Forms Of Acetylcholinesterase Research Articles

A novel genetically modified mouse seizure model for evaluating anticonvulsive and neuroprotective efficacy of an A1 adenosine receptor agonist following soman intoxication

Recently a novel humanized mouse strain has been successfully generated, in which serum carboxylesterase (CES) knock out (KO) mice (Es1−/−) were further genetically modified by knocking in (KI), or adding, the gene that encodes the human form of acetylcholinesterase (AChE). The resulting human AChE KI and serum CES KO (or KIKO) mouse strain should not only exhibit organophosphorus nerve agent (NA) intoxication in a manner more similar to humans, but also display AChE-specific treatment responses more closely mimicking those of humans to facilitate data translation to pre-clinic trials. In this study, we utilized the KIKO mouse to develop a seizure model for NA medical countermeasure investigation, and then applied it to evaluate the anticonvulsant and neuroprotectant (A/N) efficacy of a specific A1 adenosine receptor (A1AR) agonist, N-bicyclo-(2.2.1)hept-2-yl-5′-chloro-5′-deoxyadenosine (ENBA), which has been shown in a rat seizure model to be a potent A/N compound. Male mice surgically implanted with cortical electroencephalographic (EEG) electrodes a week earlier were pretreated with HI-6 and challenged with various doses (26 to 47 μg/kg, SC) of soman (GD) to determine a minimum effective dose (MED) that induced sustained status epilepticus (SSE) activity in 100% of animals while causing minimum lethality at 24 h. The GD dose selected was then used to investigate the MED doses of ENBA when given either immediately following SSE initiation (similar to wartime military first aid application) or at 15 min after ongoing SSE seizure activity (applicable to civilian chemical attack emergency triage). The selected GD dose of 33 μg/kg (1.4 x LD50) generated SSE in 100% of KIKO mice and produced only 30% mortality. ENBA at a dose as little as 10 mg/kg, IP, caused isoelectric EEG activity within minutes after administration in naïve un-exposed KIKO mice. The MED doses of ENBA to terminate GD-induced SSE activity were determined to be 10 and 15 mg/kg when treatment was given at the time of SSE onset and when seizure activity was ongoing for 15 min, respectively. These doses were much lower than in the non-genetically modified rat model, which required an ENBA dose of 60 mg/kg to terminate SSE in 100% GD-exposed rats. At MED doses, all mice survived for 24 h, and no neuropathology was observed when the SSE was stopped. The findings confirmed that ENBA is a potent A/N for both immediate and delayed (i.e., dual purposed) therapy to victims of NA exposure and serves as a promising neuroprotective antidotal and adjunctive medical countermeasure candidate for pre-clinical research and development for human application.

Anti-acetylcholinesterase activity of Corallocarpus epigaeus tuber: In vitro kinetics, in silico docking and molecular dynamics analysis

Biological activities of Corallocarpus epigaeus tuber (CET) were determined and its active constituents were identified. CET was sequentially extracted with various solvents. The methanol extract was further fractionated using silica coloumn chromatography to obtain active constituents-rich fraction called CET-MAF. The phytochemicals in CET-MAF was identified using GC-MS analysis. The in vitro antioxidant activity and inhibition against various globular forms of acetylcholinesterase (AChE) by CET-MAF were determined. In silico analysis of CET-MAF active constituents interaction with human AChE was carried out. CET-MAF showed in vitro antioxidant activity. CET-MAF inhibited various globular forms of AChE, G1 (IC50 = 147 µg/ml; Ki=13.6 µg/ml), G2 (IC50=146 µg/ml; Ki=8.6 µg/ml) and G4 (IC50 = 158 µg/ml; Ki = 9.3 µg/ml) in mixed manner. Molecular docking analysis revealed the interaction of active constituents such as 5-acetoxypentadecane (5AP) and alpha-linolenic acid (ALA) of CET-MAF with active site of AChE. Molecular dynamics simulation for 500 ns revealed the stable binding of these compounds with AChE. MMBGSA calculation revealed the binding energy of -52.95 and -55.49 kcal/mol for interaction of ALA and 5AP with AChE, respectively. Altogether, CET-MAF showed biological activities in vitro which can be used against cognitive dysfunction.

Isolation of bioactive components from Corallocarpus epigaeus tuber and inhibitory potential against various molecular forms of acetylcholinesterase

AbstractBackgroundIn human, acetylcholinesterase (AChE) is involved in cholinergic neurotransmission. AChE exhibits as different globular form such as G4 (tetramer), G2 (dimer) and G1 (monomer). For the most part of the adult brain G4 forms is present. Nevertheless, under dementia such as Alzheimer’s disease (AD) conditions, the ratio between G1 and G4 form is increased which is involved in disease progression. Inhibition of AChE is considered important therapeutic intervention to overcome the AD. Corallocarpus epigaeus comes under the family of Cucurbitacea has known for the biological properties such as anti‐snake venom, analgesic, anthelminthic properties and antidiabetic activity. Identifying natural compounds with AChE inhibition is considered important for AD treatment. In the present study, bioactive fraction extracted from tuber of C. epigaeus was investigated for its inhibition against different molecular forms of AChE in in vitro.MethodDried tuber powder of C. epigaeus was extracted using various solvents sequentially by increasing polarity in Soxhlet apparatus. Methanol extract was further purified by silica column chromatography. The partially purified fraction was studied for the inhibition against different molecular forms of AChE. Kinetic analysis was carried out to determine the type of inhibition and inhibitor constant. The active compounds in partially purified fraction were determined using gas chromatography mass spectrum analysis (GCMS).ResultPartially purified fraction of methanolic extract of C. epigaeus tuber inhibited all the molecular forms (G1, G2 and G4) of AChE at microgram per milliliter concentration. Kinetic analysis revealed the mixed type non‐competitive mode of inhibition with low Ki value. GCMS analysis 4H‐pyran‐4‐one, 2,3‐dihydro‐3,5‐dihydroxy‐6‐methyl and alpha‐linolenic acid as major constituents in the extract.ConclusionThe bioactive components in tuber of C. epigaeus inhibited AChE in in vitro. This is the first report on the inhibitory effect of C. epigaeus tuber extract against AChE. Further in vivo studies are necessary to understand the anti‐dementia effect of C. epigaeus.

Gene Expression Profile at the Motor Endplate of the Neuromuscular Junction of Fast-Twitch Muscle.

The neuromuscular junction (NMJ) is a prototypic chemical synapse between the spinal motor neuron and the motor endplate. Gene expression profiles of the motor endplate are not fully elucidated. Collagen Q (ColQ) is a collagenic tail subunit of asymmetric forms of acetylcholinesterase and is driven by two distinct promoters. pColQ1 is active throughout the slow-twitch muscle, whereas pColQ1a is active at the motor endplate of fast-twitch muscle. We made a transgenic mouse line that expresses nuclear localization signal (NLS)-attached Cre recombinase under the control of pColQ1a (pColQ1a-Cre mouse). RiboTag mouse expresses an HA-tagged ribosomal subunit, RPL22, in cells expressing Cre recombinase. We generated pColQ1a-Cre:RiboTag mouse, and confirmed that HA-tagged RPL22 was enriched at the NMJ of tibialis anterior (TA) muscle. Next, we confirmed that Chrne and Musk that are specifically expressed at the NMJ were indeed enriched in HA-immunoprecipitated (IP) RNA, whereas Sox10 and S100b, markers for Schwann cells, and Icam1, a marker for vascular endothelial cells, and Pax3, a marker for muscle satellite cells, were scarcely detected. Gene set enrichment analysis (GSEA) of RNA-seq data showed that “phosphatidylinositol signaling system” and “extracellular matrix receptor interaction” were enriched at the motor endplate. Subsequent analysis revealed that genes encoding diacylglycerol kinases, phosphatidylinositol kinases, phospholipases, integrins, and laminins were enriched at the motor endplate. We first characterized the gene expression profile under translation at the motor endplate of TA muscle using the RiboTag technique. We expect that our gene expression profiling will help elucidate molecular mechanisms of the development, maintenance, and pathology of the NMJ.

HETEROLOGOUS EXPRESSION OF TWO ACETYLCHOLINESTERASES OF COLORADO POTATO BEETLE LEPTINOTARSA DECEMLINEATA IN BACTERIA ESCHERICHIA COLI AND PRODUCTION OF FORM-SPECIFIC ANTIBODIES

The Colorado potato beetle Leptinotarsa decemlineata is a widespread pest of plants of the Solanaceae family. Huge economic loss caused by L. decemlineata around the world is multiplied by its ability to develop resistance to all major insecticide classes. Previously, such resistance was found to be associated with mutations in the target enzyme LdAChE2, orthologous to Drosophila melanogaster acetylcholinesterase. However, discovery of the second form of L. decemlineata acetylcholinesterase LdAChE1 has changed this view. In order to compare the role of two acetylcholinesterase forms in the Colorado potato beetle physiology and in pest resistance to insecticides, gene copies were cloned and their heterologous expression in bacteria E. coli was followed by production of polyclonal antibodies against the recombinant proteins. Immunoblotting with produced antibodies demonstrated the absence of cross-reactivity, a lower content of LdAChE1 in the tissues of L. decemlineata adults compared with the second form, and the association of LdAChE2 with membranes. Further immunoaffinity purification of natural enzymes from the beetle tissues as well as their heterologous expression in insect cell cultures should help to evaluate the role of each form in physiology of the pest and in its resistance to insecticides.

Biochemical adaptation in brain Acetylcholinesterase during acclimation to sub-lethal temperatures in the eurythermal fish Tilapia mossambica

Tilapia mossambica is a eurythermal tropical fish. We studied the effect of temperature on the kinetics of brain Acetylcholinesterase (AChE) during adaptation to sublethal temperatures by acclimating the fish to 37 °C, and controls to 25 °C. Electrophoresis showed the presence of two AChE bands that did not change in position or intensity with acclimation period or temperature. The apparent Km was 0.23 ± 0.01 mM ATChI and remained relatively constant over the in vitro assay temperature range 10 °C to 40 °C. Biochemical characterization suggested that the enzyme is a ‘eurytolerant protein’ in its stability of kinetic and thermal properties over a wide temperature range. Thermal stability and arrhenius plots suggested that the AChE was made up of two forms that differed in their thermal properties.The two molecular forms of acetylcholinesterase were purified from the brain of T. mossambica. Molecular weight studies revealed that the two forms were size isomers: a monomer of 59 KDa and a tetramer of 244 KDa. They differed in their Kms, thermal stabilities and energies of activation. We suggest that biochemical adaptation to temperature in the brain acetylcholinerase system of the fish Tilapia mossambica is based on the aggregation-dissociation of these size isomers.

Differentiation of erythroblast requires the dimeric form of acetylcholinesterase: Interference with erythropoietin receptor

Acetylcholinesterase (AChE) hydrolyzes acetylcholine at cholinergic synapses, and which has various isoforms of AChE, i.e. AChER, AChEH and AChET, deriving from single gene. AChEH exists as a glycophosphatidylinositol (GPI)-linked dimer (G2), presents mainly in plasma membrane of mammalian erythrocyte. Transgenic mice with ACHE gene depletion were employed here to investigate the possible role of AChE in blood cell formation. ACHE knock-out mice were found to suffer normocytic anemia. In erythrocyte of ACHE−/− mice, the amount of hemoglobin, especially α-globin, was found to be markedly reduced. In addition, the number of erythrocyte and hematocrit of ACHE−/− mice were significantly lowered. To probe the role of AChE isoforms in erythroid differentiation, erythroblast-like cells (TF-1) over-expressed with different AChE isoforms were induced to differentiate by erythropoietin (EPO): this differentiation induced the expression of each AChE isoform. Only in the TF-1 cells over-expressed with AChEH, the EPO-induced transcriptions and protein expressions of α- and β-globins could be significantly enhanced, which therefore suggested that AChEH might regulate the responsiveness of TF-1 cells to EPO. The alternation of EPO-induced downstream signaling might be accounted by association of AChE with EPO receptor in cell surface. The findings indicated the significance of AChE in erythroblast maturation, which provided an insight in elucidating possible mechanisms in regulating erythropoiesis.

Kinetics of acetylcholinesterase inhibition by hemp seed protein-derived peptides.

The aim of this work was to enhance the acetylcholinesterase (AChE)-inhibitory activity of a pepsin-produced hemp seed protein hydrolysates (HPH) through reverse-phase HPLC separation followed by identification of peptide sequences present in the most active fraction. The HPH was separated into eight fractions (F1-F8) with F7 exhibiting significantly (p<0.05) the strongest (97.5%) in vitro inhibition of electric eel AChE (eeAChE) activity in comparison to 53.8% for HPH. The HPH consisted mostly of low molecular weight peptides of<11 amino acid residues and most contained at least one hydrophobic amino acid. Kinetics of enzyme inhibition revealed a mixed-type inhibition of eeAChE activity by HPH whereas F7 acted through an uncompetitive mode; in contrast inhibition of human AChE by HPH and F7 was uncompetitive. The stronger inhibitory potency of the F7 peptides fraction against both enzymes was confirmed through reduced maximal velocity, catalytic efficiency, and inhibition constant values when compared to the HPH. PRACTICAL APPLICATIONS: The use of natural products for the prevention or treatment of human diseases continues to be an area of intense research activities. Food protein-derived peptides obtained through enzymatic hydrolysis of hemp seed proteins were shown in vitro to be strong inhibitors of activities of both the eel and human forms of acetylcholinesterase (AChE). AChE is an important therapeutic target because excessive activity of this enzyme is a causative factor of neurodegenerative diseases such as dementia and Alzheimer's. This work showed that peptides in the most active fraction are small in sizes, which may favor their absorption into blood circulation and possible permeation of the blood-brain barrier. Therefore, the hemp seed peptides are potential agents that can be used to formulate functional foods and nutraceuticals against neurodegenerative diseases.

In vivo localization of human acetylcholinesterase-derived species in a β-sheet conformation at the core of senile plaques in Alzheimer's disease

Many neurodegenerative diseases are characterized by amyloid deposition. In Alzheimer's disease (AD), β-amyloid (Aβ) peptides accumulate extracellularly in senile plaques. The AD amyloid cascade hypothesis proposes that Aβ production or reduced clearance leads to toxicity. In contrast, the cholinergic hypothesis argues for a specific pathology of brain cholinergic pathways. However, neither hypothesis in isolation explains the pattern of AD pathogenesis. Evidence suggests that a connection exists between these two scenarios: the synaptic form of human acetylcholinesterase (hAChE-S) associates with plaques in AD brains; among hAChE variants, only hAChE-S enhances Aβ fibrillization in vitro and Aβ deposition and toxicity in vivo Only hAChE-S contains an amphiphilic C-terminal domain (T40, AChE575-614), with AChE586-599 homologous to Aβ and forming amyloid fibrils, which implicates T40 in AD pathology. We previously showed that the amyloid scavenger, insulin-degrading enzyme (IDE), generates T40-derived amyloidogenic species that, as a peptide mixture, seed Aβ fibrillization. Here, we characterized 11 peptides from a T40-IDE digest for β-sheet conformation, surfactant activity, fibrillization, and seeding capability. We identified residues important for amyloidogenicity and raised polyclonal antibodies against the most amyloidogenic peptide. These new antisera, alongside other specific antibodies, labeled sections from control, hAChE-S, hAPPswe, and hAChE-S/hAPPswe transgenic mice. We observed that hAChE-S β-sheet species co-localized with Aβ in mature plaque cores, surrounded by hAChE-S α-helical species. This observation provides the first in vivo evidence of the conformation of hAChE-S species within plaques. Our results may explain the role of hAChE-S in Aβ deposition and aggregation, as amyloidogenic hAChE-S β-sheet species might seed Aβ aggregation.

Erythropoietin regulates the expression of dimeric form of acetylcholinesterase during differentiation of erythroblast.

Acetylcholinesterase (AChE; EC 3.1.1.7) is known to hydrolyze acetylcholine at cholinergic synapses. In mammalian erythrocyte, AChE exists as a dimer (G2 ) and is proposed to play role in erythropoiesis. To reveal the regulation of AChE during differentiation of erythroblast, erythroblast-like cells (TF-1) were induced to differentiate by application of erythropoietin (EPO). The expression of AChE was increased in parallel to the stages of differentiation. Application of EPO in cultured TF-1 cells induced transcriptional activity of ACHE gene, as well as its protein product. This EPO-induced event was in parallel with erythrocytic proteins, for example, α- and β-globins. The EPO-induced AChE expression was mediated by phosphorylations of Akt and GATA-1; because the application of Akt kinase inhibitor blocked the gene activation. Erythroid transcription factor also known as GATA-1, a downstream transcription factor of EPO signaling, was proposed here to account for regulation of AChE in TF-1 cell. A binding sequence of GATA-1 was identified in ACHE gene promoter, which was further confirmed by chromatin immunoprecipitation (ChIP) assay. Over-expression of GATA-1 in TF-1 cultures induced AChE expression, as well as activity of ACHE promoter tagged with luciferase gene (pAChE-Luc). The deletion of GATA-1 sequence on the ACHE promoter, pAChEΔGATA-1 -Luc, reduced the promoter activity during erythroblastic differentiation. On the contrary, the knock-down of AChE in TF-1 cultures could lead to a reduction in EPO-induced expression of erythrocytic proteins. These findings indicated specific regulation of AChE during maturation of erythroblast, which provided an insight into elucidating possible mechanisms in regulating erythropoiesis.

The Kinetics of Thermal Denaturation of Acetylcholinesterase of the Rat Red Blood Cell Membrane during Moderate Hypothermia

The thermostability of acetylcholinesterase of rat erythrocyte membranes in the norm and moderate hypothermia was studied. It is shown that the kinetics of the thermal denaturation of acetylcholinesterase is nonlinear and corresponds to a model that involves two-step denaturation, fast and slow, of the enzyme’s native form. The rate constants of the fast phase, k1, are much higher than those of the slow phase, k2, while the energy of the fast phase activation is lower by only 19.4% compared to that of the slow one. Short-term moderate hypothermia is shown to increase k1 and decrease the index of relative activity of the intermediate form of acetylcholinesterase (parameter β), leading to significant lowering of the activation energies of both stages; parameter β becomes more temperature dependent. The prolongation of hypothermia up to 3 h mainly contributes to a decrease in k1 and k2 relative to short-term hypothermia and the activation energy of denaturation increases. These data support the hypothesis according to which the structure of acetylcholinesterase is labilized at the initial stages of the development of the hypothermic state and stabilized during prolonged hypothermia.

Stereospecific cholinesterase inhibition by O,S-diethylphenylphosphonothioate

The inhibition kinetics and stereospecificity of the chiral nerve agent derivative O,S-diethylphenylphosphonothioate (DEPP) were examined for two forms of acetylcholinesterase (human and eel) and equine butyrylcholinesterase. Both S- and R-DEPP are poor inhibitors of eel AChE (IC50 150μM), consistent with a large, nondiscriminatory binding interaction in the active site of this enzyme. However, S-DEPP is active against human and equine AChE with low μM IC50s. DEPP stereospecificities (S/R) toward these enzymes are moderate (20) relative to other cholinesterase-organophosphate (OP) systems. Pralidoxime, a common rescue agent, affects a modest recovery of both hAChE and eqBChE from treatment with S-DEPP. This result is consistent with expected chemical modification by DEPP and indicates that a measurable amount of the enzyme-phosphonate adduct does not undergo aging. Kinetic analysis of inhibition of both hAChE and eqBChE by S-DEPP yields KI values near 8μM and k2 values of about 0.10min−1. In both cases, the reaction is practically irreversible. Second order rate constants calculated from these values are similar to those obtained previously using other thio-substituted OPs with bulky groups. Since BChE has a more accommodating acyl pocket than AChE, the similar behaviors of both enzymes toward S-DEPP is notable and is likely a reflection of the weakened potency of DEPP relative to chemical warfare agents.

Effect of hypoxia on cholinesterase activity in rat sensorimotor cortex

This study reports the dynamics of changes in postnatal ontogenesis of the activity of soluble and membrane-bound forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in sensorimotor cortex of rats as well as the pattern of their changes after prenatal hypoxia (E14, 7% O2, 3 h) or acute hypoxia in adult animals (4 months, 7% O2, 3 h). In normally developing rats the activity of the membrane-bound AChE form in the sensorimotor cortex gradually increased up to the end of the first month after birth and remained at this high level during all further postnatal ontogenesis, while the activity of the soluble form of AChE reached its maximum on the 10th day after birth and decreased significantly by the end of the first month. In animals exposed to prenatal hypoxia the activity both of the soluble and membrane bound forms of AChE during the first two weeks after birth was 20-25% lower, as compared to controls but increased by the end of the first month and even exceeded the control values remaining increased up to old age (1.5 years). The activity of both BChE forms in rat sensorimotor cortex at all stages of postnatal ontogenesis was significantly lower than of AChE, although the dynamics of their changes was similar to that of AChE. Prenatal hypoxia led to a decrease in the activity of the membrane-bound form of BChE, as compared to controls, practically at all developmental stages studied, but was higher at the end of the first month after birth. At the same time, the activity of the soluble form of BChE was decreased only on the 20th day of development, as compared to the control, but increased from the end of the first month of life onwards. Acute hypoxia in adult rats also led to a decrease in the activity of both forms of AChE and BChE in the sensorimotor cortex but the dynamics of these changes was different for each enzyme. Thus, insufficient oxygen supply to the nervous tissue at different stages of ontogenesis has a significant effect on the activity and ratio of various forms of cholinesterases exhibiting either growth factor or signaling properties. This may lead to changes in brain development and formation of behavioural reactions, including learning and memory, and also increase the risk of development of the sporadic form of Alzheimer's disease (AD)--one of the most common neurodegenerative diseases of advanced age. This study expands our knowledge of the properties of brain cholinesterases under normal and pathological conditions and may be useful for developing new approaches towards prevention and treatment of AD.

Dual inhibition of acetylcholinesterase and butyrylcholinesterase enzymes by allicin.

Objectives:The brain of mammals contains two major form of cholinesterase enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The dual inhibition of these enzymes is considered as a promising strategy for the treatment of neurological disorder such as Alzheimer's disease (AD), senile dementia, ataxia, and myasthenia gravis. The present study was undertaken to explore the anticholinesterase inhibition property of allicin.Materials and Methods:An assessment of cholinesterase inhibition was carried out by Ellman's assay.Results:The present study demonstrates allicin, a major ingredient of crushed garlic (Allium sativum L.) inhibited both AChE and BuChE enzymes in a concentration-dependent manner. For allicin, the IC50 concentration was 0.01 mg/mL (61.62 μM) for AChE and 0.05 ± 0.018 mg/mL (308.12 μM) for BuChE enzymes.Conclusions:Allicin shows a potential to ameliorate the decline of cognitive function and memory loss associated with AD by inhibiting cholinesterase enzymes and upregulate the levels of acetylcholine (ACh) in the brain. It can be used as a new lead to target AChE and BuChE to upregulate the level of ACh which will be useful in alleviating the symptoms associated with AD.

Collagen Q – A potential target for autoantibodies in myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies targeting proteins expressed at the neuromuscular junction (NMJ). In most cases the targets are acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), or occasionally low-density lipoprotein receptor-related protein 4 (LRP4), but there is still a group of patients, often called seronegative MG (SNMG), with unknown antibody targets. One potential target is collagen Q (COLQ), which is restricted to the NMJ and is crucial for anchoring the NMJ-specific form of acetylcholinesterase (AChE). 415 serum samples with a clinical diagnosis of MG and 43 control samples were screened for the presence of COLQ autoantibodies using a cell-based assay (CBA) with HEK293 cells overexpressing COLQ at the cell surface. COLQ antibodies were detected in 12/415 MG sera and in one/43 control samples. Five of the COLQ-Ab+individuals were also positive for AChR-Abs and 2 for MuSK-Abs. Although the COLQ antibodies were only present at low frequency, and did not differ significantly from the small control cohort, further studies could address whether they modify the clinical presentation or the benefits of anti-cholinesterase therapy.

Induction of soluble AChE expression via alternative splicing by chemical stress in Drosophila melanogaster

Various molecular forms of acetylcholinesterase (AChE) have been characterized in insects. Post-translational modification is known to be a major mechanism for the molecular diversity of insect AChE. However, multiple forms of Drosophila melanogaster AChE (DmAChE) were recently suggested to be generated via alternative splicing (Kim and Lee, 2013). To confirm alternative splicing as the mechanism for generating the soluble form of DmAChE, we generated a transgenic fly strain carrying the cDNA of DmAChE gene (Dm_ace) that predominantly expressed a single transcript variant encoding the membrane-anchored dimer. 3′ RACE (rapid amplification of cDNA ends) and western blotting were performed to compare Dm_ace transcript variants and DmAChE forms between wild-type and transgenic strains. Various Dm_ace transcripts and DmAChE molecular forms were observed in wild-type flies, whereas the transgenic fly predominantly expressed Dm_ace transcript variant encoding the membrane-anchored dimer. This supports alternative splicing as the major determinant in the generation of multiple forms of DmAChE. In addition, treatment with DDVP as a chemical stress induced the expression of the Dm_ace splice variant without the glycosylphosphatidylinositol anchor site in a dose-dependent manner and, accordingly, the soluble form of DmAChE in wild-type flies. In contrast, little soluble DmAChE was expressed in the transgenic fly upon exposure to DDVP. DDVP bioassays revealed that transgenic flies, which were unable to express a sufficient amount of soluble monomeric DmAChE, were more sensitive to DDVP compared to wild-type flies, suggesting that the soluble monomer may exert non-neuronal functions, such as chemical defense against xenobiotics.

Interaction of metal chelators with different molecular forms of acetylcholinesterase and its significance in Alzheimer's disease treatment

The peripheral anionic site (PAS) of acetylcholinesterase (AChE) is involved in amyloid beta (Aβ) peptides aggregation of Alzheimer's disease (AD). AChE exhibits an aryl acylamidase (AAA) activity along with the well known esterase activity. Numerous studies have reported the beneficiary effect of metal chelators in AD treatment. Hence, a comparative study on the effect of metal chelators on both the esterase and AAA activity of AChE globular (G4 and G1) molecular forms was performed. The inhibitory effect of 1,10-phenanthroline was high towards AChE esterase activity. The corresponding IC50 values for esterase activity of G4 and G1-form was 190 µM and 770 µM and for AAA activity it was 270 µM and 2.74 mM, respectively. Kinetic studies on both forms of AChE show that 1,10-phenanthroline inhibits esterase in competitive and AAA activity in non-competitive manner. Protection studies further revealed that the nature of 1,10-phenanthroline inhibition on AChE is through its direct binding to protein rather than its metal chelation property. Molecular docking studies shows orientation of 1,10-phenathroline in the PAS through hydrophobic interactions with the PAS residues (Trp286, Tyr124 and Tyr341) and hydrogen bonding with Phe295. Further molecular dynamics simulation of "hAChE-1,10-phenanthroline" complex revealed that both hydrogen and hydrophobic interaction contribute equally for 1,10-phenanthroline binding to hAChE. Such an interaction of 1,10-phenanthroline on PAS may hinder "AChE-Aβ peptide" complex formation. Hence, 1,10-phenanthroline can act as a lead molecule for developing drug(s) against AD ailment with dual functions namely, anti-cholinesterase and anti-amyloid aggregation potency in a single chemical entity.

Readthrough Acetylcholinesterase Is Increased in Human Liver Cirrhosis

Background & AimsThere have been many studies on plasma butyrylcholinesterase in liver dysfunction. However, no data is available about acetylcholinesterase in human cirrhosis, although profound changes have been described in cirrhotic rat models.MethodsHuman serum and liver acetylcholinesterase and its molecular forms were determined enzymatically, after butyrylcholinesterase immunodepletion. The distinct species of acetylcholinesterase, with a distinct C-terminus, were determined by western blotting, and the level of liver transcripts by real-time PCR. Liver acetylcholinesterase was also evaluated by immunocytochemistry.ResultsIn patients with liver cirrhosis, the activity of plasma acetylcholinesterase (rich in light species), appeared to be apparently unaffected. However, the levels of the soluble readthrough (R) acetylcholinesterase form, an acetylcholinesterase species usually associated with stress and pathology, was increased compared to controls. Human liver acetylcholinesterase activity levels were also unchanged, but protein levels of the acetylcholinesterase-R and other acetylcholinesterase subunit species were increased in the cirrhotic liver. This increase in acetylcholinesterase protein expression in the cirrhotic liver was confirmed by PCR analysis. Immunohistological examination confirmed that acetylcholinesterase immunoreactivity is increased in parenchymal cells of the cirrhotic liver.ConclusionsWe demonstrate significant changes in acetylcholinesterase at the protein and mRNA levels in liver cirrhosis, with no difference in enzymatic activity. The altered expression of acetylcholinesterase protein may reflect changes in its pathophysiological role.

Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction

The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission.

Distinct localization of collagen Q and PRiMA forms of acetylcholinesterase at the neuromuscular junction

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.