Gonadotropin releasing hormone (GnRH) deficiency is a disorder characterized by absent or delayed puberty, with largely unknown genetic causes. The purpose of this study was to obtain and exploit gene expression profiles of GnRH neurons during development to unveil novel biological mechanisms and genetic determinants underlying GnRH deficiency (GD). Here, we combined bioinformatic analyses of immortalized and primary embryonic GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate genes implicated in the pathogenesis of GD. Among differentially expressed and filtered transcripts, we found loss-of-function (LoF) variants of the autism-linked Neuroligin 3 (NLGN3) gene in two unrelated patients co-presenting with GD and neurodevelopmental traits. We demonstrated that NLGN3 is upregulated in maturing GnRH neurons and that NLGN3 wild type, but not mutant protein, promotes neuritogenesis when overexpressed in developing GnRH cells. Our data represent proof-of-principle that this complementary approach can identify novel candidate GD genes and demonstrate that LoF NLGN3 variants may contribute to GD. This novel genotype-phenotype correlation implies common genetic mechanisms underlying neurodevelopmental disorders, such as GD and autistic spectrum disorder.
The specification of synaptic properties is fundamental for the function of neuronal circuits. "Terminal selector" transcription factors coordinate terminal gene batteries that specify cell-type-specific properties. Moreover, pan-neuronal splicing regulators have been implicated in directing neuronal differentiation. However, the cellular logic of how splicing regulators instruct specific synaptic properties remains poorly understood. Here, we combine genome-wide mapping of mRNA targets and cell-type-specific loss-of-function studies to uncover the contribution of the RNA-binding protein SLM2 to hippocampal synapse specification. Focusing on pyramidal cells and somatostatin (SST)-positive GABAergic interneurons, we find that SLM2 preferentially binds and regulates alternative splicing of transcripts encoding synaptic proteins. In the absence of SLM2, neuronal populations exhibit normal intrinsic properties, but there are non-cell-autonomous synaptic phenotypes and associated defects in a hippocampus-dependent memory task. Thus, alternative splicing provides a critical layer of gene regulation that instructs specification of neuronal connectivity in a trans-synaptic manner.
Atypical habituation and aberrant exploration of novel stimuli have been related to the severity of autism spectrum disorders (ASDs), but the underlying neuronal circuits are unknown. Here we show that chemogenetic inhibition of dopamine (DA) neurons of the ventral tegmental area (VTA) attenuates exploration toward nonfamiliar conspecifics and interferes with the reinforcing properties of nonfamiliar conspecific interaction in mice. Exploration of nonfamiliar stimuli is associated with the insertion of GluA2-lacking AMPA receptors at excitatory synapses on VTA DA neurons. These synaptic adaptations persist upon repeated exposure to social stimuli and sustain conspecific interaction. Global or VTA DA neuron-specific loss of the ASD-associated synaptic adhesion molecule neuroligin 3 alters the behavioral response toward nonfamiliar conspecifics and the reinforcing properties of conspecific interaction. These behavioral deficits are accompanied by an aberrant expression of AMPA receptors and an occlusion of synaptic plasticity. Altogether, these findings link impaired exploration of nonfamiliar conspecifics to VTA DA neuron dysfunction in mice.
Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.
Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis.
Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring (SRM) that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands.
Title: Alternative Splicing Coupled Nonsense-Mediated Decay Generates Neuronal Cell Type-Specific Expression of SLM Proteins Traunmuller L, Bornmann C, Scheiffele P Ref: Journal of Neuroscience, 34:16755, 2014 : PubMed
The unique physiological and morphological properties of neuronal populations are crucial for the appropriate functioning of neuronal circuits. Alternative splicing represents an attractive mechanism for generating cell type-specific molecular repertoires that steer neuronal development and function. However, the mechanisms that link neuronal identity to alternative splicing programs are poorly understood. We report that cell type-specific, mutually exclusive expression of two alternative splicing regulators, SLM1 and SLM2, in the mouse hippocampus is achieved by a cross-repression mechanism. Deletion of SLM2 in vivo modifies alternative splicing of its paralog Slm1 and stabilizes its mRNA, resulting in expression of SLM1 in previously SLM2-expressing cells. Despite this ectopic upregulation of SLM1, loss of SLM2 severely disrupts the alternative splicing regulation of Nrxn1, Nrxn2, and Nrxn3, highlighting that the two SLM paralogs have partially divergent functions. Our study uncovers a hierarchical, SLM2-dependent mechanism for establishing cell type-specific expression of neuronal splicing regulators in vivo.
Despite the pivotal functions of the NMDA receptor (NMDAR) for neural circuit development and synaptic plasticity, the molecular mechanisms underlying the dynamics of NMDAR trafficking are poorly understood. The cell adhesion molecule neuroligin-1 (NL1) modifies NMDAR-dependent synaptic transmission and synaptic plasticity, but it is unclear whether NL1 controls synaptic accumulation or function of the receptors. Here, we provide evidence that NL1 regulates the abundance of NMDARs at postsynaptic sites. This function relies on extracellular, NL1 isoform-specific sequences that facilitate biochemical interactions between NL1 and the NMDAR GluN1 subunit. Our work uncovers NL1 isoform-specific cis-interactions with ionotropic glutamate receptors as a key mechanism for controlling synaptic properties.
The genetic heterogeneity of autism poses a major challenge for identifying mechanism-based treatments. A number of rare mutations are associated with autism, and it is unclear whether these result in common neuronal alterations. Monogenic syndromes, such as fragile X, include autism as one of their multifaceted symptoms and have revealed specific defects in synaptic plasticity. We discovered an unexpected convergence of synaptic pathophysiology in a nonsyndromic form of autism with those in fragile X syndrome. Neuroligin-3 knockout mice (a model for nonsyndromic autism) exhibited disrupted heterosynaptic competition and perturbed metabotropic glutamate receptor-dependent synaptic plasticity, a hallmark of fragile X. These phenotypes could be rescued by reexpression of neuroligin-3 in juvenile mice, highlighting the possibility of reverting neuronal circuit alterations in autism after the completion of development.
Pyramidal cells express various GABA(A) receptor (GABA(A)R) subtypes, possibly to match inputs from functionally distinct interneurons targeting specific subcellular domains. Postsynaptic anchoring of GABA(A)Rs is ensured by a complex interplay between the scaffolding protein gephyrin, neuroligin-2 and collybistin. Direct interactions between these proteins and GABA(A)R subunits might contribute to synapse-specific distribution of GABA(A)R subtypes. In addition, the dystrophin-glycoprotein complex, mainly localized at perisomatic synapses, regulates GABA(A)R postsynaptic clustering at these sites. Here, we investigated how the functional and molecular organization of GABAergic synapses in CA1 pyramidal neurons is altered in mice lacking the GABA(A)R alpha2 subunit (alpha2-KO). We report a marked, layer-specific loss of postsynaptic gephyrin and neuroligin-2 clusters, without changes in GABAergic presynaptic terminals. Whole-cell voltage-clamp recordings in slices from alpha2-KO mice show a 40% decrease in GABAergic mIPSC frequency, with unchanged amplitude and kinetics. Applying low/high concentrations of zolpidem to discriminate between alpha1- and alpha2/alpha3-GABA(A)Rs demonstrates that residual mIPSCs in alpha2-KO mice are mediated by alpha1-GABA(A)Rs. Immunofluorescence analysis reveals maintenance of alpha1-GABA(A)R and neuroligin-2 clusters, but not gephyrin clusters, in perisomatic synapses of mutant mice, along with a complete loss of these three markers on the axon initial segment. This striking subcellular difference correlates with the preservation of dystrophin clusters, colocalized with neuroligin-2 and alpha1-GABA(A)Rs on pyramidal cell bodies of mutant mice. Dystrophin was not detected on the axon initial segment in either genotype. Collectively, these findings reveal synapse-specific anchoring of GABA(A)Rs at postsynaptic sites and suggest that the dystrophin-glycoprotein complex contributes to stabilize alpha1-GABA(A)R and neuroligin-2, but not gephyrin, in perisomatic postsynaptic densities.
We created the Flexible Accelerated STOP Tetracycline Operator (tetO)-knockin (FAST) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A single gene targeting event yields two distinct knockin mice-STOP-tetO and tetO knockin-that permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue, 3) tetracycline-controlled transcriptional activator (tTA)-mediated misexpression, 4) tetracycline-controlled transcriptional activator (tTA)-mediated overexpression, and 5) tetracycline-controlled transcriptional silencer (tTS)-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-of-function and loss-of-function strains can therefore be generated on a time scale not previously achievable. These strains can then be screened for clinically relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor.
Title: GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development Huang ZJ, Scheiffele P Ref: Current Opinion in Neurobiology, 18:77, 2008 : PubMed
GABA-mediated synaptic inhibition is crucial in neural circuit operations. In mammalian brains, the development of inhibitory synapses and innervation patterns is often a prolonged postnatal process, regulated by neural activity. Emerging evidence indicates that gamma-aminobutyric acid (GABA) acts beyond inhibitory transmission and regulates inhibitory synapse development. Indeed, GABA(A) receptors not only function as chloride channels that regulate membrane voltage and conductance but also play structural roles in synapse maturation and stabilization. The link from GABA(A) receptors to postsynaptic and presynaptic adhesion is probably mediated, partly by neuroligin-reurexin interactions, which are potent in promoting GABAergic synapse formation. Therefore, similar to glutamate signaling at excitatory synapse, GABA signaling may coordinate maturation of presynaptic and postsynaptic sites at inhibitory synapses. Defining the many steps from GABA signaling to receptor trafficking/stability and neuroligin function will provide further mechanistic insights into activity-dependent development and possibly plasticity of inhibitory synapses.
Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.
Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3-A crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site region differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions.
Presynaptic neurexins (NRXs) bind to postsynaptic neuroligins (NLs) to form Ca(2+)-dependent complexes that bridge neural synapses. beta-NRXs bind NLs through their LNS domains, which contain a single site of alternative splicing (splice site 4) giving rise to two isoforms: +4 and Delta. We present crystal structures of the Delta isoforms of the LNS domains from beta-NRX1 and beta-NRX2, crystallized in the presence of Ca(2+) ions. The Ca(2+)-binding site is disordered in the beta-NRX2 structure, but the 1.7 A beta-NRX1 structure reveals a single Ca(2+) ion, approximately 12 A from the splice insertion site, with one coordinating ligand donated by a glutamic acid from an adjacent beta-NRX1 molecule. NMR studies of beta-NRX1+4 show that the insertion sequence is unstructured, and remains at least partially disordered in complex with NL. These results raise the possibility that beta-NRX insertion sequence 4 may function in roles independent of neuroligin binding.
Title: Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses Budreck EC, Scheiffele P Ref: European Journal of Neuroscience, 26:1738, 2007 : PubMed
Synaptic adhesion molecules are thought to play a critical role in the formation, function and plasticity of neuronal networks. Neuroligins (NL1-4) are a family of presumptive postsynaptic cell adhesion molecules. NL1 and NL2 isoforms are concentrated at glutamatergic and GABAergic synapses, respectively, but the cellular expression and synaptic localization of the endogenous NL3 and NL4 isoforms are unknown. We generated a panel of NL isoform-specific antibodies and examined the expression, developmental regulation and synaptic specificity of NL3. We found that NL3 was enriched in brain, where NL3 protein levels increased during postnatal development, coinciding with the peak of synaptogenesis. Subcellular fractionation revealed a concentration of NL3 in synaptic plasma membranes and postsynaptic densities. In cultured hippocampal neurons, endogenous NL3 was highly expressed and was localized at both glutamatergic and GABAergic synapses. Clustering of NL3 in hippocampal neurons by neurexin-expressing cells resulted in coaggregation of NL3 with glutamatergic and GABAergic scaffolding proteins. Finally, individual synapses contained colocalized NL2 and NL3 proteins, and coimmunoprecipitation studies revealed the presence of NL1-NL3 and NL2-NL3 complexes in brain extracts. These findings suggest that rodent NL3 is a synaptic adhesion molecule that is a shared component of glutamatergic and GABAergic synapses.
The structure and function of presynaptic and postsynaptic components of the synapse are highly coordinated. How such coordination is achieved and the molecules involved in this process have not been clarified. Several lines of evidence suggest that presynaptic functionalities are regulated by retrograde mechanisms from the postsynaptic side. We therefore sought postsynaptic mechanisms responsible for trans-synaptic regulation of presynaptic function at excitatory synapses in rat hippocampal CA1 pyramidal neurons. We show here that the postsynaptic complex of scaffolding protein PSD-95 and neuroligin can modulate the release probability of transmitter vesicles at synapse in a retrograde way, resulting in altered presynaptic short-term plasticity. Presynaptic beta-neurexin serves as a likely presynaptic mediator of this effect. Our results indicate that trans-synaptic protein-protein interactions can link postsynaptic and presynaptic function.
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-1beta are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1beta 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.
Title: Alternative splicing controls selective trans-synaptic interactions of the neuroligin-neurexin complex Chih B, Gollan L, Scheiffele P Ref: Neuron, 51:171, 2006 : PubMed
Formation of synapses requires specific cellular interactions that organize pre- and postsynaptic compartments. The neuroligin-neurexin complex mediates heterophilic adhesion and can trigger assembly of glutamatergic and GABAergic synapses in cultured hippocampal neurons. Both neuroligins and neurexins are encoded by multiple genes. Alternative splicing generates large numbers of isoforms, which may engage in selective axo-dendritic interactions. We explored whether alternative splicing of the postsynaptic neuroligins modifies their activity toward glutamatergic and GABAergic axons. We find that small extracellular splice insertions restrict the function of neuroligin-1 and -2 to glutamatergic and GABAergic contacts and alter interaction with presynaptic neurexins. The neuroligin isoforms associated with GABAergic contacts bind to neurexin-1alpha and a subset of neurexin-1betas. In turn, these neurexin isoforms induce GABAergic but not glutamatergic postsynaptic differentiation. Our findings suggest that alternative splicing plays a central role in regulating selective extracellular interactions through the neuroligin-neurexin complex at glutamatergic and GABAergic synapses.
Title: Control of excitatory and inhibitory synapse formation by neuroligins Chih B, Engelman H, Scheiffele P Ref: Science, 307:1324, 2005 : PubMed
The normal function of neural networks depends on a delicate balance between excitatory and inhibitory synaptic inputs. Synapse formation is thought to be regulated by bidirectional signaling between pre- and postsynaptic cells. We demonstrate that members of the Neuroligin family promote postsynaptic differentiation in cultured rat hippocampal neurons. Down-regulation of neuroligin isoform expression by RNA interference results in a loss of excitatory and inhibitory synapses. Electrophysiological analysis revealed a predominant reduction of inhibitory synaptic function. Thus, neuroligins control the formation and functional balance of excitatory and inhibitory synapses in hippocampal neurons.
The formation of neuronal synapses is thought to depend on trans-synaptic interactions between cell adhesion molecules (CAMs) on the surface of axons and dendrites. Synapses are highly asymmetric structures. Pre- and post-synaptic domains might therefore be assembled around heterophilic CAMs which are polarized to axons vs. dendrites. We here investigated the targeting of neuroligin (NLG)-1, a heterophilic CAM, which promotes synapse formation through interaction with its receptor beta-neurexin in axons. We demonstrate that NLG-1 is highly polarized to the dendritic plasma membrane. Dendritic targeting relies on a cytoplasmic amino acid motif. By expressing chimeras of NLG-1 and CD8, an unpolarized protein, we show that the cytoplasmic domain of NLG-1 is necessary and sufficient for dendritic targeting. Furthermore, by truncation analysis we isolated a 32-amino-acid targeting motif. When appended to CD8 this cytoplasmic sequence is sufficient to direct exclusively dendritic localization of the protein. Analysis of yellow fluorescent protein-tagged NLG-1 revealed that vesicular structures containing NLG-1 are excluded from the axon indicating that polarized distribution may be achieved by direct dendritic transport. We propose that the strict polarity of NLG-1 contributes to the directional assembly of synapses during development of the central nervous system.
Title: Disorder-associated mutations lead to functional inactivation of neuroligins Chih B, Afridi SK, Clark L, Scheiffele P Ref: Hum Mol Genet, 13:1471, 2004 : PubMed
Autism is a neuro-developmental syndrome that affects 0.1-0.5% of the population. It has been proposed that alterations in neuronal circuitry and/or neuronal signaling are responsible for the behavioral and cognitive aberrations in autism patients. However, the cellular basis of such alterations is unknown. Recently, point mutations in a family of neuronal cell adhesion molecules called neuroligins have been linked to autism-spectrum disorders and mental retardation. We investigated the consequences of these disease-associated mutations on neuroligin function. We demonstrate that the point mutation at arginine 451 and a nonsense mutation at aspartate 396 of neuroligin-3 and -4 (NL3 and NL4), respectively, result in intracellular retention of the mutant proteins. Over-expression of wild-type NL3 and NL4 proteins in hippocampal neurons stimulates the formation of presynaptic terminals, whereas the disease-associated mutations result in a loss of this synaptic function. Our findings suggest that the previously identified mutations in neuroligin genes are likely to be relevant for the neuro-developmental defects in autism-spectrum disorders and mental retardation since they impair the function of a synaptic cell adhesion molecule.
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.
Title: Making connections: cholinesterase-domain proteins in the CNS Scholl FG, Scheiffele P Ref: Trends in Neurosciences, 26:618, 2003 : PubMed
Recent studies have highlighted novel functions of a group of cell adhesion molecules during nervous system development. Members of this protein family are characterized by an extracellular domain with sequence homology to cholinesterases and include the neuroligins, synaptic cell adhesion molecules recently implicated in autism, and neurotactin, a cell surface receptor involved in axonal pathfinding. Although these proteins have a structural organization similar to the enzyme acetylcholinesterase, the cholinesterase domain lacks enzymatic activity and functions as a protein-protein interaction motif. This protein family provides a striking example of how the function of a catalytically active domain has evolved to mediate receptor-ligand interactions that regulate morphogenetic processes during development of the nervous system.
Most neurons form synapses exclusively with other neurons, but little is known about the molecular mechanisms mediating synaptogenesis in the central nervous system. Using an in vitro system, we demonstrate that neuroligin-1 and -2, postsynaptically localized proteins, can trigger the de novo formation of presynaptic structure. Nonneuronal cells engineered to express neuroligins induce morphological and functional presynaptic differentiation in contacting axons. This activity can be inhibited by addition of a soluble version of beta-neurexin, a receptor for neuroligin. Furthermore, addition of soluble beta-neurexin to a coculture of defined pre- and postsynaptic CNS neurons inhibits synaptic vesicle clustering in axons contacting target neurons. Our results suggest that neuroligins are part of the machinery employed during the formation and remodeling of CNS synapses.
