Mechanisms of synapse development

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The long-term goal of this project is to define molecular mechanisms that control synapse assembly, development and homeostasis. Ionotropic glutamate receptors (iGluRs) mediate neurotransmission at most excitatory synapses in the vertebrate CNS and at the neuromuscular junction (NMJ) of insects and crustaceans and include a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), N-methyl-D-aspartic acid receptors (NMDARs), and kainate receptors (KARs). Drosophila NMJ relies exclusively on KARs and utilizes at least seven distinct KAR subunits: Five subunits form two distinct postsynaptic complexes (type-A and type-B) that co-exist within individual postsynaptic densities and enable NMJ functionality; two presynaptic subunits, KaiR1D and UKAR, are required for normal basal neurotransmission. Until recently, our investigations were limited by the inability to reconstitute functional Drosophila NMJ iGluRs in heterologous systems. We solved this problem and accomplished the functional reconstitution of NMJ iGluRs in Xenopus oocytes and in HEK293 cells. Our studies have revealed that Drosophila iGluRs have unique and unexpected ligand binding profiles: the postsynaptic iGluRs have low affinity for glutamate, and no response to AMPA, kainate or NMDA, while the KaiRID-containing presynaptic receptors respond to kainate and are inhibited by NMDA. The ability to examine the functional characteristics of iGluRs in heterologous systems opens tremendous opportunities to study the modulation of iGluRs function and to identify the structural elements and the auxiliary subunits important for receptors assembly, surface delivery, synaptic recruitment and function. In flies as in vertebrates, iGluRs have conserved, dedicated auxiliary proteins which modulate receptor distribution and function and ultimately control synapse assembly, maturation, and plasticity. Our studies identified Drosophila Neto as an obligatory auxiliary subunit for the postsynaptic iGluR complexes, the type-A and type-B receptors. In the presynaptic compartment, Neto is required for the KaiR1D-controlled neurotransmitter release. Drosophila neto gene encodes two isoforms, Neto-alpha and Neto-beta, which share the highly conserved extracellular and transmembrane domains characteristics of the Neto family of proteins but have different cytoplasmic domains generated by alternative splicing. The shared domains of Neto isoforms are both required and sufficient for iGluRs clustering. The cytoplasmic domains, both rich in putative phosphorylation motifs and docking sites, are highly divergent among Neto proteins, likely reflecting cell/tissue specific roles. Our studies indicate that both Neto isoforms directly engage iGluRs as well as other intracellular and extracellular proteins to selectively regulate the distribution of iGluRs subtypes, the recruitment of postsynaptic proteins, and the organization of postsynaptic structures. Neto-beta is the predominant muscle isoform (more than 90% from total Neto) and is primarily responsible for the recruitment of postsynaptic iGluRs and other PSD components, which stabilize specific receptor subtypes. In contrast, the low abundant Neto-alpha functions to limit the size of the postsynaptic receptor fields. Using outside-out, patch clamp recordings and fast ligand application, we examined for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-alpha or Neto-beta and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-alpha and Neto-beta. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of iGluR desensitization. These functional analyses of recombinant iGluRs should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals. In the presynaptic compartment, Neto-alpha, KaiR1D and UKAR are required for normal basal neurotransmission. Our ongoing studies indicate that Neto-alpha modulate both the presynaptic distribution and the gating properties of the KaiR1D receptor channels. These studies will be expanded to investigate UKAR, a KAR subunit that appears to be part of the presynaptic autoreceptor complexes. Most synaptic proteins are present in low abundance, are heavily modified by post-translational modification and localize to crowded microenvironments, making their detection and characterization particularly challenging. Also, overexpression often produces gain-of-function phenotypes that distort the experimental results and their interpretation. To address these problems, we tested a recently described cell biology tool, the ALFA system. This system is composed of a rationally designed epitope tag of only 14 amino acids, the ALFA tag, with no homology in the main animal models, and an ALFA-Nanobody that binds to ALFA-tagged proteins with picomolar affinities. We combined the ALFA system with classic genetics, cell biology and electrophysiology to examine the distribution and function of a case study synaptic protein, the Drosophila Neurexin-1 (Nrx-1) in vivo. First, we tagged and detected Nrx-1 at synaptic terminals in one step immunohistochemistry. We edited the endogenous locus and found that in the absence of the C-terminal PDZ binding motif (PBM), an ALFA-tagged-delta-PBM variant remains confined to the ER/early secretory compartments; remarkably, a genetically encoded cytosolic Nanobody-PBM chimera can deliver the missing PDZ binding motif in trans and thus reconstitute the functional Nrx1 in vivo. This system is also amenable to live imaging and detection in specific tissues/compartments: Using a cytosolic Nanobody-mScarlet fusion, we achieved compartment-specific detection of endogenously tagged Nrx1 and tracked live Nrx1 transport along the motor neuron axons. This novel, versatile nanobody-based split system offers powerful solutions towards localizing low abundance, multidomain proteins and dissecting their functional motifs in vivo. We are now using this methodology to conduct structure-function analyses on several complex synaptic scaffolds, revealing docking motifs and regulatory mechanisms that have been eluded the field for decades.