Embryonic expression patterns of the Drosophila dystrophin-associated glycoprotein complex orthologs

Mutations in genes encoding proteins of the human dystrophin-associated glycoprotein complex (DGC) cause the Duchenne, Becker and limb-girdle muscular dystrophies. Subsets of the DGC proteins form tissue-specific complexes which are thought to play structural and signaling roles in the muscle and at the neuromuscular junction. Furthermore, mutations in the dystrophin gene that lead to Duchenne muscular dystrophy are frequently associated with cognitive and behavioral deficits, suggesting a role for dystrophin in the nervous system. Despite significant progress over the past decade, many fundamental questions about the roles played by dystrophin and the other DGC proteins in the muscle and peripheral and central nervous systems remain to be answered. Mammalian models of DGC gene function are complicated by the existence of fully or partially redundant genes whose functions can mask effects of the inactivation of a given DGC gene. The genome of the fruitfly Drosophila melanogaster encodes a single ortholog of the majority of the mammalian DGC protein subclasses, thus potentially simplifying their functional analysis. We report here the embryonic mRNA expression patterns of the individual DGC orthologs. We find that they are predominantly expressed in the nervous system and in muscle. Dystrophin, dystrobrevin-like, dystroglycan-like, syntrophin-like 1, and all three sarcoglycan orthologs are found in the brain and the ventral nerve cord, while dystrophin, dystrobrevin-like, dystroglycan-like, syntrophin-like 2, sarcoglycan alpha and sarcoglycan delta are expressed in distinct and sometimes overlapping domains of mesoderm-derived tissues, i.e. muscles of the body wall and around the gut.     

Maturation and maintenance of the neuromuscular synapse: genetic evidence for roles of the dystrophin--glycoprotein complex

The dystrophin-glycoprotein complex (DGC) links the cytoskeleton of muscle fibers to their extracellular matrix. Using knockout mice, we show that a cytoplasmic DGC component, alpha-dystrobrevin (alpha-DB), is dispensable for formation of the neuromuscular junction (NMJ) but required for maturation of its postsynaptic apparatus. We also analyzed double and triple mutants lacking other cytoskeletal DGC components (utrophin and dystrophin) and myotubes lacking a alpha-DB or a transmembrane DGC component (dystroglycan). Our results suggest that alpha-DB acts via its linkage to the DGC to enhance the stability of postsynaptic specializations following their DGC-independent formation; dystroglycan may play additional roles in assembling synaptic basal lamina. Together, these results demonstrate involvement of distinct protein complexes in the formation and maintenance of the synapse and implicate the DGC in the latter process.     

Muscle dystroglycan organizes the postsynapse and regulates presynaptic neurotransmitter release at the Drosophila neuromuscular junction

Background

The Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila.

Methodology/Principal Findings

We show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site.

Conclusion/Significance

Altogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg.     

Seeking long-term relationship: axon and target communicate to organize synaptic differentiation

Synapses form after growing axons recognize their appropriate targets. The subsequent assembly of aligned pre and postsynaptic specializations is critical for synaptic function. This highly precise apposition of presynaptic elements (i.e. active zones) to postsynaptic specializations (i.e. neurotransmitter receptor clusters) strongly suggests that communication between the axon and target is required for synaptic differentiation. What trans‐synaptic factors drive such differentiation at vertebrate synapses? First insights into the answers to this question came from studies at the neuromuscular junction (NMJ), where axon‐derived agrin and muscle‐derived laminin β2 induce post and presynaptic differentiation, respectively. Recent work has suggested that axon‐ and target‐derived factors similarly drive synaptic differentiation at central synapses. Specifically, WNT‐7a, neuroligin, synaptic cell adhesion molecule (SynCAM) and fibroblast growth factor‐22 (FGF‐22) have all been identified as target‐derived presynaptic organizers, whereas axon‐derived neuronal activity regulated pentraxin (Narp), ephrinB and neurexin reciprocally co‐ordinate postsynaptic differentiation. In addition to these axon‐ and target‐derived inducers of synaptic differentiation, factors released from glial cells have also been implicated in regulating synapse assembly. Together, these recent findings have profoundly advanced our understanding of how precise appositions are established during vertebrate nervous system development.     

Role of gelatinases in pathological and physiological processes involving the dystrophin–glycoprotein complex

Dystrophin is a cytosolic protein belonging to a membrane-spanning glycoprotein complex, called dystrophin–glycoprotein complex (DGC) that is expressed in many tissues, especially in skeletal muscle and in the nervous system. The DGC connects the cytoskeleton to the extracellular matrix and, although none of the proteins of the DGC displays kinase or phosphatase activity, it is involved in many signal transduction pathways. Mutations in some components of the DGC are linked to many forms of inherited muscular dystrophies. In particular, a mutation in the dystrophin gene, leading to a complete loss of the protein, provokes one of the most prominent muscular dystrophies, the Duchenne muscular dystrophy, which affects 1 out of 3500 newborn males. What is observed in these circumstances, is a dramatic alteration of the expression levels of a multitude of metalloproteinases (MMPs), a family of extracellular Zn²⁺-dependent endopeptidases, in particular of MMP-2 and MMP-9, also called gelatinases. Indeed, the enzymatic activity of MMP-2 and MMP-9 on dystroglycan, an important member of the DGC, plays a significant role also in physiological processes taking place in the central and peripheral nervous system. This mini-review discusses the role of MMP-2 and MMP-9, in physiological as well as pathological processes involving members of the DGC.     

The physiological and behavioral effects of carbon dioxide on Drosophila melanogaster larvae

Adult and larval insects are rapidly anesthetized by carbon dioxide (CO2); however, the mechanisms have not been addressed. In this study, we use larval Drosophila to investigate the actions of CO2 to explain the behavioral effects of rapid immobilization and cardiac arrest with acute exposure to CO2. To determine if the central nervous system (CNS) is required, studies were performed with and without the CNS. The effects of low pH induced by exposure to CO2 were also examined. An acidic saline increases the heart rate in contrast to saline containing CO2. Synaptic transmission at the skeletal neuromuscular junction (NMJ) is blocked by CO2 but not by low pH. The site of action is postsynaptic by a decreased sensitivity to glutamate, the neurotransmitter at Drosophila NMJs. The CNS remains active in synaptic transmission when exposed to CO2 which is in contrast to the synapses at the NMJ. In summary, the effects of CO2 are directly mediated on the heart to stop it and at skeletal NMJs by a reduced sensitivity to glutamate, the released neurotransmitter, from the motor nerve terminals. The rapid behavioral and physiological effects cannot be accounted for by action on the CNS within the larvae nor by a pH effect indirectly induced by CO2. The glutamate receptors in the D. melanogaster preparation are similar in function to ionotropic glutamate receptors in vertebrates which could account for the observational phenomena of CO2 not yet explained mechanistically in vertebrates.     

The nerve-muscle synapse of the garter snake

Snake nerve-muscle preparations are well-suited for study of both motor innervation patterns at the systems level and NMJ function at the cellular level. Their small size (～100 myofibers) and thinness (one fiber) allows access to all NMJs in one muscle. Snake NMJs are of three types, two twitch subtypes and a single tonic type. Properties of the NMJs supplied by a particular motor neuron, and of the motor unit fibers they innervate, are precisely regulated by the motor neuron in a manner consistent with the Henneman Size Principle. Unlike its amphibian or mammalian cousins, the snake NMJ comprises ～50 (twitch) or ～20 (tonic) individual one-bouton synapses, similar to synapses found in the central nervous system. Each bouton releases a few quanta per stimulus. Larger fibers, which require more synaptic current to initiate contraction, receive nerve terminals that contain more boutons and express receptor patches with higher sensitivity to transmitter. Quantal analysis suggests that transmitter release sites in one bouton do not behave independently; rather, they may cooperate to reduce fluctuations and enhance reliability. After release, two mechanisms coexist for retrieval and reprocessing of spent vesicles–one involving clathrin-mediated endocytosis, the other macropinocytosis. Unanswered questions include how each mechanism is regulated in a use-dependent manner.     

Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease

The kinesin, dynein, and myosin superfamily molecular motors have fundamental roles in neuronal function, plasticity, morphogenesis, and survival by transporting cargos such as synaptic vesicle precursors, neurotransmitter and neurotrophic factor receptors, and mRNAs within axons, dendrites, and synapses. Recent studies have begun to clarify the mechanisms of cargo selection and directional transport in subcellular compartments. Furthermore, molecular genetics has revealed unexpected roles for molecular motors in brain wiring, neuronal survival, neuronal plasticity, higher brain function, and control of central nervous system and peripheral nervous system development. Finally, it is also evident that molecular motors are critically involved in neuronal disease pathogenesis. Thus, molecular motor research is becoming an exciting frontier of neuroscience.     

Synapse elimination from the mouse neuromuscular junction in vitro: A non-hebbian activity-dependent process

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2–7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p< 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i. e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p< 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development. © 1993 John Wiley & Sons, Inc.     

Roles of the amyloid precursor protein family in the peripheral nervous system

Compelling evidence from in vivo model systems within the past decade shows that the APP family of proteins is important for synaptic development and function in the central and peripheral nervous systems. The synaptic role promises to be complex and multifaceted for several reasons. The three family members have overlapping and redundant functions in mammals. They have both adhesive and signaling properties and may, in principle, act as both ligands and receptors. Moreover, they bind a multitude of synapse-specific proteins, and we predict that additional interacting protein partners will be discovered. Transgenic mice with modified or abolished expression of APP and APLPs have synaptic defects that are readily apparent. Studies of the neuromuscular junction (NMJ) in these transgenic mice have revealed molecular and functional deficits in neurotransmitter release, in organization of the postsynaptic receptors, and in coordinated intercellular development. The results summarized here from invertebrate and vertebrate systems confirm that the NMJ with its accessibility, large size, and homogeneity provides a model synapse for identifying and analyzing molecular pathways of APP actions.     

Postsynaptic signaling of new players at the neuromuscular junction

The neuromuscular junction (NMJ) represents the most well studied synapse and is widely regarded as structurally and functionally less complicated than neuronal synapses in the brain. Recent studies, however, have identified the localization and function of new signaling molecules at the NMJ. Surprisingly, many synaptic proteins previously identified in the brain are indeed also concentrated on the postsynaptic muscle side of the NMJ. These include the serine/threonine kinase Cdk5, the neurotrophin receptor TrkB, Eph receptors and ephrins, NMDA receptors and nitric oxide synthase, various PDZ-domain scaffold proteins, and β-amyloid precursor protein. These observations indicate that the molecular composition of NMJ is much more intricate than we originally thought. The potential significance of these new signaling molecules at the NMJ will be discussed.     

Review. The molecular logic of sodium-coupled neurotransmitter transporters

Synaptic transmission at chemical synapses requires the removal of neurotransmitter from extracellular spaces. At synapses in the central nervous system, this is accomplished by sodium-coupled transport proteins, integral membrane proteins that thermodynamically couple the uptake of neurotransmitter to the uptake of sodium and, in some cases, the uptake and export of additional ions. Recent X-ray crystallographic studies have revealed the architecture of the two major families of neurotransmitter transporters and, together with additional biochemical and biophysical studies, have provided insights into mechanisms of ion coupling, substrate uptake, and inhibition of transport.     

Wnt Signaling in Neuromuscular Junction Development

Wnt proteins are best known for their profound roles in cell patterning, because they are required for the embryonic development of all animal species studied to date. Besides regulating cell fate, Wnt proteins are gaining increasing recognition for their roles in nervous system development and function. New studies indicate that multiple positive and negative Wnt signaling pathways take place simultaneously during the formation of vertebrate and invertebrate neuromuscular junctions. Although some Wnts are essential for the formation of NMJs, others appear to play a more modulatory role as part of multiple signaling pathways. Here we review the most recent findings regarding the function of Wnts at the NMJ from both vertebrate and invertebrate model systems.Wnt proteins are evolutionarily conserved, secreted lipo-glycoproteins involved in a wide range of developmental processes in all metazoan organisms examined to date. In addition to governing many embryonic developmental processes, Wnt signaling is also involved in nervous system maintenance and function, and deregulation of Wnt signaling pathways occurs in many neurodegenerative and psychiatric diseases (De Ferrari and Inestrosa 2000; Caricasole et al. 2005; Okerlund and Cheyette 2011). The first link between Wnt signaling and synapse development was established by Salinas and colleagues in the vertebrate nervous system (Lucas and Salinas 1997; Hall et al. 2000) and by Budnik and colleagues at the invertebrate neuromuscular junction (NMJ) (Packard et al. 2002). Since then, Wnt signaling has emerged as an essential regulator of synaptic development and function in both central and peripheral synapses. Although important roles for Wnt signaling have become known from studies in both the central and peripheral nervous system, this article is concerned with the role of Wnts at the NMJ.     

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.     

New transmitters and new targets in the autonomic nervous system

Several recent findings have made research into the autonomic nervous system even more. exciting, such as the revelation that nitric oxide is a major neurotransmitter, the delineation of the physiological roles for purines and vasoactive intestinal peptide, and the discovery that the interstitial cells of Cajal are major target cells for enteric innervation. Nitric oxide is probably the major neurotransmitter evoking inhibitory junction potentials in smooth muscle. ATP is a mediator of non-adrenergic non-cholinergic enteric innervation, as well as being a fast neurotransmitter in peripheral and autonomic neuro-neuronal synapses. The interactions between enteric nerves and both immune cells and interstitial cells of Cajal (as pacemaker cells of gut smooth muscle) are forcing a rethink of many aspects of gut physiology.     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

The dynamics of recycled acetylcholine receptors at the neuromuscular junction in vivo

At the peripheral neuromuscular junction (NMJ), a significant number of nicotinic acetylcholine receptors (AChRs) recycle back into the postsynaptic membrane after internalization to intermingle with not-yet-internalized ;pre-existing' AChRs. However, the way in which these receptor pools are maintained and regulated at the NMJ in living animals remains unknown. Here, we demonstrate that recycled receptors in functional synapses are removed approximately four times faster than pre-existing receptors, and that most removed recycled receptors are replaced by new recycled ones. In denervated NMJs, the recycling of AChRs is significantly depressed and their removal rate increased, whereas direct muscle stimulation prevents their loss. Furthermore, we show that protein tyrosine phosphatase inhibitors cause the selective accumulation of recycled AChRs in the peri-synaptic membrane without affecting the pre-existing AChR pool. The inhibition of serine/threonine phosphatases, however, has no effect on AChR recycling. These data show that recycled receptors are remarkably dynamic, and suggest a potential role for tyrosine dephosphorylation in the insertion and maintenance of recycled AChRs at the postsynaptic membrane. These findings may provide insights into long-term recycling processes at less accessible synapses in the central nervous system in vivo.     

A New Fiji-Based Algorithm That Systematically Quantifies Nine Synaptic Parameters Provides Insights into Drosophila NMJ Morphometry

The morphology of synapses is of central interest in neuroscience because of the intimate relation with synaptic efficacy. Two decades of gene manipulation studies in different animal models have revealed a repertoire of molecules that contribute to synapse development. However, since such studies often assessed only one, or at best a few, morphological features at a given synapse, it remained unaddressed how different structural aspects relate to one another. Furthermore, such focused and sometimes only qualitative approaches likely left many of the more subtle players unnoticed. Here, we present the image analysis algorithm ‘Drosophila_NMJ_Morphometrics’, available as a Fiji-compatible macro, for quantitative, accurate and objective synapse morphometry of the Drosophila larval neuromuscular junction (NMJ), a well-established glutamatergic model synapse. We developed this methodology for semi-automated multiparametric analyses of NMJ terminals immunolabeled for the commonly used markers Dlg1 and Brp and showed that it also works for Hrp, Csp and Syt. We demonstrate that gender, genetic background and identity of abdominal body segment consistently and significantly contribute to variability in our data, suggesting that controlling for these parameters is important to minimize variability in quantitative analyses. Correlation and principal component analyses (PCA) were performed to investigate which morphometric parameters are inter-dependent and which ones are regulated rather independently. Based on nine acquired parameters, we identified five morphometric groups: NMJ size, geometry, muscle size, number of NMJ islands and number of active zones. Based on our finding that the parameters of the first two principal components hardly correlated with each other, we suggest that different molecular processes underlie these two morphometric groups. Our study sets the stage for systems morphometry approaches at the well-studied Drosophila NMJ.     

Cellular mechanisms governing synapse formation: lessons from identified neurons in culture

The accessibility of embryonic and adult neurons within invertebrate nervous systems has made them excellent subjects for neurobiological study. The ability to readily identify individual neurons, together with their great capacity for regeneration, has been especially beneficial to investigations of synapse formation and the specificity of neuronal connectivity. Many invertebrate neurons survive for long periods following isolation into primary cell culture. In addition, they readily extend new neuritic arbors and form electrical and chemical connections at sites of contact. Thus, cell culture approaches have allowed neuroscientists greater access to, and resolution of, events underlying neurite outgrowth and synaptogenesis. Studies of identified neuromuscular synapses ofHelisoma have determined a number of signaling mechanisms involved in transsynaptic communication at sites of neuron-target contact. At these sites, both anterograde and retrograde signals regulate the transformation of growth cones into functional presynaptic terminals. We have found that specific muscle targets induce both global and local changes in neurotransmitter secretion and intracellular calcium handling. Here we review recent studies of culturedHelisoma synapses and discuss the mechanisms thought to govern chemical synapse formation in these identified neurons and those of other invertebrate species.