The p75NTR neurotrophin receptor is required to organize the mature neuromuscular synapse by regulating synaptic vesicle availability

Abstract The coordinated movement of organisms relies on efficient nerve-muscle communication at the neuromuscular junction. After peripheral nerve injury or neurodegeneration, motor neurons and Schwann cells increase the expression of the p75NTR pan-neurotrophin receptor. Even though p75NTR targeting has emerged as a promising therapeutic strategy to delay peripheral neuronal damage progression, the effects of long-term p75NTR inhibition at the mature neuromuscular junction have not been elucidated. We performed quantitative neuroanathomical analyses of the neuromuscular junction in p75NTR null mice by laser confocal and electron microscopy, which were complemented with electromyography, locomotor tests, and pharmacological intervention studies. Mature neuromuscular synapses of p75NTR null mice show impaired postsynaptic organization and ultrastructural complexity, which correlate with altered synaptic function at the levels of nerve activity-induced muscle responses, muscle fiber structure, force production, and locomotor performance. Our results on primary myotubes and denervated muscles indicate that muscle-derived p75NTR does not play a major role on postsynaptic organization. In turn, motor axon terminals of p75NTR null mice display a strong reduction in the number of synaptic vesicles and active zones. According to the observed pre and postsynaptic defects, pharmacological acetylcholinesterase inhibition rescued nerve-dependent muscle response and force production in p75NTR null mice. Our findings revealing that p75NTR is required to organize mature neuromuscular junctions contribute to a comprehensive view of the possible effects caused by therapeutic attempts to target p75NTR.

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Aberrant Morphology and Residual Transmitter Release at the Munc13-Deficient Mouse Neuromuscular Synapse

Molecular and Cellular Biology ◽

10.1128/mcb.25.14.5973-5984.2005 ◽

2005 ◽

Vol 25 (14) ◽

pp. 5973-5984 ◽

Cited By ~ 52

Author(s):

Frédérique Varoqueaux ◽

Michèle S. Sons ◽

Jaap J. Plomp ◽

Nils Brose

Keyword(s):

Neuromuscular Junction ◽

Synaptic Vesicle ◽

Motor Neurons ◽

Transmitter Release ◽

Hippocampal Neurons ◽

Neuromuscular Junctions ◽

Neuromuscular Synapse ◽

Synaptic Activity ◽

Neuromuscular Synaptogenesis ◽

Deficient Mice

ABSTRACT In cultured hippocampal neurons, synaptogenesis is largely independent of synaptic transmission, while several accounts in the literature indicate that synaptogenesis at cholinergic neuromuscular junctions in mammals appears to partially depend on synaptic activity. To systematically examine the role of synaptic activity in synaptogenesis at the neuromuscular junction, we investigated neuromuscular synaptogenesis and neurotransmitter release of mice lacking all synaptic vesicle priming proteins of the Munc13 family. Munc13-deficient mice are completely paralyzed at birth and die immediately, but form specialized neuromuscular endplates that display typical synaptic features. However, the distribution, number, size, and shape of these synapses, as well as the number of motor neurons they originate from and the maturation state of muscle cells, are profoundly altered. Surprisingly, Munc13-deficient synapses exhibit significantly increased spontaneous quantal acetylcholine release, although fewer fusion-competent synaptic vesicles are present and nerve stimulation-evoked secretion is hardly elicitable and strongly reduced in magnitude. We conclude that the residual transmitter release in Munc13-deficient mice is not sufficient to sustain normal synaptogenesis at the neuromuscular junction, essentially causing morphological aberrations that are also seen upon total blockade of neuromuscular transmission in other genetic models. Our data confirm the importance of Munc13 proteins in synaptic vesicle priming at the neuromuscular junction but indicate also that priming at this synapse may differ from priming at glutamatergic and γ-aminobutyric acid-ergic synapses and is partly Munc13 independent. Thus, non-Munc13 priming proteins exist at this synapse or vesicle priming occurs in part spontaneously: i.e., without dedicated priming proteins in the release machinery.

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Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Journal of Neurophysiology ◽

10.1152/jn.00431.2013 ◽

2013 ◽

Vol 110 (8) ◽

pp. 1984-1996 ◽

Cited By ~ 24

Author(s):

Kiel G. Ormerod ◽

Julia K. Hadden ◽

Lylah D. Deady ◽

A. Joffre Mercier ◽

Jacob L. Krans

Keyword(s):

Neuromuscular Junctions ◽

Neuromuscular Synapse ◽

Force Production ◽

Input Resistance ◽

Muscle Cells ◽

Excitatory Junction Potentials ◽

A Cell ◽

Chemical Synapses ◽

Stimulation Paradigm ◽

Muscle Force Production

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

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Single Cell Deconstruction of Muscle Stem Cell Heterogeneity During Aging Reveals Sensitivity to the Neuromuscular Junction

10.1101/2020.05.28.121426 ◽

2020 ◽

Author(s):

Peter J. Ulintz ◽

Jacqueline Larouche ◽

Mahir Mohiuddin ◽

Jesus Castor Macias ◽

Sarah J. Kurpiers ◽

...

Keyword(s):

Neuromuscular Junction ◽

Single Cell ◽

Motor Neurons ◽

Satellite Cells ◽

Neural Control ◽

Neuromuscular Diseases ◽

Neuromuscular Synapse ◽

List Type ◽

Genetic Rescue ◽

Before And After

AbstractDuring aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function in that impacts mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs), but the relationship between MuSCs and neural control has not been established. Herein, using a combination of single-cell transcriptomic analysis, high-resolution immunofluorescence imaging and transgenic young and aged mice as well as from mice with neuromuscular degeneration (Sod1-/-), a compensatory neuro-responsive function for a subset of MuSCs was identified. Genetic rescue of motor neurons in Sod1-/- mice reduced this subset of MuSCs and restored integrity of the neuromuscular junction (NMJ) in a manner akin to young muscle. Administration of severe neuromuscular trauma induced young MuSCs to specifically engraft in a position proximal to the NMJ but in aging, this behavior was abolished. Contrasting the expression programs of young and aged MuSCs after muscle injury at the single cell level, we observed distinctive gene expression programs between responses to neuro-muscular degeneration and muscle trauma. Collectively, these data reveal MuSCs sense synaptic perturbations during aging and neuro-muscular deterioration, and can exert support for the NMJ, particularly in young muscle.HighlightsTranscriptional landscapes of single satellite cells from different ages before and after injury as well as neurodegenerative models before and after nervous rescueA population of satellite cells reside in close proximity to neuromuscular synapse, which are lost with ageDenervation promotes satellite cell engraftment into post-synaptic regions of young as opposed to aged muscle

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Cul4 ubiquitin ligase cofactor DCAF12 promotes neurotransmitter release and homeostatic plasticity

The Journal of Cell Biology ◽

10.1083/jcb.201805099 ◽

2019 ◽

Vol 218 (3) ◽

pp. 993-1010 ◽

Cited By ~ 3

Author(s):

Lilian A. Patrón ◽

Kei Nagatomo ◽

David Tyler Eves ◽

Mays Imad ◽

Kimberly Young ◽

...

Keyword(s):

Glutamate Receptor ◽

Motor Neurons ◽

Ubiquitin Ligase ◽

Neurotransmitter Release ◽

Neuromuscular Junctions ◽

Synaptic Function ◽

Homeostatic Plasticity ◽

Independent Functions ◽

Synaptic Boutons

We genetically characterized the synaptic role of the Drosophila homologue of human DCAF12, a putative cofactor of Cullin4 (Cul4) ubiquitin ligase complexes. Deletion of Drosophila DCAF12 impairs larval locomotion and arrests development. At larval neuromuscular junctions (NMJs), DCAF12 is expressed presynaptically in synaptic boutons, axons, and nuclei of motor neurons. Postsynaptically, DCAF12 is expressed in muscle nuclei and facilitates Cul4-dependent ubiquitination. Genetic experiments identified several mechanistically independent functions of DCAF12 at larval NMJs. First, presynaptic DCAF12 promotes evoked neurotransmitter release. Second, postsynaptic DCAF12 negatively controls the synaptic levels of the glutamate receptor subunits GluRIIA, GluRIIC, and GluRIID. The down-regulation of synaptic GluRIIA subunits by nuclear DCAF12 requires Cul4. Third, presynaptic DCAF12 is required for the expression of synaptic homeostatic potentiation. We suggest that DCAF12 and Cul4 are critical for normal synaptic function and plasticity at larval NMJs.

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Targeting of the ETS Factor Gabpα Disrupts Neuromuscular Junction Synaptic Function

Molecular and Cellular Biology ◽

10.1128/mcb.00659-06 ◽

2007 ◽

Vol 27 (9) ◽

pp. 3470-3480 ◽

Cited By ~ 22

Author(s):

Debra A. O'Leary ◽

Peter G. Noakes ◽

Nick A. Lavidis ◽

Ismail Kola ◽

Paul J. Hertzog ◽

...

Keyword(s):

Skeletal Muscle ◽

Transcription Factor ◽

Neuromuscular Junction ◽

Acetylcholine Receptor ◽

Neuromuscular Junctions ◽

Binding Protein ◽

Synaptic Function ◽

Single Point Mutation ◽

Structural Formation ◽

Ga Binding Protein

ABSTRACT The GA-binding protein (GABP) transcription factor has been shown in vitro to regulate the expression of the neuromuscular proteins utrophin, acetylcholine esterase, and acetylcholine receptor subunits δ and ε through the N-box promoter motif (5′-CCGGAA-3′), but its in vivo function remains unknown. A single point mutation within the N-box of the gene encoding the acetylcholine receptor ε subunit has been identified in several patients suffering from postsynaptic congenital myasthenic syndrome, implicating the GA-binding protein in neuromuscular function and disease. Since conventional gene targeting results in an embryonic-lethal phenotype, we used conditional targeting to investigate the role of GABPα in neuromuscular junction and skeletal muscle development. The diaphragm and soleus muscles from mutant mice display alterations in morphology and distribution of acetylcholine receptor clusters at the neuromuscular junction and neurotransmission properties consistent with reduced receptor function. Furthermore, we confirmed decreased expression of the acetylcholine receptor ε subunit and increased expression of the γ subunit in skeletal muscle tissues. Therefore, the GABP transcription factor aids in the structural formation and function of neuromuscular junctions by regulating the expression of postsynaptic genes.

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ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction.

The Journal of Cell Biology ◽

10.1083/jcb.130.6.1423 ◽

1995 ◽

Vol 130 (6) ◽

pp. 1423-1434 ◽

Cited By ~ 48

Author(s):

A D Goodearl ◽

A G Yee ◽

A W Sandrock ◽

G Corfas ◽

G D Fischbach

Keyword(s):

Neuromuscular Junction ◽

Basal Lamina ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Primary Cultures ◽

Cholinergic Neurons ◽

Synaptic Cleft ◽

Motor Axons ◽

Amino Terminal

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.

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Role of ATP-Dependent Calcium Regulation in Modulation of Drosophila Synaptic Thermotolerance

Journal of Neurophysiology ◽

10.1152/jn.91209.2008 ◽

2009 ◽

Vol 102 (2) ◽

pp. 901-913 ◽

Cited By ~ 12

Author(s):

M. K. Klose ◽

G. L. Boulianne ◽

R. M. Robertson ◽

H. L. Atwood

Keyword(s):

Synaptic Transmission ◽

Motor Neurons ◽

Elevated Temperatures ◽

Neuromuscular Junctions ◽

Energy Levels ◽

Calcium Regulation ◽

Synaptic Function ◽

Calcium Stores ◽

Presynaptic Calcium ◽

Presynaptic Nerve Terminals

Maintenance of synaptic transmission requires regulation of intracellular Ca2+ in presynaptic nerve terminals; loss of this regulation at elevated temperatures may cause synaptic failure. Accordingly, we examined the thermosensitivity of presynaptic calcium regulation in Drosophila larval neuromuscular junctions, testing for effects of disrupting calcium clearance. Motor neurons were loaded with the ratiometric Ca2+ indicator Fura-dextran to monitor calcium regulation as temperature increased. Block of the Na+/Ca2+ exchanger or removal of extracellular Ca2+ prevented the normal temperature-induced increase in resting calcium. Conversely, two treatments that interfered with Ca2+ clearance—inactivation of the endoplasmic reticulum Ca2+-ATPase with thapsigargin and inhibition of the plasma membrane Ca2+-ATPase with high pH—significantly accelerated the temperature-induced rise in resting Ca2+ concentration and reduced the thermotolerance of synaptic transmission. Disrupting Ca2+-ATPase function by interfering with energy production also facilitated the temperature-induced rise in resting [Ca2+] and reduced thermotolerance of synaptic transmission. Conversely, fortifying energy levels with extra intracellular ATP extended the operating temperature range of both synaptic transmission and Ca2+ regulation. In each of these cases, Ca2+ elevations evoked by an electrical stimulation of the nerve (evoked Ca2+ responses) failed when resting Ca2+ remained >e 200 nM for several minutes. Failure of synaptic function was correlated with the release of intracellular calcium stores, and we provide evidence suggesting that release from the mitochondria disrupts evoked calcium responses and synaptic transmission. Thus the thermal limit of synaptic transmission may be directly linked to the stability of ATP-dependent mechanisms that regulate intracellular ion concentrations in the nerve terminal.

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DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION

The Journal of Cell Biology ◽

10.1083/jcb.47.2.423 ◽

1970 ◽

Vol 47 (2) ◽

pp. 423-436 ◽

Cited By ~ 17

Author(s):

Thomas L. Lentz

Keyword(s):

Neuromuscular Junction ◽

Synaptic Vesicles ◽

Neuromuscular Junctions ◽

Morphological Changes ◽

Muscle Fibers ◽

Lead Nitrate ◽

Synaptic Function ◽

Axon Termination ◽

Nerve Muscle ◽

Thiolacetic Acid

Following amputation of the limb of the newt, Triturus viridescens, muscle fibers dedifferentiate giving rise to mesenchymal cells. The earliest changes detected in neuromuscular junctions of dedifferentiating muscle fibers are the appearance of a few vacuoles and decrease in density of the terminal axoplasm. Later, synaptic vesicles become tightly clustered in the axon termination, and their content appears denser than normal. Then, vesicles diminish in number until few are seen in the ending. While these changes are occurring, the area of contact of nerve with muscle becomes smaller. Junctional folds persist only where the nerve maintains contact with muscle, but these are shorter than normal and appear as slight ridges on the muscle surface. Subsequently, the nerve withdraws from the muscle cell and is completely invested by Schwann cell cytoplasm, and all traces of junctional folds are lost at the former region of contact. Cholinesterase activity was localized with the thiolacetic acid-lead nitrate method. Even before marked morphological changes occur in the junction, DFP- and physostigmine-sensitive activity in the cleft between nerve and muscle is decreased in intensity. Activity continues to decrease as the area of nerve-muscle contact diminishes and junctional folds disappear. When the nerve has withdrawn from the muscle surface, only a few small deposits of lead are left in the intervening region. These results show that as muscle becomes less specialized during dedifferentiation, the neuromuscular junction also loses the cytological and cytochemical specializations associated with synaptic function.

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Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin

Molecules ◽

10.3390/molecules26133988 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3988

Author(s):

Ana Candalija ◽

Thomas Scior ◽

Hans-Richard Rackwitz ◽

Jordan E. Ruiz-Castelan ◽

Ygnacio Martinez-Laguna ◽

...

Keyword(s):

Motor Neurons ◽

Neuromuscular Junctions ◽

Hot Spot ◽

Potential Interaction ◽

Neurotrophin Receptor ◽

Downstream Signaling ◽

Cellular Models ◽

Tetanus Neurotoxin

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.

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

Scientific Reports ◽

10.1038/s41598-019-54900-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Mehmet Mahsum Kaplan ◽

Bernhard E. Flucher

Keyword(s):

Neuromuscular Junction ◽

Calcium Signaling ◽

Motor Neurons ◽

Motor Nerve ◽

Neuromuscular Synapse ◽

Presynaptic Differentiation ◽

Motor Nerves ◽

Muscle Calcium ◽

Activity Dependent ◽

Postsynaptic Target

AbstractProper 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.

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