Acetylcholinesterase Mutants Research Articles

The Molecular Resistance Mechanisms of European Earwigs from Apple Orchards Subjected to Different Management Strategies.

To date, apple orchards are among the most treated crops in Europe with up to 35 chemical treatments per year. Combining control methods that reduce the number of pesticide treatments is essential for agriculture and more respectful of the environment, and the use of predatory insects such as earwigs may be valuable to achieve this goal. European earwigs, Forficula auricularia (Dermaptera: Forficulidae) are considered beneficial insects in apple orchards where they can feed on many pests like aphids. The aim of this study was to investigate the potential impact of orchards' insecticide treatments on resistance-associated molecular processes in natural populations of earwigs. Because very few molecular data are presently available on earwigs, our first goal was to identify earwig resistance-associated genes and potential mutations. Using earwigs from organic, integrated pest management or conventional orchards, we identified mutations in acetylcholinesterase 2, α1 and β2 nicotinic acetylcholine receptors. In addition, the expression level of these targets and of some essential detoxification genes were monitored using RT-qPCR. Unexpectedly, earwigs collected in organic orchards showed the highest expression for acetylcholinesterase 2. Four cytochromes P450, one esterase and one glutathione S-transferases were over-expressed in earwigs exposed to various management strategies in orchards. This first study on resistance-associated genes in Forficula auricularia paves the way for future experimental studies aimed at better understanding the potential competition between natural enemies in apple orchards in order to optimize the efficiency of biocontrol.

In silico analyses of acetylcholinesterase (AChE) and its genetic variants in interaction with the anti-Alzheimer drug Rivastigmine.

Alzheimer's disease (AD) is the leading cause of dementia worldwide. Despite causing great social and economic impact, there is currently no cure for AD. The most effective therapy to manage AD symptoms is based on acetylcholinesterase inhibitors (AChEi), from which rivastigmine presented numerous benefits. However, mutations in AChE, which affect approximately 5% of the population, can modify protein structure and function, changing the individual response to Alzheimer's treatment. In this study, we performed computer simulations of AChE wild type and variants R34Q, P135A, V333E, and H353N, identified by one or more genome-wide association studies, to evaluate their effects on protein structure and interaction with rivastigmine. The functional effects of AChE variants were predicted using eight machine learning algorithms, while the evolutionary conservation of AChE residues was analyzed using the ConSurf server. Autodock4.2.6 was used to predict the binding modes for the hAChE-rivastigmine complex, which is still unknown. Molecular dynamics (MD) simulations were performed in triplicates for the AChE wild type and mutants using the GROMACS packages. Among the analyzed variants, P135A was classified as deleterious by all the functional prediction algorithms, in addition to occurring at highly conserved positions, which may have harmful consequences on protein function. The molecular docking results suggested that rivastigmine interacts with hAChE at the upper active-site gorge, which was further confirmed by MD simulations. Our MD findings also suggested that the complex hAChE-rivastigmine remains stable over time. The essential dynamics revealed flexibility alterations at the active-site gorge upon mutations P135A, V333E, and H353N, which may lead to strong and nonintuitive consequences to hAChE binding. Nonetheless, similar binding affinities were registered in the MMPBSA analysis for the hAChE wild type and variants when complexed to rivastigmine. Finally, our findings indicated that the rivastigmine binding to hAChE is an energetically favorable process mainly driven by negatively charged amino acids.

Regulation of acetylcholinesterase during the lipopolysaccharide-induced inflammatory responses in microglial cells.

The non-classical function of acetylcholine (ACh) has been reported in neuroinflammation that represents the modulating factor in immune responses via activation of α7 nicotinic acetylcholine receptor (α7 nAChR), i.e., a cholinergic anti-inflammatory pathway (CAP). Acetylcholinesterase (AChE), an enzyme for ACh hydrolysis, has been proposed to have a non-classical function in immune cells. However, the involvement of AChE in neuroinflammation is unclear. Here, cultured BV2 cell, a microglial cell line, and primary microglia from rats were treated with lipopolysaccharide (LPS) to induce inflammation and to explore the regulation of AChE during this process. The expression profiles of AChE, α7 nAChR, and choline acetyltransferase (ChAT) were revealed in BV2 cells. The expression of AChE (G4 form) was induced significantly in LPS-treated BV2 cells: the induction was triggered by NF-κB and cAMP signaling. Moreover, ACh or α7 nAChR agonist suppressed the LPS-induced production of pro-inflammatory cytokines, as well as the phagocytosis of microglia, by activating α7 nAChR and followed by the regulation of NF-κB and CREB signaling. The ACh-induced suppression of inflammation was abolished in AChE overexpressed cells, but did not show a significant change in AChE mutant (enzymatic activity knockout) transfected cells. These results indicate that the neuroinflammation-regulated function of AChE may be mediated by controlling the ACh level in the brain system.

Influence of seasonal migration on evolution of insecticide resistance in Plutella xylostella.

The diamondback moth, Plutella xylostella (L.), is one of the most destructive migratory pest species of cruciferous vegetables worldwide and has developed resistance to most of the insecticides used for its control. The migration regularity, migratory behavior, and relationship between flight and reproduction of P. xylostella have been widely reported. However, the effect of migration on insecticide resistance in this pest is still unclear. In this study, the effect of migration on P. xylostella resistance to seven insecticides was investigated using populations across the Bohai Sea that were collected in the early and late seasons during 2017-2019. The bioassay results showed that the early season populations of P. xylostella from south China possessed much higher resistance to insecticides because of intensive insecticide application; alternatively, the late season populations migrated from northeast China, where the insecticides were only used occasionally, showed much lower insecticide resistance. The genome re-sequencing results revealed that, among the eight mutations involved in insecticide resistance, the frequencies of two acetylcholinesterase mutations (A298S and G324A) responsible for organophosphorus insecticide resistance were significantly decreased in the late season populations. The results indicated that P. xylostella migration between tropical and temperate regions significantly delayed the development of insecticide resistance. These findings illustrated the effect of regional migration on the evolution of insecticide resistance in P. xylostella, and provided foundational information for further research on the relationship between migration and insecticide resistance development in other insects.

Geographical distribution and origin of acetylcholinesterase mutations conferring acaricide resistance in Tetranychus urticae populations from Turkey.

Two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a cosmopolitan pest species that can feed on more than 1000 host plant species. Historically, organophosphate (OP) and carbamate insecticides have been used to control this extremely polyphagous pest. However, its ability to develop acaricide resistance rapidly has led to failure in control. Mutations in acetylcholinesterase gene(ace), the target-site of OP and carbamate insecticides, have been reported to be one of the major mechanisms underlying this developing resistance. In this study, mutations previously associated with resistance (G119S, A201S, T280A, G328A, F331W/Y) in ace have been screened in 37 T. urticae populations collected across Turkey. All mutations were found in various populations, except G119S. Almost all populations had F331W/Y mutation (being fixed in 32 populations), whereas only two populations harboured A201S mutation, but not fixed. On the other hand, more than half of the populations contained T280A and G328A mutations. In addition, the presence of same haplotypes in populations originating from distinct geographic locations and a wide variety of ace haplotypes might indicate multiple origins of F331W and F331Y mutations; however, this needs further investigation. The results of area-wide screening showed that ace mutations are widely distributed among T. urticae populations. Therefore, the use of this group of insecticides should be limited or only rotational use might be regarded as a resistance management tool due to its different mode of action from other main acaricide groups in T. urticae control across Turkey.

Multiple Genetic Mutations Related to Insecticide Resistance are Detected in Field Kazakhstani House Flies (Muscidae: Diptera).

The house fly (Musca domestica Linnaeus) is an important disease vector. Insecticide resistance is an obstacle to effective house fly control. Previous studies have demonstrated that point mutations in acetylcholinesterase (Ace), carboxylesterase (MdαE7) and voltage-sensitive sodium channel (Vssc), and over-expression of CYP6D1v1 confer insecticide resistance in the house fly. However, information about the status and underlying mechanisms of insecticide resistance in Kazakhstani house flies is lacking. In this study, we investigated the occurrence of genetic mutations associated with insecticide resistance in field house flies collected at six different locations in southern Kazakhstan. Four mutations (V260L, G342A/V, and F407Y) in Ace and three mutations (G137D and W251L/S) in MdαE7 were detected with appreciable frequencies. Notably, haplotypes carrying triple-loci mutations in Ace and double mutations in MdαE7 were found in Kazakhstan. The L1014H and L1014F mutations in Vssc, and CYP6D1v1 resistance allele were detected at a low frequency in some of the six investigated house fly populations. Phylogenetic analyses of haplotypes supported multiple origins of resistance mutations in Ace and MdαE7. These observations suggest that house flies in southern Kazakhstan may exhibit significant resistance to organophosphates and carbamates. Regular monitoring of insecticide resistance is recommended to achieve effective house fly control by chemical agents in southern Kazakhstan.

Resistance in the Genus Spodoptera: Key Insect Detoxification Genes.

Simple SummaryThe moth larvae are among the most damaging pest species on crops worldwide. In this review, we focus on the genus Spodoptera, which can feed on many crops such as rice, cotton or corn. The massive use of insecticides to control these insects has led to the development of resistance. Here, we aim to compare the resistance mechanisms of four species (Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis and Spodoptera litura) and highlight the role of enzymes and transporters in resistance to help us understand the molecular basis of their origin.The genus Spodoptera (Lepidoptera: Noctuidae) includes species that are among the most important crop pests in the world. These polyphagous species are able to feed on many plants, including corn, rice and cotton. In addition to their ability to adapt to toxic compounds produced by plants, they have developed resistance to the chemical insecticides used for their control. One of the main mechanisms developed by insects to become resistant involves detoxification enzymes. In this review, we illustrate some examples of the role of major families of detoxification enzymes such as cytochromes P450, carboxyl/cholinesterases, glutathione S-transferases (GST) and transporters such as ATP-binding cassette (ABC) transporters in insecticide resistance. We compare available data for four species, Spodoptera exigua, S. frugiperda, S. littoralis and S. litura. Molecular mechanisms underlying the involvement of these genes in resistance will be described, including the duplication of the CYP9A cluster, over-expression of GST epsilon or point mutations in acetylcholinesterase and ABCC2. This review is not intended to be exhaustive but to highlight the key roles of certain genes.

Monitoring of Target-Site Mutations Conferring Insecticide Resistance in Spodoptera frugiperda.

Simple SummaryFall armyworm, Spodoptera frugiperda, is an invasive moth species and one of the most destructive pests of maize. It is native to the Americas but recently invaded (sub)tropical regions in Africa, Asia and Oceania. Fall armyworm larvae feeding on maize plants cause substantial economic damage and are usually controlled by the application of insecticides and genetically modified (GM) maize expressing Bacillus thuringiensis (Bt) proteins, selectively targeting fall armyworm. It has developed resistance to many different classes of insecticides and Bt proteins as well; therefore, it is important to check field populations for the presence of mutations in target proteins conferring resistance. Here, we developed molecular diagnostic tools allowing us to test the frequency of resistance alleles in field-collected populations, either alive or preserved in alcohol. We tested 34 different populations collected on four different continents for the presence of mutations conferring resistance to common classes of insecticides and Bt proteins. We detected resistance mutations which are quite widespread, whereas others are restricted to certain geographies or even completely absent. The established molecular methods show robust results in samples collected across a broad geographical range and can be used to support decisions for sustainable fall armyworm control and applied resistance management.Fall armyworm (FAW), Spodoptera frugiperda, a major pest of corn and native to the Americas, recently invaded (sub)tropical regions worldwide. The intensive use of insecticides and the high adoption of crops expressing Bacillus thuringiensis (Bt) proteins has led to many cases of resistance. Target-site mutations are among the main mechanisms of resistance and monitoring their frequency is of great value for insecticide resistance management. Pyrosequencing and PCR-based allelic discrimination assays were developed and used to genotype target-site resistance alleles in 34 FAW populations from different continents. The diagnostic methods revealed a high frequency of mutations in acetylcholinesterase, conferring resistance to organophosphates and carbamates. In voltage-gated sodium channels targeted by pyrethroids, only one population from Indonesia showed a mutation. No mutations were detected in the ryanodine receptor, suggesting susceptibility to diamides. Indels in the ATP-binding cassette transporter C2 associated with Bt-resistance were observed in samples collected in Puerto Rico and Brazil. Additionally, we analyzed all samples for the presence of markers associated with two sympatric FAW host plant strains. The molecular methods established show robust results in FAW samples collected across a broad geographical range and can be used to support decisions for sustainable FAW control and applied resistance management.

Resistance to insecticides in Anopheles gambiae complex in West Africa: A review of the current situation and the perspectives for malaria control

In West Africa, the Anopheles gambiae sensu lato (s.l.) complex mosquitoes are the main malaria vectors. Malaria control relies mainly on long-lasting insecticide-treated nets (LLINs) and to a lesser extent on indoor residual spray (IRS) of insecticides. Important progress has been made in terms of malaria morbidity and mortality in these past decades. Resistance to insecticides in An. gambiae s.l. was first attributed to their use in agriculture. Further, their use in public health increased selection pressure. Resistance has been recorded to the four main classes of insecticides used in public health in West Africa. However, the spread of insecticide resistance in West Africa is persistent due to the lack of management of resistance monitoring. Resistance to insecticides involves different mechanisms. Resistance to pyrethroids and organochlorines was caused by target modification such as some sodium channel mutations in addition to metabolic changes. Reduction of sensitivity to organophosphates and carbamates was caused by acetylcholinesterase mutations. Therefore, efforts should be made as quickly as possible to develop novel vector control techniques. Furthermore, it is also suggested to combine environmental, chemical, and biological management. In this review, we outline the insecticide resistance of Anopheles in West Africa and the current alternative tools for effective mosquito vector control.

Displaying of acetylcholinesterase mutants on surface of yeast for ultra-trace fluorescence detection of organophosphate pesticides with gold nanoclusters

Acetylcholinesterase (AChE) has been widely applied on the enzyme inhibition-based detection of organophosphate pesticides (OPs). To improve the sensitivity of fluorometric OPs assay, great efforts were made to change the fluorometric probes or analytical strategies rather than improve the sensitivity of AChE towards OPs. In this work, AChE wild-type (WT) and mutants (E69Y and E69Y/F330L) from Drosophila were successfully displayed on the surface of yeast through a-agglutinin-mediated microbial surface display system. The location of AChE on yeast surface was confirmed by immunofluorescence analysis. Further, a fluorescence OPs detection method was developed by combining yeast surface-displayed AChE mutants and protein-directed electronegative fluorescent gold nanoclusters (Au NCs). Yeast surface-displayed AChE can catalyze the hydrolysis of acetylthiocholine to produce thiocholine. The electropositive thiocholine can not only bind with AuNCs by Au–S bond but also absorb Au NCs by the electrostatic interaction, leading to the aggregation of AuNCs and corresponding fluorescence quenching. When AChE was incubated with paraoxon, a typical model of OPs, the activity of AChE was inhibited and the thiocholine-induced aggregation of AuNCs was reduced. The fluorescence assay based on Au NCs and yest-AChE-E69Y/F330L exhibited the ultra-sensitivity for ultra-trace OPs and 2–6 orders of magnitude lower detection limit (3.3 × 10−14 M) than those of AChE-WT-based method and other reported methods. In addition, the proposed method showed excellent reliability for the real samples assay. This work would provide an alternative strategy for the improvement of bio-analysis at its source.

In Silico Study of the Resistance to Organophosphorus Pesticides Associated with Point Mutations in Acetylcholinesterase of Lepidoptera: B. mandarina, B. mori, C. auricilius, C. suppressalis, C. pomonella, H. armígera, P. xylostella, S. frugiperda, and S. litura.

An in silico analysis of the interaction between the complex-ligands of nine acetylcholinesterase (AChE) structures of Lepidopteran organisms and 43 organophosphorus (OPs) pesticides with previous resistance reports was carried out. To predict the potential resistance by structural modifications in Lepidoptera insects, due to proposed point mutations in AChE, a broad analysis was performed using computational tools, such as homology modeling and molecular docking. Two relevant findings were revealed: (1) Docking results give a configuration of the most probable spatial orientation of two interacting molecules (AChE enzyme and OP pesticide) and (2) a predicted ΔGb. The mutations evaluated in the form 1 acetylcholinesterase (AChE-1) and form 2 acetylcholinesterase (AChE-2) structures of enzymes do not affect in any way (there is no regularity of change or significant deviations) the values of the binding energy (ΔGb) recorded in the AChE–OPs complexes. However, the mutations analyzed in AChE are associated with a structural modification that causes an inadequate interaction to complete the phosphorylation of the enzyme.

Counteracting tabun inhibition by reactivation by pyridinium aldoximes that interact with active center gorge mutants of acetylcholinesterase

Tabun represents the phosphoramidate class of organophosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. Currently used therapy in counteracting excessive cholinergic stimulation consists of a muscarinic antagonist (atropine) and an oxime reactivator of inhibited AChE, but the classical oximes are particularly ineffective in counteracting tabun exposure. In a recent publication (Kovarik et al., 2019), we showed that several oximes prepared by the Huisgen 1,3 dipolar cycloaddition and related precursors efficiently reactivate the tabun−AChE conjugate. Herein, we pursue the antidotal question further and examine a series of lead precursor molecules, along with triazole compounds, as reactivators of two AChE mutant enzymes. Such studies should reveal structural subtleties that reside within the architecture of the active center gorge of AChE and uncover intimate mechanisms of reactivation of alkylphosphate conjugates of AChE. The designated mutations appear to minimize steric constraints of the reactivating oximes within the impacted active center gorge. Indeed, after initial screening of the triazole oxime library and its precursors for the reactivation efficacy on Y337A and Y337A/F338A human AChE mutants, we found potentially active oxime-mutant enzyme pairs capable of degrading tabun in cycles of inhibition and reactivation. Surprisingly, the most sensitive ex vivo reactivation of mutant AChEs occurred with the alkylpyridinium aldoximes. Hence, although the use of mutant enzyme bio-scavengers in humans may be limited in practicality, bioscavenging and efficient neutralization of tabun itself or phosphoramidate mixtures of organophosphates might be achieved efficiently in vitro or ex vivo with these mutant AChE combinations.

Structure of the G119S Mutant Acetylcholinesterase of the Malaria Vector Anopheles gambiae Reveals Basis of Insecticide Resistance

Malaria is a devastating disease in sub-Saharan Africa and is transmitted by the mosquito Anopheles gambiae. While indoor residual spraying of anticholinesterase insecticides has been useful in controlling the spread of malaria, widespread application of these compounds has led to the rise of an insecticide-resistant mosquito strain that harbors a G119S mutation in the nervous system target enzyme acetylcholinesterase. We demonstrate the atomic basis of insecticide resistance through structure determination of the G119S mutant acetylcholinesterase of An. gambiae in the ligand-free state and bound to a potent difluoromethyl ketone inhibitor. These structures reveal specific features within the active-site gorge distinct from human acetylcholinesterase, including an open channel at the base of the gorge, and provide a means for improving species selectivity in the rational design of improved insecticides for malaria vector control.

Point mutations in acetylcholinesterase 1 associated with chlorpyrifos resistance in the brown planthopper, Nilaparvata lugens Stål.

Insecticide resistance frequently results from target-site insensitivity, such as point mutations in acetylcholinesterases (AChEs) for resistance to organophosphates and carbamates. From a field-originated population of Nilaparvata lugens, a major rice pest, a resistant population (R9) was obtained by nine-generation continuous selection with chlorpyrifos. From the same field population, a relatively susceptible population (S9) was also constructed through rearing without any insecticides. Compared to thesusceptible strain, Sus [medium lethal dose (LC50 ) = 0.012 mg/l], R9 had a resistance ratio (RR) of 253.08-fold, whereas the RR of S9 was only 2.25-fold. Piperonyl butoxide and triphenyl phosphate synergized chlorpyrifos in R9 less than three-fold, indicating other important mechanisms for high resistance. The target-site insensitivity was supported by the key property differences of crude AChEs between R9 and S9. Compared to S9, three mutations (G119S, F331C and I332L) were detected in NlAChE1 from individuals of the R9 and field populations, but no mutation was detected in NlAChE2. G119S and F331C could decreased insecticide sensitivities in recombinant NlAChE1, whereas I332L took effect through increasing the influence of F331C on target insensitivity. F331C might be deleterious because of its influence on the catalytic efficiency of NlAChE1, whereas I332L would decrease these adverse effects and maintain the normal functions of AChEs.

Insecticide resistance in malaria vectors in Kumasi, Ghana.

BackgroundThere have been recent reports of surge in resistance to insecticides in pocketed areas in Ghana necessitating the need for information about local vector populations and their resistance to the insecticides approved by the World Health Organization (WHO). We therefore studied a population of malaria vectors from Kumasi in the Ashanti Region of Ghana and their resistance to currently used insecticides. We conducted susceptibility tests to the four major classes of insecticides by collecting larvae of anopheline mosquitoes from several communities in the region. Surviving adults from these larvae were then subjected to the WHO-approved susceptibility tests and characterization of knockdown resistance and acetylcholinesterase mutant genes.ResultsOut of 619 Anopheles specimens sampled, 537 (87%) were identified as Anopheles gambiae (sensu stricto), which was also the species with the lowest knockdown resistance mutant gene, 61% (P = 0.017). Knockdown resistance mutant gene was as high as 91% in An. coluzzii. Mosquitoes collected showed susceptibility ranging from 98–100% to organophosphates, 38–56% to carbamates and 15–47% and 38–46% to pyrethroids and organochlorides, respectively. The knockdown resistance mutation frequency of Anopheles gambiae (sensu lato) mosquitoes that were exposed to both pyrethroids and organochlorides was 404 (65%). Acetylcholinesterase mutant gene was not found in this population of vectors.ConclusionOur study shows that pyrethroids have the highest level of resistance in the population of mosquito vectors studied probably due to their frequent use, especially in impregnation of insecticide-treated nets and in insecticides used to control pests on irrigated vegetable farms. We recommend studies to monitor trends in the use of all insecticides and of pyrethroids in particular.

Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes.

Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of the soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin, and paraoxon, inhibition. We here demonstrate that ex vivo, in whole human blood, 1 μM soman was detoxified within 30 min when supplemented with 0.5 μM Y337A/F338A AChE and 100 μM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of 42 pyridinium aldoximes, and 5 imidazole 2-aldoxime N-propylpyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2-3-fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack.

Mutation and duplication of arthropod acetylcholinesterase: Implications for pesticide resistance and tolerance

A series of common/shared point mutations in acetylcholinesterase (AChE) confers resistance to organophosphorus and carbamate insecticides in most arthropod pests. However, the mutations associated with reduced sensitivity to insecticides usually results in the reduction of catalytic efficiency and leads to a fitness disadvantage. To compensate for the reduced catalytic activity, overexpression of neuronal AChE appears to be necessary, which is achieved by a relatively recent duplication of the AChE gene (ace) as observed in the two-spotted spider mite and other insects. Unlike the cases with overexpression of neuronal AChE, the extensive generation of soluble AChE is observed in some insects either from a distinct non-neuronal ace locus or from a single ace locus via alternative splicing. The production of soluble AChE in the fruit fly is induced by chemical stress. Soluble AChE acts as a potential bioscavenger and provides tolerance to xenobiotics, suggesting its role in chemical adaptation during evolution.

Alteration in cellular acetylcholine influences dauer formation in Caenorhabditis elegans

Altered acetylcholine (Ach) homeostasis is associated with loss of viability in flies, developmental defects in mice, and cognitive deficits in human. Here, we assessed the importance of Ach in Caenorhabditis elegans development, focusing on the role of Ach during dauer formation. We found that dauer formation was disturbed in choline acetyltransferase (cha-1) and acetylcholinesterase (ace) mutants defective in Ach biosynthesis and degradation, respectively. When examined the potential role of G-proteins in dauer formation, goa-1 and egl-30 mutant worms, expressing mutated versions of mammalian Go and Gq homolog, respectively, showed some abnormalities in dauer formation. Using quantitative mass spectrometry, we also found that dauer larvae had lower Ach content than did reproductively grown larvae. In addition, a proteomic analysis of acetylcholinesterase mutant worms, which have excessive levels of Ach, showed differential expression of metabolic genes. Collectively, these results indicate that alterations in Ach release may influence dauer formation in C. elegans. [BMB Reports 2014; 47(2): 80-85]

Molecular diagnostic for detecting the acetylcholinesterase mutations in insecticide-resistant populations of Colorado potato beetle, Leptinotarsa decemlineata (Say)

Insecticide-resistant populations of Colorado potato beetle (CPB), with insensitive acetylcholinesterase (AChE) have recently been reported from commercial potato fields of Hamedan province in west of Iran. The objective of this study was to clarify the molecular mechanism of this insensitivity. The serine to glycine change at position 291 (S291G) in the AChE gene was found previously in an azinphosmethyl-resistant strain of CPB (AZ-R). PCR–RFLP assays were used to monitor the frequency of the S291G resistance mutation in resistant field populations of CPB, Aliabad, Bahar, Dehpiaz, and Yengijeh. The S291G mutation was detected in 66.6, 73.3, 53.3 and 26.6% of Bahar, Dehpiaz, Aliabad and Yengijeh populations, respectively. Moreover, only 25% of samples from the resistant field populations were homozygous for S291G mutation. There was no significant correlation between the mutation frequencies and resistance levels in the resistant populations, indicating that other mutations may contribute to this variation. PCR–SSCP method was used to find sequence variation in the AChE gene. Based on the published nucleotide sequence information on AChE gene (GenBank L41180.1), five primer pairs were designed to amplify specific PCR products of 306bp (first fragment: codons 24–142), 370bp (second fragment: codons 141–261), 403bp (third fragment: codons 248–381), 335bp (fourth fragment: codons 376–486), and 335bp (fifth fragment: codons 488–598). No specific PCR product of desirable size for either the fourth and fifth fragments could be obtained. The DNA amplification products were subjected to SSCP analysis to identify the DNA sequence variations between the susceptible strain and resistant populations. Ninety-five beetles of susceptible strain (15 beetles) and four field populations (20 of each) were screened. Allelic differences were detected by distinctive electrophoretic patterns of each single strand. Nucleotide sequence variations in the different SSCP patterns were verified by direct DNA sequencing. Alignment of the AChE gene sequences with the sequencing results revealed 16 point mutations (V44G, E128D, R140G, I143F, T248S, F250S, G251C, I261M, S265I, S291G, G353R, L356R, E366K, S378E, S378R, E382D) from the field populations of CPB, which may contribute to the AChE insensitivity. Further analysis on the RNA level is necessary for clarification and validation of this contention.

Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1.