Genetic dissection of glycan functions at the synapse

Abstract

ABSTRACT Synapse formation is driven by precisely orchestrated intercellular communication between presynaptic and postsynaptic cells. Signals traverse a complex extracellular environment, where glycans attached to glycoproteins and proteoglycans modulate trans-synaptic signaling driving synapse formation, function and plasticity. To interrogate glycan effects on synapse structure and function, I performed a Drosophila transgenic RNAi screen targeting the glycan genome, including N/O/GAG-glycan biosynthesis/modifying enzymes and glycan-binding lectins. From the screen hits, I characterized two functionally paired genes to show unique synaptic effects. The first pair comprises the heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with neurotransmission strength and postsynaptic glutamate receptor machinery decreased in hs6st but elevated in sulf1 null mutants. Consistently, hs6st and sulf1 nulls differentially misregulate WNT (Wingless) and BMP (Glass Bottom Boat) ligands, their HSPG co-receptors Dally-like Protein and Syndecan, and downstream signaling. Genetic correction of altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that the altered trans-synaptic signaling causes the functional differentiation defects. The second screen-derived functional pair is two protein α-N-acetylgalactosaminyltransferases (pgant3 and pgant35A) that regulate synaptic O-linked glycosylation (GalNAcα1-O-S/T). Loss of either pgant alone elevates presynaptic/postsynaptic molecular assembly and evoked neurotransmission strength, but synapses appear restored to normal in double mutants. Likewise, activity-dependent facilitation, augmentation and post-tetanic potentiation are all suppressively impaired in pgant mutants. I show that Position Specific 2 (αPS2) integrin receptor and transmembrane tenascin ligand are both suppressively downregulated in pgant mutant synapses. Channelrhodopsin-driven electrical activity rapidly (<1 min) drives integrin signaling in wildtype synapses, but is suppressively abolished in pgant mutants. Optogenetic stimulation alters presynaptic vesicle trafficking and postsynaptic pocket size during perturbed integrin signaling underlying synaptic plasticity defects in pgant mutants. Critically, acute blockade of integrin signaling acts synergistically with pgant mutants to eliminate all activity-dependent synaptic plasticity. Thus, I identify two O- glycosylation synaptomatrix mechanisms that regulate trans-synaptic signaling underlying synaptic transmission and activity-dependent plasticity.