• Acetylcholine receptors
• Alpha-bungarotoxin
• Dynamics
• Neuromuscular junction
• Regeneration

• NMJ
• excitability
• muscle
• neuromuscular junction
• sodium channel
• synapse

• Animals
• Neuromuscular Junction/metabolism
• Neuromuscular Junction/physiology
• Mice
• Receptors, Cholinergic/genetics
• Receptors, Cholinergic/metabolism
• Receptors, Cholinergic/physiology
• Receptor Protein-Tyrosine Kinases/genetics
• Receptor Protein-Tyrosine Kinases/metabolism
• Receptor Protein-Tyrosine Kinases/physiology
• NAV1.4 Voltage-Gated Sodium Channel/metabolism
• NAV1.4 Voltage-Gated Sodium Channel/genetics
• Action Potentials/physiology
• Male
• Female
• Mice, Knockout
• Mice, Inbred C57BL

• R01 AG067758 NIA NIH HHS
• R01 AR074985 NIAMS NIH HHS
• RF1 NS126667 NINDS NIH HHS
• R21 NS112743 NINDS NIH HHS
• S10 OD025181 NIH HHS
• R01 GM068118 NIGMS NIH HHS
• Brown University (BU)
• Ohio State University (OSU)
• Cleft Palate Foundation (CPF)

• Soman/toxicity
• Soman/poisoning
• Pyridinium Compounds/chemistry
• Pyridinium Compounds/pharmacology
• Pyridinium Compounds/therapeutic use
• Animals
• Structure-Activity Relationship
• Guinea Pigs
• Humans
• Nicotinic Antagonists/chemistry
• Nicotinic Antagonists/pharmacology
• Nicotinic Antagonists/therapeutic use
• Mice
• Acetylcholinesterase/metabolism
• Cell Line
• Drug Therapy, Combination
• Cholinesterase Inhibitors
• Oximes

• Ministry of Defence

• Cell culture
• Dystrophin complex
• Fluorescence
• MicroRNA
• Mouse
• Neuromuscular junction
• Nicotinic acetylcholine receptors
• miRNA
• nAChR

• Animals
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Receptors, Cholinergic/metabolism
• Receptors, Cholinergic/genetics
• Mice
• Neuromuscular Junction/metabolism
• Neuromuscular Junction/pathology
• Neuromuscular Junction/genetics
• Mice, Knockout
• Muscle, Skeletal/metabolism
• Muscle, Skeletal/innervation
• Agrin/metabolism
• Agrin/genetics
• Muscle Proteins/metabolism
• Muscle Proteins/genetics
• Dystrophin-Associated Proteins/metabolism
• Dystrophin-Associated Proteins/genetics
• Muscular Dystrophies/genetics
• Muscular Dystrophies/metabolism
• Muscular Dystrophies/pathology
• Muscle Denervation
• Mice, Inbred C57BL

• R01 NS111041 NINDS NIH HHS
• R01NS111041 NINDS NIH HHS
• University of Michigan
• National Institute of Neurological Disorders and Stroke

• AKAP
• PKA
• acetylcholine receptors
• cAMP
• microdomain
• myosin Va
• neuromuscular junction
• synaptic plasticity

• R41 AG073144 NIA NIH HHS
• T32 MH020068 NIMH NIH HHS
• R01 AR074985 NIAMS NIH HHS
• T32 AG041688 NIA NIH HHS
• U01 NS064295 NINDS NIH HHS
• S10 OD025181 NIH HHS
• R01 GM068118 NIGMS NIH HHS
• R21 AG073743 NIA NIH HHS
• R25 GM083270 NIGMS NIH HHS
• R21 NS112743 NINDS NIH HHS
• F99 AG079815 NIA NIH HHS

• ATP
• Adenosine
• Development
• Neurogenesis
• P1 receptors
• P2 receptors
• Purinergic signaling
• Synaptogenesis

• Caveolin 3/genetics
• Acetylcholinesterase
• Neuromuscular Junction/physiology
• Receptors, Cholinergic
• Muscles

• Research Grants Council of Hong Kong
• National Science Center, Poland grant

• Acetylcholine
• COVID-19
• brain
• heart
• pathophysiology
• post-COVID syndrome
• signaling disorder
• synapse

• maturation
• nAChR
• neuromuscular junction
• postsynaptic apparatus
• small Rho GTPases
• synaptic podosomes

• Animals
• Monomeric GTP-Binding Proteins/metabolism
• Neuromuscular Junction/metabolism
• Receptors, Nicotinic/metabolism
• Vertebrates
• ras Proteins/metabolism
• rho GTP-Binding Proteins/metabolism

• 1170614 FONDECYT
• FONDECYT

In a broad variety of species, muscle contraction is controlled at the neuromuscular junction (NMJ), the peripheral synapse composed of a motor nerve terminal, a muscle specialization, and non-myelinating terminal Schwann cells. While peripheral nerve damage leads to successful NMJ reinnervation in animal models, muscle fiber reinnervation in human patients is largely inefficient. Interestingly, some hallmarks of NMJ denervation and early reinnervation in murine species, such as fragmentation and poly-innervation, are also phenotypes of aged NMJs or even of unaltered conditions in other species, including humans. We have reasoned that rather than features of NMJ decline, such cellular responses could represent synaptic adaptations to accomplish proper functional recovery. Here, we have experimentally tackled this idea through a detailed comparative study of the short- and long-term consequences of irreversible (chronic) and reversible (partial) NMJ denervation in the convenient cranial levator auris longus muscle.

Our findings reveal that irreversible muscle denervation results in highly fragmented postsynaptic domains and marked ectopic acetylcholine receptor clustering along with significant terminal Schwann cells sprouting and progressive detachment from the NMJ. Remarkably, even though reversible nerve damage led to complete reinnervation after 11 days, we found that more than 30% of NMJs are poly-innervated and around 65% of postsynaptic domains are fragmented even 3 months after injury, whereas synaptic transmission is fully recovered two months after nerve injury. While postsynaptic stability was irreversibly decreased after chronic denervation, this parameter was only transiently affected by partial NMJ denervation. In addition, we found that a combination of morphometric analyses and postsynaptic stability determinations allows discriminating two distinct forms of NMJ fragmentation, stable-smooth and unstable-blurred, which correlate with their regeneration potential.

Together, our data unveil that reversible nerve damage imprints a long-lasting reminiscence in the NMJ that results in the rearrangement of its cellular components. Instead of being predictive of NMJ decline, these traits may represent an efficient adaptive response for proper functional recovery. As such, these features are relevant targets to be considered in strategies aimed to restore motor function in detrimental conditions for peripheral innervation.

The online version contains supplementary material available at 10.1186/s12915-022-01358-4.

• Denervation
• Fragmentation
• Neuromuscular junction
• Poly-innervation
• Postsynaptic
• Presynaptic
• Regeneration
• Terminal Schwann Cells

Lithium chloride has been widely used as a therapeutic mood stabilizer. Although cumulative evidence suggests that lithium plays modulatory effects on postsynaptic receptors, the underlying mechanism by which lithium regulates synaptic transmission has not been fully elucidated. In this work, by using the advantageous neuromuscular synapse, we evaluated the effect of lithium on the stability of postsynaptic nicotinic acetylcholine receptors (nAChRs) in vivo. We found that in normally innervated neuromuscular synapses, lithium chloride significantly decreased the turnover of nAChRs by reducing their internalization. A similar response was observed in CHO-K1/A5 cells expressing the adult muscle-type nAChRs. Strikingly, in denervated neuromuscular synapses, lithium led to enhanced nAChR turnover and density by increasing the incorporation of new nAChRs. Lithium also potentiated the formation of unstable nAChR clusters in non-synaptic regions of denervated muscle fibres. We found that denervation-dependent re-expression of the foetal nAChR γ-subunit was not altered by lithium. However, while denervation inhibits the distribution of β-catenin within endplates, lithium-treated fibres retain β-catenin staining in specific foci of the synaptic region. Collectively, our data reveal that lithium treatment differentially affects the stability of postsynaptic receptors in normal and denervated neuromuscular synapses in vivo, thus providing novel insights into the regulatory effects of lithium on synaptic organization and extending its potential therapeutic use in conditions affecting the peripheral nervous system.

• 14-3-3
• GRK2
• Src
• endocytosis
• nicotine
• α4β2 nAChR
• β-arrestin1

• AChR
• degradation
• denervation
• endocytosis
• metabolic stability
• recycling
• synaptic activity

• Animals
• Humans
• Models, Biological
• Neuromuscular Junction/metabolism
• Protein Stability
• Receptors, Nicotinic/metabolism
• Signal Transduction
• Synapses/metabolism

• R01 NS111041 NINDS NIH HHS
• R56 NS103845 NINDS NIH HHS
• R01 NS047332 NINDS NIH HHS

• Agrin
• DOK7
• MuSK
• congenital myasthenic syndromes
• myasthenia gravis
• neuromuscular junction
• sarcopenia
• spinal muscular atrophy

• MR/M006824/1 Medical Research Council

• ALS
• NMJ
• TOR
• aging
• autophagy
• mTORC1
• mTORC2
• sarcopenia

• Aging
• BMP signaling
• MuSK
• Neuromuscular junction
• Synaptic maintenance

The time window for repair of the lower trunk is shorter than that of the upper trunk in patients with obstetric brachial plexus palsy. The denervated intrinsic muscles of the hand become irreversibly atrophic much faster than the denervated biceps. However, it is unclear whether the motor endplates of the denervated interosseous muscles degenerate more rapidly than those of the denervated biceps. In this study, we used a rat model of obstetric brachial plexus palsy of the right upper limb. C5–6 was lacerated distal to the intervertebral foramina, with concurrent avulsion of C7–8 and T1, with the left upper limb used as the control. Bilateral interossei and biceps were collected at 5 and 7 weeks. Immunofluorescence was used to assess the morphology of the motor endplates. Real-time quantitative polymerase chain reaction and western blot assay were used to assess mRNA and protein expression levels of acetylcholine receptor subunits (α, β and δ), rapsyn and β-catenin. Immunofluorescence microscopy showed that motor endplates in the denervated interossei were fragmented, while those in the denervated biceps were morphologically intact with little fragmentation. The number and area of motor endplates, relative to the control side, were significantly lower in the denervated interossei compared with the denervated biceps. mRNA and protein expression levels of acetylcholine receptor subunits (α, β and δ) were significantly lower, whereas β-catenin protein expression was higher, in the denervated interossei compared with the denervated biceps. The protein expression of rapsyn was higher in the denervated biceps than in the denervated interossei at 7 weeks. Our findings demonstrate that motor endplates of interossei are destabilized, whereas those of the biceps remain stable, in the rat model of obstetric brachial plexus palsy. All procedures were approved by the Experimental Animal Ethics Committee of Fudan University, China (approval No. DF-187) in January 2016.

• acetylcholine receptor subunits
• biceps
• interossei
• motor endplates
• nerve regeneration
• obstetric brachial plexus palsy
• peripheral nerve injury

• Acetylcholine receptor
• Autophagy
• Endocytosis
• Endplate
• Neuromuscular junction
• Recycling

• Acute nicotine intoxication
• Chronic nicotine intoxication
• Fetal brain development
• Newborn behaviors
• Nicotine products
• Prenatal nicotine exposure
• Sudden infant death syndrome
• Tobacco sickness

• AChR
• NMJ
• confocal microscope
• culture myoblasts
• denervation
• rapsyn

• AChR
• DGC
• Dystrophin glycoprotein complex
• NMJ
• Neuromuscular junction

• Acetylcholine receptor
• Diacylglycerol
• Protein kinase C
• Protein kinase D

• NMJ
• imaging
• knockdown
• nAChR dymamics
• stability
• αkap

Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain.

• 2D-diffusion
• acetylcholine receptor
• cholesterol
• lateral mobility
• membrane domains
• receptor clustering

Electromyography (EMG) is used routinely to diagnose neuromuscular dysfunction in a wide range of peripheral neuropathies, myopathies, and neuromuscular degenerative diseases including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Definitive neurological diagnosis may also be indicated by the analysis of pathological neuromuscular innervation in motor-point biopsies. Our objective in this study was to preempt motor-point biopsy by combining live imaging with electrophysiological analysis of slow degeneration of neuromuscular junctions (NMJs) in vivo.

We combined conventional needle electromyography with fiber-optic confocal endomicroscopy (CEM), using an integrated hand-held, 1.5-mm-diameter probe. We utilized as a test bed, various axotomized muscles in the hind limbs of anaesthetized, double-homozygous thy1.2YFP16: Wld^S mice, which coexpress the Wallerian-degeneration Slow (Wld^S) protein and yellow fluorescent protein (YFP) in motor neurons. We also tested exogenous vital stains, including Alexa488-α-bungarotoxin; the styryl pyridinium dye 4-Di-2-Asp; and a GFP conjugate of botulinum toxin Type A heavy chain (GFP-HcBoNT/A).

We show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration. EMG amplitude declined in parallel with overt degeneration of motor nerve terminals. EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes.

Our findings constitute proof-of-concept, based on live imaging in an animal model, that combining EMG/CEM may be useful as a minimally invasive precursor or alternative to motor-point biopsy in neurological diagnosis and for monitoring local administration of potential therapeutics.

• Agrin
• Imaging
• Neuromuscular junctions
• Nicotinic acetylcholine receptors
• Recycled receptors

• Animals
• CHO Cells
• Cell Membrane/drug effects
• Cell Membrane/metabolism
• Cholesterol/metabolism
• Cholesterol/pharmacology
• Cricetulus
• Microscopy, Fluorescence
• Molecular Imaging/methods
• Protein Aggregates
• Protein Transport
• Receptors, Nicotinic/metabolism

AIM: To investigate the activity and expression of EAAT2 glutamate transporter in both in vitro and in vivo models of cholestasis.

METHODS: This study was conducted on human hepatoblastoma HepG2 cell cultures, the liver of bile duct ligated rats and human specimens from cholestatic patients. EAAT2 glutamate transporter activity and expression were analyzed using a substrate uptake assay, immunofluorescence, reverse transcription-polymerase chain reaction, and immunohistochemistry, respectively.

RESULTS: In HepG2 cells, cholestasis was mimicked by treating cells with the protein kinase C activator, phorbol 12-myristate 13-acetate. Under such conditions, EAAT2 transporter activity was decreased both at the level of substrate affinity and maximal transport velocity. The decreased uptake was correlated with intracellular translocation of EAAT2 molecules as demonstrated using immunofluorescence. In the liver of bile duct ligated rats, an increase in EAAT2 transporter protein expression in hepatocytes was demonstrated using immunohistochemistry. The same findings were observed in human liver specimens of cholestasis in which high levels of γ-glutamyl transpeptidase were documented in patients with biliary atresia and progressive familial intrahepatic cholestasis type 3.

CONCLUSION: This study demonstrates the alteration in glutamate handling by hepatocytes in liver cholestasis and suggests a potential cross-talk between glutamatergic and bile systems.

• Glutamate transport
• Hepatocyte
• Bile duct ligation
• Cholestasis
• Biliary atresia

• Animals
• Cholestasis/metabolism
• Cholestasis/surgery
• Excitatory Amino Acid Transporter 2/metabolism
• Gene Expression Regulation
• Glutamate Plasma Membrane Transport Proteins/metabolism
• Hep G2 Cells
• Humans
• Immunohistochemistry
• Liver/metabolism
• Liver Diseases/metabolism
• Male
• Microscopy, Fluorescence
• Rats
• Rats, Wistar
• Reverse Transcriptase Polymerase Chain Reaction

The steady state of the acetylcholine receptor (AChR) density at the neuromuscular junction (NMJ) is critical for efficient and reliable synaptic transmission. However, little is known about signaling molecules involved in regulating the equilibrium between the removal and insertion of AChRs that establishes a stable postsynaptic receptor density over time. In this work, we tested the effect of activities of two serine/threonine kinases, PKC and PKA, on the removal rate of AChRs from and the re-insertion rate of internalized recycled AChRs into synaptic sites of innervated and denervated NMJs of living mice. Using an in vivo time-lapse imaging approach and various pharmacological agents, we showed that PKC and PKA activities have antagonistic effects on the removal and recycling of AChRs. Inhibition of PKC activity or activation of PKA largely prevents the removal of pre-existing AChRs and promotes the recycling of internalized AChRs into the postsynaptic membrane. In contrast, stimulation of PKC or inactivation of PKA significantly accelerates the removal of postsynaptic AChRs and depresses AChR recycling. These results indicate that a balance between PKA and PKC activities may be critical for the maintenance of the postsynaptic receptor density.

• 8-Bromo Cyclic Adenosine Monophosphate/pharmacology
• Animals
• Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors
• Cyclic AMP-Dependent Protein Kinases/genetics
• Cyclic AMP-Dependent Protein Kinases/metabolism
• Enzyme Activators/pharmacology
• Female
• Isoquinolines/pharmacology
• Mice
• Muscle Denervation
• Naphthalenes/pharmacology
• Neuromuscular Junction/drug effects
• Neuromuscular Junction/metabolism
• Post-Synaptic Density
• Protein Kinase C/antagonists & inhibitors
• Protein Kinase C/genetics
• Protein Kinase C/metabolism
• Protein Kinase Inhibitors/pharmacology
• Protein Transport/drug effects
• Receptors, Cholinergic/genetics
• Receptors, Cholinergic/metabolism
• Sulfonamides/pharmacology
• Synaptic Transmission
• Tetradecanoylphorbol Acetate/pharmacology
• Time-Lapse Imaging

• R01 NS047332 NINDS NIH HHS
• NS-047332 NINDS NIH HHS

Autonomic regulation processes in striated muscles are largely mediated by cAMP/PKA-signaling. In order to achieve specificity of signaling its spatial-temporal compartmentation plays a critical role. We discuss here how specificity of cAMP/PKA-signaling can be achieved in skeletal muscle by spatio-temporal compartmentation. While a microdomain containing PKA type I in the region of the neuromuscular junction (NMJ) is important for postsynaptic, activity-dependent stabilization of the nicotinic acetylcholine receptor (AChR), PKA type I and II microdomains in the sarcomeric part of skeletal muscle are likely to play different roles, including the regulation of muscle homeostasis. These microdomains are due to specific A-kinase anchoring proteins, like rapsyn and myospryn. Importantly, recent evidence indicates that compartmentation of the cAMP/PKA-dependent signaling pathway and pharmacological activation of cAMP production are aberrant in different skeletal muscles disorders. Thus, we discuss here their potential as targets for palliative treatment of certain forms of dystrophy and myasthenia. Under physiological conditions, the neuropeptide, α-calcitonin-related peptide, as well as catecholamines are the most-mentioned natural triggers for activating cAMP/PKA signaling in skeletal muscle. While the precise domains and functions of these first messengers are still under investigation, agonists of β₂-adrenoceptors clearly exhibit anabolic activity under normal conditions and reduce protein degradation during atrophic periods. Past and recent studies suggest direct sympathetic innervation of skeletal muscle fibers. In summary, the organization and roles of cAMP-dependent signaling in skeletal muscle are increasingly understood, revealing crucial functions in processes like nerve-muscle interaction and muscle trophicity.

• AKAP
• PKA
• adrenoceptors
• dystrophy
• endplate
• metabolism
• neuromuscular junction
• skeletal muscle

Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1^−/− NMJs.

Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1^−/− mouse skeletal muscle.

Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1^−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1^−/− mouse skeletal muscle than in wild-type skeletal muscle.

EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1^−/− muscle may be due to functional compensation by other EHD paralogs.

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.

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.

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.

• Animals
• Calcitonin Gene-Related Peptide/metabolism
• Cyclic AMP/metabolism
• Cyclic AMP-Dependent Protein Kinase Type I/metabolism
• Elapid Venoms/pharmacology
• Mice
• Mice, Inbred C57BL
• Mice, Inbred mdx
• Muscle, Skeletal/drug effects
• Muscle, Skeletal/enzymology
• Muscle, Skeletal/pathology
• Muscle, Skeletal/physiopathology
• Muscular Dystrophy, Animal/pathology
• Myosin Heavy Chains/metabolism
• Myosin Type V/metabolism
• Neuromuscular Junction/drug effects
• Neuromuscular Junction/enzymology
• Neuromuscular Junction/pathology
• Neuromuscular Junction/physiopathology
• Protein Stability/drug effects
• Receptors, Cholinergic/metabolism
• Regeneration/drug effects
• Signal Transduction/drug effects
• Synapses/drug effects
• Synapses/metabolism
• Synapses/pathology

Neuregulin/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.

Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.

The turnover of acetylcholine receptors at the neuromuscular junction is regulated in an activity-dependent manner. Upon denervation and under various other pathological conditions, receptor half-life is decreased.

We demonstrate a novel approach to follow the kinetics of acetylcholine receptor lifetimes upon pulse labeling of mouse muscles with ¹²⁵I-α-bungarotoxin in vivo. In contrast to previous assays where residual activity was measured ex vivo, in our setup the same animals are used throughout the whole measurement period, thereby permitting a dramatic reduction of animal numbers at increased data quality. We identified three stability levels of acetylcholine receptors depending on the presence or absence of innervation: one pool of receptors with a long half-life of ∼13 days, a second with an intermediate half-life of ∼8 days, and a third with a short half-life of ∼1 day. Data were highly reproducible from animal to animal and followed simple exponential terms. The principal outcomes of these measurements were reproduced by an optical pulse-labeling assay introduced recently.

A novel assay to determine kinetics of acetylcholine receptor turnover with small animal numbers is presented. Our data show that nerve activity acts on muscle acetylcholine receptor stability by at least two different means, one shifting receptor lifetime from short to intermediate and another, which further increases receptor stability to a long lifetime. We hypothesize on possible molecular mechanisms.

• Algorithms
• Animals
• Bungarotoxins/metabolism
• Female
• Golgi Apparatus/metabolism
• Golgi Apparatus/ultrastructure
• Half-Life
• Iodine Radioisotopes/administration & dosage
• Iodine Radioisotopes/pharmacokinetics
• Kinetics
• Male
• Mice
• Microscopy, Electron, Transmission
• Models, Biological
• Muscle Denervation
• Muscle, Skeletal/innervation
• Muscle, Skeletal/metabolism
• Muscle, Skeletal/ultrastructure
• Neuromuscular Junction/metabolism
• Neuromuscular Junction/ultrastructure
• Receptors, Cholinergic/metabolism
• Synapses/metabolism
• Synapses/ultrastructure
• Time Factors

• Alzheimer Disease/physiopathology
• Animals
• Brain/physiology
• Disease Models, Animal
• Gene Expression Regulation/physiology
• Humans
• Parkinson Disease/physiopathology
• Receptors, Nicotinic/chemistry
• Receptors, Nicotinic/physiology

• U01 NS059344 NINDS NIH HHS
• U01 NS059344-02 NINDS NIH HHS
• P01 HL072903 NHLBI NIH HHS
• R01 NS025296-17 NINDS NIH HHS
• U01 NS059344-01 NINDS NIH HHS
• P01-HL-72903 NHLBI NIH HHS
• R01 NS025296 NINDS NIH HHS
• R01 NS025296-19 NINDS NIH HHS
• U01-NS-59344 NINDS NIH HHS
• AG-17517 NIA NIH HHS
• NS-25296 NINDS NIH HHS
• U01 NS059344-03 NINDS NIH HHS
• R01 AG017517 NIA NIH HHS
• R01 NS025296-18 NINDS NIH HHS

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).

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.

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.

• Animals
• Fluorescent Antibody Technique
• Green Fluorescent Proteins/genetics
• Green Fluorescent Proteins/metabolism
• Mice
• Mice, Inbred Strains
• Muscle Fibers, Skeletal/metabolism
• Muscle Proteins/metabolism
• Myosin Heavy Chains/metabolism
• Myosin Type V/metabolism
• Neuromuscular Junction/metabolism
• Neuronal Plasticity/physiology
• Receptors, Nicotinic/metabolism
• Transfection