Subcellular localization of the glutamate transporters GLAST and GLT at the neuromuscular junction in rodents

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.     

Abnormalities in neuromuscular junction structure and skeletal muscle function in mice lacking the P2X2 nucleotide receptor

ATP is co-released in significant quantities with acetylcholine from motor neurons at skeletal neuromuscular junctions (NMJ). However, the role of this neurotransmitter in muscle function remains unclear. The P2X2 ion channel receptor subunit is expressed during development of the skeletal NMJ, but not in adult muscle fibers, although it is re-expressed during muscle fiber regeneration. Using mice deficient for the P2X2 receptor subunit for ATP (P2X2(-/-)), we demonstrate a role for purinergic signaling in NMJ development. Whereas control NMJs were characterized by precise apposition of pre-synaptic motor nerve terminals and post-synaptic junctional folds rich in acetylcholine receptors (AChRs), NMJs in P2X2(-/-) mice were disorganized: misapposition of nerve terminals and post-synaptic AChR expression localization was common; the density of post-synaptic junctional folds was reduced; and there was increased end-plate fragmentation. These changes in NMJ structure were associated with muscle fiber atrophy. In addition there was an increase in the proportion of fast type muscle fibers. These findings demonstrate a role for P2X2 receptor-mediated signaling in NMJ formation and suggest that purinergic signaling may play an as yet largely unrecognized part in synapse formation.     

Systemic immunoregulatory and pathogenic functions of homeostatic chemokine receptors

IL-17 is a potent proinflammatory cytokine produced by activated memory T cells. The large-scale sequencing of the human and other vertebrate genomes has revealed the presence of additional genes encoding proteins clearly related to IL-17, thus defining a new family of cytokines. There are at least six members of the IL-17 family in humans and in mice. Initial characterization suggests that like IL-17, several of these newly identified molecules have the ability to modulate immune function. Neither the IL-17 family nor the cognate receptors that have been identified for these molecules bear obvious sequence similarity to other known families of proteins. Thus, they represent a distinct signaling system that appears to have been highly conserved across vertebrate evolution. The potent inflammatory actions that have been identified for several of these factors and the emerging associations with major human diseases suggest that these proteins may have significant roles in inflammatory processes.     

Proteomic analysis of NMDA receptor-adhesion protein signaling complexes

N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.     

The role of nitric oxide signaling in the formation of the neuromuscular junction

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.     

Acetylcholine receptors and nerve terminal distribution at the neuromuscular junction of non-obese diabetic mice

Skeletal muscle is one of the main targets of the metabolic alterations in diabetes, in which protein synthesis is markedly reduced followed by increased proteolysis. Ultrastructural and functional changes in the presynaptic compartment of the neuromuscular junction (NMJ) have been demonstrated, but little attention has been paid to the proteins in the postsynaptic muscle fiber membrane. In the present work, we studied the changes in acetylcholine receptors (AChRs) and nerve terminal distribution in the NMJ of non-obese diabetic (NOD) mice. The sternomastoid muscles of adult female NOD mice were double-labeled for AChR and nerve terminal observation by fluorescence and reflected light confocal microscopy. In 62.4% of the diabetic endplates, AChR branches broke apart into receptor islands that stained less than in the normal mice. These patches had regular junctional folds. At most of the endplates studied, the nerve terminals colocalized with AChRs, and sprouts were seen in 10% of the diabetic endplates. The intramuscular nerve branches and axons in the nerve to the sternomastoid muscle showed no degenerative disorders. These results suggest that metabolic alterations in the diabetic muscle fiber can affect the distribution and expression of molecules, such as AChRs, in the postsynaptic membrane of the neuromuscular junction.     

Purinergic modulation of synaptic signalling at the neuromuscular junction

Purines have physiologically important functions throughout the nervous system. In both the central (CNS) and peripheral nervous systems (PNS), purines in the form of adenosine triphosphate and adenosine can play a number of roles in neuronal activation and inhibition. In addition, purines are known to be important for glial cell signaling in both the CNS and PNS. In the PNS, the neuromuscular junction (NMJ) is an excellent model for studying simple synaptic interactions. It is well suited to investigations of neuron-glia interactions because synaptic properties are well defined and perisynaptic Schwann cells (PSCs), glial cells at the NMJ, dynamically interact with the pre- and postsynaptic elements. At the NMJ, purines are critical for presynaptic modulation but also for neuron-glia interactions. Purines signal to PSCs through metabotropic and ionotropic receptors and activation of these receptors can have both modulatory and activating functions. This review will discuss recent developments in our understanding of purinergic modulation of the NMJ with an emphasis on the involvement of purines in neuron-glia interactions at this synapse.     

Antiviral Signaling Through Retinoic Acid-Inducible Gene-I-Like Receptors

The innate immune system is essential for the first line of host defense against micropathogens. In virus-infected cells, exposed viral nucleotides are sensed by pattern recognition receptors (PRRs), resulting in the induction of type I interferon. Retinoic acid-inducible gene-I-like receptors (RLRs) are a member of PRRs and are known to be crucial molecules in innate immune responses. Upon viral recognition, RLRs recruit their specific adaptor molecules, leading to the activation of antiviral signaling molecules including interferon regulatory factor-3 and nuclear factor-κB. Mitochondrial antiviral signaling (MAVS) protein is also known as one of the adaptor molecules responsible for antiviral signaling triggered by RLRs. Recent reports have identified numerous intracellular molecules involved in the antiviral responses mediated by RLRs/MAVS. Several viral proteins interfere with the RLR/MAVS signaling, allowing the virus to evade the host defense. In this review, we comprehensively update RLR-dependent antiviral signaling with special reference to the RLRs/MAVS-mediated responses.     

Mitogen activated protein (MAP) kinases: development of ATP and non-ATP dependent inhibitors

Extracellular signals regulate most of the body's physiological functions through the MAP kinase signaling pathways. These MAP kinase signaling pathways are normally under tight regulation such that activation and inactivation occurs only when needed. However, aberrant regulation observed with naturally occurring mutations in specific signaling proteins often results in constitutive activation of the MAP kinases and is involved in several pathophysiological conditions, such as cancer, neurodegeneration, and inflammation. As such, much effort has been expended to develop inhibitory molecules of the MAP kinase signaling pathways. Several compounds have been identified that inhibit MAP kinase signaling by targeting receptors or other proteins upstream of the MAP kinases. The development of specific inhibitors of the MAP kinases themselves has been less successful and only a few compounds, which interfere with ATP binding, have been identified. A common problem with kinase inhibitors that compete with ATP binding is their lack of specificity. Thus, alternative approaches to inhibit MAP kinase function are being sought. The MAP kinase proteins contain docking domains that direct the interactions with a variety of substrate proteins. Using the 3-dimensional structure of MAP kinases and computer modeling, molecules that target specific docking domains and selectively disrupt substrate interactions are being developed. This non-ATP interfering approach may allow the selective inhibition of MAP kinase substrates involved in disease processes while preserving MAP kinase functions associated with normal cells.     

Regulation of NF-κB by the CARD proteins

Scaffold proteins play pivotal roles in the regulation of signal transduction pathways by connecting upstream receptors to downstream effector molecules. During the last decade, many scaffold proteins that contain caspase-recruitment domains (CARD) have been identified. Investigating the roles of CARD proteins has revealed that many of them play crucial roles in signaling cascades leading to activation of nuclear factor-κB (NF-κB). In this review, we discuss the contributions of CARD proteins to NF-κB activation in various signaling cascades. In particular, we share some of our personal experiences during the initial investigation of the functions of the CARMA family of CARD proteins and then summarize the roles of these proteins in signaling pathways induced by antigen receptors, G protein-coupled receptors, receptor tyrosine kinase, and C-type lectin receptors in the context of recent progress in these field.     

Acetylcholine receptors and nerve terminal distribution at the neuromuscular junction of non‐obese diabetic mice

Skeletal muscle is one of the main targets of the metabolic alterations in diabetes, in which protein synthesis is markedly reduced followed by increased proteolysis. Ultrastructural and functional changes in the presynaptic compartment of the neuromuscular junction (NMJ) have been demonstrated, but little attention has been paid to the proteins in the postsynaptic muscle fiber membrane. In the present work, we studied the changes in acetylcholine receptors (AChRs) and nerve terminal distribution in the NMJ of non‐obese diabetic (NOD) mice. The sternomastoid muscles of adult female NOD mice were double‐labeled for AChR and nerve terminal observation by fluorescence and reflected light confocal microscopy. In 62.4% of the diabetic endplates, AChR branches broke apart into receptor islands that stained less than in the normal mice. These patches had regular junctional folds. At most of the endplates studied, the nerve terminals colocalized with AChRs, and sprouts were seen in 10% of the diabetic endplates. The intramuscular nerve branches and axons in the nerve to the sternomastoid muscle showed no degenerative disorders. These results suggest that metabolic alterations in the diabetic muscle fiber can affect the distribution and expression of molecules, such as AChRs, in the postsynaptic membrane of the neuromuscular junction. Anat Rec 267:112–119, 2002. © 2002 Wiley‐Liss, Inc.     

Molecular mechanisms that underlie structural and functional changes atthe postsynaptic membrane duringsynaptic plasticity

The synaptic plasticity that is addressed in this review follows neurodegeneration in the brain and thus has both structural as well as functional components. The model of neurodegeneration that has been selected is the kainic acid lesioned hippocampus. Degeneration of the CA3 pyramidal cells results in a loss of the Schaffer collateral afferents innervating the CA1 pyramidal cells. This is followed by a period of structural plasticity where new synapses are formed. These are associated with changes in the numbers and shapes of spines as well as changes in the morphometry of the dendrites. It is suggested that this synaptogenesis is responsible for an increase in the ratio of NMDA to AMPA receptors mediating excitatory synaptic transmission at these synapses. Changes in the temporal and spatial properties of these synapses resulted in an altered balance between LTP and LTD. These properties together with a reduction in the inhibitory drive increased the excitability of the surviving CA1 pyramidal cells which in turn triggered epileptiform bursting activity. In this review we discuss the insights that may be gained from studies of the underlying molecular machinery.

Developments in one of the collections of the cogs in this machinery has been summarized through recent studies characterizing the roles of neural recognition molecules in synaptic plasticity in the adult nervous systems of vertebrates and invertebrates. Such investigations of neural cell adhesion molecules, cadherins and amyloid precursor protein have shown the involvement of these molecules on the morphogenetic level of synaptic changes, on the one hand, and signal transduction effects, on the other. Further complex cogs are found in the forms of the low-density lipoprotein receptor (LDL-R) family of genes and their ligands play pivotal roles in the brain development and in regulating the growth and remodelling of neurones. Evidence is discussed for their role in the maintenance of cognitive function as well as Alzheimer's. The molecular mechanisms responsible for the clustering and maintenance of transmitter receptors at postsynaptic sites are the final cogs in the machinery that we have reviewed.

Postsynaptic densities (PSD) from excitatory synapses have yielded many cytoskeletal proteins including actin, spectrin, tubulin, microtubule-associated proteins and calcium/calmodulin-dependent protein kinase II. Isolated PSDs have also been shown to be enriched in AMPA, kainate and NMDA receptors. However, recently, a new family of proteins, the MAGUKs (for membrane-associated guanylate kinase) has emerged. The role of these proteins in clustering different NMDA receptor subunits is discussed. The MAGUK proteins are also thought to play a role in synaptic plasticity mediated by nitric oxide (NO). Both NMDA and non-NMDA receptors are highly clustered at excitatory postsynaptic sites in cortical and hippocampal neurones but have revealed differences in their choice of molecular components. Both GABA_A and glycine (Gly) receptors mediate synaptic inhibition in the brain and spinal cord. Whilst little is known about how GABA_A receptors are localized in the postsynaptic membrane, considerable progress has been made towards the elucidation of the molecular mechanisms underlying the formation of Gly receptors. It has been shown that the peripheral membrane protein gephyrin plays a pivotal role in the formation of Gly receptor clusters most likely by anchoring the receptor to the subsynaptic cytoskeleton. Evidence for the distribution as well as function of gephyrin and Gly receptors is discussed. Postsynaptic membrane specializations are complex molecular machinery subserving a multitude of functions in the proper communication between neurones. Despite the fact that only a few key players have been identified it will be a fascinating to watch the story as to how they contribute to structural and functional plasticity unfold.     

The expanding roles of ITAM adapters FcRγ and DAP12 in myeloid cells

Summary:  The adapter proteins DAP12 and FcRγ associate with a wide spectrum of receptors in a variety of innate immune cells to mediate intracellular signaling pathways when their cognate receptor is engaged. These adapter proteins are coupled to their receptors through charged residues within the transmembrane regions of the adapter and receptor. DAP12 and FcRγ contain specific protein domains (referred to as immunoreceptor tyrosine-based activation motifs) that serve as the substrates and docking sites for kinases, allowing amplification of intracellular signaling reactions. Recent research has broadened the repertoire of receptors that utilize these adapters for signaling to include not only novel immunoglobulin superfamily members but also cytokine receptors, integrins, and other adhesion molecules. There is abundant evidence that these multifunctional signaling adapters also mediate inhibitory activity, downmodulating signaling from Toll-like receptors and other heterologous receptors. In this review, we discuss the newly described receptors that utilize DAP12 and/or FcRγ adapters to modulate innate immune responses.     

Glia cell line-derived neurotrophic factor regulates the distribution of acetylcholine receptors in mouse primary skeletal muscle cells

It was recently reported that glia cell line-derived neurotrophic factor (GDNF) facilitates presynaptic axonal growth and neurotransmitter release at neuromuscular synapses. Little is known, however, whether GDNF can also act on the postsynaptic apparatus and its underlying mechanisms. Using biochemical cold blocking of existing membrane acetylcholine receptors (AchRs) and biotinylation of newly inserted receptors we demonstrate that GDNF increases the insertion of AChRs into the surface membrane of mouse primary cultured muscle cells and that this does not require protein synthesis. Quantitative data from double-label imaging indicate that GDNF induces a quick and substantial increase in AchR insertion as well as lateral movement into AchR aggregates, relative to a weak effect on reducing the loss of receptors from pre-existing AchR aggregates, which in contrast to the effect of PMA. These effects occur in both innervated and un-innervated muscles, and GDNF affects nerve-muscle co-cultures more than it affects muscle-only cultures. Neurturin, another member of GDNF-family ligands has similar effects on AchRs as GDNF but the unrelated growth factor, EGF does not. Studies on protein phosphorylation and specific inhibitors of cell signal transduction indicate that GDNF function is mediated by receptor GFRalpha1 and involves MAPK, cAMP/cAMP responsive element-binding factor and Src kinase activities. GDNF may signal through c-Ret as well as NCAM-140 pathways since both the signaling receptors are expressed in the neuromuscular junction (NMJ). These data suggest that GDNF is an autocrine regulator of NMJ to promote the insertion and stabilization of postsynaptic AchRs. In vivo, GDNF may function as a synaptotrophic modulator for both pre- and postsynaptic differentiation to strengthen the functional and structural connections between nerve and muscle, and contribute to the synaptogenesis and plasticity of neuromuscular synapses.     

Infectious non-self recognition in invertebrates: lessons from Drosophila and other insect models

The vertebrate innate immune system recognizes infectious non-self by employing a set of germline-encoded receptors such as nucleotide-binding oligomerisation domain proteins (NODs) or Toll-like receptors (TLRs). These proteins are involved in the recognition of various microbial-derived molecules, including lipopolysaccharide (LPS), peptidoglycan (PGN) and beta1,3-glucan. Drosophila Toll receptors are not directly dedicated to non-self recognition and insect NOD orthologues have not yet been identified. Studies started more than 20 years ago and conducted on different insect models have identified other receptors on which invertebrate innate systems rely to sense invading microorganisms.     

Presynaptic calcium influx,neurotransmitter release,and neuromuscular disease

The synapse between a spinal motor neuron and a muscle cell is normally very effective at eliciting muscle contraction. A reliable connection between these two cells occurs because a single action potential reaching the motor nerve terminal normally releases hundreds of packets of transmitter containing thousands of chemical transmitter molecules, which cross the synapse and encounter a specialized region of postsynaptic muscle. Within the muscle membrane are thousands of receptor proteins specific for this transmitter. Activation of these postsynaptic receptors allows positively charged ions to cross the muscle membrane, generating a muscle cell action potential that leads to muscle contraction. Because of its size, contraction of a muscle cell requires the activation of an exceptionally large number of neurotransmitter receptors. To understand the regulation of this reliable communication and to elucidate details of pathological conditions that lead to muscle weakness, we have studied the subcellular mechanisms that govern synaptic transmission at the neuromuscular junction (NMJ). This article will review recent electron microscopic, electrophysiological, and imaging data in a discussion of the function of the motor nerve terminal in both normal and diseased states. Taken together, the existing data lead us to hypothesize that a small fraction of available calcium channels open within the transmitter releasing regions of the NMJ and that each vesicle fusion event is triggered by calcium flux through a single channel opening.     

Defects in neuromuscular junction structure in dystrophic muscle are corrected by expression of a NOS transgene in dystrophin-deficient muscles, but not in muscles lacking alpha- and beta1-syntrophins

Muscular dystrophies that arise from mutations of genes that encode proteins in the dystrophin-glycoprotein complex (DGC) frequently involve defects in the structure of neuromuscular junctions (NMJs). DGC mutations that cause NMJ defects typically cause a secondary loss of neuronal nitric oxide synthase (nNOS) from the post-synaptic membrane. We tested the hypothesis that reduction of muscle-derived NO production causes NMJ defects in DGC mutants by analyzing the effect of modulating muscle NO production on NMJ structure in mutant and wild-type muscles. We found that nNOS null mutants, dystrophin-deficient mdx mice and alpha-syntrophin null mutants showed reductions in the concentration of acetylcholine receptors (AChRs) at the post-synaptic membrane. Also, expression of a muscle-specific NOS transgene increased AChR concentration, which reflected an increase in both AChR expression and clustering. NOS transgene expression also increased the size of NMJs, and partially corrected defects in normal NMJ architecture that were observed in mdx and alpha-syntrophin null muscles. In addition, stimulation of AChR clustering in vitro by application of laminin or VVA B4 lectin induced a 3-4-fold increase in NOS activity and increased AChR clustering that could be prevented by NOS inhibition. However, the partial rescue of NMJ structure by expression of a NOS transgene required the expression of alpha- or beta1-syntrophin at the NMJ; partial NMJ rescue was seen in the muscles of alpha-syntrophin mutants that expressed beta1-syntrophin, but no rescue was observed in muscles of alpha-syntrophin mutants that also lacked beta1-syntrophin. These findings show that NO promotes AChR expression and clustering in vivo and contributes to normal NMJ architecture. The results suggest that defects in NMJ structure that occur in some DGC mutants can result from the secondary loss of NOS from muscle.     

Microbial Toll/interleukin 1 receptor proteins: A new class of virulence factors

Quite a number of microbes possess genes which encode for proteins containing a Toll/interleukin 1 receptor domain. This domain is key for the physical interaction of eukaryotic Toll-like receptors with their adaptor molecules like MyD88 enabling innate immune cells to recognize invading pathogens and to initiate appropriate defense responses. Recent findings imply that microbial Toll/interleukin 1 receptor proteins impair Toll-like receptor signaling. As a consequence, secretion of pro-inflammatory cytokines is dampened, and microbial replication is enhanced. This group of proteins can thus be classified as a new family of virulence factors able to modulate the Toll-like receptor signaling cascade. This review summarizes current knowledge of the biology of this fascinating group of molecules.