Postnatal development of Schwann cells at neuromuscular junctions,with special reference to synapse elimination

The neuromuscular junctions (NMJs) of postnatal rat soleus muscles were examined by immunohistochemical staining for S100, a marker of Schwann cells (SCs), and for protein gene product 9.5, a neuronal marker, to elucidate the involvement of SCs in synapse elimination. The morphological maturation of S100-immunoreactive terminal SCs at NMJs proceeded with the gradual increase in their number. The number of terminal SCs per NMJ was one or two at postnatal day (P) 7, reaching the adult number at P28, when it became three or four. Confocal laser scanning microscopic analysis of multi-innervated NMJs, whose number decreased between P7 and P14, revealed a change in the ratio between terminal SCs and axons with age. At P7, the ratio between axons and terminal SCs per NMJ was ≥2:1, which was exactly the reverse of that in adults, while at P14 this had changed to 2:2. A structural change appeared to occur at the same time at the preterminal region, this being prior to the establishment of a 1:1 relationship between axon and SC sheath which was detected at P14, with the ≥2:1 relationship seeming to occur at P7. Thus, synapse elimination seems to proceed, at least for one week, with the gradual loss of axons which are at different stages of maturation with respect to their spatial relationship with SCs. From our results it seems unlikely that SCs play an active role in selecting a single axon to survive.     

Lose it to use it

In this issue, Wang et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.201911114) describe a phenomenon in which neuromuscular junction synapse elimination triggers myelination of terminal motor axon branches. They propose a mechanism initiated by synaptic pruning that depends on synaptic activity, cytoskeletal maturation, and the associated anterograde transport of trophic factors including Neuregulin 1-III.

Neuromuscular junctions (NMJs) are a favorite model system to study the development, maintenance, and function of neuronal synapses because of their accessibility, size, and simplicity. Although many synaptic mechanisms discovered at the peripheral NMJ have provided important insights into synaptic mechanisms in the central nervous system (CNS), the phenomena of synapse elimination and refinement remain poorly understood in both. In the peripheral nervous system (PNS), synapse elimination is an essential developmental step that removes redundant presynaptic inputs to the muscle fiber. In addition, peripheral motor axon terminals must become myelinated to facilitate rapid and synchronized acetylcholine release to the muscle fiber. However, whether these two essential events during PNS development are coordinately regulated remains unknown.The immature rodent NMJ is first innervated by many axons which are then removed until the synapse reaches a dually innervated state (1). These two axons then further compete for synaptic territory, leaving one “winner” that eventually occupies the motor endplate by the end of the second postnatal week. To determine the relationship between synapse elimination and myelination, Wang et al. (2) used the formation of paranodal junctions between axons and Schwann cells as a surrogate for myelination and then determined whether axons that occupied NMJs in a singly or dually innervated state were more or less likely to be myelinated. They found that when the NMJ is dually innervated, myelination of the terminal axon branch is inhibited; neither synaptic occupancy of the competing axons nor axon diameter influenced myelination. However, once synapse elimination at the NMJ is complete, i.e., a single axon terminal innervates the motor endplate, the winner branch becomes myelinated. Thus, synapse elimination precedes myelination of the terminal axon branch, and competition between dually innervated NMJs restricts myelination.What mechanisms regulate the coordinated maturation of the motor neuron, Schwann cell, and muscle circuit? Since previous studies showed that synapse elimination at the NMJ depends on muscle activity (3), Wang et al. (2) inhibited synapse elimination by blocking acetylcholine receptors with α-bungarotoxin (α-Btx). This inhibition of motor endplate and muscle activity increased not only the number of dually innervated NMJs, but also significantly decreased myelination of terminal axon branches of singly innervated NMJs. Thus, neuromuscular activity must induce retrograde signaling mechanisms that promote not only synapse elimination but also myelination.During synapse elimination, the microtubule cytoskeleton of retracting axons is degraded and reduced (4). In contrast, axons that singly innervate NMJs have a higher microtubule content. α-Btx–dependent block of neuromuscular transmission reduced microtubule content in axons that singly innervate NMJs. Thus, α-Btx treatment simultaneously reduces both microtubule content and myelination.To determine if a mature microtubule-based cytoskeleton is causally related to myelination, Wang et al. used spastin knockout (spastin^KO) mice to artificially stabilize microtubules. Although spastin^KO mice had delayed axon branch removal, stabilization of the microtubule cytoskeleton increased myelination of axons that dually innervated NMJs. Thus, the brake that synaptic competition normally places on terminal branch myelination can be overcome by increasing the mass and maturity of the microtubule cytoskeleton.How does axonal microtubule stability influence terminal axon myelination? Microtubules participate in the anterograde and retrograde transport of diverse cargoes including mitochondria and growth factors. To determine if anterograde axonal transport promotes myelination of axons that singly innervate NMJs, Wang et al. used a dominant-negative mutant of kinesin-1 heavy chain which binds cargo, but lacks the protein’s motor domain, thereby impairing transport. After confirming transport inhibition by tracking impaired movement of the β1 subunit of voltage gated sodium channels, they found that myelination and node of Ranvier formation were significantly delayed in singly innervated NMJs expressing the dominant negative kinesin. Taken together, these results suggest that synapse elimination promotes maturation of the microtubule cytoskeleton which allows more efficient delivery of promyelinating signals to the terminal branch.What could these promyelinating signals be? One obvious candidate is Neuregulin 1 type III (Nrg1-III), which has long been known to promote myelination of peripheral nervous system axons (5). Consistent with this idea, conditional deletion of Nrg1-III dramatically reduced the number of myelinated axon terminals that singly innervate NMJs but did not alter the number of dually innervated NMJs. In contrast, overexpression of Nrg1-III in a transgenic mouse removed the competition-dependent block on myelination resulting in more myelination of both dually and singly innervating axon terminals. In these same transgenic Nrg1-III mice, among those NMJs that were singly innervated, their corresponding axons had higher levels of Nrg1-III. Remarkably, even in these same transgenic overexpressers, inhibition of muscle activity reduced the amount of Nrg1-III found on singly innervated axons, consistent with the observed impairment of the microtubule-based cytoskeleton after α-Btx treatment. ERK1/2 and AKT are downstream effectors of Nrg1-III in Schwann cells and implicated in the myelination pathway. Immunostaining of Schwann cells ensheathing singly innervating axon terminals revealed higher levels of pERK and pAKT.Taken together, the experiments performed by Wang et al. (2) suggest that as multiple axons actively compete for synaptic dominance at the NMJ, the myelination of their terminal branches is delayed. Upon synapse elimination, neuromuscular activity promotes a retrograde signal that increases maturity of the microtubule cytoskeleton. Maturation of the microtubule-based cytoskeleton facilitates the transport of promyelinating signals like Nrg1-III which, when presented to Schwann cells, results in myelination of the “winner” terminal axon branch of a singly innervated NMJ (Fig. 1).Open in a separate windowFigure 1.Synapse elimination promotes myelination of terminal motor axon branches. During early development, NMJs are innervated by multiple axons that compete for endplate territory. During this time, the terminal branches of the axons are not myelinated, and the tubulin cytoskeletal network remains immature. Synaptic activity induces elimination of redundant connections, which leads the winner axon’s microtubule-based cytoskeleton to mature and increase, while the microtubule cytoskeleton is degraded in the retracting axon. The maturity of the cytoskeleton allows for kinesin dependent anterograde transport of Neuregulin 1-III, which then initiates a promyelination signaling cascade via AKT and ERK activation.To the best of our knowledge, this is the first demonstration of plasticity of myelination downstream of activity and synapse refinement in the peripheral motor nervous system. Many studies in the CNS demonstrate that de novo myelination occurs in response to neuronal activity and learning paradigms (6, 7), although the mechanisms responsible remain unknown. Thus, synapse refinement and elimination-dependent myelination may be a paradigm to uncover mechanisms of learning- and activity-dependent myelination in the CNS. Functionally, the addition of myelin to the terminal motor axon branch promotes efficient neurotransmitter release through faster action potential propagation, improved metabolic support of the axon, and more efficient depolarization of the presynaptic terminal by clustered Na⁺ channels at the terminal heminode (8). Whether any or all of these benefits also exist in the CNS remains unknown.This is also the first demonstration of postsynaptic activity driving myelination of a presynaptic axon. Although it is clear that a retrograde signal from the muscle promotes the further maturation and subsequent myelination of the terminal axon, the identity of this cue is unknown. One interesting candidate for a muscle-derived competition and axonal maturation cue is the neurotrophin brain-derived neurotrophic factor (BDNF), which is released during muscle activity (9). Consistent with this idea, BDNF promotes axon maturation by stimulating both actin polymerization and microtubule assembly (10). It will be interesting to test the role of trophic factors in activity-dependent synapse elimination and subsequent myelination in both the CNS and PNS.In conclusion, Wang et al. (2) is an excellent addition to a growing body of research that demonstrates how neuronal activity promotes and modulates myelination. Furthermore, it stands as another example of how using simple model systems, such as the NMJ, may provide insights and have important implications for much more complicated biological systems.     

Congenital Myasthenic Syndrome Type 19 Is Caused by Mutations in COL13A1, Encoding the Atypical Non-fibrillar Collagen Type XIII α1 Chain

The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs^∗71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals.     

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age‐related morphological changes

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age‐related changes. Aging effects on the NMJ components—motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)—have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild‐type mice, all NMJ components showed age‐associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain‐specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic‐specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age‐related changes via sympathetic innervation.     

Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Background

Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ).

Methodology/Principal Findings

Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins.

Conclusions/Significance

In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.     

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.     

Localization and Mobility of Synaptic Vesicles in Myosin VI Mutants of Drosophila

Background

At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila.

Results

In Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching.

Conclusion

This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton.     

Assembly,plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at “non-plastic” NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Participation of myosin Va and Pka type I in the regeneration of neuromuscular junctions

Background

The unconventional motor protein, myosin Va, is crucial for the development of the mouse neuromuscular junction (NMJ) in the early postnatal phase. Furthermore, the cooperative action of protein kinase A (PKA) and myosin Va is essential to maintain the adult NMJ. We here assessed the involvement of myosin Va and PKA in NMJ recovery during muscle regeneration.

Methodology/Principal Findings

To address a putative role of myosin Va and PKA in the process of muscle regeneration, we used two experimental models the dystrophic mdx mouse and Notexin-induced muscle degeneration/regeneration. We found that in both systems myosin Va and PKA type I accumulate beneath the NMJs in a fiber maturation-dependent manner. Morphologically intact NMJs were found to express stable nicotinic acetylcholine receptors and to accumulate myosin Va and PKA type I in the subsynaptic region. Subsynaptic cAMP signaling was strongly altered in dystrophic muscle, particularly in fibers with severely subverted NMJ morphology.

Conclusions/Significance

Our data show a correlation between the subsynaptic accumulation of myosin Va and PKA type I on the one hand and NMJ regeneration status and morphology, AChR stability and specificity of subsynaptic cAMP handling on the other hand. This suggests an important role of myosin Va and PKA type I for the maturation of NMJs in regenerating muscle.     

Roles of glial cells in the formation,function, and maintenance of the neuromuscular junction

Like other vertebrate synapses, the neuromuscular junction (NMJ) has glial cells that are closely associated with the pre- and post-synaptic components. These “perisynaptic Schwann cells” (PSCs) cover nerve terminals and are in close proximity to the synapse, yet their role at the NMJ has remained mysterious for decades. In this review we explore historical perspectives on PSCs and highlight key developments in recent years that have provided novel insight into PSC functions at the NMJ. First among these developments is the generation of specific antibody probes for PSCs. Using one such antibody and the principle of complement-mediated cell lysis, we have developed a novel technique to selectively ablate PSCs en masse from frog NMJs in vivo. Applying this approach, we have shown that PSCs are essential for the long-term maintenance of synaptic structure and function. In addition, PSCs are essential for the growth and maintenance of NMJs during development. Probes for PSCs also allow us to observe in vivo that processes extended by PSCs guide nerve terminals during synapse development, remodeling, and regeneration. PSCs may therefore dictate the pattern of innervation at the NMJ. Finally, PSCs may also induce postsynaptic acetylcholine receptor expression and aggregation. This wealth of recent findings about PSCs suggests that these synapse-associated glial cells are a more integral and essential component of the NMJ than previously appreciated. New approaches currently being applied at the NMJ may further support the emerging view that glial cells help make bigger, stronger, and more stable synapses.     

Structure and activation of MuSK,a receptor tyrosine kinase central to neuromuscular junction formation

MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in the formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Inhibition of synapse assembly in mammalian muscle in vivo by RNA interference

The formation of the vertebrate neuromuscular junction (NMJ) requires the receptor tyrosine kinase MuSK and the adaptor molecule rapsyn. Here, we report that the phenotypes of mice deficient in these two molecules can be reproduced by RNA interference (RNAi) in rat muscle in vivo. Specifically, double-stranded RNA (dsRNA) targeting MuSK and rapsyn inhibited the formation of the NMJ in rat muscle fibres in vivo, while dsRNA targeting nonessential proteins did not have any effect. Moreover, plasmids that trigger RNAi to MuSK induced the disassembly of existing NMJs. These results thus demonstrate for the first time the functionality of dsRNA in silencing endogenous genes in adult mammalian muscle in vivo. Moreover, they show that MuSK is also required for the maintenance of the NMJ, offering a mechanistic explanation for the myasthenia gravis caused by auto-antibodies to MuSK.     

Triangularis sterni muscle use in supine humans

The electrical activity of the triangularis sterni (transversus thoracis) muscle was studied in supine humans during resting breathing and a variety of respiratory and nonrespiratory maneuvers known to bring the abdominal muscles into action. Twelve normal subjects, of whom seven were uninformed and untrained, were investigated. The electromyogram of the triangularis sterni was recorded using a concentric needle electrode, and it was compared with the electromyograms of the abdominal (external oblique and rectus abdominis) muscles. The triangularis sterni was usually silent during resting breathing. In contrast, the muscle was invariably activated during expiration from functional residual capacity, expulsive maneuvers, "belly-in" isovolume maneuvers, static head flexion and trunk rotation, and spontaneous events such as speech, coughing, and laughter. When three trained subjects expired voluntarily with considerable recruitment of the triangularis sterni and no abdominal muscle activity, rib cage volume decreased and abdominal volume increased. These results indicate that unlike in the dog, spontaneous quiet expiration in supine humans is essentially a passive process; the human triangularis sterni, however, is a primary muscle of expiration; and its neural activation is largely coupled with that of the abdominals. The triangularis sterni probably contributes to the deflation of the rib cage during active expiration.     

Nerve Terminal Degeneration Is Independent of Muscle Fiber Genotype in SOD1G93A Mice

Background

Motor neuron degeneration in SOD1^G93A transgenic mice begins at the nerve terminal. Here we examine whether this degeneration depends on expression of mutant SOD1 in muscle fibers.

Methodology/Principal Findings

Hindlimb muscles were transplanted between wild-type and SOD1^G93A transgenic mice and the innervation status of neuromuscular junctions (NMJs) was examined after 2 months. The results showed that muscles from SOD1^G93A mice did not induce motor terminal degeneration in wildtype mice and that muscles from wildtype mice did not prevent degeneration in SOD1^G93A transgenic mice. Control studies demonstrated that muscles transplanted from SOD1^G93A mice continued to express mutant SOD1 protein. Experiments on wildtype mice established that the host supplied terminal Schwann cells (TSCs) at the NMJs of transplanted muscles.

Conclusions/Significance

These results indicate that expression of the mutant protein in muscle is not needed to cause motor terminal degeneration in SOD1^G93A transgenic mice and that a combination of motor terminals, motor axons and Schwann cells, all of which express mutant protein may be sufficient.     

Defective Presynaptic Choline Transport Underlies Hereditary Motor Neuropathy

The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies.     

Abnormal development of the neuromuscular junction in Nedd4-deficient mice

Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5-E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.     

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.     

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.     

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.