A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to alpha- and beta-neurexins

Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic beta-neurexins, but not with presynaptic alpha-neurexins. Unexpectedly, we now find that neuroligins also bind alpha-neurexins and that alpha- and beta-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 alpha-neurexin binding but leaves beta-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 beta-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both alpha- and beta-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to alpha- and beta-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.     

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.     

Structural analysis of the synaptic protein neuroligin and its beta-neurexin complex: determinants for folding and cell adhesion

The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a beta-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an alpha/beta-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism.     

Higher-order architecture of cell adhesion mediated by polymorphic synaptic adhesion molecules neurexin and neuroligin

Polymorphic adhesion molecules neurexin and neuroligin (NL) mediate asymmetric trans-synaptic adhesion, which is crucial for synapse development and function. It is not known whether or how individual synapse function is controlled by the interactions between variants and isoforms of these molecules with differing ectodomain regions. At a physiological concentration of Ca(2+), the ectodomain complex of neurexin-1 β isoform (Nrx1β) and NL1 spontaneously assembled into crystals of a lateral sheet-like superstructure topologically compatible with transcellular adhesion. Correlative light-electron microscopy confirmed extracellular sheet formation at the junctions between Nrx1β- and NL1-expressing non-neuronal cells, mimicking the close, parallel synaptic membrane apposition. The same NL1-expressing cells, however, did not form this higher-order architecture with cells expressing the much longer neurexin-1 α isoform, suggesting a functional discrimination mechanism between synaptic contacts made by different isoforms of neurexin variants.     

Neuroligin‐1 performs neurexin‐dependent and neurexin‐independent functions in synapse validation

Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic α‐ and β‐neurexins. To test this hypothesis, we examined the functional effects of neuroligin‐1 mutations that impair only α‐neurexin binding, block both α‐ and β‐neurexin binding, or abolish neuroligin‐1 dimerization. Abolishing α‐neurexin binding abrogated neuroligin‐induced generation of neuronal synapses onto transfected non‐neuronal cells in the so‐called artificial synapse‐formation assay, even though β‐neurexin binding was retained. Thus, in this assay, neuroligin‐1 induces apparent synapse formation by binding to presynaptic α‐neurexins. In transfected neurons, however, neither α‐ nor β‐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 non‐neuronal or the neuronal synapse‐formation assay. Nevertheless, both α‐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 α‐neurexin binding and neuroligin dimerization, but extend beyond these activities.     

Synaptic arrangement of the neuroligin/beta-neurexin complex revealed by X-ray and neutron scattering

Neuroligins are postsynaptic cell-adhesion proteins that associate with their presynaptic partners, the neurexins. Using small-angle X-ray scattering, we determined the shapes of the extracellular region of several neuroligin isoforms in solution. We conclude that the neuroligins dimerize via the characteristic four-helix bundle observed in cholinesterases, and that the connecting sequence between the globular lobes of the dimer and the cell membrane is elongated, projecting away from the dimer interface. X-ray scattering and neutron contrast variation data show that two neurexin monomers, separated by 107 A, bind at symmetric locations on opposite sides of the long axis of the neuroligin dimer. Using these data, we developed structural models that delineate the spatial arrangements of different neuroligin domains and their partnering molecules. As mutations of neurexin and neuroligin genes appear to be linked to autism, these models provide a structural framework for understanding altered recognition by these proteins in neurodevelopmental disorders.     

Expression of neurexin and neuroligin in the enteric nervous system and their down-regulated expression levels in Hirschsprung disease

To investigate the expression levels of neurexins and neuroligins in the enteric nervous system (ENS) in Hirschsprung Disease (HSCR). Longitudinal muscles with adherent mesenteric plexus were obtained by dissection of the fresh gut wall of mice, guinea pigs, and humans. Double labeling of neurexin I and Hu (a neuron marker), neuroligin 1 and Hu, neurexin I and synaptophysin (a presynaptic marker), and neuroligin 1 and PSD95 (a postsynaptic marker) was performed by immunofluorescence staining. Images were merged to determine the relative localizations of the proteins. Expression levels of neurexin and neuroligin in different segments of the ENS in HSCR were investigated by immunohistochemistry. Neurexin and neuroligin were detected in the mesenteric plexus of mice, guinea pigs, and humans with HSCR. Neurexin was located in the presynapse, whereas neuroligin was located in the postsynapse. Expression levels of neurexin and neuroligin were significant in the ganglionic colonic segment of HSCR, moderate in the transitional segment, and negative in the aganglionic colonic segment. The expressions of neurexin and neuroligin in the transitional segments were significantly down-regulated compared with the levels in the normal segments (P < 0.05). Expression levels of neurexin and neuroligin in ENS are significantly down-regulated in HSCR, which may be involved in the pathogenesis of HSCR.     

Calsyntenin-3 Molecular Architecture and Interaction with Neurexin 1α

Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. Calsyntenin members play an evolutionarily conserved role in memory and learning. Cstn3 was recently shown in cell-based assays to interact with neurexin 1α (n1α), a synaptic organizer that is implicated in neuropsychiatric disease. Interaction would permit Cstn3 and n1α to form a trans-synaptic complex and promote synaptic differentiation. However, it is contentious whether Cstn3 binds n1α directly. To understand the structure and function of Cstn3, we determined its architecture by electron microscopy and delineated the interaction between Cstn3 and n1α biochemically and biophysically. We show that Cstn3 ectodomains form monomers as well as tetramers that are stabilized by disulfide bonds and Ca²⁺, and both are probably flexible in solution. We show further that the extracellular domains of Cstn3 and n1α interact directly and that both Cstn3 monomers and tetramers bind n1α with nanomolar affinity. The interaction is promoted by Ca²⁺ and requires minimally the LNS domain of Cstn3. Furthermore, Cstn3 uses a fundamentally different mechanism to bind n1α compared with other neurexin partners, such as the synaptic organizer neuroligin 2, because Cstn3 does not strictly require the sixth LNS domain of n1α. Our structural data suggest how Cstn3 as a synaptic organizer on the postsynaptic membrane, particularly in tetrameric form, may assemble radially symmetric trans-synaptic bridges with the presynaptic synaptic organizer n1α to recruit and spatially organize proteins into networks essential for synaptic function.     

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960’s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.     

Neurexin/Neuroligin Interaction Kinetics Characterized by Counting Single Cell-Surface Attached Quantum Dots

We report what to our knowledge is a new method to characterize kinetic rates between cell-surface-attached adhesion molecules. Cells expressing specific membrane receptors are surface-labeled with quantum dots coated with their respective ligands. The progressive diminution in the total number of surface-diffusing quantum dots tracked over time collectively reflects intrinsic ligand/receptor interaction kinetics. The probability of quantum dot detachment is modeled using a stochastic analysis of bond formation and dissociation, with a small number of ligand/receptor pairs, resulting in a set of coupled differential equations that are solved numerically. Comparison with the experimental data provides an estimation of the kinetic rates, together with the mean number of ligands per quantum dot, as three adjustable parameters. We validate this approach by studying the calcium-dependent neurexin/neuroligin interaction, which plays an important role in synapse formation. Using primary neurons expressing neuroligin-1 and quantum dots coated with purified neurexin-1β, we determine the kinetic rates between these two binding partners and compare them with data obtained using other techniques. Using specific molecular constructs, we also provide interesting information about the effects of neurexin and neuroligin dimerization on the kinetic rates. As it stands, this simple technique should be applicable to many types of biological ligand/receptor pairs.     

Structural basis for variant-specific neuroligin-binding by α-neurexin

Neurexins (Nrxs) are presynaptic membrane proteins with a single membrane-spanning domain that mediate asymmetric trans-synaptic cell adhesion by binding to their postsynaptic receptor neuroligins. α-Nrx has a large extracellular region comprised of multiple copies of laminin, neurexin, sex-hormone-binding globulin (LNS) domains and epidermal growth factor (EGF) modules, while that of β-Nrx has but a single LNS domain. It has long been known that the larger α-Nrx and the shorter β-Nrx show distinct binding behaviors toward different isoforms/variants of neuroligins, although the underlying mechanism has yet to be elucidated. Here, we describe the crystal structure of a fragment corresponding to the C-terminal one-third of the Nrx1α ectodomain, consisting of LNS5-EGF3-LNS6. The 2.3 Å-resolution structure revealed the presence of a domain configuration that was rigidified by inter-domain contacts, as opposed to the more common flexible “beads-on-a-string” arrangement. Although the neuroligin-binding site on the LNS6 domain was completely exposed, the location of the α-Nrx specific LNS5-EGF3 segment proved incompatible with the loop segment inserted in the B+ neuroligin variant, which explains the variant-specific neuroligin recognition capability observed in α-Nrx. This, combined with a low-resolution molecular envelope obtained by a single particle reconstruction performed on negatively stained full-length Nrx1α sample, allowed us to derive a structural model of the α-Nrx ectodomain. This model will help us understand not only how the large α-Nrx ectodomain is accommodated in the synaptic cleft, but also how the trans-synaptic adhesion mediated by α- and β-Nrxs could differentially affect synaptic structure and function.     

Characterization of the solution structure of a neuroligin/β-neurexin complex

Neuroligins are post-synaptic cell adhesion molecules that promote synaptic maturation and stabilization upon binding with pre-synaptic partners, the α- and β-neurexins. Using a combination of analytical ultracentrifugation, small angle X-ray, and neutron scattering, we have characterized the low-resolution three-dimensional structure of the extracellular domain of the neuroligins, free in solution, and in complex with β-neurexin. The globular extracellular domain of the neuroligins forms stable homodimers through a four-helix bundle typical of the cholinesterases and other members of the α/β-hydrolase fold family. The presence of the stalk region adds to the extracellular domain of neuroligin-1 an elongated structure, suggesting a rod-like nature of the stalk domain. Sedimentation equilibrium coupled with solution scattering data of the β-neurexin/neuroligin-1 complex indicated a 2:2 stoichiometry where two β-neurexin molecules bind to a neuroligin-1 dimer. Deuteration of neurexin allowed us to collect neutron scattering data that, in combination with other biochemical techniques, provide a basis for optimizing the positioning of each component in a detailed computational model of the neuroligin/neurexin complex. As several mutations of both neurexin and neuroligin genes have been linked to autism spectrum disorders and mental retardation, these new structures provide an important framework for the study of altered structure and function of these synaptic proteins.     

Characterization of the solution structure of a neuroligin/beta-neurexin complex

Neuroligins are post-synaptic cell adhesion molecules that promote synaptic maturation and stabilization upon binding with pre-synaptic partners, the alpha- and beta-neurexins. Using a combination of analytical ultracentrifugation, small angle X-ray, and neutron scattering, we have characterized the low-resolution three-dimensional structure of the extracellular domain of the neuroligins, free in solution, and in complex with beta-neurexin. The globular extracellular domain of the neuroligins forms stable homodimers through a four-helix bundle typical of the cholinesterases and other members of the alpha/beta-hydrolase fold family. The presence of the stalk region adds to the extracellular domain of neuroligin-1 an elongated structure, suggesting a rod-like nature of the stalk domain. Sedimentation equilibrium coupled with solution scattering data of the beta-neurexin/neuroligin-1 complex indicated a 2:2 stoichiometry where two beta-neurexin molecules bind to a neuroligin-1 dimer. Deuteration of neurexin allowed us to collect neutron scattering data that, in combination with other biochemical techniques, provide a basis for optimizing the positioning of each component in a detailed computational model of the neuroligin/neurexin complex. As several mutations of both neurexin and neuroligin genes have been linked to autism spectrum disorders and mental retardation, these new structures provide an important framework for the study of altered structure and function of these synaptic proteins.     

DSL-Notch signaling in the Drosophila brain in response to olfactory stimulation

Neurexin and neuroligin, which undergo heterophilic interactions with each other at the synapse, are mutated in some patients with autism spectrum disorder, a set of disorders characterized by deficits in social and emotional learning. We have explored the role of neurexin and neuroligin at sensory-to-motor neuron synapses of the gill-withdrawal reflex in Aplysia, which undergoes sensitization, a simple form of learned fear. We find that depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation and the associated presynaptic growth induced by repeated pulses of serotonin. Moreover, introduction into the motor neuron of the R451C mutation of neuroligin-3 linked to autism spectrum disorder blocks both intermediate-term and long-term facilitation. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction may govern transsynaptic signaling required for the storage of long-term memory, including emotional memory that may be impaired in autism spectrum disorder.     

Sensory Regulation of Neuroligins and Neurexin I in the Honeybee Brain

Background

Neurexins and neuroligins, which have recently been associated with neurological disorders such as autism in humans, are highly conserved adhesive proteins found on synaptic membranes of neurons. These binding partners produce a trans-synaptic bridge that facilitates maturation and specification of synapses. It is believed that there exists an optimal spatio-temporal code of neurexin and neuroligin interactions that guide synapse formation in the postnatal developing brain. Therefore, we investigated whether neuroligins and neurexin are differentially regulated by sensory input using a behavioural model system with an advanced capacity for sensory processing, learning and memory, the honeybee.

Methodology/Principal Findings

Whole brain expression levels of neuroligin 1–5 (NLG1–5) and neurexin I (NrxI) were estimated by qRT-PCR analysis in three different behavioural paradigms: sensory deprivation, associative scent learning, and lateralised sensory input. Sensory deprived bees had a lower level of NLG1 expression, but a generally increased level of NLG2–5 and NrxI expression compared to hive bees. Bees that had undergone associative scent training had significantly increased levels of NrxI, NLG1 and NLG3 expression compared to untrained control bees. Bees that had lateralised sensory input after antennal amputation showed a specific increase in NLG1 expression compared to control bees, which only happened over time.

Conclusions/Significance

Our results suggest that (1) there is a lack of synaptic pruning during sensory deprivation; (2) NLG1 expression increases with sensory stimulation; (3) concomitant changes in gene expression suggests NrxI interacts with all neuroligins; (4) there is evidence for synaptic compensation after lateralised injury.     

Synaptic modifications depend on synapse location and activity: a biophysical model of STDP

In spike-timing-dependent plasticity (STDP) the synapses are potentiated or depressed depending on the temporal order and temporal difference of the pre- and post-synaptic signals. We present a biophysical model of STDP which assumes that not only the timing, but also the shapes of these signals influence the synaptic modifications. The model is based on a Hebbian learning rule which correlates the NMDA synaptic conductance with the post-synaptic signal at synaptic location as the pre- and post-synaptic quantities. As compared to a previous paper [Saudargiene, A., Porr, B., Worgotter, F., 2004. How the shape of pre- and post-synaptic signals can influence stdp: a biophysical model. Neural Comp.], here we show that this rule reproduces the generic STDP weight change curve by using real neuronal input signals and combinations of more than two (pre- and post-synaptic) spikes. We demonstrate that the shape of the STDP curve strongly depends on the shape of the depolarising membrane potentials, which induces learning. As these potentials vary at different locations of the dendritic tree, model predicts that synaptic changes are location dependent. The model is extended to account for the patterns of more than two spikes of the pre- and post-synaptic cells. The results show that STDP weight change curve is also activity dependent.     

Human alpha‐ and beta‐NRXN1 isoforms rescue behavioral impairments of Caenorhabditis elegans neurexin‐deficient mutants

Neurexins are cell adhesion proteins that interact with neuroligin and other ligands at the synapse. In humans, mutations in neurexin or neuroligin genes have been associated with autism and other mental disorders. The human neurexin and neuroligin genes are orthologous to the Caenorhabditis elegans genes nrx‐1 and nlg‐1, respectively. Here we show that nrx‐1‐deficient mutants are defective in exploratory capacity, sinusoidal postural movements and gentle touch response. Interestingly, the exploratory behavioral phenotype observed in nrx‐1 mutants was markedly different to nlg‐1‐deficient mutants; thus, while the former had a ‘hyper‐reversal’ phenotype increasing the number of changes of direction with respect to the wild‐type strain, the nlg‐1 mutants presented a ‘hypo‐reversal’ phenotype. On the other hand, the nrx‐1‐ and nlg‐1‐defective mutants showed similar abnormal sinusoidal postural movement phenotypes. The response of these mutant strains to aldicarb (acetylcholinesterase inhibitor), levamisole (ACh agonist) and pentylenetetrazole [gamma‐aminobutyric (GABA) receptor antagonist], suggested that the varying behavioral phenotypes were caused by defects in ACh and/or GABA inputs. The defective behavioral phenotypes of nrx‐1‐deficient mutants were rescued in transgenic strains expressing either human alpha‐ or beta‐NRXN‐1 isoforms under the worm nrx‐1 promoter. A previous report had shown that human and rat neuroligins were functional in C. elegans. Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in neurological diseases for which neuroligin and neurexin are implicated in having a role.