Stoichiometry of the Heteromeric Nicotinic Receptors of the Renshaw Cell.

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers built from a variety of subunits. Some are homomeric assemblies of α subunits, others heteromeric assemblies of α and ß subunits which can adopt two stoichiometries (2α:3ß or 3α:2ß). There is evidence for the presence of heteromeric nAChRs with the two stoichiometries in the CNS, but it has not yet been possible to identify them at a given synapse. The 2α:3ß receptors are highly sensitive to agonists, whereas the 3α:2ß stoichiometric variants, initially described as low sensitivity receptors, are indeed activated by low and high concentrations of ACh. We have taken advantage of the discovery that two compounds (NS9283 and Zn) potentiate selectively the 3α:2ß nAChRs to establish (in mice of either sex) the presence of these variants at the motoneuron-Renshaw cell (MN-RC) synapse. NS9283 prolonged the decay of the two-component EPSC mediated by heteromeric nAChRs. NS9283 and Zn also prolonged spontaneous EPSCs involving heteromeric nAChRs, and one could rule out prolongations resulting from AChE inhibition by NS9283. These results establish the presence of 3α:2ß nAChRs at the MN-RC synapse. At the functional level, we had previously explained the duality of the EPSC by assuming that high ACh concentrations in the synaptic cleft account for the fast component and that spillover of ACh accounts for the slow component. The dual ACh sensitivity of 3α:2ß nAChRs now allows to attribute to these receptors both components of the EPSC.SIGNIFICANCE STATEMENT Heteromeric nicotinic receptors assemble α and ß subunits in pentameric structures, which can adopt two stoichiometries: 3α:2ß or 2α:3ß. Both stoichiometric variants are present in the CNS, but they have never been located and characterized functionally at the level of an identified synapse. Our data indicate that 3α:2ß receptors are present at the spinal cord synapses between motoneurons and Renshaw cells, where their dual mode of activation (by high concentrations of ACh for synaptic receptors, by low concentrations of ACh for extrasynaptic receptors) likely accounts for the biphasic character of the synaptic current. More generally, 3α:2ß nicotinic receptors appear unique by their capacity to operate both in the cleft of classical synapses and at extrasynaptic locations.

Sostdc1 is expressed in all major compartments of developing and adult mammalian eyes.

PURPOSE: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. METHODS: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. RESULTS: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels.  CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/ß-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.

Slow-binding inhibition of acetylcholinesterase by an alkylammonium derivative of 6-methyluracil: mechanism and possible advantages for myasthenia gravis treatment.

Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme·inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 µM; Ki'=3.17 µM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 Å resolution. The complex is stabilized by cation-π, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, τ=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.

The C5 Variant of the Butyrylcholinesterase Tetramer Includes a Noncovalently Bound 60 kDa Lamellipodin Fragment.

Humans with the C5 genetic variant of butyrylcholinesterase (BChE) have 30-200% higher plasma BChE activity, low body weight, and shorter duration of action of the muscle relaxant succinylcholine. The C5 variant has an extra, slow-moving band of BChE activity on native polyacrylamide gel electrophoresis. This band is about 60 kDa larger than wild-type BChE. Umbilical cord BChE in 100% of newborn babies has a C5-like band. Our goal was to identify the unknown, 60 kDa protein in C5. Both wild-type and C5 BChE are under the genetic control of two independent loci, the BCHE gene on Chr 3q26.1 and the RAPH1 (lamellipodin) gene on Chr 2q33. Wild-type BChE tetramers are assembled around a 3 kDa polyproline peptide from lamellipodin. Western blot of boiled C5 and cord BChE showed a positive response with an antibody to the C-terminus of lamellipodin. The C-terminal exon of lamellipodin is about 60 kDa including an N-terminal polyproline. We propose that the unknown protein in C5 and cord BChE is encoded by the last exon of the RAPH1 gene. In 90% of the population, the 60 kDa fragment is shortened to 3 kDa during maturation to adulthood, leaving only 10% of adults with C5 BChE.

Schwann cells sense and control acetylcholine spillover at the neuromuscular junction by α7 nicotinic receptors and butyrylcholinesterase.

Terminal Schwann cells (TSCs) are key components of the mammalian neuromuscular junction (NMJ). How the TSCs sense the synaptic activity in physiological conditions remains unclear. We have taken advantage of the distinct localization of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at the NMJ to bring out the function of different ACh receptors (AChRs). AChE is clustered by the collagen Q in the synaptic cleft and prevents the repetitive activation of muscle nicotinic AChRs. We found that BChE is anchored at the TSC by a proline-rich membrane anchor, the small transmembrane protein anchor of brain AChE. When BChE was specifically inhibited, ACh release was significant depressed through the activation of α7 nAChRs localized on the TSC and activated by the spillover of ACh. When both AChE and BChE were inhibited, the spillover increased and induced a dramatic reduction of ACh release that compromised the muscle twitch triggered by the nerve stimulation. α7 nAChRs at the TSC may act as a sensor for spillover of ACh adjusted by BChE and may represent an extrasynaptic sensor for homeostasis at the NMJ. In myasthenic rats, selective inhibition of AChE is more effective in rescuing muscle function than the simultaneous inhibition of AChE and BChE because the concomitant inhibition of BChE counteracts the positive action of AChE inhibition. These results show that inhibition of BChE should be avoided during the treatment of myasthenia and the pharmacological reversal of residual curarization after anesthesia.

Optimal detection of cholinesterase activity in biological samples: modifications to the standard Ellman's assay.

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

An effective solution for capturing the single twitch of muscle: application to monitor muscle relaxation.

In this study, a fast algorithm was developed to capture of train of four and to filter extra contraction and noises. A low pass filter created to filter extra contraction and high frequency noises. Then, a TCA algorithm designed to capturing of the single twitch of muscle. The algorithm updated to remove embedded extra contraction and to derive boundary values in this location from cubic spline interpolation. Efficiency of TCA and effect of extra contraction tested in time and frequency domain.

Mechanisms shaping the slow nicotinic synaptic current at the motoneuron-renshaw cell synapse.

In spinal cord slices from newborn mice we have analyzed the kinetics of the EPSCs mediated by heteromeric nicotinic receptors at the motoneuron-Renshaw cell (MN-RC) synapse. The miniature EPSCs decay with a time constant of 13.0 ± 1.1 ms whereas the decay of the evoked EPSCs (eEPSCs) is biphasic, with time constants of 15.6 ± 0.8 and 124.8 ± 9.0 ms. The slow component becomes prominent during a repetitive stimulation, but its time constant is unchanged. It is selectively reduced by the addition of acetylcholinesterase (AChE), and thus appears to involve ACh spillover. The constancy of the slow time constant during a train is best explained by a local spillover activating high-affinity receptors. In many cells a fraction of the eEPSC originates in neighboring RCs and is transmitted by the low-pass filter of the gap junctions. The component transmitted electrically can be eliminated by meclofenamic acid, a blocker of gap junctions. The local spillover produced by a repetitive stimulation was compared with the long-range spillover produced by inactivation of AChE. The pharmacological inactivation of AChE by neostigmine caused the appearance of an ultra-slow (second range) decay component in eEPSCs and also a continuous inward current interpreted as resulting from a continuous ACh presence. In animals lacking functional AChE in the CNS (PRiMA(-/-) mice) the EPSCs resembled those observed in neostigmine but the steady inward current was much smaller, suggesting an adaptation to the absence of AChE.

Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular junction via N-methyl-D-aspartate receptor activation.

Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N-methyl-d-aspartate (NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide (NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by N(x)-nitro-L-arginine methyl ester, and NMDA receptor blockade by DL-2-amino-5-phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High-frequency (10 Hz) motor nerve stimulation in a glycine-containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co-mediator of acetylcholine, is capable of triggering the NMDA receptor/NO synthase-mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well-established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity.

Protein-anchoring strategy for delivering acetylcholinesterase to the neuromuscular junction.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq(-/-) mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq(-/-) mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq(-/-) mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules.

Unsupervised classification of plethysmography signals with advanced visual representations.

Ventilation is a simple physiological function that ensures the vital supply of oxygen and the elimination of CO2. The recording of the airflow through the nostrils of a mouse over time makes it possible to calculate the position of critical points, based on the shape of the signals, to compute the respiratory frequency and the volume of air exchanged. These descriptors only account for a part of the dynamics of respiratory exchanges. In this work we present a new algorithm that directly compares the shapes of signals and considers meaningful information about the breathing dynamics omitted by the previous descriptors. The algorithm leads to a new classification of inspiration and expiration, which reveals that mice respond and adapt differently to inhibition of cholinesterases, enzymes targeted by nerve gas, pesticide, or drug intoxication.

Near-complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase.

Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA). The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE- deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete.

A new mouse model for the slow-channel congenital myasthenic syndrome induced by the AChR εL221F mutation.

We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ε subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow-channel congenital myasthenic syndrome with variable penetrance. In our mouse model we observe as in human patients prolonged endplate currents. The summation of endplate potentials may account for a depolarization block at increasing stimulus frequencies, moderate reduced muscle strength and tetanic fade. Calcium and intracellular vesicle accumulation as well as junctional fold loss and organelle degeneration underlying a typical endplate myopathy, were identified. Moreover, a remodeling of neuromuscular junctions occurs in a muscle-dependent pattern expressing variable phenotypic effects. Altogether, this mouse model provides new insight into the pathophysiology of congenital myasthenia and serves as a new tool for deciphering signaling pathways induced by excitotoxicity at peripheral synapses.

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.

Unsupervised study of plethysmography signals through DTW clustering.

The study of plethysmography time series is crucial to better understand the breathing behavior of mice, in particular the influence of neurotoxins on the respiratory system. Current approaches rely on a few respiratory descriptors computed on individual breathing cycles that fail to account for the variety of breathing habits and their evolution with time. In this paper we introduce a new procedure for the automatic analysis of plethysmography signals. Our method relies on a new and robust segmentation of respiratory cycles and a DTW-based clustering algorithm to extract the most typical respiratory cycles (called reference sequences). We can then create a symbolic representation of any new recording by matching respiratory cycles to their closest reference sequence. This new representation is a visual and quantitative tool to assess the breathing behavior of mice and its evolution with time. Our method is applied to plethysmography signals collected on mice with two different genotypes and exposed to a neurotoxin. Clinical relevance This article proposes a novel approach to study plethysmography data. Our algorithm is able to accurately extract clinically meaningful respiratory cycles and the associated ventilation patterns descriptors such as tidal volume and inhalation/exhalation duration. In addition, thanks to the associated symbolic representation of signals, the temporal evolution of respiration is easily quantified. This opens a new research path to study the often slowly evolving and subtle influence of neurotoxins on the respiratory system.

Cholinesterases in Tripartite Neuromuscular Synapse.

The neuromuscular junction (NMJ) is a tripartite synapse in which not only presynaptic and post-synaptic cells participate in synaptic transmission, but also terminal Schwann cells (TSC). Acetylcholine (ACh) is the neurotransmitter that mediates the signal between the motor neuron and the muscle but also between the motor neuron and TSC. ACh action is terminated by acetylcholinesterase (AChE), anchored by collagen Q (ColQ) in the basal lamina of NMJs. AChE is also anchored by a proline-rich membrane anchor (PRiMA) to the surface of the nerve terminal. Butyrylcholinesterase (BChE), a second cholinesterase, is abundant on TSC and anchored by PRiMA to its plasma membrane. Genetic studies in mice have revealed different regulations of synaptic transmission that depend on ACh spillover. One of the strongest is a depression of ACh release that depends on the activation of α7 nicotinic acetylcholine receptors (nAChR). Partial AChE deficiency has been described in many pathologies or during treatment with cholinesterase inhibitors. In addition to changing the activation of muscle nAChR, AChE deficiency results in an ACh spillover that changes TSC signaling. In this mini-review, we will first briefly outline the organization of the NMJ. This will be followed by a look at the role of TSC in synaptic transmission. Finally, we will review the pathological conditions where there is evidence of decreased AChE activity.

Targeting of acetylcholinesterase in neurons in vivo: a dual processing function for the proline-rich membrane anchor subunit and the attachment domain on the catalytic subunit.

Acetylcholinesterase (AChE) accumulates on axonal varicosities and is primarily found as tetramers associated with a proline-rich membrane anchor (PRiMA). PRiMA is a small transmembrane protein that efficiently transforms secreted AChE to an enzyme anchored on the outer cell surface. Surprisingly, in the striatum of the PRiMA knock-out mouse, despite a normal level of AChE mRNA, we find only 2-3% of wild type AChE activity, with the residual AChE localized in the endoplasmic reticulum, demonstrating that PRiMA in vivo is necessary for intracellular processing of AChE in neurons. Moreover, deletion of the retention signal of the AChE catalytic subunit in mice, which is the domain of interaction with PRiMA, does not restore AChE activity in the striatum, establishing that PRiMA is necessary to target and/or to stabilize nascent AChE in neurons. These unexpected findings open new avenues to modulating AChE activity and its distribution in CNS disorders.

Evidence of a dosage effect and a physiological endplate acetylcholinesterase deficiency in the first mouse models mimicking Schwartz-Jampel syndrome neuromyotonia.

Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.

MuSK is required for anchoring acetylcholinesterase at the neuromuscular junction.

At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.

Influence of differential expression of acetylcholinesterase in brain and muscle on respiration.

A mouse strain with a deleted acetylcholinesterase (AChE) gene (AChE knockout) shows a decreased inspiration time and increased tidal volume and ventilation .To investigate the respective roles of AChE in brain and muscle, we recorded respiration by means of whole-body plethysmography in knockout mice with tissue selective deletions in AChE expression. A mouse strain with the anchoring domains of AChE deleted (del E5+6 knockout mice) has very low activity in the brain and neuromuscular junction, but increased monomeric AChE in serum. A mouse strain with deletion of the muscle specific region of AChE (del i1RR knockout mice) exhibits no expression in muscle, but unaltered expression in the central nervous system. Neither strain exhibits the pronounced phenotypic traits observed in the complete AChE knockout strain. A third strain lacking the anchor molecule PRiMA, has no functional AChE and butyrylcholinesterase (BChE) in brain and an unaltered respiratory function. BChE inhibition by bambuterol decreases tidal volume and body temperature in del E5+6 and i1RR knockout strains, but not in PRiMA deletion or wild-type controls. We find that: (1) deletion of the full AChE gene is required for a pronounced alteration in respiratory phenotype, (2) BChE is involved in respiratory muscles contraction and temperature control in del E5+6 and i1RR knockout mice, and (3) AChE expression requiring a gene product splice to either exons 5 and 6 or regulated by intron1 influences temperature control.