National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

Mechanisms of Synapse Assembly, Maturation, and Growth during Development

Mihaela Serpe
  • Mihaela Serpe, PhD, Head, Unit on Cellular Communication
  • Peter Nguyen, Biological Laboratory Technician
  • Tae Hee Han, PhD, Visiting Fellow
  • Lindsey Friend, PhD, Visiting Fellow
  • Tho Huu Nguyen, PhD, Visiting Fellow
  • Rosario Vicidomini, PhD, Visiting Fellow
  • Qi Wang, PhD, Visiting Fellow

The purpose of our research is to understand the mechanisms of synapse development and homeostasis. The chemical synapse is the fundamental communication unit, connecting neurons in the nervous system to one another and to non-neuronal cells, and is designed to mediate rapid and efficient transmission of signals across the synaptic cleft. Such transmission forms the basis of the biological computations that underlie and enable our complex behavior. Crucial to this function is the ability of a synapse to change its properties, so that it can optimize its activity and adapt to the status of the cells engaged in communication and/or to the larger network comprising them. Consequently, synapse development is a highly orchestrated process coordinated by intercellular communication between the pre- and postsynaptic compartments and by neuronal activity itself. Our long-term goal is to elucidate the molecular mechanisms, particularly those involving cell-cell communication, that regulate formation of functional synapses during development and that fine-tune them during plasticity and homeostasis. We focus on three key processes in synaptogenesis: (1) trafficking of components to the proper site, (2) organizing those components to build synaptic structures, and (3) maturation and homeostasis of the synapse to optimize its activity. We address the molecular mechanisms underlying these processes using a comprehensive set of approaches that include genetics, biochemistry, molecular biology, super-resolution imaging, and electrophysiology recordings in live animals and reconstituted systems.

Because of its many advantages, we choose to study these events in a powerful genetics system, Drosophila melanogaster, and to use the neuromuscular junction (NMJ) as a model for glutamatergic synapse development and function. The fact that individual NMJs can be reproducibly identified from animal to animal and are easily accessible for electrophysiological and optical analysis makes them uniquely suited for in vivo studies on synapse assembly, growth and plasticity. In addition, the richness of genetic manipulations that can be performed in Drosophila permits independent control of individual synaptic components in distinct cellular compartments. Furthermore, the fly NMJ is a glutamatergic synapse similar in composition and physiology to mammalian central synapses. The Drosophila NMJ can thus be used to analyze and model defects in the structural and physiological plasticity of glutamatergic synapses, which are associated with a variety of human pathologies from learning and memory deficits to autism. The similarity in architecture, function, and molecular machinery supports the notion that studying the assembly and development of fly glutamatergic synapses will shed light on their human counterparts.

Synapse assembly at the Drosophila neuromuscular junction (NMJ) requires the obligatory auxiliary subunit Neto.

At the fly and vertebrate NMJ, synaptogenesis follows the arrival of a motor neuron at its target muscle. Prior to neuron arrival, the ionotropic glutamate receptors (iGluRs) form small, nascent clusters on the muscle, which are distributed in the vicinity of future synaptic sites. Neuron arrival triggers formation of large synaptic iGluRs aggregates and promotes expression of more iGluRs to permit synapse maturation and growth. The iGluR clusters interact with the local cytoskeleton and other synaptic structures to maintain local density. This involves solving two fundamental problems common to all chemical synapses: first, trafficking the components to the proper site, and second, organizing those components to build synaptic structures. Recent advances, particularly from vertebrate iGluR biology, reveal that the solution to these problems is entirely dependent on the activity of a rich array of auxiliary subunits that associate with the receptors. These highly diverse transmembrane proteins associate with iGluRs at all stages of the receptor life-cycle and mediate the delivery of receptors to the cell surface, their distribution, synaptic recruitment, association with various postsynaptic density (PSD) scaffolds, and importantly, their channel properties. As they are assembled from different subunits, iGluRs have strikingly varied biophysical properties; their association with different auxiliary subunits increases this diversity even further.

We recently discovered that an obligatory auxiliary protein, Neto, is absolutely required for iGluRs to cluster and for NMJ functionality. To date, Neto is the only auxiliary protein characterized in Drosophila. Neto belongs to a family of highly conserved auxiliary proteins that regulate glutamatergic synapses. Using Neto as our entry point, we set out to elucidate the molecular mechanisms underlying the synaptic recruitment of iGluRs and their incorporation in stable neural clusters. Given that Neto does not have any catalytic activities, we hypothesized that Neto controls synapse development by interacting with iGluRs and/or with other proteins critical for synapse development. We found that Neto (1) engages in extracellular interactions that stabilize iGluRs at synaptic sites and trigger postsynaptic differentiation, (2) mediates intracellular interactions that anchor postsynaptic density components and sculpt iGluR postsynaptic composition, and (3) modulates iGluR function but not their assembly or surface delivery. More specifically, we found that Neto activities are regulated by Furin-mediated limited proteolysis, which removes an inhibitory prodomain. When the prodomain cleavage is blocked, Neto engages iGluRs in vivo, but cannot mediate their stable incorporation/clustering at synaptic sites and fails to initiate postsynaptic differentiation.

To identify proteins that interact with Neto and provide iGluR–clustering activities at the developing NMJ, we initiated two complementary screens: a synthetic lethality screen, and mass-spectroscopy comparison of proteins present in the NMJ synaptosomal fractions isolated from control and neto mutant larval carcasses. In both cases, we took advantage of a strong neto hypomorph mutant that we isolated and characterized in our laboratory, neto109. The mutant has drastically diminished levels of synaptic iGluRs, albeit normal net levels of muscle receptors. Intriguingly, the iGluRs are expressed on the muscle surface but have a diffuse, extra-junctional distribution, indicating that neto109 has a defect in the trafficking and/or stabilization of receptors at junctional locations. Our preliminary analysis of the spectral counts in synaptosomal fractions from control and mutant larvae revealed several interesting candidates, present only in control fraction. We are in the process of validating the candidates and generating reagents to reveal their roles in synaptogenesis. In addition, 50% of neto109 hypomorphs die during development; further reduction of synaptogenic proteins in hemizygous animals should increase lethality. Using this rationale, we have started to screen the deficiencies available on chromosomes 2 and 3 and also test candidate genes previously reported to affect the NMJ function. We expect that the screen will reveal molecules that function in iGluR clustering, as well as proteins that control synaptic trafficking of Neto/iGluR complexes. In addition, the screen may identify other pre- and postsynaptic components important for synapse development.

Modulation of iGluRs function

Assembled from different subunits, iGluRs have strikingly varied biophysical properties; their association with different auxiliary subunits increases this diversity even further. Until recently, our investigations on iGluR function were limited by the inability to reconstitute functional Drosophila NMJ receptors in heterologous systems. In collaboration with Mark Mayer, we recently solved this problem by accomplishing the first functional reconstitution of NMJ iGluRs in Xenopus oocytes. Using this system, we found that Neto increases glutamate-activated currents by several orders of magnitude, but has a comparatively modest effect on the surface delivery of the iGluRs (up to a four-fold increase). Also, heterotetrameric iGluRs are absolutely required for surface expression. The Neto/iGluR complexes reconstituted in Xenopus oocytes recapitulate the properties of endogenous NMJ receptors: high permeability to Ca2+, block by polyamines, low affinity for glutamate, and no response to AMPA, kainite, or NMDA.

In flies as in humans, synapse strength and plasticity are determined by the interplay between different iGluR subtypes. At the fly NMJ, the type-A and type-B iGluRs consist of four different subunits: either GluRIIA or GluRIIB, plus GluRIIC, GluRIID, and GluRIIE. Different, genetically distinct mechanisms control the synaptic recruitment of type-A and type-B receptors at the NMJ; however, we found that both receptor subtypes absolutely require Neto for their function. Given that the synaptic recruitment of iGluRs is also influenced by receptor activity, Neto may regulate iGluR clustering partly by modulating the receptor function.

Drosophila neto codes for two isoforms (α and β), which have different intracellular domains generated by alternative splicing. Muscle expression of either Neto-α or Neto-β could rescue the embryonic lethality and iGluR clustering defects of neto null mutants, although only Neto-β appears to efficiently recruit postsynaptic components. Both isoforms increase the glutamate-activated currents of Drosophila NMJ iGluRs in Xenopus oocytes, with Neto-β having a slightly stronger effect than Neto-α. Interestingly, a truncated Neto-ΔCTD variant, which lacks any intracellular part but retains the highly conserved extracellular and transmembrane domains, could also elicit NMJ iGluR–mediated currents in Xenopus oocytes. When overexpressed in the larval muscle, Neto-ΔCTD is required and sufficient for clustering of receptors in vivo.

To further characterize the receptor properties and determine how Neto modulates iGluR function, we reconstituted iGluRs in HEK293T-17 cells and examined single-channel currents evoked repeatedly by fast glutamate applications to (outside-out) patches containing only a few recombinant receptors. We had already found that Neto is absolutely required to elicit glutamate-gated currents. As in the Xenopus system, both type-A and type-B receptors are present on the surface of the HEK293T-17 cells, but without Neto there are no detectable currents. We are currently applying this new methodology to individual type-A and -B receptors in complexes with Neto-α or Neto-β to determine subtype specific properties. These studies will allow us to directly measure mutant NMJ iGluR currents and tease apart the role of Neto in receptor function versus synaptic recruitment.

Local BMP/BMPR complexes regulate synaptic plasticity and homeostasis.

Synaptic activity and synapse development are intimately linked, but our understanding of the coupling mechanisms remains limited. Anterograde and retrograde signals together with trans-synaptic complexes enable intercellular communication. How synapse activity status is monitored and relayed across the synaptic cleft remains poorly understood. Our studies uncovered a role for Bone Morphogenetic Proteins (BMP) in sensing the activity of postsynaptic receptors across the synaptic cleft. At the Drosophila NMJ, BMP signaling is critical for NMJ growth and neurotransmitter release. It is generally thought that BMP signaling fulfills these functions via canonical and noncanonical pathways triggered primarily by muscle-secreted Glass-bottom boat (Gbb), a BMP7 homolog. Gbb signals by binding to the presynaptic BMP type-II receptor (BMPRII) Wishful thinking (Wit) and to the BMPRIs Thickveins (Tkv) and Saxophone (Sax). The canonical BMP pathway induces the accumulation of the pathway effector pMad (phosphorylated Smad) in motor neuron nuclei, which activates presynaptic transcriptional programs with distinct roles in the structural and functional development of the NMJ. Gbb and Wit signal non-canonically through the effector protein LIM kinase 1 (LIMK1) to regulate synapse stability. pMad also accumulates at synaptic locations but the biological relevance of this phenomenon remained a mystery for over a decade.

We recently found that presynaptic (neuronal) pMad correlates with postsynaptic (muscle) sensitivity and constitutes a sensor for synapse activity. Furthermore, synaptic pMad marks a novel, noncanonical BMP–signaling modality that is genetically distinguishable from all other known BMP–signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic Wit and Sax and the activity of a particular subtype of postsynaptic glutamate receptors, type-A receptors. Unlike canonical BMP signaling, synaptic pMad plays no role in the regulation of NMJ growth. Instead, we found that selective disruption of presynaptic pMad accumulation reduces postsynaptic levels of the receptor GluRIIA, revealing a positive feedback loop that appears to function to stabilize active type-A receptors at synaptic sites. Thus, the novel BMP signaling modality appears to sculpt synapse composition and maturation as a function of synapse activity. Given that synaptic pMad accumulates at the active zone, near the presynaptic membrane, in close juxtaposition with the iGluRs containing postsynaptic densities, we proposed that presynaptic pMad marks sites where active postsynaptic type-A receptors induce the assembly of trans-synaptic complexes with presynaptic BMP and BMPRs (BMP receptors).

In recent work, we started to define the composition of these local BMP/BMPR complexes using genetics and cell biology approaches. Using mutants and RNAi lines to genetically manipulate the levels of receptors, we have already established that synaptic pMad accumulation requires the type-II BMPR Wit and type-I BMPRs Tkv and Sax. In addition, we found that endogenously tagged Tkv is distributed to presynaptic aggregates that appear to co-localize with the presynaptic pMad signals. Current studies in the laboratory focus on determining the composition and regulation of the pMad–containing complexes at synaptic terminals.

It is important to recognize that all the BMP signaling modalities are coordinated by shared, limited components, in particular the BMP receptors, which are tightly regulated at transcriptional, translational, and post-translational levels. The canonical BMP signaling pathway requires endocytosis of the BMP/BMPR–signaling complexes and their retrograde transport to the motor neuron soma, whereas the noncanonical pathways rely on BMP/BMPR complexes to function at synaptic terminals. Given that the pathways share limited pools of BMPRs, the motor neurons must balance the partitioning of BMPRs among different BMP signaling modalities. Consequently, BMP signaling may monitor synapse activity and coordinate it with synapse growth and maturation.

Publications

  1. Li Y, Dharkar P, Han TH, Serpe M, Lee CH, Mayer ML. Novel functional properties of Drosophila CNS glutamate receptors. Neuron 2016 92:1036-1048.
  2. Meyerson JR, Chittori S, Merk A, Rao P, Han TH, Serpe M, Mayer ML, Subramaniam S. Structural basis of kainate subtype glutamate receptor desensitization. Nature 2016 567-571.

Collaborators

  • Chi-Hon Lee, MD, PhD, Section on Neuronal Connectivity, NICHD, Bethesda, MD
  • Mark Mayer, PhD, Laboratory of Cellular and Molecular Neurophysiology, NICHD, Bethesda, MD

Contact

For more information, email serpemih@mail.nih.gov or visit http://ucc.nichd.nih.gov.

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