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.     

The Ubiquitin Ligase Nedd4-1 Participates in Denervation-Induced Skeletal Muscle Atrophy in Mice

Skeletal muscle atrophy is a consequence of muscle inactivity resulting from denervation, unloading and immobility. It accompanies many chronic disease states and also occurs as a pathophysiologic consequence of normal aging. In all these conditions, ubiquitin-dependent proteolysis is a key regulator of the loss of muscle mass, and ubiquitin ligases confer specificity to this process by interacting with, and linking ubiquitin moieties to target substrates through protein∶protein interaction domains. Our previous work suggested that the ubiquitin-protein ligase Nedd4-1 is a potential mediator of skeletal muscle atrophy associated with inactivity (denervation, unloading and immobility). Here we generated a novel tool, the Nedd4-1 skeletal muscle-specific knockout mouse (myo^Cre;Nedd4-1^flox/flox) and subjected it to a well validated model of denervation induced skeletal muscle atrophy. The absence of Nedd4-1 resulted in increased weights and cross-sectional area of type II fast twitch fibres of denervated gastrocnemius muscle compared with wild type littermates controls, at seven and fourteen days following tibial nerve transection. These effects are not mediated by the Nedd4-1 substrates MTMR4, FGFR1 and Notch-1. These results demonstrate that Nedd4-1 plays an important role in mediating denervation-induced skeletal muscle atrophy in vivo.     

wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.     

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.     

Distinct Changes in Synaptic Protein Composition at Neuromuscular Junctions of Extraocular Muscles versus Limb Muscles of ALS Donors

The pathophysiology of amyotrophic lateral sclerosis (ALS) is very complex and still rather elusive but in recent years evidence of early involvement of the neuromuscular junctions (NMJs) has accumulated. We have recently reported that the human extraocular muscles (EOMs) are far less affected than limb muscles at the end-stage of ALS from the same donor. The present study aimed to compare the differences in synaptic protein composition at NMJ and in nerve fibers between EOM and limb muscles from ALS donors and controls. Neurofilament light subunit and synaptophysin decreased significantly at NMJs and in nerve fibers in limb muscles with ALS whereas they were maintained in ALS EOMs. S100B was significantly decreased at NMJs and in nerve fibers in both EOMs and limb muscles of ALS donors, but other markers confirmed the presence of terminal Schwann cells in these NMJs. p75 neurotrophin receptor was present in nerve fibers but absent at NMJs in ALS limb muscles. The EOMs were able to maintain the integrity of their NMJs to a very large extent until the end-stage of ALS, in contrast to the limb muscles. Changes in Ca²⁺ homeostasis, reflected by altered S100B distribution, might be involved in the breakdown of nerve-muscle contact at NMJs in ALS.     

Effects of evoked and spontaneous motoneuronal firing on synapse competition and elimination in skeletal muscle

Synapse elimination is a general feature of the development of neural connections, including the connections of motoneurons to skeletal muscle fibers. Our work addressed two questions: (1) how the action potentials generated in the set of motoneurons innervating an individual muscle (i.e., in a motor pool) are correlated in time during development in vivo; (2) what influence different firing patterns exert on the processes of polyneuronal innervation and synapse elimination which characterize the establishment of muscle innervation. We recorded the spontaneous electromyographic activity of the tibialis anterior and soleus muscles of late embryonic and neonatal rats, identifying the firing of at least two single motor unit signals in each record. We found that a striking switch occurs a few days after birth from a highly synchronous type of firing to an asynchronous one, the first thus characterizing embryonic while the second one adult motoneurons. We also investigated the effects of an evoked synchronous type of discharge on neuromuscular synapse formation, measuring polyneuronal innervation and synapse elimination. This was done in an adult in vivo model of de novo synapse formation, while a chronic TTX nerve conduction block, placed centrally with respect to the stimulating electrodes, eliminated the natural activity of motoneurons. We found that the imposed synchronous activity greatly inhibits synapse elimination, causing polyneuronal innervation to persist. We conclude that the early synchronous firing, favors the establishment of polyneuronal innervation while the subsequent switch to an asynchronous one promotes synapse elimination.     

The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses

We have identified EMS-induced mutations in Drosophila Miro (dMiro), an atypical mitochondrial GTPase that is orthologous to human Miro (hMiro). Mutant dmiro animals exhibit defects in locomotion and die prematurely. Mitochondria in dmiro mutant muscles and neurons are abnormally distributed. Instead of being transported into axons and dendrites, mitochondria accumulate in parallel rows in neuronal somata. Mutant neuromuscular junctions (NMJs) lack presynaptic mitochondria, but neurotransmitter release and acute Ca2+ buffering is only impaired during prolonged stimulation. Neuronal, but not muscular, expression of dMiro in dmiro mutants restored viability, transport of mitochondria to NMJs, the structure of synaptic boutons, the organization of presynaptic microtubules, and the size of postsynaptic muscles. In addition, gain of dMiro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs. Together, our findings suggest that dMiro is required for controlling anterograde transport of mitochondria and their proper distribution within nerve terminals.     

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.     

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro^9,5. Last, and perhaps most importantly, a small applied volume (˜50μl) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach^1,8.Download video file.^{(45M, mov)}     

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.     

Peptidergic motoneurons in the buccal ganglia of Aplysia californica Immunocytochemical,morphological, and physiological characterizations

Summary We used physiological recordings, intracellular dye injections and immunocytochemistry to further identify and characterize neurons in the buccal ganglia of Aplysia calif ornica expressing Small Cardioactive Peptide-like immunoreactivity (SCP-LI). Neurons were identified based upon soma size and position, input from premotor cells B4 and B5, axonal projections, muscle innervation patterns, and neuromuscular synaptic properties. SCP-LI was observed in several large ventral neurons including B6, B7, B9, B10, and B11, groups of s1 and s2 cluster cells, at least one cell located at a branch point of buccal nerve n2, and the previously characterized neurons B1, B2 and B15.B6, B7, B9, B10 and B11 are motoneurons to intrinsic muscles of the buccal mass, each displaying a unique innervation pattern and neuromuscular plasticity. Combined, these motoneurons innervate all major intrinsic buccal muscles (I1/I3, I2, I4, I5, I6). Correspondingly, SCP-LI processes were observed on all of these muscles. Innervation of multiple nonhomologous buccal muscles by individual motoneurons having extremely plastic neuromuscular synapses, represents a unique form of neuromuscular organization which is prevalent in this system. Our results show numerous SCPergic buccal motoneurons with widespread ganglionic processes and buccal muscle innervation, and support extensive use of SCPs in the control of feeding musculature.Abbreviations SCP-LI small cardioactive peptide-like immunoreactivity - PSC postsynaptic current - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential - FI facilitation index - TMR time to maximal response     

Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.     

The kakapo Mutation Affects Terminal Arborization and Central Dendritic Sprouting of Drosophila Motorneurons

The lethal mutation l(2)CA4 causes specific defects in local growth of neuronal processes. We uncovered four alleles of l(2)CA4 and mapped it to bands 50A-C on the polytene chromosomes and found it to be allelic to kakapo (Prout et al. 1997. Genetics. 146:275– 285). In embryos carrying our kakapo mutant alleles, motorneurons form correct nerve branches, showing that long distance growth of neuronal processes is unaffected. However, neuromuscular junctions (NMJs) fail to form normal local arbors on their target muscles and are significantly reduced in size. In agreement with this finding, antibodies against kakapo (Gregory and Brown. 1998. J. Cell Biol. 143:1271–1282) detect a specific epitope at all or most Drosophila NMJs. Within the central nervous system of kakapo mutant embryos, neuronal dendrites of the RP3 motorneuron form at correct positions, but are significantly reduced in size. At the subcellular level we demonstrate two phenotypes potentially responsible for the defects in neuronal branching: first, transmembrane proteins, which can play important roles in neuronal growth regulation, are incorrectly localized along neuronal processes. Second, microtubules play an important role in neuronal growth, and kakapo appears to be required for their organization in certain ectodermal cells: On the one hand, kakapo mutant embryos exhibit impaired microtubule organization within epidermal cells leading to detachment of muscles from the cuticle. On the other, a specific type of sensory neuron (scolopidial neurons) shows defects in microtubule organization and detaches from its support cells.     

Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

The Ras–extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice.     

Involvement of p120 catenin in myopodial assembly and nerve–muscle synapse formation

At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve‐secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin‐association of p120ctn in cell extracts. Whereas ectopic expression of wild‐type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant‐negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve‐ and agrin‐induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin‐induced myopodial assembly and suggest that myopodia increase muscle–nerve contacts and muscle's access to neural agrin to promote NMJ formation. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

BMP4 Is a Peripherally-Derived Factor for Motor Neurons and Attenuates Glutamate-Induced Excitotoxicity In Vitro

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-β) superfamily, have been shown to play important roles in the nervous system, including neuronal survival and synaptogenesis. However, the physiological functions of BMP signaling in the mammalian neuromuscular system are not well understood. In this study, we found that proteins of the type II bone morphogenetic receptors (BMPRII) were detected at the neuromuscular junction (NMJ), and one of its ligands, BMP4, was expressed by Schwann cells and skeletal muscle fibers. In double-ligated nerves, BMP4 proteins accumulated at the proximal and distal portions of the axons, suggesting that Schwann cell- and muscle fiber-derived BMP4 proteins were anterogradely and retrogradely transported by motor neurons. Furthermore, BMP4 mRNA was down-regulated in nerves but up-regulated in skeletal muscles following nerve ligation. The motor neuron-muscle interactions were also demonstrated using differentiated C2C12 muscle cells and NG108-15 neurons in vitro. BMP4 mRNA and immunoreactivity were significantly up-regulated in differentiated C2C12 muscle cells when the motor neuron-derived factor, agrin, was present in the culture. Peripherally-derived BMP4, on the other hand, promotes embryonic motor neuron survival and protects NG108-15 neurons from glutamate-induced excitotoxicity. Together, these data suggest that BMP4 is a peripherally-derived factor that may regulate the survival of motor neurons.     

神经营养因子对神经肌肉接头传递的调制作用

运动单位由运动神经元及其支配的肌纤维组成。神经肌肉接头(neuromuscular junction,NMJ)传递受到严密的调节,因而能和运动单位的活动协调一致。在NMJ,神经调制物质的释放与运动单位的活动有关,并能决定突触传递的效能。脑源性神经营养因子(brain—derived neurotrophic factor,BDNF)和神经营养因子4(neurotrophin-4,NT-4)由运动神经末梢和肌纤维产生。肌肉释放营养因子受肌肉活动调节。在NMJ,BDNF和NT-4通过激活酪氨酸激酶B受体(tyrosine kinase receptor B,TrkB),能加强自发性和诱导性的突触活动。突触前Ca^2 量的迅速增加或突触胞吐过程的易化,都能增加突触囊泡的释放,从而改善NMJ的突触传递。事实上,BDNF能促进突触前细胞内Ca^2 的释放,TrkB的激活也能通过有丝分裂活化蛋白激酶,引起突触素I(synapsinI)的磷酸化,进而增加可释放的突触囊泡的数量。在NMJ,神经营养因子还能通过影响神经调节素(neuregulin)或其他神经源性调制物质的局部释放,对接头传递进行调节。本文对近年来在NMJ突触传递的调节,运动单位的NMJ特性以及神经营养因子对突触传递效能的影响等方面的研究进展做一综述。