Acetylcholinesterase Variants Research Articles

AChE as a spark in the Alzheimer's blaze - Antagonizing effect of a cyclized variant.

The amyloid precursor protein (APP), presenilin 1 (PS1), amyloid beta (Aβ), and GSK3 are the effectors, which are significantly associated with progression of Alzheimer's Disease (AD) and its symptoms. A significant protein, acetylcholinesterase (AChE) becomes dysfunctional as a result of cholinergic neuronal loss in AD pathology. However, certain associated peptides potentiate the release of primary neuropathological hallmarks, i.e., senile plaque and neurofibrillary tangles (NFTs), by modulating the alpha 7 acetylcholinesterase receptor (α7nAChR). The AChE variants, T30 and T14 have also been found to be elevated in AD patients and mimic the toxic actions of pathological events in patients. The manuscript discusses the significance of AChE inhibitors in AD therapeutics, by indicating the disastrous role of molecular alterations and elevation of AChE, accompanied with the downstream effects instigated by the peptide, supported by clinical evidence and investigations. The cyclized variant of AChE peptide, NBP14 has been identified as a novel candidate that reverses the harmful effects of T30, T14 and Aβ, mainly calcium influx, cell viability and AChE release. The review aims to grab the attention of neuro-researchers towards the significance of triggering effectors in propagating AD and role of AChE in regulating them, which can potentially ace the development of reliable therapeutic candidates, similar to NBP14, to mitigate neurodegeneration.

Association of status of acetylcholinesterase and ACHE gene 3’ UTR variants (rs17228602, rs17228616) with drug addiction vulnerability in pakistani population

Substance addiction is a chronic, relapsing mental disorder Characterized by compulsive drug seeking, and loss of control over drug intake and relapse after prolonged abstinence. Genetics has been shown to contribute towards an individual's vulnerability to addiction. Acetylecholine (ACh), a cholinergic neurotransmitter hydrolyzed by acetylcholinesterase (AChE), is an essential neurotransmitter and neuromodulator in central and peripheral nervous system and has regulatory influence on numerous neuronal functions including addiction. The present study was carried out to investigate the role of acetylcholinesterase (AChE) in addiction through measurement of enzyme activity and to find potential association of ACHE gene 3′UTR variants rs17228602 and rs17228616 in heroin, hashish and poly drug addicts. Both SNPs are located within microRNA (miRNA) recognition sites with potential to affect miRNA/transcript interaction. A total of 122 addicts of heroin, hashish and polydrug were recruited from local rehabilitation centers to participate in this study. AChE activity was measured in blood by Ellman's method. SNP genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) and Sanger sequencing. The AChE activity was found significantly higher (p ≤ 0.005) in addicted cohort (mean ± standard error of mean 0.020 ± 0.001 μmol/L/min; 95% confidence interval (CI) 0.018–0.022) in comparison to non-addicted healthy subjects (0.011 ± 0.001 μmol/L/min; 95% confidence interval CI 0.010–0.013). A statistically significant association of ACHE rs17228602 SNP with addiction vulnerability in dominant (DM: Odd's ratio OR = 2.095, 95% CI = 1.157–3.807 p = 0.009) and allelic genetic models (OR = 1.854 95% CI = 1.082–3.187, p = 0.016) was observed. However, no statistically significant association of rs17228616 SNP with substance abuse disorder was found. The data presented here shows that AChE could play significant role in substance addiction. Further studies with larger sample size and other variants of AChE are recommended to identify novel therapeutic approaches for cholinergic based treatment of addiction.

Caligus rogercresseyi acetylcholinesterase types and variants: a potential marker for organophosphate resistance

BackgroundControl of the sea louse Caligus rogercresseyi in the Chilean salmonid industry is reliant on chemical treatments. Azamethiphos was introduced in 2013, although other organophosphates were previously used. In 2014, reduced sensitivity to azamethiphos was detected in the Los Lagos Region using bioassays. The main target of organophosphates is the enzyme acetylcholinesterase (AChE). Mutations in the AChE gene are the main cause of organophosphate resistance in arthropods, including other sea lice. In the present study, we aimed to characterize C. rogercresseyi AChE(s) gene(s) and to study the association between AChE variants and azamethiphos resistance in this sea louse species.MethodsSamples of adult male and female C. rogercresseyi were collected in the Los Lagos Region in 2014. Twenty-four hour exposure bioassays with azamethiphos were performed to select sensitive and resistant lice. The full-length cDNA coding sequences encoding for two AChEs in C. rogercresseyi were molecularly characterized. One of the AChE genes was screened by direct sequencing in the azamethiphos-selected lice to search for variants. An additional louse sampling was performed before and after an azamethiphos treatment in the field in 2017 to validate the findings.ResultsThe molecular analysis revealed two putative AChEs in C. rogercresseyi. In silico analysis and 3D modelling of the protein sequences identified both of them as invertebrate AChE type 1; they were named C. rogercresseyi AChE1a and 1b. AChE1a had the characteristics of the main synaptic AChE, while AChE1b lacked some of the important amino acids of a typical AChE. A missense change found in the main synaptic AChE (1a), F318F/V (F290 in Torpedo californica), was associated with survival of C. rogercresseyi at high azamethiphos concentrations (bioassays and field treatment). The amino acid change was located in the acyl pocket of the active-site gorge of the protein.ConclusionsThe present study demonstrates the presence of two types of AChE1 genes in C. rogercresseyi. Although enzymatic assays are needed, AChE1a is most probably the main synaptic AChE. The function of AChE1b is unknown, but evidence points to a scavenger role. The AChE1a F/V318 variant is most probably involved in organophosphate resistance, and can be a good marker for resistance monitoring.

Postnatal chlorpyrifos exposure and apolipoprotein E (APOE) genotype differentially affect cholinergic expression and developmental parameters in transgenic mice

Chlorpyrifos (CPF) is one of the most commonly used organophosphate pesticides in the world. Our previous results described that apolipoprotein E (APOE) polymorphisms are a source of individual differences in susceptibility to CPF. The aim of this study was to assess the physical and biochemical effects of postnatal exposure to CPF in the apoE targeted replacement mouse model. Mice were exposed to CPF at 0 or 1 mg/kg/day from postnatal day 10–15. Physical development, plasma and forebrain cholinesterase (ChE) activity and gene expression in liver and forebrain were evaluated. CPF exposure delays physical maturation and decreases the expression of choline acetyltransferase, α4-subunit and the α7 receptor. CPF decreases the expression of vesicular acetylcholine transporter (VAChT) mRNA in the forebrain only in apoE3 mice. The expression of paraoxonase-2 in the forebrain was also influenced by APOE genotype and CPF. Differences between genotypes were observed in litter size, ChE activity, expression of butyrylcholinesterase and paraoxonase-1 in liver and variants of acetylcholinesterase, VAChT and the α7 receptor in the forebrain. These results support that there are different vulnerabilities to postnatal CPF exposure according to the APOE polymorphism, which in turn affects the cholinergic system and defenses to oxidative stress.

SN56 neuronal cell death after 24 h and 14 days chlorpyrifos exposure through glutamate transmission dysfunction, increase of GSK-3β enzyme, β-amyloid and tau protein levels

Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75NTR and α7-nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aβ and tau proteins production and activation of GSK3β enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of Aβ and tau proteins production, activation of GSK-3β enzyme and disruption of glutamatergic transmission. We evaluated this hypothesis in septal SN56 basal forebrain cholinergic neurons, after 24 h and 14 days CPF exposure. This study shows that CPF increases glutamate levels, upregulates GSK-3β gene expression, and increases the production of Aβ and phosphorylated tau proteins and all these effects reduced cell viability. CPF increases glutaminase activity and upregulates the VGLUT1 gene expression, which could mediate the disruption of glutamatergic transmission. Our present results provide new understanding of the mechanisms contributing to the harmful effects of CPF, and its possible relevance in the pathogenesis of neurodegenerative diseases.

Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

The observation of acetylcholinesterase (AChE) type H (AChEH), which is the predominant AChE variant in visceral organs and immune cells, in lipid rafts of muscle supports functional reasons for the raft targeting of glypiated AChEH The search for these reasons revealed that liver AChE activity is mostly confined to rafts and that the liver is able to make N-extended AChE variants and target them to rafts. These results prompted us to test whether AChE and muscarinic receptors existed in the same raft. Isolation of flotillin-2-rich raft fractions by their buoyancy in sucrose gradients, followed by immunoadsorption and matrix-assisted laser desorption ionization-time of flight-mass spectrometry application, gave the following results: 1) most hepatic AChE activity emanates from AChE-H mRNA, and its product, glypiated AChEH, accumulates in rafts; 2) N-extended N-AChE readthrough variant, nonglypiated N-AChEH, and N-AChE tailed variant were all identified in liver rafts; and 3) M3 AChRs were observed in rafts, and coprecipitation of raft-confined N-AChE and M3 receptors by using anti-M3 antibodies showed that enzyme and receptor reside in the same raft unit. A raft domain that harbors tightly packed muscarinic receptor and AChE may represent a molecular device that, by means of which, the intensity and duration of cholinergic inputs are regulated.-Montenegro, M. F., Cabezas-Herrera, J., Campoy, F. J., Muñoz-Delgado, E., Vidal, C. J. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

Naturally Occurring Genetic Variants of Human Acetylcholinesterase and Butyrylcholinesterase and Their Potential Impact on the Risk of Toxicity from Cholinesterase Inhibitors.

Acetylcholinesterase (AChE) is the physiologically important target for organophosphorus toxicants (OP) including nerve agents and pesticides. Butyrylcholinesterase (BChE) in blood serves as a bioscavenger that protects AChE in nerve synapses from inhibition by OP. Mass spectrometry methods can detect exposure to OP by measuring adducts on the active site serine of plasma BChE. Genetic variants of human AChE and BChE do exist, but loss of function mutations have been identified only in the BCHE gene. The most common AChE variant, His353Asn (H322N), also known as the Yt blood group antigen, has normal AChE activity. The most common BChE variant, Ala567Thr (A539T) or the K-variant in honor of Werner Kalow, has 33% reduced plasma BChE activity. The genetic variant most frequently associated with prolonged response to muscle relaxants, Asp98Gly (D70G) or atypical BChE, has reduced activity and reduced enzyme concentration. Early studies in young, healthy males, performed at a time when it was legal to test nerve agents in humans, showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate. Additionally, animal studies indicated that BChE protects from toxicants that have a higher reactivity with AChE than with BChE (e.g., nerve agents) but not from toxicants that have a higher reactivity with BChE than with AChE (e.g., OP pesticides). As a corollary, we hypothesize that individuals with genetic variants of BChE may be at increased risk of toxicity from nerve agents but not from OP pesticides.

Increased Expression of Readthrough Acetylcholinesterase Variants in the Brains of Alzheimer's Disease Patients.

Alzheimer's disease (AD) is characterized by a decrease in the enzymatic activity of the enzyme acetylcholinesterase (AChE). AChE is expressed as multiple splice variants, which may serve both cholinergic degradative functions and non-cholinergic functions unrelated with their capacity to hydrolyze acetylcholine. We have recently demonstrated that a prominent pool of enzymatically inactive AChE protein exists in the AD brain. In this study, we analyzed protein and transcript levels of individual AChE variants in human frontal cortex from AD patients by western blot analysis using specific anti-AChE antibodies and by quantitative real-time PCR (qRT-PCR). We found similar protein and mRNA levels of the major cholinergic "tailed"-variant (AChE-T) and the anchoring subunit, proline-rich membrane anchor (PRiMA-1) in frontal cortex obtained from AD patients and non-demented controls. Interestingly, we found an increase in the protein and transcript levels of the non-cholinergic "readthrough" AChE (AChE-R) variants in AD patients compared to controls. Similar increases were detected by western blot using an antibody raised against the specific N-terminal domain, exclusive of alternative N-extended variants of AChE (N-AChE). In accordance with a subset of AChE-R monomers that display amphiphilic properties that are upregulated in the AD brain, we demonstrate that the increase of N-AChE species is due, at least in part, to N-AChE-R variants. In conclusion, we demonstrate selective alterations in specific AChE variants in AD cortex, with no correlation in enzymatic activity. Therefore, differential expression of AChE variants in AD may reflect changes in the pathophysiological role of AChE, independent of cholinergic impairment or its role in degrading acetylcholine.

Characterizations of 3′ splice variants of acetylcholinesterase (AchE) gene in rat: Implications for neurotoxicology studies

Characterizations of 3′ splice variants of acetylcholinesterase (AchE) gene in rat: Implications for neurotoxicology studies

Strain dependent effects of conditioned fear in adult C57Bl/6 and Balb/C mice following postnatal exposure to chlorpyrifos: relation to expression of brain acetylcholinesterase mRNA.

Following reports of emotional psychopathology in children and adults exposed to organophosphates, the effects of postnatal chlorpyrifos (CPF) on fear-conditioning and depression-like behaviors were tested in adult mice. Concomitant changes in expression of mRNA for synaptic and soluble splice variants of acetylcholinesterase (AChE) were examined in mouse pups and adults of the Balb/C and C57Bl/6 (B6) strains, which differ in their behavioral and hormonal stress response. Mice were injected subcutaneously with 1 mg/kg CPF on postnatal days 4–10 and tested as adults for conditioned fear, sucrose preference, and forced swim. Acetylcholinesterase activity was assessed in the brains of pups on the first and last day of treatment. Expression of soluble and synaptic AChE mRNA was assessed in brains of treated pups and fear-conditioned adults using real-time PCR. Adult Balb/C mice exposed postnatally to CPF showed exacerbated fear-conditioning and impaired active avoidance. Adult B6 mice exposed postnatally to CPF showed a more specific fear response to tones and less freezing in the inter-tone intervals, in contrast to the vehicle-pretreated mice. Chlorpyrifos also attenuated sweet preference and enhanced climbing in the forced swim test. Chlorpyrifos-treated mice had increased expression of both synaptic and readthrough AChE transcripts in the hippocampus of Balb/C mice and decreased expression in the amygdala following fear-conditioning. In conclusion, postnatal CPF had long-term effects on fear and depression, as well as on expression of AChE mRNA. These changes may be related to alteration in the interaction between hippocampus and amygdala in regulating negative emotions.

Meta-analysis of genetic and environmental Parkinson's disease models reveals a common role of mitochondrial protection pathways

Both genetic and environmental factors trigger risks of and protection from Parkinson's disease, the second most common neurodegenerative syndrome, but possible inter-relationships between these risk and protection processes were not yet explored. By examining gene expression changes in the brains of mice under multiple treatments that increase or attenuate PD symptoms we detected underlying disease and protection-associated genes and pathways.In search for potential links between these different genes and pathways, we conducted meta-analysis on 131 brain region transcriptomes from mice over-expressing native or mutated α-synuclein (SNCA) with or without the protective HSP70 chaperone, or exposed to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), with or without the protective acetylcholinesterase (AChE-R) variant. All these models showed shared risk-inducible and protection-suppressible transcript modifications. Self-organized map (SOM) classification revealed risk- and protection-associated alterations in nuclear and mitochondrial metal ion-regulated transcripts, respectively; Gene Ontology based analysis validated these pathways. To complement this approach, and identify potential outcome damages, we further searched for shared functional enrichments in the lists of genes detected in young SNCA mutant or in old SNCA mutants and MPTP-exposed mice. This post-hoc functional analysis identified early-onset changes in Parkinsonian, immune and alternative splicing pathways which shifted into late-onset or exposure-associated NFkB-mediated neuro-inflammation. Our study suggests metal ions-mediated cross-talk between nuclear and mitochondrial pathways by both environmental and genetic risk and protective factors involved in Parkinson's disease, which eventually culminates in neuro-inflammation. Together, these findings offer new insights and novel targets for therapeutic interference with the gene–environment interactions underlying sporadic PD.

Acetylcholinesterase inhibitor pretreatment alters stress-induced expression of acetylcholinesterase transcripts in the mouse brain

The role of the rare soluble splice variant of acetylcholinesterase (AChE-R) in anxiety behavior was assayed using the elevated plus maze (EPM). The effects of pretreatment with restraint stress and the acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP) were tested, as these treatments are known to enhance the expression of AChE-R in several brain regions. Mice from the CD-1 outbred and C57BL/6 inbred strains were randomly assigned to seven treatment groups: homecage control, elevated plus maze without pretreatment, 3 days restraint stress or 3 days pretreatment with saline or one of three doses of DFP, for a total of 14 groups. All mice, except homecage controls, were tested twice on the elevated plus maze. AChE-R transcript expression was increased following elevated plus maze stress in hippocampus and amygdala, but not in the prefrontal cortex of CD-1 and not in C57BL/6 mice. Saline-injected C57BL/6 mice had reduced expression of AChE-R transcripts compared to untreated C57 BL/6 mice. DFP pretreatment reversed the stress-induced changes, increased AChE-R transcripts in CD-1 mice. AChE-R expression in the striatum and amygdala were positively correlated with anxiety in the EPM.

Therapeutic potential of some stress mediators in early Alzheimer's disease

Lifestyle factors emerge as key modifiers of the risk and progression of Alzheimer's disease (AD), the most prevalent senile dementia. Stress accelerates, whereas positive lifestyle factors such as physical and mental exercise, slow the progression of AD symptoms and neuropathological hallmarks in humans and genetic mouse models of the condition. Positive lifestyle factors are thought to improve brain function and delay ageing through the induction of a mild stress response as opposed to excessive stress reactions causing the harmful effects of adverse stressors. This suggests that stress mediators can be targeted for the development of improved treatment for AD. This review discussed the involvement of glucocorticoids, corticotrophin-releasing hormone (CRH) and cholinergic stress reactions, through the induction of a protective splice variant of synaptic acetylcholinesterase (AChE-R), in the progression of AD symptoms and neuropathological hallmarks and the impact of lifestyle factors. It is concluded that CRH is likely to play a critical, dose-dependent role in AD progression, protective at low doses but harmful at high doses, whereas an excess of glucocorticoids may only exacerbate some aspects of the disease. AChE-R shows a promising therapeutic potential which needs to be further investigated.

Strain and regional dependence of alternate splicing of acetylcholinesterase in the murine brain following stress or treatment with diisopropylfluorophosphate

Induction of the rare readthrough variant of acetylcholinesterase (AChE-R) by an acetylcholinesterase (AChE) inhibitor or by stress was tested in four mouse strains that differ in their behavioural profiles on tests of anxiety and depression. BALB/C, C57Bl/6, C3H/He and CD-1 mouse strains were tested in the elevated plus maze in two sessions, separated by 48h. All strains, except CD-1, showed the expected reduction in open arm exploration on the second session. BALB/C and C3H mice spent a greater proportion of the time in the open arms on the first exposure, but spent more time immobile in the maze compared to the CD1 and C57 strains. Immobility was attenuated upon the second exposure in all strains, except the BALB/C mice. Real-time PCR was used to investigate regional and strain differences in induction of AChE-R mRNA following four daily injections of diisopropylfluorophosphate (DFP) (.1mg/kg). AChE-R induction was found in the frontal cortex, but not in amygdala, hippocampus or striatum of CD-1 mice. Nor was there AChE-R induction in the brains of the inbred strains. Four daily sessions of swim stress were used to investigate stress-induced induction of AChE-R. BALB/C mice showed significantly more immobility in the forced swim test (FST) compared to the other strains. FST did not induce AChE-R mRNA in any brain region tested; however, AChE-R mRNA expression in the frontal cortex was negatively correlated with immobility in the FST.

Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase

Butyrylcholinesterase (BChE) and the T splice variant of acetylcholinesterase that is predominant in mammalian brain and muscles (AChE(T)) possess a characteristic C-terminal tail (t) peptide. This t peptide allows their assembly into tetramers associated with the anchoring proteins ColQ and PRiMA. Although the t peptides of all vertebrate cholinesterases are remarkably similar and, in particular, contain seven strictly conserved aromatic residues, these enzymes differ in some of their oligomerization properties. To explore these differences, we studied human AChE (Aa) and BChE (Bb), and chimeras in which the t peptides (a and b) were exchanged (Ab and Ba). We found that secretion was increased by deletion of the t peptides, and that it was more efficient with a than with b. The patterns of oligomers were similar for Aa and Ab, as well as for Ba and Bb, indicating a predominant influence of the catalytic domains. However, addition of a cysteine within the aromatic-rich segment of the t peptides modified the oligomeric patterns: with a cysteine at position 19, the proportion of tetramers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesser extent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms, from monomers to hexamers. These results indicate that both the catalytic domains and the C-terminal t peptides contribute to the capacity of cholinesterases to form and secrete various oligomers. Sequence comparisons show that the differences between the t peptides of AChE and BChE are remarkably conserved among all vertebrates, suggesting that they reflect distinct functional adaptations.

Nicotine Relieves Anxiogenic-Like Behavior in Mice that Overexpress the Read-Through Variant of Acetylcholinesterase but Not in Wild-Type Mice

Stress increases vulnerability and causes relapse to drugs of abuse. The usually rare read-through variant of acetylcholinesterase (AChE-R) is causally involved in stress-related behaviors, and transgenic mice constitutively overexpressing AChE-R (TgR) show behaviors characteristic of chronic stress. We measured anxiety-like behavior on TgR and control mice under normal conditions and under long-term nicotine treatment. In addition, we measured epibatidine binding in the brain and transcription status in the striatum, using microarrays, in wild-type and TgR mice. TgR mice behaved as more anxious than controls, an effect normalized by long-term nicotine intake. In control mice, long-term nicotine augmented epibatidine binding in several areas of the brain, including the hippocampus and striatum. In TgR transgenics, long-term nicotine increased epibatidine binding in some areas but not in the hippocampus or the striatum. Because the striatum is involved in the mechanisms of drug addiction, we studied how the transgene affected striatal gene expression. Whole-genome DNA microarray showed that 23 transcripts were differentially expressed in TgR mouse striata, including 15 known genes, 7 of which are anxiety-related. Subsequent reverse-transcriptase polymerase chain reaction validated changes in 7 of those 15 genes, confirmed the increase trend in 5 more transcripts, and further revealed changes in 5 genes involved in cholinergic signaling. In summary, we found that nicotine acts as an anxiolytic in TgR mice but not in control mice and that continuously overexpressed AChE-R regulates striatal gene expression, modulating cholinergic signaling and stress-related pathways.

N-Acetylcholinesterase-Induced Apoptosis in Alzheimer's Disease

BackgroundAlzheimer's disease (AD) involves loss of cholinergic neurons and Tau protein hyper-phosphorylation. Here, we report that overexpression of an N-terminally extended “synaptic” acetylcholinesterase variant, N-AChE-S is causally involved in both these phenomena.Methodology and Principal FindingsIn transfected primary brain cultures, N-AChE-S induced cell death, morphological impairments and caspase 3 activation. Rapid internalization of fluorescently labeled fasciculin-2 to N-AChE-S transfected cells indicated membranal localization. In cultured cell lines, N-AChE-S transfection activated the Tau kinase GSK3, induced Tau hyper-phosphorylation and caused apoptosis. N-AChE-S-induced cell death was suppressible by inhibiting GSK3 or caspases, by enforced overexpression of the anti-apoptotic Bcl2 proteins, or by AChE inhibition or silencing. Moreover, inherent N-AChE-S was upregulated by stressors inducing protein misfolding and calcium imbalances, both characteristic of AD; and in cortical tissues from AD patients, N-AChE-S overexpression coincides with Tau hyper-phosphorylation.ConclusionsTogether, these findings attribute an apoptogenic role to N-AChE-S and outline a potential value to AChE inhibitor therapeutics in early AD.

The role of the read through variant of acetylcholinesterase in anxiogenic effects of predator stress in mice

This study examined the role of the read through variant of acetylcholinesterase (AChE-R) in lasting changes in murine affective behavior produced by a brief predator stress. AChE-R is elevated by stress in limbic cholinergic circuits implicated in anxiogenic effects of predator stress. The expression of AChE-R was blocked with a systemically administered central acting antisense oligonucleotide for AChE-R (EN101). EN101 was injected at multiple points prior to and after a predator stress in male C57 mice. Seven days after the last injection, behavior was tested. Predator stress caused a significant increase in startle amplitude, which EN101 blocked. This effect was specific to EN101, as the negative control inactive form of EN101, INVEN101 was without effect on stress effects on startle. Neither EN101 nor INVEN101 altered the anxiogenic effects of predator stress on behavior in the elevated plus maze, and both drugs partially reduced stress suppression of time active in the hole board. In the light dark box test, INVEN101 exhibited a weak block of stress effects on behavior for reasons which are unclear. Taken together, findings support the view that multiple neural systems are responsible for the different changes in behavior produced by predator stress. Present findings also suggest a role for AChE-R in specific anxiogenic (hyperarousal) effects following predator stress. Since AChE-R manipulations took place starting 23 h prior to predator stress and continued 48 h after predator stress, further research is necessary to determine the role of AChE-R in initiation and/or consolidation of hyperarousal effects of predator stress.

Habituation, discrimination and anxiety in transgenic mice overexpressing acetylcholinesterase splice variants

TgS and TgR transgenic mice overexpress different splice variants of acetylcholinesterase and serve as models for genetic disruption of the cholinergic system. Whereas the TgS mouse overexpresses synaptic AChE, the TgR mouse overexpresses the rare readthrough variant whose C-terminal lacks the cysteine residue which permits adherence to the membrane. The two genotypes were compared to the parent strain, FVB/N mice on locomotion, discrimination learning and anxiety behavior following two exposures to the elevated plus maze. Male TgS mice were slower to acquire a simple odor discrimination, failed to habituate to a novel environment but were not impaired on reversal or set shifting compared to the FVB/N or TgR mice. In addition, TgS mice showed less avoidance behavior on the first exposure and but less exploration on the second exposure to the EPM. TgR mice were not impaired on discrimination learning; however, the females showed excessive running in circles in the activity meter. The findings suggest that the effects of overexpression of AChE are unique to different splice variants and may be sex-dependent.

Translational control of recombinant human acetylcholinesterase accumulation in plants

BackgroundCodon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content.ResultsWe demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA.ConclusionOur findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well.