Postsynaptic Muscle Fiber Research Articles

Neuronal signaling modulates protein homeostasis in Caenorhabditis elegans post-synaptic muscle cells

Protein homeostasis maintains proper intracellular balance by promoting protein folding and clearance mechanisms while minimizing the stress caused by the accumulation of misfolded and damaged proteins. Chronic expression of aggregation-prone proteins is deleterious to the cell and has been linked to a wide range of conformational disorders. The molecular response to misfolded proteins is highly conserved and generally studied as a cell-autonomous process. Here, we provide evidence that neuronal signaling is an important modulator of protein homeostasis in post-synaptic muscle cells. In a forward genetic screen in Caenorhabditis elegans for enhancers of polyglutamine aggregation in muscle cells, we identified unc-30, a neuron-specific transcription factor that regulates the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We used additional sensors of protein conformational states to show that defective GABA signaling or increased acetylcholine (ACh) signaling causes a general imbalance in protein homeostasis in post-synaptic muscle cells. Moreover, exposure to GABA antagonists or ACh agonists has a similar effect, which reveals that toxins that act at the neuromuscular junction are potent modifiers of protein conformational disorders. These results demonstrate the importance of intercellular communication in intracellular homeostasis.

Derailed regulates development of the Drosophila neuromuscular junction.

Neural function is dependent upon the proper formation and development of synapses. We show here that Wnt5 regulates the growth of the Drosophila neuromuscular junction (NMJ) by signaling through the Derailed receptor. Mutations in both wnt5 and drl result in a significant reduction in the number of synaptic boutons. Cell-type specific rescue experiments show that wnt5 functions in the presynaptic motor neuron while drl likely functions in the postsynaptic muscle cell. Epistatic analyses indicate that drl acts downstream of wnt5 to promote synaptic growth. Structure-function analyses of the Drl protein indicate that normal synaptic growth requires the extracellular Wnt inhibitory factor domain and the intracellular domain, which includes an atypical kinase. Our findings reveal a novel signaling mechanism that regulates morphology of the Drosophila NMJ.

Regulation of the Intermediate Filament Protein Nestin at Rodent Neuromuscular Junctions by Innervation and Activity

The intermediate filament nestin is localized postsynaptically at rodent neuromuscular junctions. The protein forms a filamentous network beneath and between the synaptic gutters, surrounds myofiber nuclei, and is associated with Z-discs adjacent to the junction. In situ hybridization shows that nestin mRNA is synthesized selectively by synaptic myonuclei. Although weak immunoreactivity is present in myelinating Schwann cells that wrap the preterminal axon, nestin is not detected in the terminal Schwann cells (tSCs) that cover the nerve terminal branches. However, after denervation of muscle, nestin is upregulated in tSCs and in SCs within the nerve distal to the lesion site. In contrast, immunoreactivity is strongly downregulated in the muscle fiber. Transgenic mice in which the nestin neural enhancer drives expression of a green fluorescent protein (GFP) reporter show that the regulation in SCs is transcriptional. However, the postsynaptic expression occurs through enhancer elements distinct from those responsible for regulation in SCs. Application of botulinum toxin shows that the upregulation in tSCs and the loss of immunoreactivity in muscle fibers occurs with blockade of transmitter release. Extrinsic stimulation of denervated muscle maintains the postsynaptic expression of nestin but does not affect the upregulation in SCs. Thus, a nestin-containing cytoskeleton is promoted in the postsynaptic muscle fiber by nerve-evoked muscle activity but suppressed in tSCs by transmitter release. Nestin antibodies and GFP driven by nestin promoter elements serve as excellent markers for the reactive state of SCs. Vital imaging of GFP shows that SCs grow a dynamic set of processes after denervation.

Neuregulins at the neuromuscular synapse: Past, present, and future

At the developing vertebrate neuromuscular junction, neuregulins are growth/differentiation factors essential for terminal Schwann cell survival. Neuregulins have also been thought as the critical signals responsible for the increased transcription of acetylcholine receptor subunit genes at the neuromuscular synapse. This latter role is now highly controversial. This article reviews the evidence that has shaped the views of the neuregulins and how these views have been challenged. The most recent experiments indicate that neuregulin signaling to postsynaptic muscle fibers may modulate, rather than determine, acetylcholine receptor expression at the neuromuscular junction. Based on findings from my lab and those of others, I propose that this modulation might involve novel posttranscriptional molecular mechanisms. Finally, I also suggest that neuregulin signaling may have an important role to play in mediating the response of adult terminal Schwann cells to denervation.

Bernard Katz, quantal transmitter release and the foundations of presynaptic physiology

Bernard Katz, quantal transmitter release and the foundations of presynaptic physiology

Neural agrin: A synaptic stabiliser

Neural agrin is a heparan sulphate proteoglycan first defined by its ability to induce the clustering of acetylcholine receptors (AChRs) on cultured muscle cells. Neural agrin activates the transmembrane Muscle Specific Kinase (MuSK) on the postsynaptic muscle cell to stabilise the developing neuromuscular synapse. Three biological mechanisms for agrin/MuSK signalling are briefly discussed: selective transcription of synaptic genes such as MuSK itself, to reinforce developing postsynaptic clusters of AChRs; initiation of second messenger signalling pathways that can induce the formation of AChR clusters and retrograde signalling downstream of agrin/MuSK that may transform the growth cone of the motor axon into a stable differentiated nerve terminal, specialised for regulated exocytosis of neurotransmitter. Here we briefly review some key mechanisms through which neural agrin acts to foster the formation of mature neuromuscular synapses.

Dystrophin Is Required for Appropriate Retrograde Control of Neurotransmitter Release at theDrosophilaNeuromuscular Junction

Mutations in the human dystrophin gene cause the Duchenne and Becker muscular dystrophies. The Dystrophin protein provides a structural link between the muscle cytoskeleton and extracellular matrix to maintain muscle integrity. Recently, Dystrophin has also been found to act as a scaffold for several signaling molecules, but the roles of dystrophin-mediated signaling pathways remain unknown. To further our understanding of this aspect of the function of dystrophin, we have generated Drosophila mutants that lack the large dystrophin isoforms and analyzed their role in synapse function at the neuromuscular junction. In expression and rescue studies, we show that lack of the large dystrophin isoforms in the postsynaptic muscle cell leads to elevated evoked neurotransmitter release from the presynaptic apparatus. Overall synapse size, the size of the readily releasable vesicle pool as assessed with hypertonic shock, and the number of presynaptic neurotransmitter release sites (active zones) are not changed in the mutants. Short-term synaptic facilitation of evoked transmitter release is decreased in the mutants, suggesting that the absence of dystrophin results in increased probability of release. Absence of the large dystrophin isoforms does not lead to changes in muscle cell morphology or alterations in the postsynaptic electrical response to spontaneously released neurotransmitter. Therefore, postsynaptic glutamate receptor function does not appear to be affected. Our results indicate that the postsynaptically localized scaffolding protein Dystrophin is required for appropriate control of neuromuscular synaptic homeostasis.

Signaling at the vertebrate synapse: New roles for embryonic morphogens?

The formation of synapses is critical for functional neuronal connectivity. The coordinated assembly at both sides of the synapse is fundamental for the proper apposition of the neurotransmitter release machinery on the presynaptic neuron and the clustering of neurotransmitter receptors and ion channels on the receptive postsynaptic cell. This process requires bidirectional communication between the presynaptic neuron and its postsynaptic target, another neuron, or muscle fiber. Extracellular signals such as WNT, TGF-beta, and FGF factors are emerging as key target-derived signals required for the initial stages of synaptic assembly. Studies in invertebrates are also providing new insights into the function of these signals in synaptic growth and homeostasis. During early embryonic patterning, WNT, TGF-beta, and FGF factors function as typical morphogens in a concentration-dependent manner to regulate cell fate decisions. This mode of action raises the provocative idea that these same morphogens might also provide a coordinate system for axons to establish the distance to their targets during axon guidance and synapse formation.

Organization of synaptic myonuclei by Syne proteins and their role during the formation of the nerve–muscle synapse

In most textbooks, the nucleus is drawn as a circle in the center of a cell. However, this is far from the truth, as nuclei can occupy many different locations in a given cell depending on its stage during cell division, development, and differentiation. In polarized cells, nuclei are often positioned at the cell boundary (reviewed in ref. 1). Positioning of nuclei and their differential gene expression profile is also a source of cellular differentiation. One of the prominent examples is the Drosophila embryo, where differential gene expression in the nuclei of the syncytial blastoderm is the basis for the segmentation of the developing embryo (2). In mammalian muscle, myonuclei are localized at the periphery of individual muscle fibers. At the neuromuscular junction (NMJ), myonuclei are aggregated underneath the site of contact between the presynaptic motor nerve terminal and the postsynaptic muscle fiber (Fig. 1 A ). These subsynaptic myonuclei are functionally specialized as they synthesize proteins localized to the postsynaptic apparatus (black nuclei in Fig. 1 A ), whereas gene expression for these proteins is silenced in the myonuclei outside of the NMJ (white nuclei in Fig. 1 A ). Thus, it is hypothesized that the aggregation of myonuclei is essential for the proper development of the NMJ. In a recent issue of PNAS, Grady et al. (3) provide strong evidence that the myonuclei-associated protein Syne-1 (for synaptic nuclear envelope-1) is a structural component that is important for the tethering of myonuclei to the NMJ. However, the blocking of this aggregation does not impair the formation and the function of the NMJ. Fig. 1. Development of the NMJ and the role of Syne-1. ( A Left ) Motor axons approach muscle fibers with myonuclei (gray) expressing high levels of clustered AChRs (green). ( A Right ) Mature NMJ with the presynaptic terminal containing aggregates of synaptic vesicles (white) …

DrosophilaAmphiphysin Functions during Synaptic Fasciclin II Membrane Cycling

Recent studies have revealed that endocytosis and exocytosis of postsynaptic receptors play a major role in the regulation of synaptic function, particularly during long-term potentiation and long-term depression. Interestingly, many of the proteins implicated in exocytosis and endocytosis of synaptic vesicles are also involved in postsynaptic protein cycling. In vertebrates, Amphiphysin is postulated to function during endocytosis in nerve terminals; however, several recent reports using a Drosophila amphiphysin (damph) null mutant have failed to substantiate such a role at fly synapses. In addition, Damph is surprisingly enriched at the postsynapse. Here we used the glutamatergic larval neuromuscular junction to study the synaptic role of Damph. By selectively labeling internal and external pools of the cell adhesion molecule Fasciclin II (FasII), and by using a novel in vivo surface FasII immunocapture protocol, we show that the level of external FasII is decreased in damph mutants although the total level of FasII remains constant. In vivo FasII internalization assays indicate that the reincorporation of FasII molecules into the cell surface is severely inhibited in damph mutants. Moreover, we show that blocking soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function in postsynaptic muscle cells interferes with FasII exocytosis. These experiments suggest that in Drosophila, Damph functions during SNARE-dependent postsynaptic FasII membrane cycling. This study challenges the notion that synaptic Amphiphysin is involved exclusively in endocytosis and suggests a novel role for this protein in postsynaptic exocytosis.

Regulation of AChR Clustering by Dishevelled Interacting with MuSK and PAK1

An important aspect of synapse development is the clustering of neurotransmitter receptors in the postsynaptic membrane. Although MuSK is required for acetylcholine receptor (AChR) clustering at the neuromuscular junction (NMJ), the underlying molecular mechanisms remain unclear. We report here that in muscle cells, MuSK interacts with Dishevelled (Dvl), a signaling molecule important for planar cell polarity. Disruption of the MuSK-Dvl interaction inhibits Agrin- and neuron-induced AChR clustering. Expression of dominant-negative Dvl1 in postsynaptic muscle cells reduces the amplitude of spontaneous synaptic currents at the NMJ. Moreover, Dvl1 interacts with downstream kinase PAK1. Agrin activates PAK, and this activation requires Dvl. Inhibition of PAK1 activity attenuates AChR clustering. These results demonstrate important roles of Dvl and PAK in Agrin/MuSK-induced AChR clustering and reveal a novel function of Dvl in synapse development.

Improvement of RNA Aptamer Activity against Myasthenic Autoantibodies by Extended Sequence Selection

Myasthenia gravis (MG) is mainly engendered by autoantibodies directed against acetylcholine receptors (AChRs) located in the postsynaptic muscle cell membrane. Previously, we isolated an RNA aptamer with 2′-amino pyrimidines using in vitro selection techniques that acted as a decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and patient autoantibodies with MG (1). However, low affinity of this RNA to mAb198 relative to that of AChR might limit potential of the RNA as an inhibitor of the autoantibodies. To improve decoy activity of the RNA aptamer against autoantibodies, here we employed in vitro selection methods with RNA libraries containing extra random nucleotides extended to the 3′ end of previously selected RNA sequences. RNAs isolated in this study showed significant increases in the binding affinities to mAb198 as well as bioactivities protecting AChRs on human cells from both mAb198 and patient autoantibodies, compared with the previous RNA aptamers. These results have important implications for the development of antigen-specific modulation of autoimmune diseases including MG.

Synaptic activity modulates presynaptic excitability.

Synaptic activity modulates synaptic efficacy and is important in learning and development. Here we show that development of excitability in presynaptic motor neurons required synaptic activation of postsynaptic muscle cells. Synaptic blockade broadened action potentials and decreased repetitive firing of presynaptic neurons. Consistent with these findings, synaptic blockade also decreased potassium-current density in the presynaptic cell. Application of neurotrophin-3, but not related neurotrophins, prevented these changes. Recordings from patches of somatic membrane indicated that modifications of presynaptic potassium and sodium currents occurred in a remote, nonsynaptic compartment. Thus, activity-dependent postsynaptic signals modulated presynaptic excitability, potentially regulating transmission at all synapses of the presynaptic cell.

Nitric oxide and cyclic GMP induce vesicle release at Drosophila neuromuscular junction.

Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO-induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae.

Dopaminergic modulation of motor neuron activity and neuromuscular function in Drosophila melanogaster

Dopamine is found in both neuronal and non-neuronal tissues in the larval stage of the fruit fly, Drosophila melanogaster, and functions as a signaling molecule in the nervous system. Although dopaminergic neurons in the central nervous system (CNS) were previously thought solely to be interneurons, recent studies suggest that dopamine may also act as a neuromodulator in humoral pathways. We examined both application of dopamine on intact larval CNS-segmental preparations and isolated neuromuscular junctions (NMJs). Dopamine rapidly decreased the rhythmicity of the CNS motor activity. Application of dopamine on neuromuscular preparations of the segmental muscles 6 and 7 resulted in a dose-responsive decrease in the excitatory junction potentials (EJPs). With the use of focal, macro-patch synaptic current recordings the quantal evoked transmission showed a depression of vesicular release at concentrations of 10 μM. Higher concentrations (1 mM) produced a rapid decrement in evoked vesicular release. Dopamine did not alter the shape of the spontaneous synaptic currents, suggesting that dopamine does not alter the postsynaptic muscle fiber receptiveness to the glutaminergic motor nerve transmission. The effects are presynaptic in causing a reduction in the number of vesicles that are stimulated to be released due to neural activity.

Commissureless endocytosis is correlated with initiation of neuromuscular synaptogenesis.

We show that the Commissureless (COMM) transmembrane protein is required at neuromuscular synaptogenesis. All muscles in the Drosophila embryo express COMM during the period of motoneuron-muscle interaction. It is endocytosed into muscles before synaptogenesis. In comm loss-of-function mutants, motoneuron growth cones fail to initiate synaptogenesis at target muscles. This stall phenotype is rescued by supplying wild-type COMM to the muscles. Cytoplasmically truncated COMM protein fails to internalize. Expressing this mutant protein in muscles phenocopies the synaptogenesis defects of comm mutants. Thus, synaptogenesis initiation is positively correlated with endocytosis of COMM in postsynaptic muscle cells. We propose that COMM is an essential part of the dynamic cell surface remodeling needed by postsynaptic cells in coordinating synaptogenesis initiation.

Cloning of cDNAs encoding Xenopus neuregulin: expression in myotomal muscle during embryo development

Neuregulin has diverse functions in neural development, and one of them is the up regulation of acetylcholine receptors (AChRs) at the muscle fiber during the formation of neuromuscular junctions. Although the primary source of neuregulin is derived from motor neuron, the expression in muscle has also been demonstrated. The precise role of neuron-derived and muscle-derived neuregulin during the early stages of development is not known. In order to study the role of neuregulin during early embryo development, we isolated the cDNAs encoding Xenopus neuregulin by cross-hybridization with its chick homologue. The amino acid sequence of Xenopus protein is 50 to 70% identical to members of the neuregulin family. The cDNAs encoding different isoforms of Xenopus neuregulin were identified, and these isoforms have two variation sites: (i) the spacer domain with either 0 or 43 amino acid insertion; and (ii) the C-terminus of EGF-like domain to derive either α or β isoform. When the EGF-like domain of Xenopus neuregulin was expressed in mammalian cells, the recombinant protein was able to induce the expression of AChR and the tyrosine phosphorylation of erbB receptors in cultured myotubes. An ∼6.5 kb transcript corresponding to neuregulin was detected in RNA isolated from brain and muscle. Various splicing variants were expressed in different Xenopus tissues. In situ hybridization showed a strong expression of neuregulin in developing brain and spinal cord of Xenopus embryo. In addition, it was also prominently expressed in the myotomal muscle. These data suggest that in addition to motor neurons, the postsynaptic muscle cells can also contribute neuregulin for synaptogenesis.

Denervation decreases the ipsilateral expression of AChE in chick lumbaric motor neurons

In vertebrate neuromuscular junctions, acetylcholinesterase (AChE; EC 3.l.1.7) is highly concentrated at the synaptic basal lamina and the postsynaptic muscle fiber. The postsynaptic muscle cell is the primary source of AChE. However, several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE monoclonal antibody in immunohistochemical staining, we found that the AChE-positive cells were labeled only at the motor neurons of the chick spinal cords. When the protein extract of chick spinal cords was analyzed by a Western blot analysis, a protein band of ~105 kDa was recognized. In denervated chicks, the expression of motor neuron AChE, as recognized on a Western blot, decreased by ~50% 4 days after denervation. The AChE expression in denervated chick spinal cords, however, was restored to control level 10 days after denervation. The decreased AChE expression was restricted to the ipsilateral side of the denervated chick spinal cord while the contralateral side was relatively unchanged. In comparison with the contralateral side, the level of AChE protein and enzymatic activity expressed in the ipsilateral spinal cord was ~50% lower. This is the first demonstration to show that the ipsilateral and contralateral sides of chick spinal cords respond differently after nerve denervation.

Regulation of Quantal Secretion from Developing Motoneurons by Postsynaptic Activity-Dependent Release of NT-3

Neurotrophic factors derived from postsynaptic muscle cells may play important roles in the development of presynaptic neuronal functions. In 3-d-old Xenopus nerve-muscle cultures, embryonic spinal neurons that had made natural contact with co-cultured myocytes exhibited spontaneous release of larger packets of acetylcholine (ACh) quanta than those released by the isolated neurons having no contact with any myocyte. Treatment of isolated neurons with neurotrophin-3 (NT-3) for 2 d increased the average sizes of quantal ACh packets at newly formed nerve-muscle synapses, whereas treatment with antibody against NT-3 or with K252a, a specific inhibitor of tyrosine kinase receptors, decreased the quantal size at existing synapses, which suggests that NT-3 supplied by the postsynaptic muscle cell may be responsible for the development and maintenance of the quantal packets. The muscle effect seems to depend on synaptic activities mediated by postsynaptic ACh receptor channels, because chronic treatment of the culture with D-tubocurarine (D-Tc) for 2 d resulted in a marked reduction of the quantal sizes, when assayed after extensive washing of the culture with Ringer's solution. The curare treatment did not affect the postsynaptic ACh receptor sensitivity, because iontophoretically applied ACh induced current responses similar to those of control. Finally, co-treatment of the culture with NT-3 and D-Tc reversed the effect of D-Tc on the quantal size, and this reversal effect was abolished when K252a was also applied concomitantly. Our results suggest that muscle-derived NT-3 participates in the maturation of normal transmitter packets in developing neurons, and the secretion of NT-3 depends on spontaneous synaptic activity.

Chick Muscle Expresses Various ARIA Isoforms: Regulation during Development, Denervation, and Regeneration

Acetylcholine receptor inducing activity (ARIA) is a glycoprotein released from the motor neuron to stimulate the synthesis of acetylcholine receptors (AChRs) on the postsynaptic muscle fiber. Transcripts encoding ARIA were detected not only in brain but also in muscle, and immunohistochemical staining showed that muscle-derived ARIA was restricted to the neuromuscular junctions. RT-PCR analysis revealed three biological active isoforms of ARIA in chick muscle, namely ARIAβ1, ARIAα2, and ARIAβ2that were classified based on their variation in the carboxyl-terminus of the EGF-like domain. The expression of these ARIA isoforms in muscle changed during development, denervation, and nerve regeneration. ARIAβ1, ARIAα2, and ARIAβ2were expressed in embryonic and young chick muscles, while ARIAβ1was the major isoform expressed in adult chicken. The embryonic-like expression of ARIAα2and ARIAβ2was induced after nerve injury in adult chicken. However, the prominent expression of ARIAβ1in adult-like profile was restored after nerve regeneration. A splicing variation in the region between Ig-like and EGF-like domains of ARIA was also revealed; a zero-amino acid insertion (ARIASP0), a 17-amino acid insertion (ARIASP17), or a 34-amino acid insertion (ARIASP34) were identified. Unlike ARIASP0, the expression of ARIASP17and ARIASP34was found in muscle and sciatic nerve only. The expression of ARIASP0, ARIASP17, and ARIASP34in chick muscle remained unchanged during development and after nerve injury. Moreover, the specific expression of these ARIA isoforms in cultured myotubes was not affected by drug treatments or by coculturing with neurons. Our findings provide strong evidence that muscle ARIA may play an important role in the formation of neuromuscular junctions.