PKA and PKC Balance in Synapse Elimination during Neuromuscular Junction Development.

During the development of the nervous system, synaptogenesis occurs in excess though only the appropriate connections consolidate. At the neuromuscular junction, competition between several motor nerve terminals results in the maturation of a single axon and the elimination of the others. The activity-dependent release of transmitter, cotransmitters, and neurotrophic factors allows the direct mutual influence between motor axon terminals through receptors such as presynaptic muscarinic ACh autoreceptors and the tropomyosin-related kinase B neurotrophin receptor. In previous studies, we investigated the synergistic and antagonistic relations between these receptors and their downstream coupling to PKA and PKC pathways and observed a metabotropic receptor-driven balance between PKA (stabilizes multinnervation) and PKC (promotes developmental axonal loss). However, how much does each kinase contribute in the developmental synapse elimination process? A detailed statistical analysis of the differences between the PKA and PKC effects in the synapse elimination could help to explore this point. The present short communication provides this analysis and results show that a similar level of PKA inhibition and PKC potentiation would be required during development to promote synapse loss.

Roles of the synaptic molecules Hevin and SPARC in mouse neuromuscular junction development and repair.

Hevin and secreted protein acidic and rich in cysteine (SPARC) are highly homologous matricellular proteins that function in concert to guide the formation of brain synapses. Here, we investigated the role of these glycoproteins in neuromuscular junction (NMJ) maturation, stability, and repair following injury. Hevin and SPARC mRNA levels in developing (postnatal day 9), adult (postnatal days 90 and 120), and injured (fibular nerve crush) skeletal muscles were assessed with qPCR. Muscle fiber size was analyzed in developing (P9) mice lacking SPARC, Hevin, and both SPARC and Hevin. NMJ morphology was assessed in developing (P9), adult (P90) and injured (fibular nerve crush) mice lacking SPARC, Hevin, and both SPARC and Hevin skeletal muscle. Hevin and SPARC are expressed in skeletal muscles and are upregulated following nerve injury. Hevin-/- mice exhibited delayed NMJ and muscle fiber development but displayed normal NMJ morphology in adulthood and accelerated NMJ reinnervation following nerve injury. Mice lacking SPARC displayed normal NMJ and muscle fiber development but exhibited smaller NMJs with fewer acetylcholine receptor islands in adulthood. Further, SPARC deletion did not result in overt changes in NMJ reformation following nerve injury. The combined deletion of Hevin and SPARC had little effect on NMJ phenotypes observed in single knockouts, however deletion of SPARC in combination with Hevin reversed deficiencies in muscle fiber maturation observed in Hevin-/- muscle. These results identify SPARC and Hevin as extracellular matrix proteins with roles in NMJ development and repair.

Postsynaptic cAMP signalling regulates the antagonistic balance of Drosophila glutamate receptor subtypes.

The balance among different subtypes of glutamate receptors (GluRs) is crucial for synaptic function and plasticity at excitatory synapses. However, the mechanisms balancing synaptic GluR subtypes remain unclear. Herein, we show that the two subtypes of GluRs (A and B) expressed at Drosophila neuromuscular junction synapses mutually antagonize each other in terms of their relative synaptic levels and affect subsynaptic localization of each other, as shown by super-resolution microscopy. Upon temperature shift-induced neuromuscular junction plasticity, GluR subtype A increased but subtype B decreased with a timecourse of hours. Inhibition of the activity of GluR subtype A led to imbalance of GluR subtypes towards more GluRIIA. To gain a better understanding of the signalling pathways underlying the balance of GluR subtypes, we performed an RNA interference screen of candidate genes and found that postsynaptic-specific knockdown of dunce, which encodes cAMP phosphodiesterase, increased levels of GluR subtype A but decreased subtype B. Furthermore, bidirectional alterations of postsynaptic cAMP signalling resulted in the same antagonistic regulation of the two GluR subtypes. Our findings thus identify a direct role of postsynaptic cAMP signalling in control of the plasticity-related balance of GluRs.

Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction Development.

Neuromuscular junctions (NMJs) govern efficient neuronal communication with muscle cells, relying on proper architecture of specialized postsynaptic compartments. However, the intrinsic mechanism in muscle cells contributing to NMJ development remains unclear. In this study, we reveal that dynamin-2 (Dyn2) is involved in postsynaptic development of NMJs. Mutations of Dyn2 have been linked to human muscular disorder and centronuclear myopathy (CNM), as well as featured with muscle atrophy and defective NMJs, yet the function of Dyn2 at the postsynaptic membrane is largely unknown. We demonstrate that Dyn2 is enriched at the postsynaptic membrane and regulates NMJ development via actin remodeling. Dyn2 functions as an actin-bundling GTPase to regulate podosome turnover and cytoskeletal organization of the postsynaptic apparatus, and CNM-Dyn2 mutations display abnormal actin remodeling and electrophysiological activity of fly NMJs. Altogether, Dyn2 primarily regulates actin cytoskeleton remodeling and NMJ morphogenesis at the postsynaptic membrane, which is distinct from its endocytosis regulatory role at the presynaptic membrane.

Synapse development and maturation at the drosophila neuromuscular junction.

Synapses are the sites of neuron-to-neuron communication and form the basis of the neural circuits that underlie all animal cognition and behavior. Chemical synapses are specialized asymmetric junctions between a presynaptic neuron and a postsynaptic target that form through a series of diverse cellular and subcellular events under the control of complex signaling networks. Once established, the synapse facilitates neurotransmission by mediating the organization and fusion of synaptic vesicles and must also retain the ability to undergo plastic changes. In recent years, synaptic genes have been implicated in a wide array of neurodevelopmental disorders; the individual and societal burdens imposed by these disorders, as well as the lack of effective therapies, motivates continued work on fundamental synapse biology. The properties and functions of the nervous system are remarkably conserved across animal phyla, and many insights into the synapses of the vertebrate central nervous system have been derived from studies of invertebrate models. A prominent model synapse is the Drosophila melanogaster larval neuromuscular junction, which bears striking similarities to the glutamatergic synapses of the vertebrate brain and spine; further advantages include the simplicity and experimental versatility of the fly, as well as its century-long history as a model organism. Here, we survey findings on the major events in synaptogenesis, including target specification, morphogenesis, and the assembly and maturation of synaptic specializations, with a emphasis on work conducted at the Drosophila neuromuscular junction.

Morphological variability is greater at developing than mature mouse neuromuscular junctions.

The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post-synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, thereby enabling the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, how synaptic morphology changes during development, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi-automated workflow called NMJ-morph to quantify features of pre- and post-synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild-type mice were dissected and compared at immature (post-natal day, P7) and early adult (P31-32) timepoints. The inter-muscular variability was greater in mature post-synaptic AChR morphology than that of the pre-synaptic motor neuron terminal. Moreover, the developing NMJ showed greater differences across muscles than the mature synapse, perhaps due to the observed distinctions in synaptic growth between muscles. Nevertheless, the amount of nerve to muscle contact was consistent, suggesting that pathological denervation can be reliably compared across different muscles in mouse models of neurodegeneration. Additionally, mature post-synaptic endplate diameters correlated with fibre type, independently of muscle fibre diameter. Altogether, this work provides detailed information on healthy pre- and post-synaptic NMJ morphology from five anatomically and functionally distinct mouse muscles, delivering useful reference data for future comparison with neuromuscular disease models.

Developmental arrest of Drosophila larvae elicits presynaptic depression and enables prolonged studies of neurodegeneration.

Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. We have investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions in which Drosophila larvae are terminally arrested, we have characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). Although wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which time NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.

The conserved microRNA miR-34 regulates synaptogenesis via coordination of distinct mechanisms in presynaptic and postsynaptic cells.

Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, however, relatively few specific miRNAs have been identified that control synapse formation. Using a conditional transgenic toolkit for competitive inhibition of miRNA function in Drosophila, we performed an unbiased screen for novel regulators of synapse morphogenesis at the larval neuromuscular junction (NMJ). From a set of ten new validated regulators of NMJ growth, we discovered that miR-34 mutants display synaptic phenotypes and cell type-specific functions suggesting distinct downstream mechanisms in the presynaptic and postsynaptic compartments. A search for conserved downstream targets for miR-34 identified the junctional receptor CNTNAP4/Neurexin-IV (Nrx-IV) and the membrane cytoskeletal effector Adducin/Hu-li tai shao (Hts) as proteins whose synaptic expression is restricted by miR-34. Manipulation of miR-34, Nrx-IV or Hts-M function in motor neurons or muscle supports a model where presynaptic miR-34 inhibits Nrx-IV to influence active zone formation, whereas, postsynaptic miR-34 inhibits Hts to regulate the initiation of bouton formation from presynaptic terminals.

MMP-mediated modulation of ECM environment during axonal growth and NMJ development.

Motor neurons, skeletal muscles, and perisynaptic Schwann cells interact with extracellular matrix (ECM) to form the tetrapartite synapse in the peripheral nervous system. Dynamic remodeling of ECM composition is essential to diversify its functions for distinct cellular processes during neuromuscular junction (NMJ) development. In this review, we give an overview of the proteolytic regulation of ECM proteins, particularly by secreted and membrane-type matrix metalloproteinases (MMPs), in axonal growth and NMJ development. It is suggested that MMP-2, MMP-9, and membrane type 1-MMP (MT1-MMP) promote axonal outgrowth and regeneration upon injury by clearing the glial scars at the lesion site. In addition, these MMPs also play roles in neuromuscular synaptogenesis, ranging from spontaneous formation of synaptic specializations to activity-dependent synaptic elimination, via proteolytic cleavage or degradation of growth factors, neurotrophic factors, and ECM molecules. For instance, secreted MMP-3 has been known to cleave agrin, the main postsynaptic differentiation inducer, further highlighting the importance of MMPs in NMJ formation and maintenance. Furthermore, the increased level of several MMPs in myasthenia gravis (MG) patient suggest the pathophysiological mechanisms of MMP-mediated proteolytic degradation in MG pathogenesis. Finally, we speculate on potential major future directions for studying the regulatory functions of MMP-mediated ECM remodeling in axonal growth and NMJ development.

Drosophila ammonium transporter Rh50 is required for integrity of larval muscles and neuromuscular system.

Rhesus glycoproteins (Rh50) have been shown to be ammonia transporters in many species from bacteria to human. They are involved in various physiological processes including acid excretion and pH regulation. Rh50 proteins can also provide a structural link between the cytoskeleton and the plasma membranes that maintain cellular integrity. Although ammonia plays essential roles in the nervous system, in particular at glutamatergic synapses, a potential role for Rh50 proteins at synapses has not yet been investigated. To better understand the function of these proteins in vivo, we studied the unique Rh50 gene of Drosophila melanogaster, which encodes two isoforms, Rh50A and Rh50BC. We found that Drosophila Rh50A is expressed in larval muscles and enriched in the postsynaptic regions of the glutamatergic neuromuscular junctions. Rh50 inactivation by RNA interference selectively in muscle cells caused muscular atrophy in larval stages and pupal lethality. Interestingly, Rh50-deficiency in muscles specifically increased glutamate receptor subunit IIA (GluRIIA) level and the frequency of spontaneous excitatory postsynaptic potentials. Our work therefore highlights a new role for Rh50 proteins in the maintenance of Drosophila muscle architecture and synaptic physiology, which could be conserved in other species.

Postsynaptic CaV1.1-driven calcium signaling coordinates presynaptic differentiation at the developing neuromuscular junction.

Proper formation of neuromuscular synapses requires the reciprocal communication between motor neurons and muscle cells. Several anterograde and retrograde signals involved in neuromuscular junction formation are known. However the postsynaptic mechanisms regulating presynaptic differentiation are still incompletely understood. Here we report that the skeletal muscle calcium channel (CaV1.1) is required for motor nerve differentiation and that the mechanism by which CaV1.1 controls presynaptic differentiation utilizes activity-dependent calcium signaling in muscle. In mice lacking CaV1.1 or CaV1.1-driven calcium signaling motor nerves are ectopically located and aberrantly defasciculated. Axons fail to recognize their postsynaptic target structures and synaptic vesicles and active zones fail to correctly accumulate at the nerve terminals opposite AChR clusters. These presynaptic defects are independent of aberrant AChR patterning and more sensitive to deficient calcium signals. Thus, our results identify CaV1.1-driven calcium signaling in muscle as a major regulator coordinating multiple aspects of presynaptic differentiation at the neuromuscular synapse.

Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation.

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl-deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, the growth of an identified motor axon beyond an "en passant" synapse and later axon branching from synaptic points are impaired, leading to functional deficits. Mechanistically, motor-neuron-autonomous Chodl function depends on its intracellular domain and on binding muscle-derived collagen XIXa1 by its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a "synapse-first" scenario of motor axon growth in zebrafish.

Nesprin-1-alpha2 associates with kinesin at myotube outer nuclear membranes, but is restricted to neuromuscular junction nuclei in adult muscle.

Nesprins, nuclear envelope spectrin-repeat proteins encoded by the SYNE1 and SYNE2 genes, are involved in localization of nuclei. The short isoform, nesprin-1-alpha2, is required for relocation of the microtubule organizer function from centromeres to the nuclear rim during myogenesis. Using specific antibodies, we now show that both nesprin-1-alpha2 and nesprin-1-giant co-localize with kinesin at the junctions of concatenated nuclei and at the outer poles of nuclear chains in human skeletal myotubes. In adult muscle, nesprin-1-alpha2 was found, together with kinesin, only on nuclei associated with neuromuscular junctions, whereas all adult cardiomyocyte nuclei expressed nesprin-1-alpha2. In a proteomics study, kinesin heavy and light chains were the only significant proteins in myotube extracts pulled down by nesprin-1-alpha2, but not by a mutant lacking the highly-conserved STAR domain (18 amino-acids, including the LEWD motif). The results support a function for nesprin-1-alpha2 in the specific localization of skeletal muscle nuclei mediated by kinesins and suggest that its primary role is at the outer nuclear membrane.

iPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases.

The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.

Two Pathways for the Activity-Dependent Growth and Differentiation of Synaptic Boutons in Drosophila.

Synapse formation can be promoted by intense activity. At the Drosophila larval neuromuscular junction (NMJ), new synaptic boutons can grow acutely in response to patterned stimulation. We combined confocal imaging with electron microscopy and tomography to investigate the initial stages of growth and differentiation of new presynaptic boutons at the Drosophila NMJ. We found that the new boutons can form rapidly in intact larva in response to intense crawling activity, and we observed two different patterns of bouton formation and maturation. The first pathway involves the growth of filopodia followed by a formation of boutons that are initially devoid of synaptic vesicles (SVs) but filled with filamentous matrix. The second pathway involves rapid budding of synaptic boutons packed with SVs, and these more mature boutons are sometimes capable of exocytosis/endocytosis. We demonstrated that intense activity predominantly promotes the second pathway, i.e., budding of more mature boutons filled with SVs. We also showed that this pathway depends on synapsin (Syn), a neuronal protein which reversibly associates with SVs and mediates their clustering via a protein kinase A (PKA)-dependent mechanism. Finally, we took advantage of the temperature-sensitive mutant sei to demonstrate that seizure activity can promote very rapid budding of new boutons filled with SVs, and this process occurs at scale of minutes. Altogether, these results demonstrate that intense activity acutely and selectively promotes rapid budding of new relatively mature presynaptic boutons filled with SVs, and that this process is regulated via a PKA/Syn-dependent pathway.

Glial response to hypoxia in mutants of NPAS1/3 homolog Trachealess through Wg signaling to modulate synaptic bouton organization.

Synaptic structure and activity are sensitive to environmental alterations. Modulation of synaptic morphology and function is often induced by signals from glia. However, the process by which glia mediate synaptic responses to environmental perturbations such as hypoxia remains unknown. Here, we report that, in the mutant for Trachealess (Trh), the Drosophila homolog for NPAS1 and NPAS3, smaller synaptic boutons form clusters named satellite boutons appear at larval neuromuscular junctions (NMJs), which is induced by the reduction of internal oxygen levels due to defective tracheal branches. Thus, the satellite bouton phenotype in the trh mutant is suppressed by hyperoxia, and recapitulated in wild-type larvae raised under hypoxia. We further show that hypoxia-inducible factor (HIF)-1α/Similar (Sima) is critical in mediating hypoxia-induced satellite bouton formation. Sima upregulates the level of the Wnt/Wingless (Wg) signal in glia, leading to reorganized microtubule structures within presynaptic sites. Finally, hypoxia-induced satellite boutons maintain normal synaptic transmission at the NMJs, which is crucial for coordinated larval locomotion.

NAD+ improves neuromuscular development in a zebrafish model of FKRP-associated dystroglycanopathy.

BACKGROUND: Secondary dystroglycanopathies are muscular dystrophies that result from mutations in genes that participate in Dystroglycan glycosylation. Glycosylation of Dystroglycan is essential for muscle fibers to adhere to the muscle extracellular matrix (myomatrix). Although the myomatrix is disrupted in a number of secondary dystroglycanopathies, it is unknown whether improving the myomatrix is beneficial for these conditions. We previously determined that either NAD+ supplementation or overexpression of Paxillin are sufficient to improve muscle structure and the myomatrix in a zebrafish model of primary dystroglycanopathy. Here, we investigate how these modulations affect neuromuscular phenotypes in zebrafish fukutin-related protein (fkrp) morphants modeling FKRP-associated secondary dystroglycanopathy. RESULTS: We found that NAD+ supplementation prior to muscle development improved muscle structure, myotendinous junction structure, and muscle function in fkrp morphants. However, Paxillin overexpression did not improve any of these parameters in fkrp morphants. As movement also requires neuromuscular junction formation, we examined early neuromuscular junction development in fkrp morphants. The length of neuromuscular junctions was disrupted in fkrp morphants. NAD+ supplementation prior to neuromuscular junction development improved length. We investigated NMJ formation in dystroglycan (dag1) morphants and found that although NMJ morphology is disrupted in dag1 morphants, NAD+ is not sufficient to improve NMJ morphology in dag1 morphants. Ubiquitous overexpression of Fkrp rescued the fkrp morphant phenotype but muscle-specific overexpression only improved myotendinous junction structure. CONCLUSIONS: These data indicate that Fkrp plays an early and essential role in muscle, myotendinous junction, and neuromuscular junction development. These data also indicate that, at least in the zebrafish model, FKRP-associated dystroglycanopathy does not exactly phenocopy DG-deficiency. Paxillin overexpression improves muscle structure in dag1 morphants but not fkrp morphants. In contrast, NAD+ supplementation improves NMJ morphology in fkrp morphants but not dag1 morphants. Finally, these data show that muscle-specific expression of Fkrp is insufficient to rescue muscle development and homeostasis.

Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

Tao Negatively Regulates BMP Signaling During Neuromuscular Junction Development in Drosophila.

The coordinated growth and development of synapses is critical for all aspects of neural circuit function and mutations that disrupt these processes can result in various neurological defects. Several anterograde and retrograde signaling pathways, including the canonical Bone Morphogenic Protein (BMP) pathway, regulate synaptic development in vertebrates and invertebrates. At the Drosophila larval neuromuscular junction (NMJ), the retrograde BMP pathway is a part of the machinery that controls NMJ expansion concurrent with larval growth. We sought to determine whether the conserved Hippo pathway, critical for proportional growth in other tissues, also functions in NMJ development. We found that neuronal loss of the serine-threonine protein kinase Tao, a regulator of the Hippo signaling pathway, results in supernumerary boutons which contain a normal density of active zones. Tao is also required for proper synaptic function, as reduction of Tao results in NMJs with decreased evoked excitatory junctional potentials. Surprisingly, Tao function in NMJ growth is independent of the Hippo pathway. Instead, our experiments suggest that Tao negatively regulates BMP signaling as reduction of Tao leads to an increase in pMad levels in motor neuron nuclei and an increase in BMP target gene expression. Taken together, these results support a role for Tao as a novel inhibitor of BMP signaling in motor neurons during synaptic development and function.

Glial cells maintain synapses by inhibiting an activity-dependent retrograde protease signal.

Glial cells regulate multiple aspects of synaptogenesis. In the absence of Schwann cells, a peripheral glial cell, motor neurons initially innervate muscle but then degenerate. Here, using a genetic approach, we show that neural activity-regulated negative factors produced by muscle drive neurodegeneration in Schwann cell-deficient mice. We find that thrombin, the hepatic serine protease central to the hemostatic coagulation cascade, is one such negative factor. Trancriptomic analysis shows that expression of the antithrombins serpin C1 and D1 is significantly reduced in Schwann cell-deficient mice. In the absence of peripheral neuromuscular activity, neurodegeneration is completely blocked, and expression of prothrombin in muscle is markedly reduced. In the absence of muscle-derived prothrombin, neurodegeneration is also markedly reduced. Together, these results suggest that Schwann cells regulate NMJs by opposing the effects of activity-regulated, muscle-derived negative factors and provide the first genetic evidence that thrombin plays a central role outside of the coagulation system.