National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2018 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, Research Fellow
  • Saumitra Dey Choudhury, 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, whose purpose is to mediate rapid and efficient transmission of signals across the synaptic cleft. Synaptic 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 post-synaptic 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.

Neto, an essential protein that recruits neurotransmitter receptors and organizes post-synaptic densities at the Drosophila NMJ

Many neurological disorders are linked to defects in synaptogenesis. The initial clustering functions of receptors in synaptogenesis are poorly understood. Prior to motor neuron arrival at its target muscle, 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, which 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 post-synaptic density (PSD) scaffolds, and importantly, their channel properties. iGluRs assembled from different subunits have strikingly different biophysical properties; their association with different auxiliary subunits increases this diversity even further. In flies, as in humans, synapse strength and plasticity are determined by the interplay between different iGluRs 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.

We previously reported that the Drosophila Neto, a transmembrane protein, plays a key role in the initial clustering of the iGluRs at nascent synapses. First, in the absence of Neto, iGluRs are not recruited at synaptic locations and remain scattered as small, nascent clusters away from the presynaptic arbor. In fact, Neto is limiting for iGluRs clustering: Reducing the Neto levels, by RNAi-mediated knock down or in a strong neto hypomorph (neto109), drastically diminishes the synaptic iGluRs levels. The net levels of muscle iGluRs remain normal, indicating a redistribution of receptors from junctional to extrajunctional locations. Second, masking the extracellular structural motifs of Neto called CUB domains does not prevent Neto from engaging iGluRs and forming functional receptors, but effectively blocks Neto’s ability to mediate iGluRs clustering. Post-synaptic differentiation is not even initiated at these synapses, indicating that clustering of iGluRs and not synapse activity triggers synapse development. Thus, self-aggregation and/or interactions with other extracellular cues enable the Neto–dependent clustering of iGluRs. Third, our recent results indicate that the intracellular domain of Neto-β directly engages iGluRs as well as other intracellular proteins to selectively regulate the distribution of iGluR subtypes, the recruitment of post-synaptic proteins, and the organization of post-synaptic structures. Fourth, our studies on functional receptors reconstituted in heterologous systems revealed that Neto modulates the function of iGluRs but not their assembly or surface delivery. Given that iGluR gating properties control the distribution and trafficking of these receptors in vivo, Neto could influence the synaptic recruitment of iGluRs by simultaneously controlling several steps in receptor trafficking and clustering and/or receptor function.

To further characterize the functional domains of Neto, we generated truncated Neto variants and tested their cellular distribution and ability to rescue Neto function during development. Neto is expressed in and apically targeted in several epithelia, permitting us to distinguish between surface delivery defects and synaptic recruitment. We found that the extracellular part of Neto is required for apical targeting as well as for clustering of Neto/iGluR complexes at the NMJ. The intracellular domains of Neto could be completely removed without preventing targeting and clustering at the NMJ. We also determined that Neto binds to iGluRs via its LDLa (low-density lipoprotein class A) motif and transmembrane domain (Tm). Our studies indicate that: (1) that the extracellular and Tm part of Neto are both required and sufficient to (a) interact with iGluRs, (b) target Neto to post-synaptic sites, and (c) stabilize the synaptic iGluRs/Neto clusters; and that (2) the intracellular domains regulate the synaptic targeting of Neto/iGluR complexes.

Our current efforts focus on identifying proteins that interact with Neto and provide iGluR–clustering activities at the developing NMJ. To this end, we initiated several complementary screens: (1) pull-down and mass-spectroscopy comparisons of proteins interacting with the intracellular domains of the fly Neto proteins (α and β); and (2) a synthetic lethality screen (below).

Tenectin, an integrin ligand critical for structural and functional integrity of the fly NMJ

To search for novel extracellular matrix (ECM) proteins important for NMJ development, we took advantage of neto109, a strong neto hypomorph mutant that we isolated and characterized in our laboratory. The mutant has drastically diminished levels of synaptic iGluRs, but normal net levels of muscle receptors, indicating a defect in the trafficking and/or stabilization of receptors at junctional locations. Given that 50% of neto109 hypomorphs die during development, further reduction of synaptogenic proteins in hemizygous animals should increase lethality. Using this rationale, we set up a synthetic lethality screen to identify proteins that interact genetically with Neto and that control the development of NMJ. In a pilot, proof-of-concept screen of candidate interactors, we confirmed known NMJ modulators, such as Glass bottom boat (Gbb), a BMP ligand with critical roles during NMJ development, and Mind the gap (Mtg), a neuronal secreted protein previously implicated in the assembly of functional post-synaptic domain. In a search for ECM candidates, we identified a set of overlapping deficiencies that dramatically increased the lethality of neto109 hemizygotes. Among the common loci disrupted by these deficiencies was tenectin, a gene encoding a large, secreted protein conserved in many insects but with no obvious mammalian homolog.

Previous studies indicated that Tenectin is a large secreted mucin that confers structural integrity to tubular structures. Also, reduction of Tenectin via RNAi produced flightless adults with locomotor defects. We found that Tenectin is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Using genetics, biochemistry, electrophysiology, histology, and electron microscopy, we found that Tenectin recruits the αPS2/βPS integrin (a transmembrane protein that flexibly links the inside to the outside surface of cells) at synaptic terminals and forms pre- and post-synaptic biologically active cis complexes with distinct functions: The presynaptic Tenectin/integrin complexes control neurotransmitter release, while the post-synaptic complexes ensure the architectural integrity of synaptic boutons. Interestingly, removal of Tenectin disrupted integrin recruitment selectively at synaptic locations, without affecting integrin anchoring at muscle attachment sites. Also, excess Tenectin in the striated muscle depleted the synaptic pool of integrin, presumably by sequestering integrin at ectopic locations. We exploited the remarkable features of this selective integrin ligand to reveal an unprecedented role for integrin in connecting the ECM of the synaptic cleft with the cytoskeletal protein spectrin, in particular to the spectrin-based membrane skeleton. These Tenectin/integrin/spectrin complexes are crucial for the integrity and function of synaptic structures. Our identification and genetic manipulation of a highly selective ligand, such as Tenectin, opens the door for similar strategies to deplete integrins locally and parse out compartment-specific functions for integrins, and thus reveal hidden aspects of the integrin/ECM biology and their functions at synapses.

Local BMP/BMPR complexes regulate synaptic plasticity.

Synaptic activity and synapse development are intimately linked, but our understanding of the coupling mechanisms is limited. In particular, how synapse activity status is monitored and communicated across the synaptic cleft remains poorly understood. Our studies uncovered a role for bone morphogenetic proteins (BMPs) in sensing the activity of post-synaptic receptors and relaying this information across the synaptic cleft.

At the Drosophila NMJ, BMP signaling is critical for NMJ growth and neurotransmitter release. BMP signaling fulfills these functions via a canonical signaling pathway triggered primarily by muscle-secreted Glass-bottom boat (Gbb), a BMP7 homolog. Gbb binding to the presynaptic BMP type-II receptor (BMPRII) Wishful thinking (Wit) and to the BMPRIs Thickveins (Tkv) and Saxophone (Sax) induces formation of BMP signaling complexes that are retrogradely transported from the synaptic terminals to the motor-neuron soma, which resides in the ventral ganglia. The BMP/BMPR signaling complexes phosphorylate the pathway effector Mad, which associates with a co-Smad and translocates into the motor-neuron nuclei. The nuclear pMad/co-Smad complexes activate transcriptional programs with distinct roles in the structural and functional development of the NMJ.

Interestingly, pMad also accumulates at synaptic locations. We recently demonstrated that synaptic pMad localizes presynaptically at the active zone and constitutes a sensor for synapse activity because it correlates with the post-synaptic iGluRs activity. Furthermore, synaptic pMad marks a novel BMP signaling modality that is genetically distinct 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 post-synaptic glutamate receptors, the type-A iGluRs. 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 the post-synaptic levels of 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 post-synaptic densities, we proposed that presynaptic pMad marks sites where active post-synaptic type-A iGluRs induce the assembly of trans-synaptic complexes with presynaptic BMP/BMPRs. In this model, the BMP signaling modalities are coordinated by shared, limited components, in particular the BMPRs, which are tightly regulated at transcriptional, translational, and post-translational levels. The canonical BMP signaling pathway requires endocytosis of the BMP/BMPR complexes and their retrograde transport to the motor neuron soma, whereas the novel pathway requires that BMP/BMPRs 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. Thus, BMP signaling may monitor synapse activity and coordinate it with synapse growth and maturation.

Our current efforts focus on elucidating the composition, regulation, and function of the synaptic BMP/BMPR complexes, using genetics and cell-biology approaches. We had already established genetically that the accumulation of synaptic pMad requires the BMPRII Wit and the BMPRIs Tkv and Sax. In addition, we found that Tkv tagged with the red fluorescent tracer mCherry is distributed to presynaptic aggregates that appear to co-localize with the synaptic pMad at active zones. In recent studies, we screened the entire collection of Mad mutants for variant(s) that uncouple the accumulation of synaptic vs. nuclear pMad. We found a mutation that appears to selectively impair the accumulation of synaptic but not nuclear pMad. Importantly, this Mad mutant also has reduced GluRIIA synaptic levels, as indicated by immunohistochemistry and electrophysiological assays. Modeling the interaction between this Mad variant and the BMP/BMPR complexes should provide insights into the structural elements required for the deployment and function of this new BMP signaling modality.

Publications

  1. 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.
  2. Wang Q, Han TH, Nguyen P, Jarnik M, Serpe M. Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction. eLife 2018;7:e35518.

Collaborators

  • Chi-Hon Lee, MD, PhD, Academia Sinica, Taipei, Taiwan
  • Gregory T. Macleod, PhD, Florida Atlantic University, Jupiter, FL
  • Mark Mayer, PhD, Scientist Emeritus, NINDS, Bethesda, MD
  • Thomas B. Thompson, PhD, University of Cincinnati, Cincinnati, Ohio

Contact

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

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