N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn⁶⁰. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn⁷⁰/Asn¹⁰⁴ flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn⁶⁰ reduces adhesion, N-glycans at Asn⁷⁰/Asn¹⁰⁴ of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.     

The novel synaptogenic protein Farp1 links postsynaptic cytoskeletal dynamics and transsynaptic organization

Synaptic adhesion organizes synapses, yet the signaling pathways that drive and integrate synapse development remain incompletely understood. We screened for regulators of these processes by proteomically analyzing synaptic membranes lacking the synaptogenic adhesion molecule SynCAM 1. This identified FERM, Rho/ArhGEF, and Pleckstrin domain protein 1 (Farp1) as strongly reduced in SynCAM 1 knockout mice. Farp1 regulates dendritic filopodial dynamics in immature neurons, indicating roles in synapse formation. Later in development, Farp1 is postsynaptic and its 4.1 protein/ezrin/radixin/moesin (FERM) domain binds SynCAM 1, assembling a synaptic complex. Farp1 increases synapse number and modulates spine morphology, and SynCAM 1 requires Farp1 for promoting spines. In turn, SynCAM 1 loss reduces the ability of Farp1 to elevate spine density. Mechanistically, Farp1 activates the GTPase Rac1 in spines downstream of SynCAM 1 clustering, and promotes F-actin assembly. Farp1 furthermore triggers a retrograde signal regulating active zone composition via SynCAM 1. These results reveal a postsynaptic signaling pathway that engages transsynaptic interactions to coordinate synapse development.     

SynCAM 1 adhesion dynamically regulates synapse number and impacts plasticity and learning

Synaptogenesis is required for wiring neuronal circuits in the developing brain and continues to remodel adult networks. However, the molecules organizing synapse development and maintenance in?vivo remain incompletely understood. We now demonstrate that the immunoglobulin adhesion molecule SynCAM 1 dynamically alters synapse number and plasticity. Overexpression of SynCAM 1 in transgenic mice promotes excitatory synapse number, while loss of SynCAM 1 results in fewer excitatory synapses. By turning off SynCAM 1 overexpression in transgenic brains, we show that it maintains the newly induced synapses. SynCAM 1 also functions at mature synapses to alter their plasticity by regulating long-term depression. Consistent with these effects on neuronal connectivity, SynCAM 1 expression affects spatial learning, with knock-out mice learning better. The reciprocal effects of increased SynCAM 1 expression and loss reveal that this adhesion molecule contributes to the regulation of synapse number and plasticity, and impacts how neuronal networks undergo activity-dependent changes.     

Bioinformatic characterization of the SynCAM family of immunoglobulin-like domain-containing adhesion molecules

SynCAM 1 (synaptic cell adhesion molecule 1, alternatively named Tslc1 and nectin-like protein 3) belongs to the immunoglobulin superfamily and is an adhesion molecule that operates in a variety of important contexts. Exemplary are its roles in adhesion at synapses in the central nervous system and as tumor suppressor. Here, I describe a family of genes homologous to SynCAM 1 comprising four genes found solely in vertebrates. All SynCAM genes encode proteins with three immunoglobulin-like domains of the V-set, C1-set, and I-set subclasses. Comparison of genomic with cDNA sequences provides their exon-intron structure. Alternative splicing generates isoforms of SynCAM proteins, and diverse SynCAM 1 and 2 isoforms are created in an extracellular region rich in predicted O-glycosylation sites. Protein interaction motifs in the cytosolic sequence are highly conserved among all four SynCAM proteins, indicating their critical functional role. These findings aim to facilitate the understanding of SynCAM genes and provide the framework to examine the physiological functions of this family of vertebrate-specific adhesion molecules.     

Astrocyte-specific disruption of SynCAM1 signaling results in ADHD-like behavioral manifestations

SynCAM1 is an adhesion molecule involved in synaptic differentiation and organization. SynCAM1 is also expressed in astroglial cells where it mediates astrocyte-to astrocyte and glial-neuronal adhesive communication. In astrocytes, SynCAM1 is functionally linked to erbB4 receptors, which are involved in the control of both neuronal/glial development and mature neuronal and glial function. Here we report that mice carrying a dominant-negative form of SynCAM1 specifically targeted to astrocytes (termed GFAP-DNSynCAM1 mice) exhibit disrupted diurnal locomotor activity with enhanced and more frequent episodes of activity than control littermates during the day (when the animals are normally sleeping) accompanied by shorter periods of rest. GFAP-DNSynCAM1 mice also display high levels of basal activity in the dark period (the rodent's awake/active time) that are attenuated by the psychostimulant D,L-amphetamine, and reduced anxiety levels in response to both avoidable and unavoidable provoking stimuli. These results indicate that disruption of SynCAM1-dependent astroglial function results in behavioral abnormalities similar to those described in animals model of attention-deficit hyperactive disorder (ADHD), and suggest a hitherto unappreciated contribution of glial cells to the pathophysiology of this disorder.     

Structure and interactions of NCAM Ig1-2-3 suggest a novel zipper mechanism for homophilic adhesion

The neural cell adhesion molecule, NCAM, mediates Ca(2+)-independent cell-cell and cell-substratum adhesion via homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. NCAM plays a key role in neural development, regeneration, and synaptic plasticity, including learning and memory consolidation. The crystal structure of a fragment comprising the three N-terminal Ig modules of rat NCAM has been determined to 2.0 A resolution. Based on crystallographic data and biological experiments we present a novel model for NCAM homophilic binding. The Ig1 and Ig2 modules mediate dimerization of NCAM molecules situated on the same cell surface (cis interactions), whereas the Ig3 module mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans interactions) through simultaneous binding to the Ig1 and Ig2 modules. This arrangement results in two perpendicular zippers forming a double zipper-like NCAM adhesion complex.     

N-Cadherin Relocalizes from the Periphery to the Center of the Synapse after Transient Synaptic Stimulation in Hippocampal Neurons

N-cadherin is a cell adhesion molecule which is enriched at synapses. Binding of N-cadherin molecules to each other across the synaptic cleft has been postulated to stabilize adhesion between the presynaptic bouton and the postsynaptic terminal. N-cadherin is also required for activity-induced changes at synapses, including hippocampal long term potentiation and activity-induced spine expansion and stabilization. We hypothesized that these activity-dependent changes might involve changes in N-cadherin localization within synapses. To determine whether synaptic activity changes the localization of N-cadherin, we used structured illumination microscopy, a super-resolution approach which overcomes the conventional resolution limits of light microscopy, to visualize the localization of N-cadherin within synapses of hippocampal neurons. We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft. Furthermore, the N-cadherin localization pattern within synapses changes during KCl depolarization and after transient synaptic stimulation. During KCl depolarization, N-cadherin relocalizes away from the central region of the synaptic cleft to the periphery of the synapse. In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells. This indicates that transient synaptic stimulation modulates N-cadherin localization within the synapse. These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.     

More than just glue: The diverse roles of cell adhesion molecules in the Drosophila nervous system

Cell adhesion is the fundamental driving force that establishes complex cellular architectures, with the nervous system offering a striking, sophisticated case study. Developing neurons adhere to neighboring neurons, their synaptic partners, and to glial cells. These adhesive interactions are required in a diverse array of contexts, including cell migration, axon guidance and targeting, as well as synapse formation and physiology. Forward and reverse genetic screens in the fruit fly Drosophila have uncovered several adhesion molecules that are required for neural development, and detailed cell biological analyses are beginning to unravel how these factors shape nervous system connectivity. Here we review our current understanding of the most prominent of these adhesion factors and their modes of action.Key words: drosophila, cell adhesion, nervous system, glia, axon, synapse     

IGSF4: a new intercellular adhesion molecule that is called by three names,TSLC1, SgIGSF and SynCAM,by virtue of its diverse function

Members of the immunoglobulin superfamily often play key roles in intercellular adhesion. IGSF4 is a novel immunoglobulin (Ig)-like intercellular adhesion molecule. Three Ig-like domains are included in the extracellular domain of IGSF4 and mediate homophilic or heterophilic interactions independently of Ca2+. The cytoplasmic domain of IGSF4 contains the binding motifs that connect to actin fibers. Since IGSF4 has been characterized by several independent research groups, this molecule is called by three names, TSLC1, SgIGSF and SynCAM. IGSF4 was first characterized as a tumor suppressor of non-small cell lung cancer and termed TSLC1, although how IGSF4 suppresses tumor growth remains unknown. Silencing of the IGSF4 gene was primarily achieved by allelic loss and promoter methylation in this type of cancers. Soon after this discovery, IGSF4 was found to have roles in adhesion of spermatogenic cells to Sertoli cells and mast cells to fibroblasts and termed SgIGSF. Other researchers revealed that IGSF4 drives synaptic formation of neural cells and termed it SynCAM.     

Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.     

The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that—like SynCAM 1—MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)_A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABA_A receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABA_A receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.

This study shows that the MAGUK scaffold protein MPP2 is located at the periphery of postsynaptic densities in excitatory neurons, where it interacts with GABA-A receptors, thereby serving as a functional adaptor that links excitatory and inhibitory components of synaptic transmission at glutamatergic synapses.     

Complex interactions amongst N-cadherin, DLAR, and Liprin-α regulate Drosophila photoreceptor axon targeting

The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-α play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone and are themselves regulated remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, we evaluate genetic and physical interactions among these three proteins. We demonstrate that DLAR function in this context is independent of phosphatase activity but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-α function in a complex to regulate adhesive interactions between pre- and post-synaptic cells and provide a novel mechanism for controlling the activity of Liprin-α in the developing growth cone.     

Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts

Transformation of a contact between axon and dendrite into a synapse is accompanied by accumulation of the synaptic machinery at this site, being delivered in intracellular organelles mainly of TGN origin. Here, we report that in cultured hippocampal neurons, TGN organelles are linked via spectrin to clusters of the neural cell adhesion molecule (NCAM) in the plasma membrane. These complexes are translocated along neurites and trapped at sites of initial neurite-to-neurite contacts within several minutes after initial contact formation. The accumulation of TGN organelles at contacts with NCAM-deficient neurons is reduced when compared with wild-type cells, suggesting that NCAM mediates the anchoring of intracellular organelles in nascent synapses.     

The Extent of Synaptic Stripping of Motoneurons after Axotomy Is Not Correlated to Activation of Surrounding Glia or Downregulation of Postsynaptic Adhesion Molecules

Synapse elimination in the adult central nervous system can be modelled by axotomy of spinal motoneurons which triggers removal of synapses from the cell surface of lesioned motoneurons by processes that remain elusive. Proposed candidate mechanisms are removal of synapses by reactive microglia and astrocytes, based on the remarkable activation of these cell types in the vicinity of motoneurons following axon lesion, and/or decreased expression of synaptic adhesion molecules in lesioned motoneurons. In the present study, we investigated glia activation and adhesion molecule expression in motoneurons in two mouse strains with deviant patterns of synapse elimination following axotomy. Mice deficient in complement protein C3 display a markedly reduced loss of synapses from axotomized motoneurons, whereas mice with impaired function of major histocompatibility complex (MHC) class Ia display an augmented degree of stripping after axotomy. Activation of microglia and astrocytes was assessed by semiquantative immunohistochemistry for Iba 1 (microglia) and GFAP (astrocytes), while expression of synaptic adhesion molecules was determined by in situ hybridization. In spite of the fact that the two mouse strains display very different degrees of synapse elimination, no differences in terms of glial activation or in the downregulation of the studied adhesion molecules (SynCAM1, neuroligin-2,-3 and netrin G-2 ligand) could be detected. We conclude that neither glia activation nor downregulation of synaptic adhesion molecules are correlated to the different extent of the synaptic stripping in the two studied strains. Instead the magnitude of the stripping event is most likely a consequence of a precise molecular signaling, which at least in part is mediated by immune molecules.     

Synaptic adhesion molecules

Formation, differentiation and plasticity of synapses, the specialized cell-cell contacts through which neurons communicate, all require interactions between pre- and post-synaptic partners. Several synaptically localized adhesion molecules potentially capable of mediating these interactions have been identified recently. Functional studies suggest roles for some of them in target recognition (e.g. SYG-1 and sidekicks), formation and alignment of synaptic specializations (e.g. SynCAM, neuroligin and neurexin), and regulation of synaptic structure and function (e.g. cadherins and syndecan).     

Imaging activity-dependent regulation of neurexin-neuroligin interactions using trans-synaptic enzymatic biotinylation

The functions of trans-synaptic adhesion molecules, such as neurexin and neuroligin, have been difficult to study due to the lack of methods to directly detect their binding in living neurons. Here, we use biotin labeling of intercellular contacts (BLINC), a method for imaging protein interactions based on interaction-dependent biotinylation of a peptide by E. coli biotin ligase, to visualize neurexin-neuroligin trans-interactions at synapses and study their role in synapse development. We found that both developmental maturation and acute synaptic activity stimulate the growth of neurexin-neuroligin adhesion complexes via a combination of neurexin and neuroligin surface insertion and internalization arrest. Both mechanisms require NMDA receptor activity. We also discovered that disruption of activity-induced neurexin-neuroligin complex growth prevents recruitment of the AMPA receptor, a hallmark of mature synapses. Our results provide support for neurexin-neuroligin function in synapse maturation and introduce a general method to study intercellular protein-protein interactions.     

Linkage of N-cadherin to multiple cytoskeletal elements revealed by a proteomic approach in hippocampal neurons

The CNS synapse is an adhesive junction differentiated for chemical neurotransmission and is equipped with presynaptic vesicles and postsynaptic neurotransmitter receptors. Cell adhesion molecule cadherins not only maintain connections between pre- and postsynaptic membranes but also modulate the efficacy of synaptic transmission. Although the components of the cadherin-mediated adhesive apparatus have been studied extensively in various cell systems, the complete picture of these components, particularly at the synaptic junction, remains elusive. Here, we describe the proteomic assortment of the N-cadherin-mediated synaptic adhesion apparatus in cultured hippocampal neurons. N-cadherin immunoprecipitated from Triton X-100-solubilized neuronal extract contained equal amounts of β- and α-catenins, as well as F-actin-related membrane anchor proteins such as integrins bridged with α-actinin-4, and Na(+)/K(+)-ATPase bridged with spectrins. A close relative of β-catenin, plakoglobin, and its binding partner, desmoplakin, were also found, suggesting that a subset of the N-cadherin-mediated adhesive apparatus also anchors intermediate filaments. Moreover, dynein heavy chain and LEK1/CENPF/mitosin were found. This suggests that internalized pools of N-cadherin in trafficking vesicles are conveyed by dynein motors on microtubules. In addition, ARVCF and NPRAP/neurojungin/δ2-catenin, but not p120ctn/δ1-catenin or plakophilins-1, -2, -3, -4 (p0071), were found, suggesting other possible bridges to microtubules. Finally, synaptic stimulation by membrane depolarization resulted in an increased 93-kDa band, which corresponded to proteolytically truncated β-catenin. The integration of three different classes of cytoskeletal systems found in the synaptic N-cadherin complex may imply a dynamic switching of adhesive scaffolds in response to synaptic activity.     

Cell adhesion molecules in Drosophila synapse development and function

Synapse is a highly specialized inter-cellular structure between neurons or between a neuron and its target cell that mediates cell-cell communications. Ample results indicate that synaptic adhesion molecules are critically important in modulating the complexity and specificity of the synapse. And disruption of adhesive properties of synapses may lead to neurodevelopmental or neurodegenerative diseases. In this review, we will use the Drosophila NMJ as a model system for glutamatergic synapses to discuss the structure and function of homophilic and heterophilic synaptic adhesion molecules with special focus on recent findings in neurexins and neuroligins in Drosophila.