Direct Visualization of Trans-Synaptic Neurexin-Neuroligin Interactions during Synapse Formation (original) (raw)
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Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation
Embo Journal, 2009
Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic aand b-neurexins. To test this hypothesis, we examined the functional effects of neuroligin-1 mutations that impair only a-neurexin binding, block both aand b-neurexin binding, or abolish neuroligin-1 dimerization. Abolishing a-neurexin binding abrogated neuroligin-induced generation of neuronal synapses onto transfected non-neuronal cells in the so-called artificial synapse-formation assay, even though b-neurexin binding was retained. Thus, in this assay, neuroligin-1 induces apparent synapse formation by binding to presynaptic a-neurexins. In transfected neurons, however, neither anor b-neurexin binding was essential for the ability of postsynaptic neuroligin-1 to dramatically increase synapse density, suggesting a neurexin-independent mechanism of synapse formation. Moreover, neuroligin-1 dimerization was not required for either the nonneuronal or the neuronal synapse-formation assay. Nevertheless, both a-neurexin binding and neuroligin-1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin-1 in transfected neurons. Thus, neuroligin-1 performs diverse synaptic functions by mechanisms that include as essential components of a-neurexin binding and neuroligin dimerization, but extend beyond these activities. EMBO THE EMBO JOURNAL THE EMBO JOURNAL Neurexin-dependent functions of neuroligin-1 J Ko et al
Structural insights into the exquisite selectivity of neurexin/neuroligin synaptic interactions
The EMBO Journal, 2010
The extracellular domains of neuroligins and neurexins interact through Ca 2 þ to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic maturation and differentiation, is regulated by gene selection, alternative mRNA splicing and post-translational modifications. A new, 2.6 Å-resolution crystal structure of a soluble neurexin-1b-neuroligin-4 (Nrx1b-NL4) complex permits a detailed description of the Ca 2 þcoordinated interface and unveils concerted positional rearrangements of several residues of NL4, not observed in neuroligin-1, associated with Nrx1b binding. Surface plasmon resonance analysis of the binding of structureguided Nrx1b mutants towards NL4 and neuroligin-1 shows that flexibility of the Nrx1b-binding site in NL4 is reflected in a greater dissociation constant of the complex and higher sensitivity to ionic strength and pH variations. Analysis of neuroligin mutants points to critical functions for two respective residues in neuroligin-1 and neuroligin-2 in governing the affinity of the complexes. Although neuroligin-1 and neuroligin-2 have pre-determined conformations that respectively promote and prevent Nrx1b association, unique conformational reshaping of the NL4 surface is required to permit Nrx1b association.
Neurexin Iβ and neuroligin are localized on opposite membranes in mature central synapses
Journal of Neurochemistry, 2007
Synaptogenesis requires formation of trans-synaptic complexes between neuronal cell-adhesion receptors. Heterophilic receptor pairs, such as neurexin Iβ and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre-and postsynaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the postsynaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Iβ and neuroligin in mature cerebrocortical synapses, we have developed a novel method for physical separation of junctional membranes and their direct analysis by western blotting. Using urea and DTT, we disrupted trans-synaptic protein links, without dissolving the lipid phase, and fractionated the pre-and postsynaptic membranes. The purity of these fractions was validated by electron microscopy and western blotting using multiple synaptic markers. A quantitative analysis has confirmed that neuroligin is localized strictly in the postsynaptic membrane. We have also demonstrated, that neurexin Iβ is largely (96%) presynaptic. Thus, neurexin Iβ and neuroligin normally form trans-synaptic complexes and can transduce bidirectional signals.
Proceedings of the National Academy of Sciences, 2011
Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation. hippocampus | neuropsychiatric disorders T he enormous processing power of the mammalian brain is the result of a vast network of precise synaptic connections, where functionally diverse presynaptic neurons establish synapses with specific properties onto select populations of postsynaptic cells. Neuroligins (NLs) and neurexins (NRXs) are a prototypical transsynaptic adhesion pair (1, 2) that is ideally situated to play important roles in such synaptic processes. Interactions between the four NLs (NL1-4) and the three NRXs are highly regulated at the level of alternative mRNA splicing, generating an intricate code that regulates both the affinity of interactions and the consequences on synapse specification (3, 4). Given the complexity of NL-NRX interactions, it was surprising to find that leucine-rich repeat transmembrane proteins (LRRTMs) are also high-affinity receptors for NRXs and share many of the binding characteristics of NLs .
Silencing of Neuroligin Function by Postsynaptic Neurexins
Journal of Neuroscience, 2007
The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin-neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1 are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1 with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.
Neurexin mediates the assembly of presynaptic terminals
Nature Neuroscience, 2003
Neurexins are a large family of proteins that act as neuronal cell-surface receptors. The function and localization of the various neurexins, however, have not yet been clarified. Beta-neurexins are candidate receptors for neuroligin-1, a postsynaptic membrane protein that can trigger synapse formation at axon contacts. Here we report that neurexins are concentrated at synapses and that purified neuroligin is sufficient to cluster neurexin and to induce presynaptic differentiation. Oligomerization of neuroligin is required for its function, and we find that beta-neurexin clustering is sufficient to trigger the recruitment of synaptic vesicles through interactions that require the cytoplasmic domain of neurexin. We propose a two-step model in which postsynaptic neuroligin multimers initially cluster axonal neurexins. In response to this clustering, neurexins nucleate the assembly of a cytoplasmic scaffold to which the exocytotic apparatus is recruited. Synapses are highly specialized cellular junctions that join individual neurons into a functional network. The appropriate differentiation of pre-and postsynaptic terminals is controlled by bidirectional signaling between the synaptic partners 1-3. One family of postsynaptic proteins that might contribute to the induction of synapse formation between CNS neurons are the neuroligins 4 , 5. Non-neuronal HEK293 cells that ectopically express neuroligin-1 or neuroligin-2 trigger the formation of functional presynaptic elements in contacting axons in vitro 6. The extracellular domain of neuroligin presented at the cell surface of the non-neuronal cells is sufficient for this activity, suggesting that it may signal through an axonal receptor that triggers the induction of presynaptic differentiation. The identity of this axonal receptor is unknown. Leading candidates are the beta-neurexins, which are neuronal plasma membrane proteins that interact with neuroligins in vitro 4 , 7. Consistent with a role for beta-neurexins in neuroligin-induced synapse formation, recombinant beta-neurexin can block the synapse-inducing activity of neuroligin-expressing HEK293 cells 6 , though a similar blocking effect would be expected for any protein interacting with the extracellular domain of neuroligin. A major concern regarding the role of neurexins as presynaptic neuroligin receptors is that there is currently no direct evidence for a synaptic function or localization of neurexins in the vertebrate CNS. Moreover, it is unclear how neuroligin-binding to beta-neurexin could trigger the recruitment of the presynaptic exocytotic machinery to the cell-cell contact sites. An
Neuron, 2005
Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic b-neurexins, but not with presynaptic a-neurexins. Unexpectedly, we now find that neuroligins also bind a-neurexins and that a-and b-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines a-neurexin binding but leaves b-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only b-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both a-and b-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to a-and b-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.
Membrane-Tethered Monomeric Neurexin LNS-Domain Triggers Synapse Formation
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2013
Neurexins are presynaptic cell-adhesion molecules that bind to postsynaptic cell-adhesion molecules such as neuroligins and leucinerich repeat transmembrane proteins (LRRTMs). When neuroligins or LRRTMs are expressed in a nonneuronal cell, cocultured neurons avidly form heterologous synapses onto that cell. Here we show that knockdown of all neurexins in cultured hippocampal mouse neurons did not impair synapse formation between neurons, but blocked heterologous synapse formation induced by neuroligin-1 or LRRTM2. Rescue experiments demonstrated that all neurexins tested restored heterologous synapse formation in neurexin-deficient neurons. Neurexin-deficient neurons exhibited a decrease in the levels of the PDZ-domain protein CASK (a calcium/calmodulin-activated serine/ threonine kinase), which binds to neurexins, and mutation of the PDZ-domain binding sequence of neurexin-3 blocked its transport to the neuronal surface and impaired heterologous synapse formation. However, replacement of the C-terminal neurexin sequence with an unrelated PDZ-domain binding sequence that does not bind to CASK fully restored surface transport and heterologous synapse formation in neurexin-deficient neurons, suggesting that no particular PDZ-domain protein is essential for neurexin surface transport or heterologous synapse formation. Further mutagenesis revealed, moreover, that the entire neurexin cytoplasmic tail was dispensable for heterologous synapse formation in neurexin-deficient neurons, as long as the neurexin protein was transported to the neuronal cell surface. Furthermore, the single LNS-domain (for laminin/neurexin/sex hormone-binding globulin-domain) of neurexin-1 or neurexin-3, when tethered to the presynaptic plasma membrane by a glycosylinositolphosphate anchor, was sufficient for rescuing heterologous synapse formation in neurexin-deficient neurons. Our data suggest that neurexins mediate heterologous synapse formation via an extracellular interaction with presynaptic and postsynaptic ligands without the need for signal transduction by the neurexin cytoplasmic tail.
Neuron, 2009
Neurexins and neuroligins provide trans-synaptic connectivity by the Ca 2+ -dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1b. Neuroligin-1 forms a constitutive dimer, and two neurexin-1b monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1b complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca 2+ . Alternatively spliced sites in neurexin-1b and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1b, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1b complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.
Neuron, 2007
Neurexins and neuroligins provide trans-synaptic connectivity by the Ca 2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1b. Neuroligin-1 forms a constitutive dimer, and two neurexin-1b monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1b complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca 2+. Alternatively spliced sites in neurexin-1b and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1b, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1b complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.