Postsynaptic Adhesion Molecules Research Articles

The loss of βΙ spectrin alters synaptic size and composition in the ja/ja mouse.

Deletion or mutation of members of the spectrin gene family contributes to many neurologic and neuropsychiatric disorders. While each spectrinopathy may generate distinct neuropathology, the study of βΙ spectrin's role (Sptb) in the brain has been hampered by the hematologic consequences of its loss. Jaundiced mice (ja/ja) that lack βΙ spectrin suffer a rapidly fatal hemolytic anemia. We have used exchange transfusion of newborn ja/ja mice to blunt their hemolytic pathology, enabling an examination of βΙ spectrin deficiency in the mature mouse brain by ultrastructural and biochemical analysis. βΙ spectrin is widely utilized throughout the brain as the βΙΣ2 isoform; it appears by postnatal day 8, and concentrates in the CA1,3 region of the hippocampus, dentate gyrus, cerebellar granule layer, cortical layer 2, medial habenula, and ventral thalamus. It is present in a subset of dendrites and absent in white matter. Without βΙ spectrin there is a 20% reduction in postsynaptic density size in the granule layer of the cerebellum, a selective loss of ankyrinR in cerebellar granule neurons, and a reduction in the level of the postsynaptic adhesion molecule NCAM. While we find no substitution of another spectrin for βΙ at dendrites or synapses, there is curiously enhanced βΙV spectrin expression in the ja/ja brain. βΙΣ2 spectrin appears to be essential for refining postsynaptic structures through interactions with ankyrinR and NCAM. We speculate that it may play additional roles yet to be discovered.

Essential Role of Latrophilin-1 Adhesion GPCR Nanoclusters in Inhibitory Synapses.

Latrophilin-1 (Lphn1, aka CIRL1 and CL1; gene symbol Adgrl1) is an adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knock-in/knock-out mice for Lphn1 that contain an extracellular myc epitope tag. Mice of both sexes were used in all experiments. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but is required for some inhibitory synaptic connections.

Region-Specific Phosphorylation Determines Neuroligin-3 Localization to Excitatory Versus Inhibitory Synapses

BackgroundNeuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. MethodsWe investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. ResultsNeuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. ConclusionsThese data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.

Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism.

Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.

Neuroligin-3 confines AMPA receptors into nanoclusters, thereby controlling synaptic strength at the calyx of Held synapses.

The subsynaptic organization of postsynaptic neurotransmitter receptors into nanoclusters that are aligned with presynaptic release sites is essential for the high fidelity of synaptic transmission. However, the mechanisms controlling the nanoscale organization of neurotransmitter receptors in vivo remain incompletely understood. Here, we deconstructed the role of neuroligin-3 (Nlgn3), a postsynaptic adhesion molecule linked to autism, in organizing AMPA-type glutamate receptors in the calyx of Held synapse. Deletion of Nlgn3 lowered the amplitude and slowed the kinetics of AMPA receptor–mediated synaptic responses. Super-resolution microscopy revealed that, unexpectedly, these impairments in synaptic transmission were associated with an increase in the size of postsynaptic PSD-95 and AMPA receptor nanoclusters but a decrease of the densities in these clusters. Modeling showed that a dilution of AMPA receptors into larger nanocluster volumes decreases synaptic strength. Nlgn3, likely by binding to presynaptic neurexins, thus is a key organizer of AMPA receptor nanoclusters that likely acts via PSD-95 adaptors to optimize the fidelity of synaptic transmission.

Deletion of Calsyntenin-3, an atypical cadherin, suppresses inhibitory synapses but increases excitatory parallel-fiber synapses in cerebellum.

Cadherins contribute to the organization of nearly all tissues, but the functions of several evolutionarily conserved cadherins, including those of calsyntenins, remain enigmatic. Puzzlingly, two distinct, non-overlapping functions for calsyntenins were proposed: As postsynaptic neurexin ligands in synapse formation, or as presynaptic kinesin adaptors in vesicular transport. Here, we show that, surprisingly, acute CRISPR-mediated deletion of calsyntenin-3 in mouse cerebellum in vivo causes a large decrease in inhibitory synapse, but a robust increase in excitatory parallel-fiber synapses in Purkinje cells. As a result, inhibitory synaptic transmission was suppressed, whereas parallel-fiber synaptic transmission was enhanced in Purkinje cells by the calsyntenin-3 deletion. No changes in the dendritic architecture of Purkinje cells or in climbing-fiber synapses were detected. Sparse selective deletion of calsyntenin-3 only in Purkinje cells recapitulated the synaptic phenotype, indicating that calsyntenin-3 acts by a cell-autonomous postsynaptic mechanism in cerebellum. Thus, by inhibiting formation of excitatory parallel-fiber synapses and promoting formation of inhibitory synapses in the same neuron, calsyntenin-3 functions as a postsynaptic adhesion molecule that regulates the excitatory/inhibitory balance in Purkinje cells.

Neuroligin-1 plays an important role in methamphetamine-induced hippocampal synaptic plasticity

The neurotoxic effects of methamphetamine (METH) include not only neuronal apoptosis and autophagy, but also lead to substance use disorder and have become increasingly prominent. Studies suggest that synaptic plasticity may be the structural basis of METH-induced neurological impairment. Neuroligins are postsynaptic adhesion molecules involved in the regulation of synaptic organization and function. Animal studies have shown that neuroligin (NLG)− 1 is involved in memory formation; however, its role in METH-induced neurotoxicity is not clear. In the present study, we used 1 mM METH in vitro; mice in the acute and subacute exposure groups received intraperitoneal injections of 30 mg/kg METH (1 injection) or 15 mg/kg METH (8 separate injections at 12-h intervals). We found that the expression of NLG-1, Synapsin-1, and postsynaptic density-95 were increased after METH exposure. We further observed that METH-induced inhibition of long-term potentiation and spatial memory loss could be alleviated when mice were pretreated with NLG-1 small interfering RNA. Therefore, our study provides evidence that NLG-1 is involved in METH-induced hippocampal synaptic plasticity and may be a potential target for the treatment of METH-induced neurotoxicity.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

SUMMARYGABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

The role of NMDA receptor and neuroligin rare variants in synaptic dysfunction underlying neurodevelopmental disorders.

Many genes encoding synaptic proteins are associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorders (ASDs), intellectual disability (ID), and epilepsy. Here we review recent studies on the synaptic effects of disease-associated rare variants identified in two families of synaptic proteins: NMDA receptors (NMDARs) and the postsynaptic adhesion molecules neuroligins (NLGNs). Many NMDAR subunit genes (GRINs) are highly intolerant to variation, and both gain-of-function (GOF) and loss-of-function (LOF) variants are implicated in disease. NLGN genes are also associated with ASDs, and in some cases, contribute to the male bias identified in these patients. Understanding the molecular basis of synaptic dysfunction of rare variants in these genes will help the design of new therapeutic approaches.

Neuroligin1 Contributes to Neuropathic Pain by Promoting Phosphorylation of Cofilin in Excitatory Neurons.

Neuropathic pain is a kind of chronic pain that remains difficult to treat due to its complicated underlying mechanisms. Accumulating evidence has indicated that enhanced synaptic plasticity of nociceptive interneurons in the superficial spinal dorsal horn contributes to the development of neuropathic pain. Neuroligin1 (NL1) is a type of excitatory postsynaptic adhesion molecule, which can mediate excitatory synaptic activity, hence promoting neuronal activation. Vglut2 is the most common marker of excitatory glutamatergic neurons. To explore the role of NL1 in excitatory neurons in nociceptive regulation, we used transgenic mice with cre recombinase expression driven by the Vglut2 promoter combined with viral vectors to knockdown the expression of NL1 in excitatory neurons in the spinal dorsal horn. We found that NL1 was upregulated in the L4–L6 spinal dorsal horn in Vglut2-cre+/– mouse subjected to spared nerve injury (SNI). Meanwhile, the expression of phosphorylated cofilin (p-cofilin) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 1 (GluR1) was also increased. Spinal microinjection of a cre-dependent NL1-targeting RNAi in Vglut2-cre+/– mouse alleviated the neuropathic pain-induced mechanical hypersensitivity and reduced the increase in p-cofilin and GluR1 caused by SNI. Taken together, NL1 in excitatory neurons regulates neuropathic pain by promoting the SNI-dependent increase in p-cofilin and GluR1 in the spinal dorsal horn. Our study provides a better understanding of the role of NL1 in excitatory neurons, which might represent a possible therapeutic target for alleviating neuropathic pain.

Neuroligins and Neurodevelopmental Disorders: X-Linked Genetics.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that results in social-communication impairments, as well as restricted and repetitive behaviors. Moreover, ASD is more prevalent in males, with a male to female ratio of 4 to 1. Although the underlying etiology of ASD is generally unknown, recent advances in genome sequencing have facilitated the identification of a host of associated genes. Among these, synaptic proteins such as cell adhesion molecules have been strongly linked with ASD. Interestingly, many large genome sequencing studies exclude sex chromosomes, which leads to a shift in focus toward autosomal genes as targets for ASD research. However, there are many genes on the X chromosome that encode synaptic proteins, including strong candidate genes. Here, we review findings regarding two members of the neuroligin (NLGN) family of postsynaptic adhesion molecules, NLGN3 and NLGN4. Neuroligins have multiple isoforms (NLGN1-4), which are both autosomal and sex-linked. The sex-linked genes, NLGN3 and NLGN4, are both on the X chromosome and were among the first few genes to be linked with ASD and intellectual disability (ID). In addition, there is a less studied human neuroligin on the Y chromosome, NLGN4Y, which forms an X-Y pair with NLGN4X. We will discuss recent findings of these neuroligin isoforms regarding function at the synapse in both rodent models and human-derived differentiated neurons, and highlight the exciting challenges moving forward to a better understanding of ASD/ID.

Calsyntenin-3 interacts with both α- and β-neurexins in the regulation of excitatory synaptic innervation in specific Schaffer collateral pathways

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive β-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.

Neuroligin3 splice isoforms shape inhibitory synaptic function in the mouse hippocampus

Synapse formation is a dynamic process essential for the development and maturation of the neuronal circuitry in the brain. At the synaptic cleft, trans-synaptic protein-protein interactions are major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity, and dysregulation of the E-I balance has been implicated in neurodevelopmental disorders, including autism spectrum disorders. However, the molecular mechanisms underlying the E-I balance remain to be elucidated. Here, using single-cell transcriptomics, immunohistochemistry, and electrophysiology approaches to murine CA1 pyramidal neurons obtained from organotypic hippocampal slice cultures, we investigate neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that the NLGN3 protein differentially regulates inhibitory synaptic transmission in a splice isoform-dependent manner at hippocampal CA1 synapses. We also found that distinct subcellular localizations of the NLGN3 isoforms contribute to the functional differences observed among these isoforms. Finally, results from single-cell RNA-Seq analyses revealed that Nlgn1 and Nlgn3 are the major murine Nlgn genes and that the expression levels of the Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons. Our results delineate isoform-specific effects of Nlgn genes on the E-I balance in the murine hippocampus.

LAR receptor phospho-tyrosine phosphatases regulate NMDA-receptor responses.

LAR-type receptor phosphotyrosine-phosphatases (LAR-RPTPs) are presynaptic adhesion molecules that interact trans-synaptically with multitudinous postsynaptic adhesion molecules, including SliTrks, SALMs, and TrkC. Via these interactions, LAR-RPTPs are thought to function as synaptogenic wiring molecules that promote neural circuit formation by mediating the establishment of synapses. To test the synaptogenic functions of LAR-RPTPs, we conditionally deleted the genes encoding all three LAR-RPTPs, singly or in combination, in mice before synapse formation. Strikingly, deletion of LAR-RPTPs had no effect on synaptic connectivity in cultured neurons or in vivo, but impaired NMDA-receptor-mediated responses. Deletion of LAR-RPTPs decreased NMDA-receptor-mediated responses by a trans-synaptic mechanism. In cultured neurons, deletion of all LAR-RPTPs led to a reduction in synaptic NMDA-receptor EPSCs, without changing the subunit composition or the protein levels of NMDA-receptors. In vivo, deletion of all LAR-RPTPs in the hippocampus at birth also did not alter synaptic connectivity as measured via AMPA-receptor-mediated synaptic responses at Schaffer-collateral synapses monitored in juvenile mice, but again decreased NMDA-receptor mediated synaptic transmission. Thus, LAR-RPTPs are not essential for synapse formation, but control synapse properties by regulating postsynaptic NMDA-receptors via a trans-synaptic mechanism that likely involves binding to one or multiple postsynaptic ligands.

NGL-1/LRRC4C-Mutant Mice Display Hyperactivity and Anxiolytic-Like Behavior Associated With Widespread Suppression of Neuronal Activity.

Netrin-G ligand-1 (NGL-1), encoded by Lrrc4c, is a post-synaptic adhesion molecule implicated in various brain disorders, including bipolar disorder, autism spectrum disorder, and developmental delay. Although previous studies have explored the roles of NGL-1 in the regulation of synapse development and function, the importance of NGL-1 for specific behaviors and the nature of related neural circuits in mice remain unclear. Here, we report that mice lacking NGL-1 (Lrrc4c–/–) show strong hyperactivity and anxiolytic-like behavior. They also display impaired spatial and working memory, but normal object-recognition memory and social interaction. c-Fos staining under baseline and anxiety-inducing conditions revealed suppressed baseline neuronal activity as well as limited neuronal activation in widespread brain regions, including the anterior cingulate cortex (ACC), motor cortex, endopiriform nucleus, bed nuclei of the stria terminalis, and dentate gyrus. Neurons in the ACC, motor cortex, and dentate gyrus exhibit distinct alterations in excitatory synaptic transmission and intrinsic neuronal excitability. These results suggest that NGL-1 is important for normal locomotor activity, anxiety-like behavior, and learning and memory, as well as synapse properties and excitability of neurons in widespread brain regions under baseline and anxiety-inducing conditions.

TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering

SummaryTetraspanins are a class of evolutionarily conserved transmembrane proteins with 33 members identified in mammals that have the ability to organize specific membrane domains, named tetraspanin-enriched microdomains (TEMs). Despite the relative abundance of different tetraspanins in the CNS, few studies have explored their role at synapses. Here, we investigate the function of TSPAN5, a member of the tetraspanin superfamily for which mRNA transcripts are found at high levels in the mouse brain. We demonstrate that TSPAN5 is localized in dendritic spines of pyramidal excitatory neurons and that TSPAN5 knockdown induces a dramatic decrease in spine number because of defects in the spine maturation process. Moreover, we show that TSPAN5 interacts with the postsynaptic adhesion molecule neuroligin-1, promoting its correct surface clustering. We propose that membrane compartmentalization by tetraspanins represents an additional mechanism for regulating excitatory synapses.

ELFN2 is a postsynaptic cell adhesion molecule with essential roles in controlling group III mGluRs in the brain and neuropsychiatric behavior.

The functional characterization of the GPCR interactome has predominantly focused on intracellular binding partners; however, the recent emergence of trans-synaptic GPCR complexes represents an additional dimension to GPCR function that has previously been unaccounted for in drug discovery. Here, we characterize ELFN2 as a novel post-synaptic adhesion molecule with a distinct expression pattern throughout the brain and a selective binding with group III metabotropic glutamate receptors (mGluRs) in trans. Using a transcellular GPCR signaling platform, we report that ELFN2 critically alters group III mGluR secondary messenger signaling by directly altering G protein coupling kinetics and efficacy. Loss of ELFN2 in mice results in the selective downregulation of group III mGluRs and dysregulated glutamatergic synaptic transmission. Elfn2 knockout (Elfn2 KO) mice also feature a range of neuropsychiatric manifestations including seizure susceptibility, hyperactivity, and anxiety/compulsivity which can be rescued by pharmacological augmentation of group III mGluRs. Thus, we conclude that extracellular trans-synaptic scaffolding by ELFN2 in the brain is a cardinal organizational feature of group III mGluRs essential for their signaling properties and brain function.

Possible Implication of the CA2 Hippocampal Circuit in Social Cognition Deficits Observed in the Neuroligin 3 Knock-Out Mouse, a Non-Syndromic Animal Model of Autism

Autism spectrum disorders (ASDs) comprise a heterogeneous group of neuro-developmental abnormalities with a strong genetic component, characterized by deficits in verbal and non-verbal communication, impaired social interactions, and stereotyped behaviors. In a small percentage of cases, ASDs are associated with alterations of genes involved in synaptic function. Among these, relatively frequent are mutations/deletions of genes encoding for neuroligins (NLGs). NLGs are postsynaptic adhesion molecules that, interacting with their presynaptic partners neurexins, ensure the cross talk between pre- and postsynaptic specializations and synaptic stabilization, a condition needed for maintaining a proper excitatory/inhibitory balance within local neuronal circuits. We have focused on mice lacking NLG3 (NLG3 knock-out mice), animal models of a non-syndromic form of autism, which exhibit deficits in social behavior reminiscent of those found in ASDs. Among different brain areas involved in social cognition, the CA2 region of the hippocampus has recently emerged as a central structure for social memory processing. Here, in vivo recordings from anesthetized animals and ex vivo recordings from hippocampal slices have been used to assess the dynamics of neuronal signaling in the CA2 hippocampal area. In vivo experiments from NLG3-deficient mice revealed a selective impairment of spike-related slow wave activity in the CA2 area and a significant reduction in oscillatory activity in the theta and gamma frequencies range in both CA2 and CA3 regions of the hippocampus. These network effects were associated with an increased neuronal excitability in the CA2 hippocampal area. Ex vivo recordings from CA2 principal cells in slices obtained from NLG3 knock-out animals unveiled a strong excitatory/inhibitory imbalance in this region accompanied by a strong reduction of perisomatic inhibition mediated by CCK-containing GABAergic interneurons. These data clearly suggest that the selective alterations in network dynamics and GABAergic signaling observed in the CA2 hippocampal region of NLG3 knock-out mice may account for deficits in social memory reminiscent of those observed in autistic patients.

NGL-3 in the regulation of brain development, Akt/GSK3b signaling, long-term depression, and locomotive and cognitive behaviors.

Netrin-G ligand-3 (NGL-3) is a postsynaptic adhesion molecule known to directly interact with the excitatory postsynaptic scaffolding protein postsynaptic density-95 (PSD-95) and trans-synaptically with leukocyte common antigen-related (LAR) family receptor tyrosine phosphatases to regulate presynaptic differentiation. Although NGL-3 has been implicated in the regulation of excitatory synapse development by in vitro studies, whether it regulates synapse development or function, or any other features of brain development and function, is not known. Here, we report that mice lacking NGL-3 (Ngl3−/− mice) show markedly suppressed normal brain development and postnatal survival and growth. A change of the genetic background of mice from pure to hybrid minimized these developmental effects but modestly suppressed N-methyl-D-aspartate (NMDA) receptor (NMDAR)-mediated synaptic transmission in the hippocampus without affecting synapse development, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR)-mediated basal transmission, and presynaptic release. Intriguingly, long-term depression (LTD) was near-completely abolished in Ngl3−/− mice, and the Akt/glycogen synthase kinase 3β (GSK3β) signaling pathway, known to suppress LTD, was abnormally enhanced. In addition, pharmacological inhibition of Akt, but not activation of NMDARs, normalized the suppressed LTD in Ngl3−/− mice, suggesting that Akt hyperactivity suppresses LTD. Ngl3−/− mice displayed several behavioral abnormalities, including hyperactivity, anxiolytic-like behavior, impaired spatial memory, and enhanced seizure susceptibility. Among them, the hyperactivity was rapidly improved by pharmacological NMDAR activation. These results suggest that NGL-3 regulates brain development, Akt/GSK3β signaling, LTD, and locomotive and cognitive behaviors.

Presynaptic Expression of LRIT3 Transsynaptically Organizes the Postsynaptic Glutamate Signaling Complex Containing TRPM1

SUMMARYThroughout the CNS, interactions between pre- and postsynaptic adhesion molecules establish normal synaptic structure and function. Leucine-rich repeat (LRR) domain-containing proteins are a large family that has a diversity of ligands, and their absence can cause disease. At the first retinal synapse, the absence of LRIT3 expression leads to the disassembly of the postsynaptic glutamate signaling complex (signalplex) expressed on depolarizing bipolar cell (DBC) dendrites. The prevalent view is that assembly of the signalplex results from direct post-synaptic protein:protein interactions. In contrast, we demonstrate that LRIT3 is expressed presynaptically, in rod photoreceptors (rods), and when we restore LRIT3 expression in Lrit3−/− rods, we restore expression of the postsynaptic glutamate signalplex and rod-driven vision. Our results demonstrate that, in the retina, the LRR-containing protein LRIT3 acts as a transsynaptic organizer of the postsynaptic complex required for normal synaptic function.