Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3'-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.     

High-resolution localization of acetylcholinesterase at the rat neuromuscular junction

To overcome the limited ultrastructural resolution of conventional acetylcholinesterase (AChE) ultrahistochemistry, acetylcholine (ATCh) was used to reduce the rate of enzymic thiocholine liberation. The conventionally limited resolution is mainly due to the high focal activity of the enzyme in neural structures, because cleavage of substrate is faster than histochemical trapping reactions. Therefore, using the copper-thiocholine method, we investigated the reduction of thiocholine liberation by acetylcholine (ACh). As examined biochemically, the apparent Ki for ACh was close to the Km for ATCh. The ACh/ATCh ratio, therefore, determined the reduction of thiocholine production in histochemical experiments. In addition, the morphological appearance of the precipitated reaction product after its changes during the histochemical procedure was monitored using electric eel AChE immobilized on Sepharose 4B. The improved fine structural resolution at 40- to 100-fold excess of ACh over ATCh is demonstrated at the neuromuscular junction of rat lumbricalis muscle. The highest focal enzyme activity is found at the presynaptic membrane and in the secondary cleft, but not on top of the junctional folds, indicating the separation of esterase and nicotinic receptors. The physiological events during neuromuscular transmission are discussed on the basis of the new "gradient switch hypothesis" suggested in this report.     

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.     

In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction

Recently, it has become possible to directly detect changes in neuropeptide vesicle dynamics in nerve terminals in vivo and to measure the release of neuropeptides induced experimentally or evoked by normal behavior. These results were obtained with the use of transgenic fruit flies that express a neuropeptide tagged with green fluorescent protein. Here, we describe how vesicle movement and neuropeptide release can be studied in the larval Drosophila neuromuscular junction using fluorescence microscopy. Analysis methods are described for quantifying movement based on time lapse and fluorescence recovery after photobleaching data. Specific approaches that can be applied to nerve terminals include single particle tracking, correlation and Fourier analysis. Utilization of these methods led to the first detection of vesicle mobilization in nerve terminals and the discoveries of activity-dependent capture of transiting vesicles and post-tetanic potentiation of neuropeptide release. Overall, this protocol can be carried out in an hour with ready Drosophila.     

Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction

Endplate 16S acetylcholinesterase (16S-AChE) from rat anterior gracilis muscle was assessed, 6 hr to 10 days after denervation, by velocity sedimentation analysis on linear sucrose gradients. The innervating obturator nerve was transected either close (1-2 mm, short stump) or far (35-40 mm, long stump) from the muscle. In both instances, the activity of 16S-AChE gradually decreased and reached approximately the same level (10%-20% of control) by 6 days after denervation. However, enzymatic decay started considerably earlier in short stump (12-24 hr) as compared to long stump (4-5 days) preparations, i.e., the time of onset of 16S-AChE loss depended on the length of nerve that remained attached to the muscle. Whether this result extended to other AChE molecular forms (10S, 4S) in muscle endplates could not be determined because, in contrast to 16S-AChE, these forms were also detected in red blood cells (4S) and plasma (10S). Only small amounts of 16S-AChE were found in intact obturator nerves (1/100 of that in gracilis endplate regions). Thus a faster depletion of enzyme from shorter nerve stumps after axotomy could not entirely account for the substantial effect of nerve stump length on 16S-AChE. Since muscle contraction ceases immediately following nerve transection, regardless of nerve stump length, the results can be ascribed to the lack of some neural influence other than nerve-evoked muscle activity. The present findings are consistent with the view that maintenance of 16S-AChE at neuromuscular junctions primarily depends on regulatory substances which are conveyed by axonal transport and released from nerve terminals.     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.     

Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction.

Endplate 16S acetylcholinesterase (16S-AChE) from rat anterior gracilis muscle was assessed, 6 hr to 10 days after denervation, by velocity sedimentation analysis on linear sucrose gradients. The innervating obturator nerve was transected either close (1--2 mm, short stump) or far (35--40 mm, long stump) from the muscle. In both instances, the activity of 16S-AChE gradually decreased and reached approximately the same level (10%--20% of control) by 6 days after denervation. However, enzymatic decay started considerably earlier in short stump (12--24 hr) as compared to long stump (4--5 days preparations, i.e., the time of onset of 16S-AChE loss depended on the length of nerve that remained attached to the muscle. Whether this result extended to other AChE molecular forms (10S, 4S) in muscle endplates could not be determined because, in contrast to 16S-AChE, these forms were also detected in red blood cells (4S) and plasma (10S). Only small amounts of 16S-AChE were found in intact obturator nerves (1/100 of that in gracilis endplate regions). Thus a faster depletion of enzyme from shorter nerve stumps after axotomy could not entirely account for the substantial effect of nerve stump length on 16S-AChE. Since muscle contraction ceases immediately following nerve transection, regardless of nerve stump length, the results can be ascribed to the lack of some neural influence other than nerve-evoked muscle activity. The present findings are consistent with the view that maintenance of 16SAChE at neuromuscular junctions primarily depends on regulatory substances which are conveyed by axonal transport and released from nerve terminals.     

Simultaneous labelling of basal lamina components and acetylcholinesterase at the neuromuscular junction

Summary A double labelling technique has been developed which permits the concomitant localization of basal lamina constituents together with acetylcholinesterase in mouse skeletal muscles. First, using the protein A-gold technique, type IV collagen and laminin were revealed on basal laminae ensheathing skeletal muscle fibres. The immunolabelling for both proteins was higher in synaptic than extrasynaptic regions. At synaptic sites the anti-type IV collagen immunolabelling exhibited an asymmetry; it was more intense on the portion of basal lamina closest to the postsynaptic membrane, whereas the anti-laminin immunolabelling was more uniformly distributed. It was also observed that the laminin immunoreactivity associated with Schwann and perineural cells was higher than that of skeletal muscle fibres. Secondly, the two basal lamina antigens were revealed simultaneously with another synaptic protein, acetylcholinesterase, using a refined cytochemical technique prior to the immunolabelling. The cytochemical reaction, which facilitates the location of endplates, did not alter the immunolabelling pattern. This double labelling procedure permits ready comparison of the distributions of type IV collagen and laminin with that of acetylcholinesterase, and may prove to be a useful approach in studies on synaptic components in developing and diseased muscle.     

Heparin solubilizes asymmetric acetylcholinesterase from rat neuromuscular junction

Citrate synthase from Acinetobacter calcoaceticus was subjected to proteolysis with subtilisin. Although the enzyme proved relatively resistant to inactivation by this treatment, SDS-polyacrylamide gel electrophoresis clearly revealed breakdown of the citrate synthase to smaller fragments. The regulatory responses of the native enzyme to inhibition by NADH and re-activation by AMP were retained on proteolysis, indicating that the fragments bind tightly to each other and preserve the overall cooperative molecular interactions.     

The dynamics of recycled acetylcholine receptors at the neuromuscular junction in vivo

At the peripheral neuromuscular junction (NMJ), a significant number of nicotinic acetylcholine receptors (AChRs) recycle back into the postsynaptic membrane after internalization to intermingle with not-yet-internalized ;pre-existing' AChRs. However, the way in which these receptor pools are maintained and regulated at the NMJ in living animals remains unknown. Here, we demonstrate that recycled receptors in functional synapses are removed approximately four times faster than pre-existing receptors, and that most removed recycled receptors are replaced by new recycled ones. In denervated NMJs, the recycling of AChRs is significantly depressed and their removal rate increased, whereas direct muscle stimulation prevents their loss. Furthermore, we show that protein tyrosine phosphatase inhibitors cause the selective accumulation of recycled AChRs in the peri-synaptic membrane without affecting the pre-existing AChR pool. The inhibition of serine/threonine phosphatases, however, has no effect on AChR recycling. These data show that recycled receptors are remarkably dynamic, and suggest a potential role for tyrosine dephosphorylation in the insertion and maintenance of recycled AChRs at the postsynaptic membrane. These findings may provide insights into long-term recycling processes at less accessible synapses in the central nervous system in vivo.     

Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction

Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.     

Electron cytochemistry of acetylcholinesterase in the neuromuscular junction during ontogenesis.

Electron histochemical studies were performed on neuromuscular junctions of the diaphragm in early postnatal states of development in rat by means of the acetylthiocholine technique. Presynaptic AChE appears to be derived from perikaryal sources, carried along by means of axonal transport mechanisms. Postsynaptic AChE is synthesized in the sarcotubular system of the underlying muscle fiber and within the perinuclear and endoplasmic reticulum of fundamental cells. Distribution of AChE synthesizing loci parallels with that of acetylcholine sensitive areas.     

N-CAM at the vertebrate neuromuscular junction

We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.     

Effects of diazepam at the neuromuscular junction

Diazepam (Valieum, Roche) is a centrally-acting drug belonging to the benzodiazepine group of tranquillisers with anxiolytic, hypnotic, anti-convulsant and myorelaxant properties (1). It has been reported that in addition to its central effects (1), diazepam also produces relaxation of the skeletal muscle (2, 3). The myorelaxation produced by diazepam is thought to be of central origin (2), although at least some of the effects is due to a peripheral effect of diazepam, i.e. at the neuromuscular junction.Although the effects and interactions of diazepam with neuromuscular blocking agents have been studied by many workers (2–12), the results reported are somehow are controversial (4–8). In sum, diazepam can either enhance or depress neuromuscular transmission, the effect being dependent on the concentration and the type of the preparation used. A multi-site of action of diazepam may provide an explanation for some of the anomalies reported in the literature.