Search

Two near infra-red (NIR) fluorescent probes HupNIR1 and HupNIR2 based on the huprine scaffold and cyanine 5.0 dye have been synthesised and evaluated in situ for the detection of acetylcholinesterases in different tissues. As anticipated by the initial properties of huprine, both probes displayed a high affinity and selectivity for AChE toward BChE, with IC50 values in the nanomolar range and without any non-specific binding in the tissues. HupNIR2 appears the best probe for AChE with a great selectivity and sensitivity for AChE even in the brain region displaying a low AChE concentration as striatum. Moreover, the binding of HupNIR2 is affected when AChE is inhibited with toxic molecules such as organophosphates. This work provides a new tool to visualize active AChE in biological applications.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

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.

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.

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.

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.

At the vertebrate skeletal neuromuscular junction (NMJ), two closely related enzymes can hydrolyze acetylcholine (ACh): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Advances in mouse genomics offer new approaches to assess the role of specific cholinesterases involved in neuromuscular transmission (Minic et al., 2003). AChE knockout mice provide a valuable tool for examining the effects of long-term complete and selective abolition of AChE activity (Xie et al., 2000). AChE and BChE genes encode two functional domains--the catalytic domain (exons 2, 3, and 4 of AChE, or exon 2 of BChE) and a C-terminal domain (exon 5 or 6 of AChE, or exon 3 of BChE)--that dictate the targeting of the enzymes (Massoulié, 2002). In mammals, the AChE gene produces three types of coding regions by deleting 5'- splice acceptor sites, which generate proteins; these proteins possess the same catalytic domain associated with distinct C-terminal peptides. AChE subunits of type R (readthrough) produce soluble monomers; they are expressed during development and are thought to be induced in the mouse brain by stress (Kaufer et al., 1998). AChE subunits of type H (hydrophobic) produce GPI-anchored dimers, mainly in blood cells. Subunits of type T (tailed) exist for both AChE and BChE. They represent the predominant AChE variant expressed in cholinergically innervated tissues (muscle and nerve). These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins ColQ (Krejci et al., 1997) and PRiMA (Perrier et al., 2002). At the NMJ, AChE is clustered by the interaction of the coding sequence of exon 6 with ColQ (Feng et al., 1999). The deletion of exons 5 and 6 in the AChE gene transforms anchored AChE into a soluble enzyme (Camp et al., 2004). The present study was designed to evaluate neuromuscular transmission and nicotinic ACh receptor (nAChR) distribution in muscles from mutant mice with deletions of these two spliced exons (AChE-del-exons-5+6-/-).

We have studied the consequences of the constitutive acetylcholinesterase (AChE) deficiency in knockout mice for the AChE gene on the subcellular localization of the m2 receptor (m2R) and the regulation of its intraneuronal compartmentalization by the cholinergic environment, using immunohistochemistry at light and electron microscopic levels. (1) In AChE +/+ mice in vivo, m2R is mainly located at the neuronal membrane in striatum, hippocampus, and cortex. In AChE -/- mice, m2R is almost absent at the membrane but is accumulated in the endoplasmic reticulum and Golgi complex. (2) In vivo and in vitro (organotypic culture) dynamic studies demonstrate that the balance between membrane and intracytoplasmic m2R can be regulated by the cholinergic influence: In AChE -/- mice, m2R is translocated from the cytoplasm to the cell surface after (1) blockade of muscarinic receptors by atropine, (2) supplementation of AChE -/- neurons with AChE in vitro, and (3) disruption of the cortical and hippocampal cholinergic afferents in vitro. Our results suggest that the neurochemical environment may contribute to the control of the abundance and availability of cell surface receptors, and consequently to the control of neuronal sensitivity to neurotransmitters or drugs, by regulating their delivery from the endoplasmic reticulum and Golgi complex.

Acetylcholinesterase inhibitors (AChE-I) of various pharmacological classes have been used to provoke acute and chronic hypercholinergy in brain. Each condition induces a dramatic decrease of the abundance of muscarinic receptors at the membrane of neurons with simultaneous increase of these receptors in the cytoplasm in association with different subcellular organelles with characteristics depending on the duration of the treatment (short-term versus long term treatment). Each condition also induces a dramatic increase of cytoplasmic receptors associated with endosomes and multivesicular bodies. Chronic treatment with MTF induces a general decrease of m4R in the striatum without modification of the mRNA level but with an exaggerated abundance of muscarinic receptors in the cytoplasm at the sites of synthesis and maturation, i.e., endoplasmic reticulum, nuclear membrane and Golgi apparatus. These results suggest that the membrane abundance and intraneuronal distribution of neurotransmitter receptors are modified following drug treatment with specificity depending on the nature and the duration of treatment., ((c) 2002 Elsevier Science (USA).)

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. [ABSTRACT FROM AUTHOR]

Abstract: Schwartz-Jampel syndrome (SJS) is a recessive disorder with muscle hyperactivity that results from hypomorphic mutations in the perlecan gene, a basement membrane proteoglycan. Analyses done on a mouse model have suggested that SJS is a congenital form of distal peripheral nerve hyperexcitability resulting from synaptic acetylcholinesterase deficiency, nerve terminal instability with preterminal amyelination, and subtle peripheral nerve changes. We investigated one adult patient with SJS to study this statement in humans. Perlecan deficiency due to hypomorphic mutations was observed in the patient biological samples. Electroneuromyography showed normal nerve conduction, neuromuscular transmission, and compound nerve action potentials while multiple measures of peripheral nerve excitability along the nerve trunk did not detect changes. Needle electromyography detected complex repetitive discharges without any evidence for neuromuscular transmission failure. The study of muscle biopsies containing neuromuscular junctions showed well-formed post-synaptic element, synaptic acetylcholinesterase deficiency, denervation of synaptic gutters with reinnervation by terminal sprouting, and long nonmyelinated preterminal nerve segments. These data support the notion of peripheral nerve hyperexcitability in SJS, which would originate distally from synergistic actions of peripheral nerve and neuromuscular junction changes as a result of perlecan deficiency. [Copyright &y& Elsevier]