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. Drosophila NMJ is a glutamatergic synapse, similar in structure and physiology to mammalian central excitatory synapses. In flies each NMJ is unique and identifiable, synapses are large and accessible for electrophysiological and optical analysis, making the Drosophila NMJ a powerful genetic system to study synapse development. Drosophila NMJ utilizes at least six ionotropic glutamate receptor (iGluR) 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 plasticity 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. The iGluR recruitment mechanism has been a long-standing question in the field. Our studies identified Drosophila Neto as an obligatory subunit of 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. Structure-function studies revealed that various Neto activities map to different protein domains. The highly conserved extracellular and transmembrane domains of Neto are both required and sufficient for iGluRs clustering. Muscle expression of a Neto variant with no intracellular domain (Neto-deltaCTD) can rescue the iGluRs synaptic recruitment in neto-null mutants. In flies, Neto activities are further restricted by an inhibitory prodomain which must be removed by Furin-mediated proteolysis. When the prodomain cleavage is blocked, Neto is properly targeted to the muscle membrane and engages the iGluR complexes in vivo but fails to enable the incorporation of iGluRs in stable synaptic clusters. Drosophila neto encodes two isoforms, Neto-alpha and Neto-beta, with different cytoplasmic domains generated by alternative splicing. The cytoplasmic domains, both rich in putative phosphorylation motifs and docking sites, are highly divergent among Neto proteins across species, 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, loss of Neto-alpha has no detectable effect on the postsynaptic iGluRs accumulation. Instead, we found that Neto-alpha functions to limit the size of the postsynaptic receptor fields. In recent studies we found that Neto-alpha also functions in the presynaptic compartment of the Drosophila NMJ to modulate basal neurotransmission and to enable a presynaptic homeostatic response. Since presynaptic KaiRID has been implicated in the control of basal neurotransmission and the expression of homeostatic potentiation, we asked whether Neto-alpha modulates the presynaptic distribution and function of KaiRID. Our results demonstrate that Neto-alpha controls neurotransmitter release in a KaiRID-dependent manner, presumably by modulating the gating properties of this channel. However, Neto-alpha is both required and sufficient for the presynaptic homeostatic response: Overexpression of Neto-alpha in motor neurons 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.