InteractiveFly: GeneBrief

mind the gap: Biological Overview | References


Gene name - mind the gap

Synonyms -

Cytological map position - 84F11-84F6

Function - carbohydrate-binding protein

Keywords - synapse assembly; neuromuscular synaptic transmission; regulation of synapse organization, N-glycosaminoglycan-binding protein, modifier of signaling by the ligand Jelly belly and its receptor Alk, CNS

Symbol - mtg

FlyBase ID: FBgn0260386

Genetic map position - chr3R:4076220-4086440

Classification - Chitin binding Peritrophin-A domain

Cellular location - secreted



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Formation and regulation of excitatory glutamatergic synapses is essential for shaping neural circuits throughout development. A genetic screen for synaptogenesis mutants in Drosophila identified mind the gap (mtg), which encodes a secreted, extracellular N-glycosaminoglycan-binding protein. Mtg is expressed neuronally and detected in the synaptic cleft, and is required to form the specialized transsynaptic matrix that links the presynaptic active zone with the post-synaptic glutamate receptor (GluR) domain. Null mtg embryonic mutant synapses exhibit greatly reduced GluR function, and a corresponding loss of localized GluR domains. All known post-synaptic signaling/scaffold proteins functioning upstream of GluR localization are also grossly reduced or mislocalized in mtg mutants, including the dPix-dPak-Dock cascade and the Dlg/PSD-95 scaffold. Ubiquitous or neuronally targeted mtg RNA interference (RNAi) similarly reduce post-synaptic assembly, whereas post-synaptically targeted RNAi has no effect, indicating that presynaptic MTG induces and maintains the post-synaptic pathways driving GluR domain formation. These findings suggest that MTG is secreted from the presynaptic terminal to shape the extracellular synaptic cleft domain, and that the cleft domain functions to mediate transsynaptic signals required for post-synaptic development (Rohrbough, 2007).

Glutamatergic synapse formation and maturation is critical for sculpting neural circuits. Synaptogenesis defects cause crippling neurological disabilities ranging from motor ataxias to profound mental retardation, and subsequent modulation of glutamatergic synapses is a lifelong dynamic process underlying the ability to learn and remember. A critical hypothesis, developed largely from the vertebrate neuromuscular junction (NMJ) model, is that presynaptic signals trigger post-synaptic assembly and modulation. Proposed secreted signals include Agrin, WNTs, FGFs and Narp (see O'Brien, 2002). Transmembrane synaptic signaling proteins include SynCAM, EphrinB-EphB, and β-neurexin/neuroligin. Both classes of signals are proposed to interact intimately with a specialized synaptic cleft extracellular matrix (ECM), which is molecularly distinct from nonsynaptic ECM (Rohrbough, 2007).

Synaptic cleft ECM components include heterotrimeric (α/β/γ) laminin glycoproteins and heparan sulfate proteoglycans (HSPGs), which bind extracellular glucuronic acid and N-acetyl glucosamine (GlcNAc) polysaccharides. The secreted HSPGs Agrin and Perlecan are established regulators of NMJ synaptogenesis. The HSPG Syndecan (Sdc)-2 is similarly implicated in hippocampal synapse formation and plasticity. ECM signaling at the mammalian NMJ also acts via the α/β-dystroglycan-glycoprotein complex (DGC). Numerous integrin receptors localize to mammalian NMJs and central glutamatergic synapses, and integrin-ECM interactions regulate aspects of synaptic development and modulation, including glutamatergic transmission and plasticity (Rohrbough, 2007).

The Drosophila glutamatergic NMJ contains an ultrastructurally distinctive synaptic cleft ECM present only between pre- and post-synaptic densities, but little is known of its molecular composition. HSPGs, including laminin-binding Sdc and GPI-anchored Dallylike (Dlp), and the secreted Hikaru Genki (HIG) are localized to the cleft ECM and regulate synaptic differentiation. A post-synaptic Dystrophin scaffold has recently been shown to regulate NMJ maturation. Drosophila WNT Wingless (Wg) is a secreted anterograde synaptic maturation signal that acts via its pre/post-synaptic receptor Frizzled (Dfz2). Integrin ECM receptors and laminin are localized to NMJ synapses and regulate synapse formation, and both structural and functional development (Rohrbough, 2007).

The Drosophila NMJ post-synaptic domain contains two subclasses of tetrameric AMPA/kainate-like glutamate receptors (GluRs), composed of either IIA (A-class) or IIB (B-class) subunits, in combination with common required IIC, IID, and IIE subunits. The PDZ-domain scaffold Discs Large (Dlg), a PSD-95/SAP70 homolog, plays a key role in localizing post-synaptic proteins including B-class GluRs. The Drosophila p21-activated kinase (dPak), a serine threonine kinase activated by GTPases Rac and Cdc42, and its localizing Rho-type GEF, dPix, also play essential roles in the post-synaptic domain. dPAK interacts directly via its kinase domain with Dlg, and is required for Dlg synaptic expression. In a second pathway, dPak binding to the adaptor Dreadlocks (Dock), a Src homology (SH3 and SH2)-containing Nck homolo, regulates A-class GluR abundance. The dPix-dPak-Dock and dPak-Dlg pathways therefore converge to regulate localization of both GluR classes in the post-synaptic domain (Rohrbough, 2007).

Extensive work in this model system has established that the presynaptic neuron induces post-synaptic differentiation, inducing development and modulation of GluR domains. GluR domain formation involves lateral membrane receptor diffusion, receptor sequestration/anchoring mechanisms, regulated GluR subunit transcription, and local post-synaptic translation. To define the molecular mechanisms regulating functional post-synaptic differentiation, a systematic mutagenesis screen was undertaken to isolate mutants with defective post-synaptic assembly. This approach has revealed mind the gap (mtg), which encodes a secreted protein required for the formation of the synaptic cleft matrix, as well as a localization of the signaling pathways regulating GluR domains. Presynaptic mtg knockdown inhibits post-synaptic differentiation, indicating that presynaptically secreted MTG organizes the extracellular cleft domain and is critically required for transsynaptic signaling that induces post-synaptic differentiation (Rohrbough, 2007).

The mind the gap (mtg) gene was isolated in an unbiased forward Drosophila genetic screen for novel mutants blocking functional differentiation of the glutamatergic NMJ synapse. This screen has now revealed numerous novel genes and mechanisms regulating pre- or post-synaptic development, including GluR subunits and genes regulating functional receptor expression. Loss of mtg results in a severe, ~80% loss of GluR function at the NMJ. Notably, this phenotype represents the most severe mutant GluR impairment ever reported, with the exception of genetic mutants for the requisite GluR subunits (IIC-IIE) themselves . MTG is expressed neuronally and localized synaptically, contains a well-conserved secretory signal sequence, and is secreted and binds GlcNAc in vitro. These findings support the working hypothesis that MTG is secreted into the synaptic cleft to bind ECM glycosaminoglycans (GAG) or proteoglycans (PG) during glutamatergic synaptogenesis (Rohrbough, 2007).

The mtg developmental expression profile closely parallels the timing of NMJ synapse formation and functional differentiation. Expression increases sharply with initial nerve-muscle contact (12-13 h after fertilization), and peaks at 16-17 h, correlating with post-synaptic GluR domain assembly (15-17 h). MTG protein is concentrated in embryonic neurons, and becomes increasingly localized with development to NMJ synaptic domains, and to other cell adhesion sites such as muscle attachment sites, where many synaptic signaling (Pix, Pak, integrins) and scaffold proteins (Dlg) are colocalized. MTG is found in the presynaptic terminal and post-synaptic SSR, clearly detected within the extracellular synaptic cleft domain, consistent with presynaptic secretion as well as a transsynaptic regulatory role. In the absence of MTG, post-synaptic GluR puncta apposed to the presynaptic terminal are lost, and GluRs are dispersed in the nonsynaptic membrane, resulting in profound functional transmission loss. The mislocalized GluRs appear nonfunctional based on the muscle response to exogenously applied glutamate, which demonstrates a dramatic overall loss of functional cell surface GluRs. All PSD proteins known to act in the upstream GluR regulatory pathways (dPix, dPak, Dlg, Dock) are severely reduced or mislocalized in mtg mutants, indicating that MTG acts at an upstream organizing step required to establish this cascade of post-synaptic interactions. The loss of synaptic cleft matrix material at mtg mutant synapses indicates first, that MTG is required for this signaling domain to be established, and second, suggests that this domain has an important inductive, instructive role in post-synaptic assembly (Rohrbough, 2007).

Genetically rescuing the mtg mutant has not been accomplished with a wild-type copy of the mtg gene. Since mtg is present at low overall levels during much of development, it is likely that mtg expression timing and/or level must be precisely regulated for normal protein function and animal viability. Transgenic rescue with tissue-targeted Gal4-driven mtg expression may therefore result in a deleterious overexpression condition. Transgenically expressed mtg RNAi, however, phenocopies mtg mutant phenotypes. Ubiquitous mtg RNAi causes early lethality and defective post-synaptic assembly, with reduced synaptic localization of GluRs and other upstream regulatory proteins. These phenotypes are more severe in the mtg1 mutant, which exhibits lethality at the hatching stage, and also nonsynaptic mislocalization of post-synaptic proteins. Therefore, the findings are consistent with the expected effects of RNAi as a partial loss-of-function condition. Most conclusively, targeted RNAi knockdown of MTG in the presynaptic neuron impairs post-synaptic differentiation and the assembly of GluR domains with a similar severity to the ubiquitous RNAi condition. In contrast, muscle-targeted mtg RNAi has no detectable effect on movement or animal viability, and does not cause any detectable synaptic impairment or defects in post-synaptic assembly. Taken together, these results support the identity of the mtg gene and suggest that presynaptically secreted MTG protein is required for post-synaptic development. It is concluded that MTG is a critical element in the presynaptic inductive mechanism (Rohrbough, 2007).

MTG has a cysteine-rich carbohydrate-binding module (CBM) with homology with ChtBDs found in peritrophic matrix proteins, lectins, and other known ECM proteins. This domain contains six conserved cysteines predicted to form three disulfide bridges within a β-folded carbohydrate-binding structure that binds GlcNAc moieties. Lectins that specifically bind GlcNAc (WGA) and GalNAc (DBA, VVA) are commonly used synaptic cleft or post-synaptic markers at the vertebrate NMJ (Martin 2003). This study confirms that WGA-binding targets are localized to Drosophila NMJ boutons, and that extracted MTG protein binds GlcNAc in vitro, suggesting that MTG recognizes GlcNAc-containing target(s) in the synaptic cleft. Drosophila S2 cells transformed with MTG-GFP secrete the protein, which accumulates both on the outer surface of the cells and in the medium, supporting its extracellular localization. However, it has not yet been possible to replicate the GlcNAc binding with purified MTG protein, the ideal experiment to confirm and further probe the binding specificity. GlcNAc-containing carbohydrate and GAG scaffolds (e.g., chitin, hyaluronic acid [HA], heparin) and PG (e.g., heparin sulfate, chondroitin sulfate) are components of neuronal and muscle ECM, and concentrated within the specialized synaptic cleft matrix at the NMJ and other synapses in multiple species. It was recently shown that loss of GlcNAc transferase alters In Drosophila NMJ synaptic structure, function, and locomotory behavior, independently demonstrating GlcNAc-mediated interactions have roles in synaptic maturation (Rohrbough, 2007).

MTG does not share significant overall whole-sequence homology with an identified vertebrate protein. It is increasingly recognized that many Drosophila proteins are conserved at a structural level to serve identical functions with mammalian functional homologs; among many examples are neurotrophin-like proteins and their receptors and olfactory receptors. Several vertebrate protein families contain cysteine-rich domains with predicted structural homology with MTG, including the TGF-β, GPH, PDGF, and NGF growth factor families. CDM/ChtBD-related domains are common in other secreted protein families, including knottins, mucins and lectins. These domains contain six to 10 cysteine residues predicted to form disulfide bridges, which mediate homo- or heterodimer formation, carbohydrate binding, and extracellular ligand-target/receptor interactions. Laminin integrin ligands contain the structurally related type-1 EGF domain, which is a site of receptor recognition and ECM binding. The consistent extracellular function of these related disulfide-forming protein domains is to mediate ECM protein interactions and ligand-receptor intercellular signaling. A similar function is proposed for MTG in organizing the synaptic cleft matrix and mediating transsynaptic signaling critical for synaptogenesis (Rohrbough, 2007).

Null mtg mutants show a reduction or complete absence of electron-dense synaptic matrix, suggesting a loss or gross disorganization of multiple synaptic ECM components and binding proteins. This is the first report of such a cleft phenotype reported for a functional synaptic mutant. Vertebrate neuronal and perisynaptic ECM consists of a GAG scaffold matrix (e.g., heparin sulfate, chondroitin sulfate, HA), numerous bound proteins, laminins, and transmembrane molecules/receptors interacting with the matrix, including NCAM family proteins and integrins. Much less is known, however, about the mechanisms linking the synaptic cleft matrix structural and signaling environment to post-synaptic assembly. The regulation of acetylcholine receptor (AChR) expression/localization at vertebrate NMJs by the agrin-agrin receptor (MusK)-rapsyn pathway provides an obvious framework for comparison, although the role of agrin in post-synaptic receptor maintenance versus domain assembly has recently been redefined (Misgeld, 2005; Kummer, 2006). The vertebrate cholinergic NMJ is concentrated in GlcNAc- and GalNAc-containing GAG and PG, in particular secreted (agrin, perlecan) and transmembrane (syndecan) heparin sulfate proteoglycans (HSPG); laminins, which act as integrin ligands and interact directly with other membrane proteins including Ca2+ channels (Nishimune. 2004); and ECM transmembrane receptors, including receptor tyrosine kinases (RTK) and integrins, activated by binding of ECM ligands. Integrins represent an appealing ECM receptor-mediated link to Pix-Pak pathway activation in vertebrates, and hippocampal synapse formation and plasticity in the hippocampus (Rohrbough, 2007).

Three integrin receptor subtypes (αPS1/βPS, αPS2/βPS, αPS3/βPS) localize to the Drosophila NMJ, and regulate synaptic structural development and functional transmission properties, including activity-dependent plasticity. Integrins, RGD domain-containing laminins, and the secreted synaptic cleft protein Hikaru Genki regulate synapse formation and ultrastructure. More recently, two synaptic HSPGs, Syndecan (Sdc) and Dallylike (Dlp), were shown to localize to the NMJ and regulate presynaptic terminal growth and AZ formation, respectively. The receptor tyrosine phosphatase dLAR is a receptor for both Sdc and Dlp and interacts with these ligands via their GAG chains. Drosophila dystrophin has also recently been shown to localize post-synaptically, and form a synaptic glycoprotein complex with extracellular dystroglycan. Surprisingly, Drosophila dystrophin regulates presynaptic properties, but not post-synaptic GluR expression. These studies do not suggest that MTG is a major structural component of the synaptic cleft matrix, but rather that MTG has a necessary role in organizing the broader structure and transsynaptic signaling capabilities of the synaptic cleft ECM. If this hypothesis is correct, the severity of the mtg mutant phenotypes may be due to a disruption of multiple transsynaptic signaling pathways (Rohrbough, 2007).

The findings of this study suggest that interactions between MTG and its GAG/PG-binding partner(s) in the synaptic cleft matrix are linked to the activation and localization of PSD signaling pathways. Protein localization/binding studies and three-dimensional structural models suggest that the cleft domain is a dense GAG scaffold extensively linked by secreted matrix PG, including chondroitin sulfate PG (tenascins, lecticans, phosphacans), and HSPGs (perlecan, β-glycans, agrins), as well as by transmembrane HSPGs (syndecans, systroglycans), GPI-linked membrane-bound proteins (glypicans), NCAMs, and integrins. This matrix could act in part to sterically trap or limit lateral movement of transmembrane proteins to maintain them in the synapse. Such a mechanism could preferentially or selectively serve to localize a key upstream signaling molecule, such as dPix, thus localizing the downstream dPak-Dlg-Dock cascade necessary for GluR aggregation. Similarly, the synaptic matrix may directly inhibit GluR lateral membrane diffusion, effectively ensnaring GluRs at post-synaptic sites. In the absence of the matrix, GluR dispersal could prevent accumulation of functional puncta. Alternatively, MTG may be more directly involved in forming a synaptic cleft signaling environment that allows signal molecules and/or receptors to be properly presented or anchored. It is also possible that MTG may function directly as an inductive signal by binding to an unidentified receptor. For example, synaptic localization of Dock requires its interaction with an unidentified SH2 domain-containing RTK, but the identity of this RTK or its effectors is unknown (Rohrbough, 2007).

MTG is required for the post-synaptic localization/activation of the dPix-dPak-Dock-Dlg pathways. dPix (Rho-type guanine exchange factor) binds to and is required to localize dPak; dpix and dpak mutants equally reduce synaptic Dlg level, and essentially eliminate formation of the post-synaptic SSR domain where these proteins reside (Albin, 2004). A dPak-Dock interaction is required to regulate synaptic levels of A-class GluRs (Albin, 2004). The GluR phenotypes of dpix, dpak, and dock mutants are all similar, reducing A-class GluRs by ~50%. These mutants are nevertheless viable through larval development in the near absence of dPix, dPak, or Dock, and NMJ synaptic transmission strength in basal evoked recordings is normal, due in large part to compensatory mechanisms leading to increased transmitter release (Albin, 2004). In studies in mature larvae, dpix, dpak, and dock mutations cause decreased expression level of post-synaptic proteins, without the dramatic mislocalization phenotypes characteristic of mtg functional null mutants at the embryonic NMJ. However, this study shows that strong MTG knockdown by RNAi in embryos and larvae causes a loss of post-synaptic protein levels that appears comparable with the phenotype in dpix and dpak mutant larvae. These findings suggest that the greater severity of the mtg1 mutant phenotype is likely due to a more severe loss of MTG function in the null condition, rather than simply due to a developmental disruption during the embryonic synaptogenesis period. Alternatively, since loss of MTG affects synaptic localization of both dPak and Dock individually, the full loss-of-function mtg phenotype severity may result from an additive block of several branches of the intertwined post-synaptic differentiation pathways (Rohrbough, 2007).

It is hypothesized that presynaptically secreted MTG establishes the synaptic cleft matrix signaling environment required for transsynaptic ligand-receptor pathways inducing post-synaptic differentiation. Several questions must next be addressed to test this hypothesis. One goal is to thoroughly test the GlcNAc binding specificity of MTG using purified protein and competitive binding assays. The future task will be to identify GlcNAc-containing GAG- or PG-binding target(s) of MTG resident in the synaptic cleft. Synaptic labeling with carbohydrate-specific lectins and matrix-specific antibodies, and genetically/pharmacologically altering synaptic protein glycosylation, will identify predicted glycosylated matrix components and potential targets. Site-directed mutational analysis of the key disulfide-forming cysteines in the GlcNAc-binding domain will allow testing this domain's role in MTG function. Another major goal will be to test other known transsynaptic signal/receptor pathways include Wg/Frz, Gbb/Wit, Syndecan/dLAR, DG/Dystrophin and integrin ligands (Hig, laminin)/integrins. Future studies will test whether these pathway components are lost/mislocalized in mtg mutants, as predicted by the model. Genetic interactions should exist between established signaling pathways and MTG, which can be tested in double mutant combinations to determine whether mtg phenotypes result from the additive disruption of multiple signaling pathways, as predicted. Finally, MTG may itself be an anterograde transsynaptic signaling molecule acting through a post-synaptic receptor. The potential receptor identities and assay possible signal-receptor interaction mechanisms during synaptogenesis will be the subject of future studies (Rohrbough, 2007).

Presynaptic secretion of Mind-the-gap organizes the synaptic extracellular matrix-integrin interface and postsynaptic environments

Mind-the-Gap (MTG) is required during synaptogenesis of the Drosophila glutamatergic neuromuscular junction (NMJ) to organize the postsynaptic domain. In this study MTG::GFP transgenic animals were generated to demonstrate MTG is synaptically targeted, secreted, and localized to punctate domains in the synaptic extracellular matrix (ECM). Drosophila NMJs form specialized ECM carbohydrate domains, with carbohydrate moieties and integrin ECM receptors occupying overlapping territories. Presynaptically secreted MTG recruits and reorganizes secreted carbohydrates, and acts to recruit synaptic integrins and ECM glycans. Transgenic MTG::GFP expression rescues hatching, movement, and synaptogenic defects in embryonic-lethal mtg null mutants. Targeted neuronal MTG expression rescues mutant synaptogenesis defects, and increases rescue of adult viability, supporting an essential neuronal function. These results indicate that presynaptically secreted MTG regulates the ECM-integrin interface, and drives an inductive mechanism for the functional differentiation of the postsynaptic domain of glutamatergic synapses. It is suggested that MTG pioneers a novel protein family involved in ECM-dependent synaptic differentiation (Rushton, 2009).

It is hypothesized that presynaptically secreted Mind-the-Gap (MTG) binds within the synaptic cleft extra-cellular matrix (ECM) to establish the synaptic cleft environment required for inductive signaling pathways driving postsynaptic assembly, including glutamate receptor (GluR) localization/maintenance. It has been shown previously that the MTG protein contains a predicted secretion signal peptide, and a 6-cysteine domain related to “cysteine-knot” domains, with high homology to the carbohydrate-binding domain (CBD) in ECM-binding protein families (Rohrbough, 2007). Endogenous MTG expression peaks sharply during late embryonic stages (16 – 8 hr), corresponding to the period of functional synapse differentiation. The key features of the mtg null mutant phenotype include loss of the electron-dense ECM material characterizing the synaptic cleft, a matrix of unknown composition and function; and the reduction/mislocalization of multiple postsynaptic density (PSD) proteins, including dPix, dPak, DLG, and Dock/Dreadlocks, which function in the pathway(s) regulating GluR localization and abundance (Rohrbough, 2007). Loss of MTG prevents GluR accumulation at the synapse, resulting in a severe (~80%) loss of the functional postsynaptic glutamate response. Neuronally-targeted mtg RNAi knockdown partially phenocopies these defects, supporting the hypothesis of an inductive, presynaptic requirement for MTG in postsynaptic assembly (Rushton, 2009).

Several key questions that were not adequately answered in earlier studies have been thoroughly addressed in this study. mtg gene identity was conclusively confirmed by demonstrating rescue of mtg null mutant viability with a single copy of a CG7549 cDNA GFP fusion construct. It was further shown that mtg overexpression is deleterious, demonstrating the need for precise regulation of MTG function, consistent with the sharp temporal regulation of endogenous mtg developmental expression levels (Rohrbough, 2007). In mtg null animals, it was first shown that the MTG::GFP transgene rescues MTG function. It was then shown that neuronally expressed MTG is subcellularly trafficked to synapses both in the CNS and at the NMJ. Cell-specific targeting of the MTG::GFP transgene using the GAL4-UAS approach was critical for demonstrating specific neuronal requirements, including presynaptic targeting and secretion of MTG, as well as non-neuronal requirements (Rushton, 2009).

The embryonic and larval central synaptic neuropil contains multiple classes of chemical synapses, including predominantly excitatory cholinergic connections driving glutamatergic motor output, as well as GABAergic and glutamatergic connections and neurosecretory terminals. The entire neuropil contains concentrated synaptic connections, as evident from the dense presentation of presynaptic active-zone proteins (BRP), synaptic vesicle-associated proteins and vesicular neurotransmitter transporters, and postsynaptic proteins (e.g., DLG). MTG::GFP is prominently localized in similar punctae distributed primarily along longitudinal axon tracts in central and medial regions of the ventral neuropil. When viewed in thin transverse or longitudinal neuropil sections, most MTG punctae appear closely adjacent to or surrounded by BRP punctae, rather than obviously co-localized with BRP. This finding is consistent with MTG functional synaptic localization both presynaptically near active zones and in secreted extracellular aggregates closely opposed to presynaptic boutons. Since MTG is targeted to and secreted at glutamatergic NMJ terminals, an attractive possibility is that MTG also has a specific parallel function at central glutamatergic synapses. A recent study suggested glutamatergic central synapses are primarily concentrated in dorsal neuropil regions in the larval ventral nerve cord (Daniels, 2008). Future localization studies, in combination with glutamatergic- and cholinergic-specific synaptic markers, are needed to identify whether MTG is localized to a particular subclass of chemical synapse (Rushton, 2009).

Utilizing the salivary gland (SG) as an accessible, specialized secretory tissue, this study showed that transgenically expressed MTG is prominently targeted to SG vesicles, subsequently secreted, and strongly accumulated in the lumen, providing in vivo demonstration of predicted MTG secretory function and validating earlier in vitro studies (Rohrbough, 2007). Secreted MTG remains closely associated with the external secretory cell membrane, revealing the profiles of fused vesicles, suggesting that the secreted protein remains bound to ECM. Neuronally expressed MTG::GFP is prominently contained within punctate aggregates both in the central neuropil and at NMJ synaptic boutons. Using detergent-free conditions to directly test secretion at NMJ synapses, this study showed that MTG aggregates are extracellularly localized immediately surrounding presynaptic boutons. The source of targeted MTG::GFP is entirely presynaptic, showing this externally localized MTG to be secreted from presynaptic terminals (Rushton, 2009).

The results indicate that secreted MTG occupies subregions of synaptic ECM. Using detergent-free lectin-labeling and immunostaining assays to isolate the extracellular domain, it was shown that the Drosophila NMJ synaptic ECM represents a specialized carbohydrate- and receptor-containing matrix domain, bearing many similarities, but also differences, compared to the vertebrate cholinergic NMJ. A punctate βPS integrin distribution defines a synaptic ECM subdomain surrounding type I synaptic boutons. ECM glycans, revealed by VVA and WGA lectins, occupy characteristic but overlapping subdomains with integrin receptors broadly surrounding the synaptic terminal. Other lectin probes (e.g., DBA, PNA) show no synaptic localization, underscoring the important fact that the synaptic ECM is defined by the exclusion of certain extracellular components, as well as the inclusion of synapse-specific molecules. Importantly, it was shown that secreted MTG::GFP aggregates localize within this broader ECM environment, overlapping with βPS integrins and VVA distribution, showing that MTG is positioned to interact with integrins and other ECM molecules. One potential interpretation is that MTG has a signaling function via integrins and/or specific glycans, and that these interactions occur in discrete extracellular synaptic signaling domains (Rushton, 2009).

A critical finding is that presynaptic MTG is necessary for normal synaptic integrin localization. βPS is a required subunit in all Drosophila synaptic integrin receptor subclasses; thus, the MTG-dependent reduction of βPS predicts a concurrent loss of α-integrin proteins and functional synaptic integrin receptors. The importance of bidirectional integrin-ligand interactions and patterning in other tissues suggests that synaptic PS integrins may have a major role in shaping synaptic cleft ECM organization and composition. Since secreted MTG occupies a subset of the βPS synaptic domain, another possibility is that MTG regulates an ECM integrin ligand, such as laminin-A, which in turn regulates integrin localization or maintenance within the synapse. It is difficult to assess the absolute requirement for synaptic integrins in ECM regulation because Drosophila βPS null mutants (myospheroid; mys) are 100% early embryonic lethal, with severely abnormal muscle patterning and altered NMJ morphological differentiation. A residual (~20% of normal) level of NMJ-localized βPS in mysxg43/mysts1 hypomorphs is sufficient for relatively normal synaptic composition, although mutant NMJ morphology and synaptic function are significantly perturbed. In these same mutants, nonsynaptic VVA labeling is abnormally elevated, showing an altered synapse-specificity in ECM glycans with reduced levels of the βPS synaptic integrin receptors (Rushton, 2009).

A second series of critical findings is that the MTG level regulates the lectin-defined carbohydrate distribution in the salivary gland, and that presynaptically targeted MTG modifies NMJ synaptic carbohydrate ECM domains. MTG::GFP strongly overlaps with VVA and WGA fluorescence within SG cells and at the synapse, suggesting that MTG and lectin probes recognize accumulations of similar carbohydrates. Lectins are not primarily recognizing MTG itself, as MTG::GFP and lectin signals can be spatially separated, and where they tightly overlap, their fluorescence intensities are not proportional. Thus, MTG overexpression results in increased recruitment or accumulation of carbohydrates, even in regions where MTG itself is only weakly localized. It is of particular interest to consider the in vivo synaptic glycoproteins and/or glycolipids that are recognized by VVA and WGA, and modulated by MTG expression level. In the synaptic ECM, candidate targets for VVA and WGA include the integrin receptors, which are glycosylated and have a distribution overlapping that of both lectins. However, synaptic VVA labeling persists in βPS mys mutants with greatly reduced synaptic integrin levels, suggesting that integrins do not carry the glycans recognized by VVA. An alternative possibility is that integrin glycans are lectin targets, but in mys mutants the ECM is remodeled in such a way as to restore these glycan moieties (Rushton, 2009).

One potential synaptic target of Vicia villosa agglutinin (VVA) lectins is O-linked glycans on dystroglycan (DG), an important postsynaptic ECM receptor linked to the muscle cytoskeleton (Haines, 2007). DG has roles in regulating quantal content and vesicle release probability in the presynaptic bouton, and also in recruiting GluRIIB to the postsynaptic domain. Haines (2007) recently reported that NMJ VVA staining is increased by DG overexpression, but not by overexpression of DG lacking the extracellular mucin domain. If VVA labeling at the NMJ is primarily recognizing DG, alterations in VVA resulting from changes in MTG or βPS integrin expression may be mediated in part through changes in the synaptic localization or modification of the DG receptor. Alternatively, DG may itself be recruiting VVA targets to the NMJ ECM. These possibilities will be addressed in future studies. It is stressed that no assumptions can be made about which specific glycoproteins or glycolipids are recognized in vivo by a given lectin. Indeed, it is recognized that lectins may bind other glycans at lower affinity than their preferred substrate target. However, the inhibition of WGA and VVA in vivo labeling by preincubation with their preferred sugars strongly suggests that these lectins recognize these same carbohydrates at the synapse. Given the possible range of synaptic lectin targets, it is of great interest that MTG is able to significantly regulate the entire lectin-labeled glycan pool (Rushton, 2009).

Finally, this study has demonstrated that transgenic MTG expression confers rescue of the GluR, dPak, and DLG punctate postsynaptic domains that are severely disrupted in mtg null synapses (Rohrbough, 2007). This restoration of postsynaptic differentiation occurs in parallel with the demonstrated central neuropil and NMJ MTG synaptic targeting, localized punctate presynaptic expression, and secreted external localization in the ECM, and with restored mutant movement and viability. Together, these results indicate a synaptic requirement for functional rescue. Evidence for clear postsynaptic rescue with neuronal-specific presynaptic MTG expression supports this conclusion, and is consistent with previous and present results showing a specific presynaptic requirement for MTG in PSD/GluR and βPS localization. The mechanism for the MTG inductive requirement in postsynaptic assembly remains unknown. If MTG acts through integrins in this pathway, then null βPS mutants would be predicted to show loss or mislocalization phenotypes for at least a subset of PSD and GluR. This hypothesis cannot be rigorously tested owing to the essential, pleiotropic requirements for mys during embryogenesis. Alternatively, MTG may act through unidentified matrix or postsynaptic signaling proteins, perhaps including the dystroglycan complex. Several important questions remain to be addressed in future work, including determining the in vivo binding target(s) of MTG, which additional matrix proteins may interact with or be regulated by secreted MTG, and whether MTG directly or indirectly governs the composition/function of the specialized synaptic cleft microdomain (Rushton, 2009).

Anterograde Jelly belly ligand to Alk receptor signaling at developing synapses is regulated by Mind the gap

Bidirectional trans-synaptic signals induce synaptogenesis and regulate subsequent synaptic maturation. Presynaptically secreted Mind the gap (Mtg) molds the synaptic cleft extracellular matrix, leading to a hypothesis that Mtg functions to generate the intercellular environment required for efficient signaling. In Drosophila secreted Jelly belly (Jeb) and its receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) are localized to developing synapses. Jeb localizes to punctate aggregates in central synaptic neuropil and neuromuscular junction (NMJ) presynaptic terminals. Secreted Jeb and Mtg accumulate and colocalize extracellularly in surrounding synaptic boutons. Alk concentrates in postsynaptic domains, consistent with an anterograde, trans-synaptic Jeb-Alk signaling pathway at developing synapses. Jeb synaptic expression is increased in Alk mutants, consistent with a requirement for Alk receptor function in Jeb uptake. In mtg null mutants, Alk NMJ synaptic levels are reduced and Jeb expression is dramatically increased. NMJ synapse morphology and molecular assembly appear largely normal in jeb and Alk mutants, but larvae exhibit greatly reduced movement, suggesting impaired functional synaptic development. jeb mutant movement is significantly rescued by neuronal Jeb expression. jeb and Alk mutants display normal NMJ postsynaptic responses, but a near loss of patterned, activity-dependent NMJ transmission driven by central excitatory output. It is concluded that Jeb-Alk expression and anterograde trans-synaptic signaling are modulated by Mtg and play a key role in establishing functional synaptic connectivity in the developing motor circuit (Rohrbough, 2010).

Jeb and Alk are localized to pre- and postsynaptic junctions during embryonic synaptogenesis, predicting an inductive anterograde synaptic signaling role. Jeb-Alk RTK signaling at embryonic somatic-visceral mesoderm junctions similarly directs visceral muscle specification and differentiation. Jeb is the only identified Alk ligand, and Alk is the only identified Jeb receptor. It was recently shown that the C. elegans Alk ortholog SCD-2 is similarly neuronally expressed and activated by a Jeb-like secreted ligand, HEN-1, which contains an LDLa domain (Reiner, 2008). Jeb-Alk anterograde signaling has recently been shown to regulate circuit formation in the Drosophila developing optic lobe (Bazigou, 2007; Rohrbough, 2010 and references therein).

Jeb-Alk NMJ and neuropil expression patterns indicate that anterograde signaling occurs at both peripheral and central synapses. Jeb localizes to NMJ presynaptic terminals and is secreted extracellularly, whereas Alk localizes to opposing postsynaptic membranes. The Jeb neuronal expression/trafficking profile suggests transport to the NMJ, rather than neuronal Jeb uptake from muscle, as previously suggested. Jeb and Alk display reciprocal expression levels at NMJ synapses, with lower Jeb levels at boutons expressing highest postsynaptic Alk levels. Jeb is also strongly increased at Alk mutant synapses, suggesting that internalization of secreted Jeb in postsynaptic cells requires Alk receptor function. This predicted synaptic signaling cascade therefore parallels the mechanism in mesoderm development (Rohrbough, 2010).

A working hypothesis predicts that the ECM environment modulates trans-synaptic ligand-receptor interactions. A key finding, therefore, is that the Jeb-Alk pathway is regulated by Mtg, a presynaptically secreted glycoprotein crucial for synaptic cleft ECM formation (Rohrbough, 2007; Rushton, 2009). In the absence of Mtg, postsynaptic Alk is strongly reduced and secreted Jeb is dramatically accumulated at NMJ synapses. Maintenance of Alk might be part of a larger role for Mtg in postsynaptic differentiation, as numerous postsynaptic components are lost/mislocalized in mtg mutants (Rohrbough, 2007). Alternatively, Mtg might more directly regulate Alk, possibly by ECM tethering/anchoring of the Alk receptor. The Jeb upregulation should be partly attributable to the Mtg-dependent reduction in postsynaptic Alk. However, synaptic Jeb is upregulated to a much greater degree, despite a less severe downregulation of Alk, in mtg than in Alk null mutants. Jeb NMJ expression is also modulated independently of Alk by targeted neuronal or muscle Mtg overexpression, indicating that Mtg regulates Jeb independently of Alk. It is concluded that Mtg expression and function are highly likely to regulate developmental Jeb-Alk synaptic signaling. However, this interpretation must be verified in future studies by demonstrating a regulatory function for Mtg in previously established Jeb-Alk RTK molecular signaling pathways (Rohrbough, 2010).

Mtg and Jeb are co-expressed in developing NMJ presynaptic boutons, and are secreted to occupy largely overlapping domains within the synaptomatrix. The current findings suggest that Mtg normally acts at NMJ synapses to restrict localized Jeb accumulation within the synaptomatrix. It is suggested that the Mtg-dependent ECM might function as a barrier to maintain localized Jeb pools and/or as a scaffold that is required to appropriately present or proteolytically remove Jeb in the extracellular signaling space. It is presently unclear whether Mtg has a parallel regulatory role at developing central synapses, where Mtg is expressed in a more limited neuronal subset. Changes in central Jeb/Alk expression might be indirectly related to Mtg loss or overexpression in the CNS. Alternatively, changes in neuronal Mtg level might have greater effects on Jeb/Alk NMJ expression. Mammalian Alk candidate ligands, such as pleiotrophin, heparin affinity regulatory peptide (HARP), heparin-binding neurotrophic factor (HBNF), and midkine, are heparin-binding growth factors, further highlighting that Alk activation occurs via ligands that function within the complex and dynamic glycomatrix. It is proposed that Mtg-dependent modulation of extracellular space is critical for the signaling activity of multiple trans-synaptic signals (Rohrbough, 2010).

The Jeb-Alk pathway is not detectably required for embryonic axonal pathfinding, synapse morphogenesis or molecular assembly during synaptogenesis, including the proper localized expression of pre- and postsynaptic proteins. Likewise, Jeb-Alk function is not required for establishing functional NMJ synapses, including postsynaptic GluR domains. Jeb-Alk signaling is likely to have a role(s) during postembryonic NMJ development. The Alk receptor is required for expression and signaling of the TGFβ signaling component Dpp in developing endoderm, and Alk is similarly suggested to modulate a TGFβ pathway in C. elegans (Reiner, 2008). Therefore, Alk potentially regulates the TGFβ-dependent retrograde signaling pathway(s) involved in synaptic plasticity and function during larval NMJ development (Rohrbough, 2010).

The results indicate that Jeb and Alk have a role in the development of locomotion behavior. Jeb-Alk signaling regulates somatic as well as visceral muscle differentiation, with similar defects resulting from Alk removal or ectopic overexpression in muscle. Likewise, this study found that either muscle or neuronal Alk overexpression impairs locomotion and results in early larval lethality. However, jeb and Alk mutant muscle responds to direct stimulation and evoked NMJ transmission is normal, indicating that the primary locomotory impairment is not defective muscle or NMJ function. Moreover, jeb mutant locomotion is significantly rescued by neuronal, but not muscle, Jeb expression, consistent with a requirement for Jeb signaling from central neurons. Importantly, loss of Jeb-Alk signaling severely reduces endogenous NMJ neurotransmission by effectively reducing the occurrence of centrally generated, patterned synaptic output to the NMJ. The underlying excitatory synaptic drive onto motoneurons parallels the development of locomotion behavior. Central neuron recordings show functional excitatory synaptic input to jeb/Alk and mtg mutant motoneurons, which surprisingly show no significant loss of activity that might be suggested by the severe locomotion impairments. CNS dissection/recording conditions may effectively re-excite depressed motor activity, similar to the effect of direct stimulation in provoking mutant movement (Rohrbough, 2010).

The current results indicate that anterograde Jeb-Alk synaptic signaling is crucial for the maturation of locomotory behavior, and that Mtg regulatory activity intersects with the Jeb-Alk pathway during NMJ synaptic differentiation. It is proposed that Jeb-Alk signaling is essential for the functional differentiation of the central synaptic connections that drive motor circuit activity. Loss of Jeb-Alk signaling function impairs central excitatory synaptic transmission, resulting in a loss of endogenous central pattern generator activity driving motor output to the NMJ. Future studies will be directed towards dissecting the intersecting roles of Mtg and Jeb secreted signals in the functional differentiation of central motor circuits (Rohrbough, 2010).

Structure-function analysis of endogenous lectin mind-the-gap in synaptogenesis

Mind-the-Gap (MTG) is required for neuronal induction of Drosophila neuromuscular junction (NMJ) postsynaptic domains, including glutamate receptor (GluR) localization. It has previously been hypothesized that MTG is secreted from the presynaptic terminal to reside in the synaptic cleft, where it binds glycans to organize the heavily glycosylated, extracellular synaptomatrix required for transsynaptic signaling between neuron and muscle. This study tests this hypothesis with MTG structure-function analyses of predicted signal peptide (SP) and carbohydrate-binding domain (CBD), by introducing deletion and point-mutant transgenic constructs into mtg null mutants. The SP is shown to be required for MTG secretion and localization to synapses in vivo. It is further shown that the CBD is required to restrict MTG diffusion in the extracellular synaptomatrix and for postembryonic viability. However, CBD mutation results in elevation of postsynaptic GluR localization during synaptogenesis, not the mtg null mutant phenotype of reduced GluRs as predicted by the hypothesis, suggesting that proper synaptic localization of MTG limits GluR recruitment. In further testing CBD requirements, it was shown that MTG binds N-acetylglucosamine (GlcNAc) in a Ca(2+)-dependent manner, and thereby binds HRP-epitope glycans, but that these carbohydrate interactions do not require the CBD. It is concluded that the MTG lectin has both positive and negative binding interactions with glycans in the extracellular synaptic domain, which both facilitate and limit GluR localization during NMJ embryonic synaptogenesis (Rushton, 2012).

The core hypothesis for the role of MTG in synaptogenesis has proven to be wrong. It was postulated that MTG is secreted from the presynaptic neuron to bind extracellular glycans via a well-conserved CBD, to build an extracellular synaptomatrix required for the anterograde transsynaptic signaling inducing postsynaptic GluR domains (Rohrbough, 2007; Rushton, 2009; Rohrbough, 2010; Rushton, 2012 and references therein).

In this study a systematic test of this hypothesis was undertaken with structure-function analyses of SP and CBD requirements in vivo. It was confirmed that the SP is required for secretion, and MTG was discovered to be strongly down regulated when introduction into the secretory pathway is prevented. However, contrary to our hypothesis, the CBD is not required for binding glycans (as shown in vitro on GlcNAc-conjugated beads), glycoproteins (as shown by IP of HRP-epitope proteins), or binding to the extracellular membrane and/or pericellular matrix (as shown by in vivo imaging). The CBD does play an important regulatory role in the localization/anchoring of MTG in the synaptic ECM, since δCBD MTG migrates much further from the secretory synaptic boutons. This function may well be dependent on glycan binding, as indicated by the alteration in glycoprotein binding in δCBD MTG IP experiments. Moreover, contrary to the hypothesis, the CBD is not required for the MTG role in functional GluR postsynaptic localization during embryonic synaptogenesis. Rather, the CBD plays an unexpected role in limiting GluR recruitment to the developing NMJ synapse, and is essential for postembryonic viability (Rushton, 2012).

Although the CBD requirement is clearly fundamentally different from the previous hypothesis, this structure-function analysis reveals intriguing CBD functions. The MTG CBD has a strong homology to CBD14 (Rohrbough, 2007), yet it is not required for binding to GlcNAc, GlcNAc polymer, or other glycans, showing that another cryptic CBD must be present that is sufficient to mediate this carbohydrate binding. Candidate carbohydrate-binding regions include the glutamine-rich region near the N-terminus, and the coiled-coil domain near the C terminus. The glutamine-rich region is a particularly intriguing candidate. This domain bears a striking resemblance to the prion-like domains of Aplysia CPEB (Si, 2010) and Drosophila CPEB (Orb2) (Keleman, 2007), as well as Drosophila fragile X mental retardation protein (FMRP; Banerjee, 2010), all of which are necessary for synaptic mechanisms underlying learning and memory. Interestingly, the prion-like domain of Aplysia CPEB causes mouse CPEB (which lacks this domain) to aggregate into puncta, very similar to MTG puncta (Si, 2010). Similarly, the FMRP prion-like domain drives aggregation and puncta formation (Banerjee, 2010). It would therefore be of great interest to extend the structure-function analysis to the MTG glutamine-rich region, specifically to investigate whether this domain is involved in carbohydrate-binding and/or extracellular puncta formation (Rushton, 2012).

Endogenous animal lectins are extremely diverse, and are organized into many families, including C-type, R-type, siglecs, galectins, and chitinase-like lectins, among others. Each lectin family has characteristic carbohydrate- binding-fold consensus sequences, which differ greatly from one family to another. Within several families, examples exist of lectins that have the characteristic disulfide-bonding fold domain, but do not bind predicted carbohydrate substrates. For example, the C-lectin family includes several members with canonical C-type lectin domains (CTLD) that do not bind carbohydrates, nor is calcium always required for their carbohydrate binding. Specifically, collectin- like tetranectin binds calcium and the protein kringle 4 via its CTLD, yet binds the carbohydrate heparin in a separate, non-CTLD domain. Likewise, while lecticans bind tenascin- R in a Ca2+-dependent manner, this binding does not require tenascin-linked carbohydrates, but rather appears to be a protein-protein interaction. Thus, the relationship between lectins and their carbohydrate-binding partners is not simple or easily defined, and it is obvious that CBDs acquire new functions and new binding properties during evolution. This certainly appears to be the case for the MTG CBD, which is not required for carbohydrate to binding, yet regulates MTG mobility/anchoring at the NMJ, and regulates the recruitment of postsynaptic GluRs (Rushton, 2012).

It is particularly intriguing that MTG carbohydrate binding is Ca2+-dependent, and it is tempting to place MTG in the C-type lectin family based on this characteristic. However, MTG lacks a canonical CTLD, and does not appear to resemble any of the very diverse but well-characterized C-type lectin families. Nor does it resemble the pentraxin domain, another Ca2+-dependent lectin domain. Rather, MTG has the CBD14 domain of the peritrophin lectin family. Invertebrate lectins of this family have not been reported to require calcium for carbohydrate binding, although the related Clostridium endo beta-1.3-glucanase Lic16A binds GlcNAc polymer far more strongly in the presence of calcium and the related mammalian FIBCD1 also has a Ca2+-dependent binding requirement. It is speculated that the calcium requirement for MTG binding to synaptic carbohydrates may be physiologically important, since extracellular calcium concentration in the synaptic cleft and surrounding synaptomatrix is modulated by synaptic activity, involving presynaptic and postsynaptic calcium influx and calcium exchange (Rushton, 2012).

Although the MTG CBD has homology to chitin-binding domains, there is no indication that MTG binds chitin in vivo. MTG does not localize in chitin-rich tissues and when exogenously expressed in these tissues, it is not retained to any detectable degree. When MTG:GFP is expressed ubiquitously, the protein does not localize in the trachea, nor in other chitinous structures such as the external cuticle. In contrast, Drosophila Serpentine and Vermiform chitin deacetylases with canonical chitin-binding domains strongly colocalize with chitin in the trachea, and a Serp N-terminus construct with the SP and CBD fused to GFP likewise strongly co-localizes with chitin in the trachea. Indeed, it is very striking that MTG is strongly downregulated outside nervous tissue when driven ubiquitously, and does not detectably accumulate in epidermis or muscle, except immediately surrounding the NMJ terminal. Thus, there must be a mechanism to specifically retain and preserve MTG in neurons, particularly in synaptic domains. Likewise, within the embryonic CNS, expression of MTG is virtually identical whether it is driven by a neural-specific or a ubiquitous GAL4 driver, indicating that MTG:GFP is accumulated at synapses in a very specific manner and down-regulated elsewhere. The one notable exception is the salivary gland, a tissue specialized for secretion, but it appears even the salivary gland cannot maintain MTG without the SP required for secretion into the extracellular lumenal domain. These data suggest that MTG likely binds GlcNAc inside the chains of N-glycans, O-glycans, and/or GAGs within the pericellular matrix and particularly within the specialized extracellular synaptomatrix (Rushton, 2012).

How might the MTG CBD affect synaptic functional development? It is well established that ECM glycoproteins and proteoglycans are essential for the organization of synaptic components. Lectins that bind selectively to the carbohydrate component of these molecules can regulate, modulate, stabilize, or sequester their activities. At the vertebrate NMJ, the Agrin lectin is required for AChR cluster maintenance in a fashion similar to the MTG requirement for GluR localization at the Drosophila NMJ. In the vertebrate CNS, lecticans brevican and neurocan bind to tenascin and hyaluronic acid to stabilize the extracellular synaptomatrix lattice, and have been implicated in affecting synaptic development and plasticity. Digestion of this lattice by hyaluronidase causes increased lateral diffusion of AMPA GluRs, suggesting the matrix acts as an important diffusion/ mobility barrier. Similarly, the NP lectin family has been implicated in AMPA GluR trafficking: NP-1 and NP-2 (also known as Narp) form a complex with the NP receptor to colocalize with and trigger clustering of AMPA GluRs at postsynaptic sites. Specifically, this lectin mechanism mediates postsynaptic recruitment of the AMPA GluRs with GluR1 and GluR4 subunits. The results presented in this study show that the CBD of MTG is similarly important for regulating GluR trafficking and postsynaptic maintenance at the Drosophila developing embryonic NMJ (Rushton, 2012).

In conclusion, this study has shown that MTG is a secreted, Ca2+-dependent carbohydrate-binding protein resident in the extracellular matrix surrounding synapses. The predicted SP is required for the secretion of MTG, but the CBD is not demonstrably required for glycan interaction, indicating that a cryptic CBD must also be present within MTG. The CBD appears to regulate binding affinity of MTG to the ECM, and is clearly required to anchor properly MTG close to the synaptic interface. In the absence of the CBD, excess GluRs are recruited to the embryonic NMJ postsynaptic domain. This is the opposite consequence to the loss of postsynaptic GluRs occurring with complete removal of MTG. It is concluded that the MTG lectin has both positive and negative roles regulating GluR recruitment during synaptogenesis (Rushton, 2012).

N-glycosylation requirements in neuromuscular synaptogenesis

Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:alpha-3-D-mannoside-beta1,2-N-acetylglucosaminyl-transferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid N-glycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre- and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)gl] scaffolds. It is concluded that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis (Parkinson, 2013).

This study began with the hypothesis that disruption of synaptomatrix N-glycosylation would alter trans-synaptic signaling underlying NMJ synaptogenesis (Dani, 2012). MGAT1 loss transforms the synaptomatrix glycan environment. Complete absence of the HRP epitope, α1-3-fucosylated N-glycans, is expected to require MGAT1 activity: key HRP epitope synaptic proteins include fasciclins, Neurotactin and Neuroglian, among others. This study shows that HRP epitope modification of the key synaptogenic regulator Fasciclin 2 is not required for stabilization or localization, suggesting a role in protein function. However, complete loss of Vicia villosa (VVA) lectin reactivity synaptomatrix labeling is surprising because the epitope is a terminal β-GalNAc. This result suggests that the N-glycan LacdiNAc is enriched at the NMJ, and that the terminal GalNAc expected on O-glycans/glycosphingolipids may be present on N-glycans in this synaptic context. Importantly, VVA labels Dystroglycan and loss of Dystroglycan glycosylation blocks extracellular ligand binding and complex formation in Drosophila, and causes muscular dystrophies in humans. This study shows that VVA-recognized Dystroglycan glycosylation is not required for protein stabilization or synaptic localization, but did not test functionality or complex formation, which probably requires MGAT1-dependent modification. Conversely, the secreted endogenous lectin MTG is highly elevated in Mgat1 null synaptomatrix, probably owing to attempted compensation for complex and hybrid N-glycan losses that serve as MTG binding sites. MTG binds GlcNAc in a calcium-dependent manner and pulls down a number of HRP-epitope proteins by immunoprecipitation (Rushton, 2012), although the specific proteins have not been identified. It will be of interest to perform immunoprecipitation on Mgat1 samples to identify changes in HRP bands. Importantly, MTG is crucial for synaptomatrix glycan patterning and functional synaptic development. MTG regulates VVA synaptomatrix labeling, suggesting a mechanistic link between the VVA and MTG changes in Mgat1 mutants. The MTG elevation observed in Mgat1 nulls provides a plausible causative mechanism for strengthened functional differentiation (Parkinson, 2013).

Consistent with recent glycosylation gene screen findings (Dani, 2012), Mgat1 nulls exhibit increased synaptic growth and structural overelaboration. Therefore, complex and hybrid N-glycans overall provide a brake on synaptic morphogenesis, although individual N-glycans may provide positive regulation. Likely players include MGAT1-dependent HRP-epitope proteins (e.g., fasciclins, Neurotactin, Neuroglian), and position-specific (PS) integrin receptors and their ligands, all of which are heavily glycosylated and have well-characterized roles regulating synaptic architecture. An alternative hypothesis is that Mgat1 phenotypes may result from the presence of high-mannose glycans on sites normally carrying complex/hybrid structures, suggesting possible gain of function rather than loss of function of specific N-glycan classes. NMJ branch and bouton number play roles in determining functional strength, although active zones and GluRs are also regulated independently. Thus, the increased functional strength could be caused by increased structure at Mgat1 null NMJs. However, muscle-targeted UAS-Mgat1 rescues otherwise Mgat1 null function, but has no effect on structural defects, demonstrating that these two roles are separable. Presynaptic Mgat1 RNAi also causes strong functional defects, showing there is additionally a presynaptic requirement in functional differentiation. Neuron-targeted Mgat1 causes lethality, indicating that MGAT1 levels must be tightly regulated, but preventing independent assessment of Mgat1 presynaptic rescue of synaptogenesis defects (Parkinson, 2013).

Presynaptic glutamate release and postsynaptic glutamate receptor responses drive synapse function. Using lipophilic dye to visualize SV cycling, this study found Mgat1 null mutants endogenously cycle less than controls, but have greater cycling capacity upon depolarizing stimulation. The endogenous cycling defect is consistent with the sluggish locomotion of Mgat1 mutants, whereas the elevated stimulation-evoked cycling is consistent with electrophysiological measures of neurotransmission. Similarly, mutation of dPOMT1, which glycosylates VVA-labeled Dystroglycan, decreases SV release probability (Wairkar, 2008), although dPOMT1 adds mannose not GalNAc. Null Mgat1 mutants display no change in SV cycle components (e.g. Synaptobrevin, Synaptotagmin, Synaptogyrin, etc.), but exhibit reduced expression of the key active zone component Bruchpilot. Other examples of presynaptic glycosylation requirements include the Drosophila Fuseless (FUSL) glycan transporter, which is critical for Cacophony (CAC) voltage-gated calcium channel recruitment to active zones, and the mammalian GalNAc transferase (GALGT2), whose overexpression causes decreased active zone assembly. Postsynaptically, Mgat1 nulls show specific loss of GLURIIB-containing receptors. Similarly, dPOMT1 mutants exhibit specific GLURIIB loss (Wairkar, 2008), although dystroglycan nulls display GLURIIA loss. Selective GLURIIB loss in Mgat1 nulls may drive increased neurotransmission owing to channel kinetics differences in GLURIIA versus GLURIIB receptors (Parkinson, 2013).

Bidirectional trans-synaptic signaling regulates NMJ structure, function and pre/postsynaptic composition. This intercellular signaling requires ligand passage through, and containment within, the heavily glycosylated synaptomatrix, which is strongly compromised in Mgat1 mutants. In testing three well-characterized signaling pathways, this study found that Wingless (Wg) accumulates, whereas both GBB and JEB are reduced in the Mgat1 null synaptomatrix. WG has two N-glycosylation sites, but these do not regulate ligand expression, suggesting WG build-up occurs owing to lost synaptomatrix N-glycosylation. Importantly, WG overexpression increases NMJ bouton formation similarly to the phenotype of Mgat1 nulls, suggesting a possible causal mechanism. GBB is predicted to be N-glycosylated at four sites, but putative glycosylation roles have not yet been tested. Importantly, GBB loss impairs presynaptic active zone development similarly to Mgat1 nulls, suggesting a separable causal mechanism. JEB is not predicted to be N-glycosylated, indicating that JEB loss is caused by lost synaptomatrix N-glycosylation. Importantly, it has been shown that loss of JEB signaling increases functional synaptic differentiation similarly to Mgat1 nulls (Rohrbough, 2013). In addition, jeb mutants exhibit strongly suppressed NMJ endogenous activity, similarly to the reduced endogenous SV cycling in Mgat1 nulls. Moreover, the MTG lectin negatively regulates JEB accumulation in NMJ synaptomatrix, consistent with elevated MTG causing JEB downregulation in Mgat1 nulls (Parkinson, 2013).

Trans-synaptic signaling drives recruitment of scaffolds that, in turn, recruit pre- and postsynaptic molecular components. Specifically, DLG1 and L(2)GL scaffolds regulate the distribution and density of both active zone components (e.g. BRP) and postsynaptic GluRs, and both of these scaffolds are reduced at Mgat1 null NMJs. Importantly, dlg1 mutants display selective loss of GLURIIB, with GLURIIA unchanged, similar to Mgat1 nulls, suggesting a causal mechanism. Moreover, l(2)gl mutants display both a selective GLURIIB impairment as well as reduction of BRP aggregation in active zones, similarly to Mgat1 nulls, suggesting a separable involvement for this synaptic scaffold. DLG1 and L(2)GL are known to interact in other developmental contexts, indicating a likely interaction at the developing synapse. Although synaptic ultrastructure has not been examined in l(2)gl mutants, dlg1 mutants exhibit impaired NMJ development, including a deformed SSR. These synaptogenesis requirements predict similar ultrastructural defects in Mgat1 mutants, albeit presumably due to the combined loss of both DLG1 and L(2)GL scaffolds. Future work will focus on electron microscopy analyses to probe N-glycosylation mechanisms of synaptic development (Parkinson, 2013).


REFERENCES

Search PubMed for articles about Drosophila Mind the gap

Albin, S. D. and Davis, G. W. (2004). Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology. J Neurosci 24: 6871-6879. PubMed ID:15295021

Banerjee, P., Schoenfeld, B. P., Bell, A. J., Choi, C. H., Bradley, M. P., Hinchey, P., Kollaros, M., Park, J. H., McBride, S. M. and Dockendorff, T. C. (2010). Short- and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein. J Neurosci 30: 6782-6792. PubMed ID:20463240

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Dani, N. and Broadie, K. (2012). Glycosylated synaptomatrix regulation of trans-synaptic signaling. Dev Neurobiol 72: 2-21. PubMed ID: 21509945

Daniels, R. W., Gelfand, M. V., Collins, C. A. and DiAntonio, A. (2008). Visualizing glutamatergic cell bodies and synapses in Drosophila larval and adult CNS. J Comp Neurol 508: 131-152. PubMed ID:18302156

Haines, N., Seabrooke, S. and Stewart, B. A. (2007). Dystroglycan and protein O-mannosyltransferases 1 and 2 are required to maintain integrity of Drosophila larval muscles. Mol Biol Cell 18: 4721-4730. PubMed ID:17881734

Keleman, K., Kruttner, S., Alenius, M. and Dickson, B. J. (2007). Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nat Neurosci 10: 1587-1593. PubMed ID:17965711

Kummer, T. T., Misgeld, T. and Sanes, J. R. (2006). Assembly of the postsynaptic membrane at the neuromuscular junction: paradigm lost. Curr Opin Neurobiol 16: 74-82. PubMed ID:16386415

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Misgeld, T., Kummer, T. T., Lichtman, J. W. and Sanes, J. R. (2005). Agrin promotes synaptic differentiation by counteracting an inhibitory effect of neurotransmitter. Proc Natl Acad Sci U S A 102: 11088-11093. PubMed ID:16043708

Nishimune, H., Sanes, J. R. and Carlson, S. S. (2004). A synaptic laminin-calcium channel interaction organizes active zones in motor nerve terminals. Nature 432: 580-587. PubMed ID:15577901

O'Brien, R., Xu, D., Mi, R., Tang, X., Hopf, C. and Worley, P. (2002). Synaptically targeted narp plays an essential role in the aggregation of AMPA receptors at excitatory synapses in cultured spinal neurons. J Neurosci 22: 4487-4498. PubMed ID:12040056

Parkinson, W., Dear, M. L., Rushton, E. and Broadie, K. (2013). N-glycosylation requirements in neuromuscular synaptogenesis. Development 140(24): 4970-81. PubMed ID: 24227656

Reiner, D. J., Ailion, M., Thomas, J. H. and Meyer, B. J. (2008). C. elegans anaplastic lymphoma kinase ortholog SCD-2 controls dauer formation by modulating TGF-beta signaling. Curr Biol 18: 1101-1109. PubMed ID:18674914

Rohrbough, J., Rushton, E., Woodruff, E., Fergestad, T., Vigneswaran, K. and Broadie, K. (2007). Presynaptic establishment of the synaptic cleft extracellular matrix is required for post-synaptic differentiation. Genes Dev 21: 2607-2628. PubMed ID:17901219

Rohrbough, J. and Broadie, K. (2010). Anterograde Jelly belly ligand to Alk receptor signaling at developing synapses is regulated by Mind the gap. Development 137: 3523-3533. PubMed ID:20876658

Rohrbough, J., Kent, K. S., Broadie, K. and Weiss, J. B. (2013). Jelly Belly trans-synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture. Dev Neurobiol 73: 189-208. PubMed ID: 22949158

Rushton, E., Rohrbough, J. and Broadie, K. (2009). Presynaptic secretion of mind-the-gap organizes the synaptic extracellular matrix-integrin interface and postsynaptic environments. Dev Dyn 238: 554-571. PubMed ID:19235718

Rushton, E., Rohrbough, J., Deutsch, K. and Broadie, K. (2012). Structure-function analysis of endogenous lectin mind-the-gap in synaptogenesis. Dev Neurobiol 72: 1161-1179. PubMed ID:22234957

Si, K., Choi, Y. B., White-Grindley, E., Majumdar, A. and Kandel, E. R. (2010). Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation. Cell 140: 421-435. PubMed ID:20144764

Wairkar, Y. P., Fradkin, L. G., Noordermeer, J. N. and DiAntonio, A. (2008). Synaptic defects in a Drosophila model of congenital muscular dystrophy. J Neurosci 28: 3781-3789. PubMed ID: 18385336


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