Neuron-glia interactions: the roles of Schwann cells in neuromuscular synapse formation and function

The NMJ (neuromuscular junction) serves as the ultimate output of the motor neurons. The NMJ is composed of a presynaptic nerve terminal, a postsynaptic muscle and perisynaptic glial cells. Emerging evidence has also demonstrated an existence of perisynaptic fibroblast-like cells at the NMJ. In this review, we discuss the importance of Schwann cells, the glial component of the NMJ, in the formation and function of the NMJ. During development, Schwann cells are closely associated with presynaptic nerve terminals and are required for the maintenance of the developing NMJ. After the establishment of the NMJ, Schwann cells actively modulate synaptic activity. Schwann cells also play critical roles in regeneration of the NMJ after nerve injury. Thus, Schwann cells are indispensable for formation and function of the NMJ. Further examination of the interplay among Schwann cells, the nerve and the muscle will provide insights into a better understanding of mechanisms underlying neuromuscular synapse formation and function.     

Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.     

Muscle specific kinase: organiser of synaptic membrane domains

Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ.     

Glutamatergic innervation of rat skeletal muscle by supraspinal neurons: a new paradigm in spinal cord injury repair

Acetylcholine is the specific chemical code of spinal nerve terminal transmission at the mammalian neuromuscular junction (NMJ), whereas nicotinic acetylcholine receptors inserted into the membrane of muscle fibres mediate signalling for the muscle response. Glutamate has a primary role in neuromuscular transmission of organisms that are phylogenetically distant from mammals, the invertebrates, including insect and molluscs. Recent research has shown that diverting descending glutamatergic fibres in the spinal cord to rat skeletal muscle by means of a peripheral nerve graft causes the cholinergic synapse to switch to the glutamatergic type. These data demonstrate that under appropriate surgical manipulation supraspinal neurons can directly target muscle fibres and specify the postsynaptic receptors to achieve a functional glutamatergic NMJ.     

Motor nerve terminal loss from degenerating muscle fibers

The fate of nerve terminals following elimination of postsynaptic target cells was studied in living mouse muscle. Several days after muscle fiber damage, observations of previously identified neuromuscular junctions showed that motor nerve terminal branches had rapidly disappeared from degenerating muscle fibers. Following muscle fiber regeneration, loss of terminal branches ceased and nerve terminals regrew, reestablishing some of the original sites and adding new branches. The distribution of acetylcholine receptors reorganized in the regenerated muscle so that perfect alignment was reestablished with the newly configured nerve terminals. These results argue that the maintenance of the full complement of nerve terminal branches at a neuromuscular junction is dependent on the presence of a healthy muscle fiber. Similarly, regenerating muscle is dependent on the nerve terminal for the organization and maintenance of postsynaptic receptors.     

The Neuromuscular Junction: Measuring Synapse Size,Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.     

Formation of the neuromuscular junction: molecules and mechanisms

The vertebrate skeletal neuromuscular junction is the site at which motor neurons communicate with their target muscle fibers. At this synapse, as at synapses throughout the nervous system, efficient and appropriate communication requires the formation and precise alignment of specializations for transmitter release in the axon terminal with those for transmitter detection in the postsynaptic cell. Classical developmental studies demonstrate that synapse formation at the neuromuscular junction is a mutually inductive event; neurons induce postsynaptic differentiation in muscle cells and myofibers induce presynaptic differentiation in motor axon terminals. More recent experiments indicate that Schwann cells, which cap axon terminals, also play an active role in the formation and maintenance of the neuromuscular junction. Here, we review recent advances in the identification of molecules mediating such inductive interactions and the mechanisms by which they produce their effects. Although our discussion concerns events at developing neuromuscular junctions, it seems likely that similar molecules and mechanisms may act at neuron–neuron synapses in the peripheral as well as the central nervous system. BioEssays 20 :819–829, 1998. © 1998 John Wiley & Sons, Inc.     

Mesdc2 plays a key role in cell-surface expression of Lrp4 and postsynaptic specialization in myotubes

Low-density lipoprotein receptor-related protein 4 (Lrp4) is essential for pre- and post-synaptic specialization at the neuromuscular junction (NMJ), an indispensable synapse between a motor nerve and skeletal muscle. Muscle-specific receptor tyrosine kinase MuSK must form a complex with Lrp4 to organize postsynaptic specialization at NMJs. Here, we show that the chaperon Mesdc2 binds to the intracellular form of Lrp4 and promotes its glycosylation and cell-surface expression. Furthermore, knockdown of Mesdc2 suppresses cell-surface expression of Lrp4, activation of MuSK, and postsynaptic specialization in muscle cells. These results suggest that Mesdc2 plays an essential role in NMJ formation by promoting Lrp4 maturation.     

The signaling pathways mediated by P2Y nucleotide receptors in the formation and maintenance of the skeletal neuromuscular junction

The motor neuron, the Schwann cell and the muscle cell are highly specialized at the vertebrate skeletal neuromuscular junction (NMJ). The muscle cell surface contains a high local density of acetylcholine (ACh) receptors (AChRs), acetylcholinesterase (AChE) and their interacting macromolecules at the NMJ, forming the postsynaptic specializations. During the early stages of development, the incoming nerve terminal induces the formation of these postsynaptic specializations; the nerve secretes agrin and neuregulin (NRG), which are known to aggregate existing AChRs and to increase the expression of AChR at the synaptic region, respectively. In addition, adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is coreleased with ACh during muscle contraction. Recent evidence suggests that ATP can play a role in forming and maintaining the postsynaptic specializations by activating its corresponding receptors. In particular, one of the nucleotide receptor subtypes, the P2Y(1) receptor, is specifically localized at the NMJs. The gene expression of AChR and AChE is upregulated after the activation of P2Y(1) receptors. Thus, the synaptic ATP together with agrin and NRG can act as a synapse-organizing factor to induce the expression of postsynaptic functional effectors.     

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.     

Postsynaptic signaling of new players at the neuromuscular junction

The neuromuscular junction (NMJ) represents the most well studied synapse and is widely regarded as structurally and functionally less complicated than neuronal synapses in the brain. Recent studies, however, have identified the localization and function of new signaling molecules at the NMJ. Surprisingly, many synaptic proteins previously identified in the brain are indeed also concentrated on the postsynaptic muscle side of the NMJ. These include the serine/threonine kinase Cdk5, the neurotrophin receptor TrkB, Eph receptors and ephrins, NMDA receptors and nitric oxide synthase, various PDZ-domain scaffold proteins, and β-amyloid precursor protein. These observations indicate that the molecular composition of NMJ is much more intricate than we originally thought. The potential significance of these new signaling molecules at the NMJ will be discussed.     

Processing of ARIA and release from isolated nerve terminals

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage-gated Na+ channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage-gated Na+ channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo. ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.     

Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations

Synapse formation requires the coordination of pre- and postsynaptic differentiation. An unresolved question is which steps in the process require interactions between pre- and postsynaptic cells, and which proceed cell-autonomously. One current model is that factors released from presynaptic axons organize postsynaptic differentiation directly beneath the nerve terminal. Here, we used neuromuscular junctions (NMJs) of the zebrafish primary motor system to test this model. Clusters of neurotransmitter (acetylcholine) receptors (AChRs) formed in the central region of the myotome, destined to be synapse-rich, before axons extended and even when axon extension was prevented. Time-lapse imaging revealed that pre-existing clusters on early-born slow (adaxial) muscle fibers were incorporated into NMJs as axons advanced. Axons were, however, required for the subsequent remodeling and selective stabilization of synaptic clusters that precisely appose post- to presynaptic elements. Thus, motor axons are dispensable for the initial stages of postsynaptic differentiation but are required for later stages. Moreover, many AChR clusters on later-born fast muscle fibers formed at sites that had already been contacted by axons, suggesting heterogeneity in the signaling mechanisms leading to synapse formation by a single axon.     

Glycosylated synaptomatrix regulation of trans-synaptic signaling

Synapse formation is driven by precisely orchestrated intercellular communication between the presynaptic and the postsynaptic cell, involving a cascade of anterograde and retrograde signals. At the neuromuscular junction (NMJ), both neuron and muscle secrete signals into the heavily glycosylated synaptic cleft matrix sandwiched between the two synapsing cells. These signals must necessarily traverse and interact with the extracellular environment, for the ligand-receptor interactions mediating communication to occur. This complex synaptomatrix, rich in glycoproteins and proteoglycans, comprises heterogeneous, compartmentalized domains where specialized glycans modulate trans-synaptic signaling during synaptogenesis and subsequent synapse modulation. The general importance of glycans during development, homeostasis and disease is well established, but this important molecular class has received less study in the nervous system. Glycan modifications are now understood to play functional and modulatory roles as ligands and co-receptors in numerous tissues; however, roles at the synapse are relatively unexplored. We highlight here properties of synaptomatrix glycans and glycan-interacting proteins with key roles in synaptogenesis, with a particular focus on recent advances made in the Drosophila NMJ genetic system. We discuss open questions and interesting new findings driving this investigation of complex, diverse, and largely understudied glycan mechanisms at the synapse.     

Dynactin is necessary for synapse stabilization

We present evidence that synapse retraction occurs during normal synaptic growth at the Drosophila neuromuscular junction (NMJ). An RNAi-based screen to identify the molecular mechanisms that regulate synapse retraction identified Arp-1/centractin, a subunit of the dynactin complex. Arp-1 dsRNA enhances synapse retraction, and this effect is phenocopied by a mutation in P150/Glued, also a dynactin component. The Glued protein is enriched within the presynaptic nerve terminal, and presynaptic expression of a dominant-negative Glued transgene enhances retraction. Retraction is associated with a local disruption of the synaptic microtubule cytoskeleton. Electrophysiological, ultrastructural, and immunohistochemical data support a model in which presynaptic retraction precedes disassembly of the postsynaptic apparatus. Our data suggests that dynactin functions locally within the presynaptic arbor to promote synapse stability.     

Regulation and restoration of motoneuronal synaptic transmission during neuromuscular regeneration in the pulmonate snail Helisoma trivolvis

Regeneration of motor systems involves reestablishment of central control networks, reinnervation of muscle targets by motoneurons, and reconnection of neuromodulatory circuits. Still, how these processes are integrated as motor function is restored during regeneration remains ill defined. Here, we examined the mechanisms underlying motoneuronal regeneration of neuromuscular synapses related to feeding movements in the pulmonate snail Helisoma trivolvis. Neurons B19 and B110, although activated during different phases of the feeding pattern, innervate similar sets of muscles. However, the percentage of muscle fibers innervated, the efficacy of excitatory junction potentials, and the strength of muscle contractions were different for each cell's specific connections. After peripheral nerve crush, a sequence of transient electrical and chemical connections formed centrally within the buccal ganglia. Neuromuscular synapse regeneration involved a three-phase process: the emergence of spontaneous synaptic transmission (P1), the acquisition of evoked potentials of weak efficacy (P2), and the establishment of functional reinnervation (P3). Differential synaptic efficacy at muscle contacts was recapitulated in cell culture. Differences in motoneuronal presynaptic properties (i.e., quantal content) were the basis of disparate neuromuscular synapse function, suggesting a role for retrograde target influences. We propose a homeostatic model of molluscan motor system regeneration. This model has three restoration events: (1) transient central synaptogenesis during axonal outgrowth, (2) intermotoneuronal inhibitory synaptogenesis during initial neuromuscular synapse formation, and (3) target-dependent regulation of neuromuscular junction formation.     

Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures

Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest "fractionation of movement" capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.     

Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.     

β-Catenin stabilization in skeletal muscles, but not in motor neurons, leads to aberrant motor innervation of the muscle during neuromuscular development in mice

β-Catenin, a key component of the Wnt signaling pathway, has been implicated in the development of the neuromuscular junction (NMJ) in mice, but its precise role in this process remains unclear. Here we use a β-catenin gain-of-function mouse model to stabilize β-catenin selectively in either skeletal muscles or motor neurons. We found that β-catenin stabilization in skeletal muscles resulted in increased motor axon number and excessive intramuscular nerve defasciculation and branching. In contrast, β-catenin stabilization in motor neurons had no adverse effect on motor innervation pattern. Furthermore, stabilization of β-catenin, either in skeletal muscles or in motor neurons, had no adverse effect on the formation and function of the NMJ. Our findings demonstrate that β-catenin levels in developing muscles in mice are crucial for proper muscle innervation, rather than specifically affecting synapse formation at the NMJ, and that the regulation of muscle innervation by β-catenin is mediated by a non-cell autonomous mechanism.     

Therapeutic Potential of CKD-504, a Novel Selective Histone Deacetylase 6 Inhibitor,in a Zebrafish Model of Neuromuscular Junction Disorders

The neuromuscular junction (NMJ), which is a synapse for signal transmission from motor neurons to muscle cells, has emerged as an important region because of its association with several peripheral neuropathies. In particular, mutations in GARS that affect the formation of NMJ result in Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. These disorders are mainly considered to be caused by neuronal axon abnormalities; however, no treatment is currently available. Therefore, in order to determine whether the NMJ could be targeted to treat neurodegenerative disorders, we investigated the NMJ recovery effect of HDAC6 inhibitors, which have been used in the treatment of several peripheral neuropathies. In the present study, we demonstrated that HDAC6 inhibition was sufficient to enhance movement by restoring NMJ impairments observed in a zebrafish disease model. We found that CKD-504, a novel HDAC6 inhibitor, was effective in repairing NMJ defects, suggesting that treatment of neurodegenerative diseases via NMJ targeting is possible.