Super-Resolution Microscopy Reveals a Nanoscale Organization of Acetylcholine Receptors for Trans-Synaptic Alignment at Neuromuscular Synapses.

The neuromuscular junction (NMJ) is a chemical synapse formed between motoneurons and skeletal muscle fibers. The vertebrate NMJ uses acetylcholine (ACh) as the neurotransmitter and features numerous invaginations of the postsynaptic muscle membrane termed junctional folds. ACh receptors (AChRs) are believed to be concentrated on the crest of junctional folds but their spatial organization remains to be fully understood. In this study, we utilized super-resolution microscopy to examine the nanoscale organization of AChRs at NMJ. Using Structured Illumination Microscopy, we found that AChRs appear as stripes within the pretzel-shaped mouse NMJs, which however, do not correlate with the size of the crests of junctional folds. By comparing the localization of AChRs with several pre- and postsynaptic markers of distinct compartments of NMJs, we found that AChRs are not distributed evenly across the crest of junctional folds as previously thought. Instead, AChR stripes are more closely aligned with the openings of junctional folds as well as with the presynaptic active zone. Using Stochastic Optical Reconstruction Microscopy (STORM) for increased resolution, we found that each AChR stripe contains an AChR-poor slit at the center that is equivalent to the size of the opening of junctional folds. Together, these findings indicate that AChRs are largely localized to the edges of crests surrounding the opening of folds to align with the presynaptic active zones. Such a nanoscale organization of AChRs potentially enables trans-synaptic alignment for effective synaptic transmission of NMJs.

[Influence of modeling of gravitational unloading on the postsynaptic acetylcholine receptor organization and acetylcholinesterase activity in neuromuscular synapses of rat fast and slow muscles].

Using immunofluorescent techniques, we have revealed that, after 35 days of rats hindlimb unloading, neuromuscular synapses of fast and slow muscles show enhanced fluorescence intensity and decreased area of fluorescent staining of acetylcholine receptors; increased fluorescent intensity and area of fluorescent staining for acetylcholinesterase. The ratio of the number of postsynaptic acetylcholine receptors and the amount of acetylcholinesterase changed as well as their spatial position in relation to each other. These rearrangements correspond to electrophysiological data on the reduction of the amplitude of the miniature endplate currents in both muscles. Identified synapses restructuring accompanied by a decrease in the volume of muscle fibers. Hindlimb unloading (simulation of hypogravity) leads to an increase in functional activity of acetylcholinesterase on the background of reduced postsynaptic membrane area occupied by acetylcholine receptors. This leads to a decrease in the amplitude of excitatory postsynaptic potentials thereby reducing the nerve-muscle excitation transmission safety factor.

Might prepatterned acetylcholine-receptor clusters on surface myotubes be a sign of neuromuscular-junction maturation failure?

INTRODUCTION: During human myogenesis and synaptogenesis, the first contact between multiaxonal nerve terminals and the primary myotube occurs at an early stage of gestation, then monoaxonal nerve terminals form and postsynaptic clusters of acetylcholine-receptor are modified and redistributed to the site of muscle-nerve contact. The aim of this study is to present the ultrastructural features of muscle and motor-junction immaturity severe enough to lead to death in the first months of life. MATERIAL AND METHODS: Ultrastructural-level analysis was carried out on the quadriceps femoris muscle of an infant born at full term with severe respiratory distress but with normal SMN1 and IGHMBP2 genes. RESULTS: Arrested muscle maturation was manifested in the presence of primary and mature myotubes, prepatterned acetylcholine-receptor clusters devoid of terminal axons, lack of synapses and multiaxonal unmyelinated intramuscular nerves. CONCLUSION: The "naked" prepatterned clusters observed on the surface of myotubes normally never observed in neonates might be a sign of a new genetic defect in innervation.

GFPT1-myasthenia: clinical, structural, and electrophysiologic heterogeneity.

OBJECTIVE: To identify patients with GFPT1-related limb-girdle myasthenia and analyze phenotypic consequences of the mutations. METHODS: We performed genetic analysis, histochemical, immunoblot, and ultrastructural studies and in vitro electrophysiologic analysis of neuromuscular transmission. RESULTS: We identified 16 recessive mutations in GFPT1 in 11 patients, of which 12 are novel. Ten patients had slowly progressive limb-girdle weakness responsive to cholinergic agonists with onset between infancy and age 19 years. One patient (no. 6) harbored a nonsense mutation and a second mutation that disrupts the muscle-specific GFPT1 exon. This patient never moved in utero, was apneic and arthrogrypotic at birth, and was bedfast, tube-fed, and barely responded to therapy at age 6 years. Histochemical studies in 9 of 11 patients showed tubular aggregates in 6 and rimmed vacuoles in 3. Microelectrode studies of intercostal muscle endplates in 5 patients indicated reduced synaptic response to acetylcholine in 3 and severely reduced quantal release in patient 6. Endplate acetylcholine receptor content was moderately reduced in only one patient. The synaptic contacts were small and single or grape-like, and quantitative electron microscopy revealed hypoplastic endplate regions. Numerous muscle fibers of patient 6 contained myriad dilated and degenerate vesicular profiles, autophagic vacuoles, and bizarre apoptotic nuclei. Glycoprotein expression in muscle was absent in patient 6 and reduced in 5 others. CONCLUSIONS: GFPT1-myasthenia is more heterogeneous than previously reported. Different parameters of neuromuscular transmission are variably affected. When disruption of muscle-specific isoform determines the phenotype, this has devastating clinical, pathologic, and biochemical consequences.

Gating movement of acetylcholine receptor caught by plunge-freezing.

The nicotinic acetylcholine (ACh) receptor converts transiently to an open-channel form when activated by ACh released into the synaptic cleft. We describe here the conformational change underlying this event, determined by electron microscopy of ACh-sprayed and freeze-trapped postsynaptic membranes. ACh binding to the α subunits triggers a concerted rearrangement in the ligand-binding domain, involving an ~1-Å outward displacement of the extracellular portion of the ß subunit where it interacts with the juxtaposed ends of α-helices shaping the narrow membrane-spanning pore. The ß-subunit helices tilt outward to accommodate this displacement, destabilising the arrangement of pore-lining helices, which in the closed channel bend inward symmetrically to form a central hydrophobic gate. Straightening and tangential motion of the pore-lining helices effect channel opening by widening the pore asymmetrically and increasing its polarity in the region of the gate. The pore-lining helices of the α(γ) and δ subunits, by flexing between alternative bent and straight conformations, undergo the greatest movements. This coupled allosteric transition shifts the structure from a tense (closed) state toward a more relaxed (open) state.

The absence of dystrophin rather than muscle degeneration causes acetylcholine receptor cluster defects in dystrophic muscle.

Duchenne muscular dystrophy is the most common genetic muscle disease. Affected muscles are characterized by abnormal acetylcholine receptor (AChR) clustering. Some studies have suggested that changes in AChR clusters are secondary to degenerative processes. In this study, we demonstrate that AChR cluster fragmentation and muscle degeneration are separate events. We compared AChR clusters and pathological features in mdx mice (mutated dystrophin) and dko mice (mutated dystrophin and utrophin). AChR clusters were identified by binding with α-bungarotoxin, and pathological features were observed by classical immunohistochemical techniques. AChR clusters in mdx and dko mice were reduced in number and exhibited structural fragmentation. However, AChR cluster fragmentation was not significantly different in mdx and dko mice, although more severe inflammatory infiltration and degeneration were observed in dko mice. Furthermore, neuronal nitric oxide synthase, which interacts with dystrophin to anchor itself at the sarcolemma, was notably reduced in mdx and dko mice. Fragmentation of AChR and muscle degeneration are separate events, and both are secondary results of destabilization on the sarcolemma and the cytoskeleton.

LRP4 serves as a coreceptor of agrin.

Neuromuscular junction (NMJ) formation requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. We report that LRP4, a low-density lipoprotein receptor (LDLR)-related protein, is expressed specifically in myotubes and binds to neuronal agrin. Its expression enables agrin binding and MuSK signaling in cells that otherwise do not respond to agrin. Suppression of LRP4 expression in muscle cells attenuates agrin binding, agrin-induced MuSK tyrosine phosphorylation, and AChR clustering. LRP4 also forms a complex with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations indicate that LRP4 is a coreceptor of agrin that is necessary for MuSK signaling and AChR clustering and identify a potential target protein whose mutation and/or autoimmunization may cause muscular dystrophies.

Rapid and modifiable neurotransmitter receptor dynamics at a neuronal synapse in vivo.

Synaptic plasticity underlies the adaptability of the mammalian brain, but has been difficult to study in living animals. Here we imaged the synapses between pre- and postganglionic neurons in the mouse submandibular ganglion in vivo, focusing on the mechanisms that maintain and regulate neurotransmitter receptor density at postsynaptic sites. Normally, synaptic receptor densities were maintained by rapid exchange of receptors with nonsynaptic regions (over minutes) and by continual turnover of cell surface receptors (over hours). However, after ganglion cell axons were crushed, synaptic receptors showed greater lateral mobility and there was a precipitous decline in insertion. These changes led to near-complete loss of synaptic receptors and synaptic depression. Disappearance of postsynaptic spines and presynaptic terminals followed this acute synaptic depression. Therefore, neurotransmitter receptor dynamism associated with rapid changes in synaptic efficacy precedes long-lasting structural changes in synaptic connectivity.

Glial imaging during synapse remodeling at the neuromuscular junction.

Glia are an indispensable structural and functional component of the synapse. They modulate synaptic transmission and also play important roles in synapse formation and maintenance. The vertebrate neuromuscular junction (NMJ) is a classic model synapse. Due to its large size, simplicity and accessibility, the NMJ has contributed greatly to our understanding of synapse development and organization. In the past decade, the NMJ has also emerged as an effective model for studying glia-synapse interactions, in part due to the development of various labeling techniques that permit NMJs and associated Schwann cells (the glia at NMJs) to be visualized in vitro and in vivo. These approaches have demonstrated that Schwann cells are actively involved in synapse remodeling both during early development and in post-injury reinnervation. In vivo imaging has also recently been combined with serial section transmission electron microscopic (ssTEM) reconstruction to directly examine the ultrastructural organization of remodeling NMJs. In this review, we focus on the anatomical studies of Schwann cell dynamics and their roles in formation, maturation and remodeling of vertebrate NMJs using the highest temporal and spatial resolution methods currently available.

Involvement of nitric oxide in depolarization-induced suppression of inhibition in hippocampal pyramidal cells during activation of cholinergic receptors.

Several types of neurons are able to regulate their synaptic inputs via releasing retrograde signal molecules, such as endocannabinoids or nitric oxide (NO). Here we show that, during activation of cholinergic receptors, retrograde signaling by NO controls CB1 cannabinoid receptor (CB1R)-dependent depolarization-induced suppression of inhibition (DSI). Spontaneously occurring IPSCs were recorded in CA1 pyramidal neurons in the presence of carbachol, and DSI was induced by a 1-s-long depolarization step. We found that, in addition to the inhibition of CB1Rs, blocking the NO signaling pathway at various points also disrupted DSI. Inhibitors of NO synthase (NOS) or NO-sensitive guanylyl cyclase (NO-sGC) diminished DSI, whereas a cGMP analog or an NO donor inhibited IPSCs and partially occluded DSI in a CB1R-dependent manner. Furthermore, an NO scavenger applied extracellularly or postsynaptically also decreased DSI, whereas L-arginine, the precursor for NO, prolonged it. DSI of electrically evoked IPSCs was also blocked by an inhibitor of NOS in the presence, but not in the absence, of carbachol. In line with our electrophysiological data, double immunohistochemical staining revealed an NO-donor-induced cGMP accumulation in CB1R-positive axon terminals. Using electron microscopy, we demonstrated the postsynaptic localization of neuronal NOS at symmetrical synapses formed by CB1R-positive axon terminals on pyramidal cell bodies, whereas NO-sGC was found in the presynaptic terminals. These electrophysiological and anatomical results in the hippocampus suggest that NO is involved in depolarization-induced CB1R-mediated suppression of IPSCs as a retrograde signal molecule and that operation of this cascade is conditional on cholinergic receptor activation.

Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays.

Intact acetylcholine receptors have been purified on a novel affinity resin from three electric fish endemic to Australian waters. Their binding properties and morphology are compared with those of their northern hemisphere homolog, Torpedo marmorata. All four exhibit apparent dissociation constants, Kd, in the nanomolar range for the snake neurotoxin alpha-bungarotoxin and have a distinctive rosette-like appearance when viewed in negative stain under the electron microscope. Furthermore, these rosettes are paired, indicating that acetylcholine receptors from southern ocean electric fish exist as dimers, in the same fashion as their northern hemisphere counterparts. The cDNAs of the receptor's four subunits were sequenced from Hypnos monopterigium and the northern hemisphere counterpart, Torpedo marmorata, while cDNAs from only two subunits, alpha and delta, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the delta subunit of each of the newly sequenced fish species is a cysteine residue. Its conservation suggests that the mechanism for the observed dimerization of acetylcholine receptors is disulfide bond formation between the delta subunit of adjacent receptors, analogous to acetylcholine receptor dimers observed in other electric fish. It appears that this mechanism for receptor clustering is unique to acetylcholine receptors packed and organized in the specialized organs of electric fish. Alignment of the deduced protein sequences with the equivalent sequences from Torpedo californica and humans reveals a high degree of homology.

Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid-detergent matrices.

Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions. The crystallization of the AChR from Torpedo marmorata electric organs is challenged here in lipidic-detergent matrices. Detergent-soluble AChR complexed with alpha-bungarotoxin (alphaBTx), a polypeptidic competitive antagonist, was purified. The AChR-alphaBTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The alphaBTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor-toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor-toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor-toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.

Rapsyn mutations in myasthenic syndrome due to impaired receptor clustering.

Rapsyn, a 43-kDa postsynaptic protein, is essential for anchoring and clustering acetylcholine receptors (AChRs) at the endplate (EP). Mutations in the rapsyn gene have been found to cause a postsynaptic congenital myasthenic syndrome (CMS). We detected six patients with CMS due to mutations in the rapsyn gene (RAPSN). In vitro studies performed in the anconeus muscle biopsies of four patients showed severe reduction of miniature EP potential amplitudes. Electron microscopy revealed various degrees of impaired development of postsynaptic membrane folds. All patients carried the N88K mutation. Three patients were homozygous for N88K and had less severe phenotypes and milder histopathologic abnormalities than the three patients who were heterozygous and carried a second mutation (either L14P, 46insC, or Y269X). Surprisingly, two N88K homozygous patients had one asymptomatic relative each who carried the same genotype, suggesting that additional genetic factors to RAPSN mutations are required for disease expression.

Structure and gating mechanism of the acetylcholine receptor pore.

The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.

Tyrosine-phosphorylated and nonphosphorylated isoforms of alpha-dystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions.

alpha-Dystrobrevin (DB), a cytoplasmic component of the dystrophin-glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking alphaDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main alphaDB isoforms, alphaDB1 and alphaDB2, with common NH2-terminal but distinct COOH-terminal domains. alphaDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. alphaDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in alphaDB-/- mice prevented muscle fiber degeneration; however, only alphaDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of alphaDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, alphaDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, alphaDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Ultrastructure of acetylcholine receptor aggregates parallels mechanisms of aggregation.

BACKGROUND: Acetylcholine receptors become aggregated at the developing neuromuscular synapse shortly after contact by a motorneuron in one of the earliest manifestations of synaptic development. While a major physiological signal for receptor aggregation (agrin) is known, the mechanism(s) by which muscle cells respond to this and other stimuli have yet to be worked out in detail. The question of mechanism is addressed in the present study via a quantitative examination of ultrastructural receptor arrangement within aggregates. RESULTS: In receptor rich cell membranes resulting from stimulation by agrin or laminin, or in control membrane showing spontaneous receptor aggregation, receptors were found to be closer to neighboring receptors than would be expected at random. This indicates that aggregation proceeds heterogeneously: nanoaggregates, too small for detection in the light microscope, underlie developing microaggregates of receptors in all three cases. In contrast, the structural arrangement of receptors within nanoaggregates was found to depend on the aggregation stimulus. In laminin induced nanoaggregates receptors were found to be arranged in an unstructured manner, in contrast to the hexagonal array of about 10 nm spacing found for agrin induced nanoaggregates. Spontaneous aggregates displayed an intermediate amount of order, and this was found to be due to two distinct population of nanoaggregates. CONCLUSIONS: The observations support earlier studies indicating that mechanisms by which agrin and laminin-1 induced receptor aggregates form are distinct and, for the first time, relate mechanisms underlying spontaneous aggregate formation to aggregate structure.

Receptores nicotínicos neuronales / Neuronal nicotinic receptors

Desde hace varios siglos se conoce el potente efecto de la nicotina sobre los procesos cognitivos y el comportamiento. En la actualidad, la familia de los canales iónicos activados por ligandos y particularmente los receptores nicotínicos de acetilcolina son extensamente estudiados, pues la comprensión de su farmacología y fisiología puede ayudar a decifrar la clave de muchos procesos del cerebro (regulación de neurotransmisores y nocicepción) y también de varias enfermedades en las que se han demostrado su participación (miastenia gravis, Parkinson, Alzheimer, ezquizofrenia). En este artículo, se hace una primera aproximación a la estructura, clasificación y funcionamiento de los receptores nicotínicos neuronales, con el objeto de sistematizar la muy extensa información existente sobre el tema y permitir una mejor comprensión sobre su participación en diferentes eventos que ocurren en el sistema nervioso y las posibilidades terapéuticas actuales. sináptica química es el proceso funcional mediante el cual se desarrolla la comunicación entre células excitables (neurona-neurona o neurona-músculo). El proceso de la neurotransmisión química se inicia en la célula presináptica mediante eventos de despolarización que activan los canales iónicos en la membrana del terminal axónico y permiten que los iones adecuados penetren. El aumento intracelular de los iones inicia la exocitosis de las vesículas presentes en la terminal sináptica, que contienen los La transmisión neurotransmisores, así estos son vertidos en la hendidura sináptica y luego difunden hasta la célula postsináptica, en donde se unen a sus receptores específicos localizados en la membrana celular, generando la respuesta de la célula postsináptica. El tipo de respuesta de la neurona al neurotransmisor está determinada por el receptor o receptores hacia los cuales va dirigido. En algunas ocasiones el receptor al ser estimulado por el neurotransmisor desencadena el desarrollo de ciertas acciones enzimáticas como por ejemplo la modulación de la concentración de metabolitos intracelulares, estos receptores son los llamados metabotrópicos...

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice.

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.