Glycosylated synaptomatrix regulation of trans-synaptic signaling

Abstract

Synapse formation is driven by precisely orchestrated intercellular communication between the presynaptic and the postsynaptic cell, involving a cascade of anterograde and retrograde signals. At the neuromuscular junction (NMJ), both neuron and muscle secrete signals into the heavily glycosylated synaptic cleft matrix sandwiched between the two synapsing cells. These signals must necessarily traverse and interact with the extracellular environment, for the ligand-receptor interactions mediating communication to occur. This complex synaptomatrix, rich in glycoproteins and proteoglycans, comprises heterogeneous, compartmentalized domains where specialized glycans modulate trans-synaptic signaling during synaptogenesis and subsequent synapse modulation. The general importance of glycans during development, homeostasis and disease is well established, but this important molecular class has received less study in the nervous system. Glycan modifications are now understood to play functional and modulatory roles as ligands and co-receptors in numerous tissues; however, roles at the synapse are relatively unexplored. We highlight here properties of synaptomatrix glycans and glycan-interacting proteins with key roles in synaptogenesis, with a particular focus on recent advances made in the Drosophila NMJ genetic system. We discuss open questions and interesting new findings driving this investigation of complex, diverse, and largely understudied glycan mechanisms at the synapse.

Figure 1. Glycan and glycan-interacting lectin expression domains at the Drosophila NMJ

The Drosophila wandering third instar NMJ probed with a range of lectins and antibodies in detergent-free conditions to maintain synaptomatrix integrity. A) NMJ probed with WGA lectin (red) and anti-HRP (green), which recognizes glycans associated exclusively with the presynaptic neuronal membrane. The inset shows WGA domains in the synaptomatrix surrounding a single NMJ bouton. B) VVA lectin (red) and HRP (green). The VVA labeling occupies a different domain than WGA labeling, and is very highly enriched in the NMJ synaptomatrix. C) PNA lectin (red); HRP (green). D) DBA lectin (red); HRP (green). Note that both PNA and DBA lectins do not detectably label the NMJ synaptomatrix, although strong labeling is present in adjacent tissues (not shown). E) The MTG:GFP fusion protein (green) co-labeled with anti-HRP (blue). The MTG lectin localizes to synaptomatrix punctae (arrows) surround NMJ synaptic boutons. F) Triple labeling of MTG:GFP (green), βPS integrins (red) and HRP (blue). Note the three overlapping domains in the synaptomatrix. Scale bars = 5 μm.

Figure 2. Diagram of trans-synaptic signaling pathways at the Drosophila NMJ

Presynaptic: The active zone (AZ) is indicated by a T-bar. Cell membrane components include PS integrins (α3 and βPS subunits), homophilic CAM Fasciclin II (FasII) and cacophony calcium channels (Cac). Cytoplasmic proteins include kinases JNK and Shaggy, and the MAP1B Futsch. Postsynaptic: The glutamate receptor (GluR) domain includes two GluR classes (GluRIIA and B) and potassium channels (K+). Cell membrane components include PS integrins (α1/2 and βPS) and FasII. Membrane associated and cytoplasmic proteins include scaffolding proteins Discs large (DLG) and Dock, kinase PAK and regulators PIX and GIT1, and calmodulin kinase II (CamKII). Trans-synaptic pathways: Secreted signals Wingless (Wg), Glass bottom boat (Gbb) and Jelly belly (Jeb), and their respective membrane cognate receptors Frizzled 2 (Fz2), Thickveins (Tkv)/Wishful Thinking (Wit)/Saxophone (Sax) and Anaplastic lymphoma kinase (Alk). The Frizzled nuclear import pathway is indicated as FNI. The known downstream transcription factor for Gbb is Mothers against decapentaplegic (Mad; P-Mad indicating phosphorylated form), and for Jeb is ERK (P-ERK indicating phosphorylated form). Extracellular synaptomatrix components are indicated as ECM between the presynaptic neuron and postsynaptic muscle cells.