Neuroligin Research Articles

An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons.

Neuroligins (NLGNs) are a class of postsynaptic cell adhesion molecules that interact with presynaptic neurexins (NRXNs) and regulate synapse function. NLGN4 is a member of the NLGN family and consists of a unique amino acid sequence in humans that is not evolutionarily well conserved in rodents. The human-specific NLGN4 gene has been reported to be mutated in many patients with autism and other neurodevelopmental disorders. However, it remained unclear how these mutations might alter the molecular properties of NLGN4 and affect synaptic transmission in human neurons. Here, we describe a severely autistic male patient carrying a single amino acid substitution (R101Q) in the NLGN4 gene. When expressed in HEK293 cells, the R101Q mutation in NLGN4 did not affect its binding affinity for NRXNs or its capacity to form homodimers. This mutation, however, impaired the maturation of NLGN4 protein by inhibiting N-linked glycosylation at an adjacent residue (N102), which is conserved in all NLGNs. As a result, the R101Q substitution significantly decreased the surface trafficking of NLGN4 and increased its retention in the endoplasmic reticulum and Golgi apparatus. In human neurons derived from male stem cell lines, the R101Q mutation also similarly reduced the synaptic localization of NLGN4, resulting in a loss-of-function phenotype. This mutation-induced trafficking defect substantially diminished the ability of NLGN4 to form excitatory synapses and modulate their functional properties. Viewed together, our findings suggest that the R101Q mutation is pathogenic for NLGN4 and can lead to synaptic dysfunction in autism.

Autism-associated variants of neuroligin 4X impair synaptogenic activity by various molecular mechanisms

BackgroundSeveral genetic alterations, including point mutations and copy number variations in NLGN genes, have been associated with psychiatric disorders, such as autism spectrum disorder (ASD) and X-linked mental retardation (XLMR). NLGN genes encode neuroligin (NL) proteins, which are adhesion molecules that are important for proper synaptic formation and maturation. Previously, we and others found that the expression level of murine NL1 is regulated by proteolytic processing in a synaptic activity-dependent manner.MethodsIn this study, we analyzed the effects of missense variants associated with ASD and XLMR on the metabolism and function of NL4X, a protein which is encoded by the NLGN4X gene and is expressed only in humans, using cultured cells, primary neurons from rodents, and human induced pluripotent stem cell-derived neurons.ResultsNL4X was found to undergo proteolytic processing in human neuronal cells. Almost all NL4X variants caused a substantial decrease in the levels of mature NL4X and its synaptogenic activity in a heterologous culture system. Intriguingly, the L593F variant of NL4X accelerated the proteolysis of mature NL4X proteins located on the cell surface. In contrast, other variants decreased the cell-surface trafficking of NL4X. Notably, protease inhibitors as well as chemical chaperones rescued the expression of mature NL4X.LimitationsOur study did not reveal whether these dysfunctional phenotypes occurred in individuals carrying NLGN4X variant. Moreover, though these pathological mechanisms could be exploited as potential drug targets for ASD, it remains unclear whether these compounds would have beneficial effects on ASD model animals and patients.ConclusionsThese data suggest that reduced amounts of the functional NL4X protein on the cell surface is a common mechanism by which point mutants of the NL4X protein cause psychiatric disorders, although different molecular mechanisms are thought to be involved. Furthermore, these results highlight that the precision medicine approach based on genetic and cell biological analyses is important for the development of therapeutics for psychiatric disorders.

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.

Targeting QKI-7 in vivo restores endothelial cell function in diabetes

Vascular endothelial cell (EC) dysfunction plays a key role in diabetic complications. This study discovers significant upregulation of Quaking-7 (QKI-7) in iPS cell-derived ECs when exposed to hyperglycemia, and in human iPS-ECs from diabetic patients. QKI-7 is also highly expressed in human coronary arterial ECs from diabetic donors, and on blood vessels from diabetic critical limb ischemia patients undergoing a lower-limb amputation. QKI-7 expression is tightly controlled by RNA splicing factors CUG-BP and hnRNPM through direct binding. QKI-7 upregulation is correlated with disrupted cell barrier, compromised angiogenesis and enhanced monocyte adhesion. RNA immunoprecipitation (RIP) and mRNA-decay assays reveal that QKI-7 binds and promotes mRNA degradation of downstream targets CD144, Neuroligin 1 (NLGN1), and TNF-α-stimulated gene/protein 6 (TSG-6). When hindlimb ischemia is induced in diabetic mice and QKI-7 is knocked-down in vivo in ECs, reperfusion and blood flow recovery are markedly promoted. Manipulation of QKI-7 represents a promising strategy for the treatment of diabetic vascular complications.

Neuroligin 2 regulates absence seizures and behavioral arrests through GABAergic transmission within the thalamocortical circuitry

Epilepsy and autism spectrum disorders (ASD) are two distinct brain disorders but have a high rate of co-occurrence, suggesting shared pathogenic mechanisms. Neuroligins are cell adhesion molecules important in synaptic function and ASD, but their role in epilepsy remains unknown. In this study, we show that Neuroligin 2 (NLG2) knockout mice exhibit abnormal spike and wave discharges (SWDs) and behavioral arrests characteristic of absence seizures. The anti-absence seizure drug ethosuximide blocks SWDs and rescues behavioral arrests and social memory impairment in the knockout mice. Restoring GABAergic transmission either by optogenetic activation of the thalamic reticular nucleus (nRT) presynaptic terminals or postsynaptic NLG2 expression in the thalamic neurons reduces the SWDs and behavioral arrests in the knockout mice. These results indicate that NLG2-mediated GABAergic transmission at the nRT-thalamic circuit represents a common mechanism underlying both epileptic seizures and ASD.

Environmental enrichment modulates affiliative and aggressive social behaviour in the neuroligin-3 R451C mouse model of autism spectrum disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by impairments in social communication and the presence of restrictive and repetitive behaviours. A mouse model expressing an autism-associated R451C mutation in the gene encoding the synaptic adhesion protein neuroligin-3 (NL3) has been extensively characterised and shows altered behaviour relevant to core traits observed in ASD. Reported impairments in social behaviours in NL3R451C mice however remain controversial due to inconsistent findings in various assays across different laboratories. Such inconsistencies could plausibly be explained by an increased susceptibility of the NL3R451C mouse social phenotype to environmental modulation. To address this, NL3R451C mice were housed in standard or enriched housing from 4 weeks of age prior to behavioural testing. Enrichment rearing enhanced direct interactions with the stranger mouse in all mice in the three-chamber social interaction test however, NL3R451C mice did not show impairment in social interaction in the three-chamber test, in contrast with previous reports. Environmental enrichment enhanced aggressive behaviour in all mice, and did not specifically alter the heightened aggressive phenotype previously described in NL3R451C mice. Specific genotype effects of enrichment included reduced anxiety-like behaviour in WT mice, and lower locomotor activity levels in NL3 mice. While genotype-specific effects of enrichment were not seen on social behaviour, the general increase in affiliative social interaction and aggression seen in all mice, indicates that these behaviours, are vulnerable to change based on housing condition. Mouse models expressing ASD-associated mutations have great utility in elucidating the neurobiology underling development of core traits and it is crucial that efforts are focussed on those models exhibiting robust phenotypes. In light of the findings in the present study, we suggest approaches to improve replicability and reproducibility in mouse models of ASD.

Altered Caecal Neuroimmune Interactions in the Neuroligin-3R451C Mouse Model of Autism.

The intrinsic nervous system of the gut interacts with the gut-associated lymphoid tissue (GALT) via bidirectional neuroimmune interactions. The caecum is an understudied region of the gastrointestinal (GI) tract that houses a large supply of microbes and is involved in generating immune responses. The caecal patch is a lymphoid aggregate located within the caecum that regulates microbial content and immune responses. People with Autism Spectrum Disorder (ASD; autism) experience serious GI dysfunction, including inflammatory disorders, more frequently than the general population. Autism is a highly prevalent neurodevelopmental disorder defined by the presence of repetitive behavior or restricted interests, language impairment, and social deficits. Mutations in genes encoding synaptic adhesion proteins such as the R451C missense mutation in neuroligin-3 (NL3) are associated with autism and impair synaptic transmission. We previously reported that NL3R451C mice, a well-established model of autism, have altered enteric neurons and GI dysfunction; however, whether the autism-associated R451C mutation alters the caecal enteric nervous system and immune function is unknown. We assessed for gross anatomical changes in the caecum and quantified the proportions of caecal submucosal and myenteric neurons in wild-type and NL3R451C mice using immunofluorescence. In the caecal patch, we assessed total cellular density as well as the density and morphology of Iba-1 labeled macrophages to identify whether the R451C mutation affects neuro-immune interactions. NL3R451C mice have significantly reduced caecal weight compared to wild-type mice, irrespective of background strain. Caecal weight is also reduced in mice lacking Neuroligin-3. NL3R451C caecal ganglia contain more neurons overall and increased numbers of Nitric Oxide (NO) producing neurons (labeled by Nitric Oxide Synthase; NOS) per ganglion in both the submucosal and myenteric plexus. Overall caecal patch cell density was unchanged however NL3R451C mice have an increased density of Iba-1 labeled enteric macrophages. Macrophages in NL3R451C were smaller and more spherical in morphology. Here, we identify changes in both the nervous system and immune system caused by an autism-associated mutation in Nlgn3 encoding the postsynaptic cell adhesion protein, Neuroligin-3. These findings provide further insights into the potential modulation of neural and immune pathways.

Effect of Neuroligin1 and Neurexin1 on the Colonic Motility in a Mouse Model of Neuronal Intestinal Dysplasia.

Aim To investigate the expressions of neuroligin1 (NL1) and neurexin1 (NX1) in a mouse model of neuronal intestinal dysplasia (Tlx2−/− mice) and to explore their effects on colonic motility. Methods Immunohistochemistry staining was employed to explore the histological appearances of NL1, NX1, the presynaptic marker of glutamatergic synapses VGLUT1, and the subunit of NMDA receptors of NR1 in the colon of mice with or without Tlx2 mutation. Western blotting and qRT-PCR were performed to detect their relative expressions in the colon. Colonic motility was measured by a glass bead technique. Then, the Tlx2−/− mice were intervened by Huperzine A. Variations on expressions of NL1, NX1, VGLUT1, and NR1 and variations on colonic motility were measured. Additionally, serum concentrations of Glu were measured by ELISA. Results Immunohistochemistry staining reveals that NL1, NX1, VGLUT1, and NR1 were mainly concentrated in the myenteric plexus of ENS. Compared to those in WT and Tlx2+/- mice, expressions of NL1 and NX1 in colon of Tlx2−/− mice were upregulated with increased VGLUT1 and NR1 abundances and impaired colonic motility (P < 0.05). After intervention, the upregulated expressions of NL1 and NX1 were decreased with a correlated reduce of VGLUT1 and NR1 and a recovery of the impaired colonic motility (P < 0.05). After intervention, the upregulated expressions of NL1 and NX1 were decreased with a correlated reduce of VGLUT1 and NR1 and a recovery of the impaired colonic motility (P < 0.05). After intervention, the upregulated expressions of NL1 and NX1 were decreased with a correlated reduce of VGLUT1 and NR1 and a recovery of the impaired colonic motility (Conclusion NL1 and NX1 are closely related to the colonic motility through their effects of targeting the formation of glutamatergic synapses and may be involved in the pathogenesis of NID. The variations of serum Glu seem to be a potential and less painful auxiliary measure for colonic motility and NID.

Molecular Analysis of Neuronal Cell Surface Protein gene; Neuroligin 3 in Autism Spectrum Disorder

Introduction: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder that manifests before the age of three. ASD is characterized by a triad of limited or absent verbal communication, a lack of reciprocal social interaction or responsiveness, and restricted, stereotypic patterns of interests and behavior. More than 1 million cases per year are reported in India. It is now believed that ASD is due to complex genetic and environmental interactions. Changes in over 1,000 genes have been reported to be associated with ASD. Many of the associated genes are involved in the development and functions of the brain. The proposed candidate gene for ASD includes the Neuroligin (NLGN). It is a cell adhesion protein on the postsynaptic membrane involved in Synaptogenesis. Objective: To screen the variants in NLGN3 gene associated with Autism Spectrum Disorder. Methods: 1821 mentally ill children from the North Karnataka region of Karnataka, India were screened as per DSM-V Criteria and 108 autistic were included in the study. Genomic DNA was extracted and the candidate gene was amplified using standard PCR condition. The product was sequenced for genetic variation. Result: one 5' UTR variant, one missense variant, and four 3' UTR variants were recorded. The novel Coding sequence variant c.551 T>C transition was observed in 16 autistics (14.81%). This transition changes Valine to Alanine (p.Val184Ala). This may lead to abnormal neuronal cell surface Protein. Conclusion: This was the first study in Karnataka on genetic aspects of autism. Only one coding sequence variant was observed in 14.81% of our population. This variant may lead to potentially abnormal Neuroligin for the etiology of autism in north Karnataka.

Rational Design and In Vitro Evaluation of Novel Peptides Binding to Neuroligin-1 for Synaptic Targeting.

Neuroligin-1 (NL1) is a postsynaptic cell adhesion protein that plays a crucial role in synapsis and signaling between neurons. Due to its clustered distribution in synaptic clefts, NL1 appears as a novel potential site for synaptic targeting purposes. In this work, in silico protein topography analysis was employed to identify two prospective binding sites on the NL1 dimer surface in the 2:2 synaptic adhesion complex with β-neurexin (PDB code 3B3Q ). Receptor-based rational design, cell-penetrating capability prediction, molecular docking, molecular dynamics simulations, and binding free energy calculations were used to identify five heptapeptides candidates with favorable predicted profiles as non cell-penetrating NL1-binding agents. Preliminary in vitro colocalization assays with NL1-transfected HEK 293 cells confirmed that peptides remain in the extracellular space without inducing detectable changes in cell morphology. The highest NL1-colocatization capability was attained by the peptide ADEAIVA, which appears as a promising candidate for the future development of specific NL1-targeting systems as part of synapse-directed therapies against central nervous system diseases.

Neuroligin 3 Regulates Dendritic Outgrowth by Modulating Akt/mTOR Signaling.

Neuroligins (NLs) are a group of postsynaptic cell adhesion molecules that function in synaptogenesis and synaptic transmission. Genetic defects in neuroligin 3 (NL3), a member of the NL protein family, are associated with autism. Studies in rodents have revealed that mutations of NL3 gene lead to increased growth and complexity in dendrites in the central nervous system. However, the detailed mechanism is still unclear. In our study, we found that deficiency of NL3 led to morphological changes of the pyramidal neurons in layer II/III somatosensory cortex in mice, including enlarged somata, elongated dendritic length, and increased dendritic complexity. Knockdown of NL3 in cultured rat neurons upregulated Akt/mTOR signaling, resulting in both increased protein synthesis and dendritic growth. Treating neurons with either rapamycin to inhibit the mTOR or LY294002 to inhibit the PI3K/Akt activity rescued the morphological abnormalities resulting from either NL3 knockdown or knockout (KO). In addition, we found that the hyperactivated Akt/mTOR signaling associated with NL3 defects was mediated by a reduction in phosphatase and tensin (PTEN) expression, and that MAGI-2, a scaffold protein, interacted with both NL3 and PTEN and could be a linker between NL3 and Akt/mTOR signaling pathway. In conclusion, our results suggest that NL3 regulates neuronal morphology, especially dendritic outgrowth, by modulating the PTEN/Akt/mTOR signaling pathway, probably via MAGI-2. Thereby, this study provides a new link between NL3 and neuronal morphology.

Neurobehavioral Comorbidities in Pentylenetetrazol-kindled Mice and Role of the α4β2 Nicotinic Acetylcholine Receptor

Patients with epilepsy are often affected by not only seizures but also a variety of cognitive and psychiatric comorbidities that further impair their quality of life. However, it is unclear whether epilepsy is associated with psychic function. The aim of the present study was to clarify the effects of kindling-induced epileptic seizures on psychic functioning, using behavioral pharmacological tests. Pentylenetetrazol (PTZ)-kindled mice displayed impaired motor coordination (in the rotarod test), and social approach impairment (in the three-chamber social test) compared with vehicle mice. Intraperitoneal ABT-418 treatment (0.05 mg/kg) alleviated these behavioral abnormalities in PTZ-kindled mice. Immunolabeling of tissue sections demonstrated that expression of the α4 subunit of the α4β2 nicotinic acetylcholine receptor in the piriform cortex was significantly decreased in PTZ-kindled mice. In contrast, expression of the synaptic adhesion molecule neuroligin 3 (NLG3) was significantly higher in the piriform cortex of PTZ-kindled mice compared with vehicle mice. Collectively, our findings suggest that attention deficit/hyperactivity disorder (ADHD)-like or autistic-like behavioral abnormalities associated with epilepsy are closely linked to downregulation of the α4 subunit of the α4β2 receptor and upregulation of NLG3 in the mouse piriform cortex. In summary, this study indicates that ABT-418 is a good candidate for the treatment of patients with epilepsy complicated by psychiatric symptoms such as autism and ADHD.

A Conserved Tyrosine Residue in Slitrk3 Carboxyl-Terminus Is Critical for GABAergic Synapse Development

Single-passing transmembrane protein, Slitrk3 (Slit and Trk-like family member 3, ST3), is a synaptic cell adhesion molecule highly expressed at inhibitory synapses. Recent studies have shown that ST3, through its extracellular domain, selectively regulates inhibitory synapse development via the trans-synaptic interaction with presynaptic cell adhesion molecule, receptor protein tyrosine phosphatase δ (PTPδ) and the cis-interaction with postsynaptic cell adhesion molecule, Neuroligin 2 (NL2). However, little is known about the physiological function of ST3 intracellular, carboxyl (C)-terminal region. Here we report that in heterologous cells, ST3 C-terminus is not required for ST3 homo-dimerization and trafficking to the cell surface. In contrast, in hippocampal neurons, ST3 C-terminus, more specifically, the conserved tyrosine Y969 (in mice), is critical for GABAergic synapse development. Indeed, overexpression of ST3 Y969A mutant markedly reduced the gephyrin puncta density and GABAergic transmission in hippocampal neurons. In addition, single-cell genetic deletion of ST3 strongly impaired GABAergic transmission. Importantly, wild-type (WT) ST3, but not the ST3 Y969A mutant, could fully rescue GABAergic transmission deficits in neurons lacking endogenous ST3, confirming a critical role of Y969 in the regulation of inhibitory synapses. Taken together, our data identify a single critical residue in ST3 C-terminus that is important for GABAergic synapse development and function.

Fluorescence-Based Quantitative Synapse Analysis for Cell Type-Specific Connectomics

Anatomical methods for determining cell type-specific connectivity are essential to inspire and constrain our understanding of neural circuit function. We developed genetically-encoded reagents for fluorescence-synapse labeling and connectivity analysis in brain tissue, using a fluorogen-activating protein (FAP)-coupled or YFP-coupled, postsynaptically-localized neuroligin-1 (NL-1) targeting sequence (FAP/YFPpost). FAPpost expression did not alter mEPSC or mIPSC properties. Sparse AAV-mediated expression of FAP/YFPpost with the cell-filling, red fluorophore dTomato (dTom) enabled high-throughput, compartment-specific detection of putative synapses across diverse neuron types in mouse somatosensory cortex. We took advantage of the bright, far-red emission of FAPpost puncta for multichannel fluorescence alignment of dendrites, FAPpost puncta, and presynaptic neurites in transgenic mice with saturated labeling of parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP)-expressing neurons using Cre-reporter driven expression of YFP. Subtype-specific inhibitory connectivity onto layer 2/3 (L2/3) neocortical pyramidal (Pyr) neurons was assessed using automated puncta detection and neurite apposition. Quantitative and compartment-specific comparisons show that PV inputs are the predominant source of inhibition at both the soma and the dendrites and were particularly concentrated at the primary apical dendrite. SST inputs were interleaved with PV inputs at all secondary-order and higher-order dendritic branches. These fluorescence-based synapse labeling reagents can facilitate large-scale and cell-type specific quantitation of changes in synaptic connectivity across development, learning, and disease states.

Role of NL-1 in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horn during remifentanil-induced hyperalgesia in mice with incisional pain

Objective To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain. Methods Forty SPF healthy male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 5 groups (n=8 each) using a random number table method: control group (group C), NL1-shRNA plasmid group (group NL), incisional pain plus remifentanil group (group I+ R), incisional pain plus remifentanil plus blank vector group (group I+ R+ B), and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+ R+ NL). Negative lentivirus was intrathecally injected in group I+ R+ B.In NL and I+ R+ NL groups, 10 μl NL-1-shRNA lentivirus at 1×108 IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+ R groups.After transfection was stable, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+ R, I+ R+ B and I+ R+ NL groups, 0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals, and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3, 6, 24 and 48 h after the end of administration (T1-4). The animals were sacrificed after measurement of pain threshold at T4, and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors, and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated. Results Compared with group C, the MWT was significantly decreased, and TFL was shortened at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated, and m/t ratio was increased in I+ R and I+ R+ B groups (P<0.05). Compared with I+ R and I+ R+ B groups, the MWT was significantly increased and TFL was prolonged at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated, and m/t ratio was decreased in group I+ R+ NL (P<0.05). Conclusion NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane, which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain. Key words: Connexins; Pain, postopertive; Piperidines; Hyperalgesia; Spinal cord dorsal horn; Receptors, glutamate; Receptors, AMPA

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.

Down-Regulation of Essential Synaptic Components by GI-Tract Microbiome-Derived Lipopolysaccharide (LPS) in LPS-Treated Human Neuronal-Glial (HNG) Cells in Primary Culture: Relevance to Alzheimer's Disease (AD).

Trans-synaptic neurotransmission of both electrical and neurochemical information in the central nervous system (CNS) is achieved through a highly interactive network of neuron-specific synaptic proteins that include pre-synaptic and post-synaptic elements. These elements include a family of several well-characterized integral- and trans-membrane synaptic core proteins necessary for the efficient operation of this complex signaling network, and include the pre-synaptic proteins: (i) neurexin-1 (NRXN-1); (ii) the synaptosomal-associated phosphoprotein-25 (SNAP-25); (iii) the phosphoprotein synapsin-2 (SYN-2); and the post-synaptic elements: (iv) neuroligin (NLGN), a critical cell adhesion protein; and (v) the SH3-ankyrin repeat domain, proline-rich cytoskeletal scaffolding protein SHANK3. All five of these pre- and post-synaptic proteins have been found to be significantly down-regulated in primary human neuronal-glial (HNG) cell co-cultures after exposure to Bacteroides fragilis lipopolysaccharide (BF-LPS). Interestingly, LPS has also been reported to be abundant in Alzheimer’s disease (AD) affected brain cells where there are significant deficits in this same family of synaptic components. This “Perspectives” paper will review current research progress and discuss the latest findings in this research area. Overall these experimental results provide evidence (i) that gastrointestinal (GI) tract-derived Gram-negative bacterial exudates such as BF-LPS express their neurotoxicity in the CNS in part through the directed down-regulation of neuron-specific neurofilaments and synaptic signaling proteins; and (ii) that this may explain the significant alterations in immune-responses and cognitive deficits observed after bacterial-derived LPS exposure to the human CNS.

The structure of Neurexin 1α (n1α) and its role as synaptic organizer

α - and b-neurexins (NRXNs) are transmembrane adhesion protein complexes localized in presynaptic membranes into neurons and interact with the postsynaptic neuroligins (NLGNs). Our findings indicate that the neurexin 1α (n1α) is a synaptic organizer that directs postsynaptic development in neurons, evidenced in GABAergic neurons and trials with Knock-out Mice. Also, the interactions between hypervariable surfaces of n1α and ligands (neurexophilin, a-dystroglycan, and GABAA) promotes a proper protein-binding recognition, and consequently, a better synaptic adhesion. There is a direct relationship between mental disorders and the n1α assemblage because NRXN1 gene encodes for n1α proteins which are involved in the transmission of information into the brain. For this reason, damage in this complex-protein or some neurexin gene variations causes pathological abnormalities and neuropsychiatric diseases such as schizophrenia, autism spectrum disorders, and intellectual disabilities.

Neuroligins Differentially Mediate Subtype-Specific Synapse Formation in Pyramidal Neurons and Interneurons.

Neuroligins (NLs) are postsynaptic cell-adhesion proteins that play important roles in synapse formation and the excitatory-inhibitory balance. They have been associated with autism in both human genetic and animal model studies, and affect synaptic connections and synaptic plasticity in several brain regions. Yet current research mainly focuses on pyramidal neurons, while the function of NLs in interneurons remains to be understood. To explore the functional difference among NLs in the subtype-specific synapse formation of both pyramidal neurons and interneurons, we performed viral-mediated shRNA knockdown of NLs in cultured rat cortical neurons and examined the synapses in the two major types of neurons. Our results showed that in both types of neurons, NL1 and NL3 were involved in excitatory synapse formation, and NL2 in GABAergic synapse formation. Interestingly, NL1 affected GABAergic synapse formation more specifically than NL3, and NL2 affected excitatory synapse density preferentially in pyramidal neurons. In summary, our results demonstrated that different NLs play distinct roles in regulating the development and balance of excitatory and inhibitory synapses in pyramidal neurons and interneurons.

Sorting nexin 27 rescues neuroligin 2 from lysosomal degradation to control inhibitory synapse number.

Retromer is an evolutionarily conserved endosomal trafficking complex that mediates the retrieval of cargo proteins from a degradative pathway for sorting back to the cell surface. To promote cargo recycling, the core retromer trimer of VPS (vacuolar protein sorting)26, VPS29 and VPS35 recognises cargo either directly, or through an adaptor protein, the most well characterised of which is the PDZ [postsynaptic density 95 (PSD95), disk large, zona occludens] domain-containing sorting nexin SNX27. Neuroligins (NLGs) are postsynaptic trans-synaptic scaffold proteins that function in the clustering of postsynaptic proteins to maintain synaptic stability. Here, we show that each of the NLGs (NLG1-3) bind to SNX27 in a direct PDZ ligand-dependent manner. Depletion of SNX27 from neurons leads to a decrease in levels of each NLG protein and, for NLG2, this occurs as a result of enhanced lysosomal degradation. Notably, while depletion of the core retromer component VPS35 leads to a decrease in NLG1 and NLG3 levels, NLG2 is unaffected, suggesting that, for this cargo, SNX27 acts independently of retromer. Consistent with loss of SNX27 leading to enhanced lysosomal degradation of NLG2, knockdown of SNX27 results in fewer NLG2 clusters in cultured neurons, and loss of SNX27 or VPS35 reduces the size and number of gephyrin clusters. Together, these data indicate that NLGs are SNX27-retromer cargoes and suggest that SNX27-retromer controls inhibitory synapse number, at least in part through trafficking of NLG2.