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 utilizes at least six distinct KAR subunits: They form two distinct postsynaptic complexes (type-A and type-B) that co-exist within individual postsynaptic densities (PSDs) and enable NMJ functionality and a presynaptic KaiRID-containing complex that modulates basal neurotransmission. The postsynaptic iGluRs are heterotetrameric complexes composed of three shared subunits, GluRIIC, GluRIID and GluRIIE, and either GluRIIA (type-A receptors) or GluRIIB (type-B). The shared subunits are essential for viability and for the iGluRs synaptic recruitment. Until recently, our investigations were limited by the inability to reconstitute functional Drosophila NMJ iGluRs in heterologous systems. We have recently solved this problem by accomplishing the first functional reconstitution of NMJ iGluRs in Xenopus oocytes. 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. Furthermore, we have succeeded in expressing functional receptors in HEK293 cells, where the biophysical properties of these receptors can be further dissected using fast glutamate applications. The ability to examine the functional characteristics of iGluRs in heterologous systems opens up 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 and vertebrates, iGluRs have conserved, dedicated auxiliary proteins which modulate receptor distribution and function, ultimately controlling 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 absence of Neto, these receptors fail to cluster at synaptic sites and the animals die as completely paralyzed embryos. Neto belongs to a family of highly conserved proteins sharing an ancestral role in formation and modulation of glutamatergic synapses. Our investigations uncovered essential roles for Neto during synapse development and strongly support the notion that trafficking of both iGluR subtypes on the muscle membrane, their synaptic recruitment and stabilization, and their function are tightly regulated by Neto. Our results also suggest that the fly Neto isoforms (alpha and beta) 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. Since iGluRs gating properties control the distribution and trafficking of these receptors in vivo, Neto could influence the synaptic recruitment of iGluRs by simultaneously controlling multiple steps in receptor trafficking and clustering and/or receptor function. Drosophila neto 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, probably reflecting cell/tissue specific roles. We found that 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. In recent studies we found that Neto-alpha functions primarily in the presynaptic compartment, in motor neurons terminals, to modulate basal neurotransmission and to enable a presynaptic homeostatic response. At the fly NMJ, presynaptic KaiRID-containing autoreceptor complexes control basal neurotransmission; as expected, Neto-alpha controls neurotransmitter release in a KaiRID-dependent manner. Our ongoing studies demonstrate that Neto-alpha modulate both the presynaptic distribution and the gating properties of this channel. The fly NMJ is also a powerful model system to study homeostatic plasticity: Manipulations that decrease the responsiveness of postsynaptic iGluRs (leading to a decrease in quantal size) trigger a robust compensatory increase in presynaptic neurotransmitter release which restores the evoked muscle responses to normal levels. Both KaiRID and Neto-alpha are required for the expression of this homeostatic response. However, only Neto-alpha is both required and sufficient for the presynaptic homeostatic response: Neuronal overexpression of KaiRID cannot compensate for loss of Neto-alpha, whereas overexpression of Neto-alpha renders KaiRID dispensable for the presynaptic potentiation response, indicating that KaiRID functions to localize Neto-alpha activities at synaptic terminals. These findings challenge our current thinking that auxiliary subunits assist iGluRs and provide an example of an auxiliary protein that performs a key synaptic function with assistance from iGluRs.