterase activity. Biochemical tests are not always able to differentiate between pathological and normal sera; hence in some cases unanticipated complications can still occur during anesthesia even after measurements of enzyme activity and dibucaine numbers within the normal range. Therefore, molecular genetic testing is required for the accurate diagnosis of this deficiency. Here we present a study of plasma cholinesterase activity and BCHE genotyping of patients with a history of prolonged neuromuscular block and most of their pedigrees. All four exons of the BCHE gene were directly sequenced from samples and a number of mutations responsible for the reduction of plasma cholinesterase activity were identified. In most cases the atypical mutation in exon 2 (nt 209A → G, Asp70 → Gly) was found together with the K-variant mutation in exon 4 (nt 1615G → A, Ala539 → Thr), which is in good agreement with previous data suggesting that these mutations along with two others (at nt −116 and nt 1914) are in linkage disequilibrium.