Neuroligin Research Articles

Modulation of heteromeric glycine receptor function through high concentration clustering.

Ion channels are targeted by many drugs for treating neurological, musculoskeletal, renal and other diseases. These drugs bind to and alter the function of individual channels to achieve desired therapeutic effects. However, many ion channels function in high concentration clusters in their native environment. It is unclear if and how clustering modulates ion channel function. Human heteromeric glycine receptors (GlyRs) are the major inhibitory neurotransmitter receptors in the spinal cord and are active targets for developing chronic pain medications. We show that the α2β heteromeric GlyR assembles with the master postsynaptic scaffolding gephyrin (GPHN) into micron-sized clustered at the plasma membrane after heterologous expression. The inhibitory trans- synaptic adhesion protein neuroligin-2 (NL2) further increases both the cluster sizes and GlyR concentration. The apparent glycine affinity increases monotonically as a function of GlyR concentration but not with cluster size. We also show that ligand re-binding to adjacent GlyRs alters kinetics but not chemical equilibrium. A positively charged N- terminus sequence of the GlyR β subunit was further identified essential for glycine affinity modulation through clustering. Taken together, we propose a mechanism where clustering enhances local electrostatic potential, which in turn concentrates ions and ligands, modulating the function of GlyR. This mechanism is likely universal across ion channel clusters found ubiquitously in biology and provides new perspectives in possible pharmaceutical development.

Weaker neuroligin 2 - neurexin 1β interaction tethers membranes and signal synaptogenesis through clustering.

Single-pass transmembrane proteins neuroligin (NL) and neurexin (NRX) constitute a pair of synaptic adhesion molecules (SAMs) that are essential for the formation of functional synapses. Binding affinities vary by ∼ 1000 folds between arrays of NL and NRX subtypes, which contribute to chemical and spatial specificities. Current structures are obtained with truncated extracellular domains of NL and NRX and are limited to the higher-affinity NL1/4-NRX complexes. How NL-NRX interaction leads to functional synapses remains unknown. Here we report structures of full-length NL2 alone, and in complex with NRX1β in several conformations, which has the lowest affinity among major NL-NRX subtypes. We show how conformational flexibilities may help in adapting local membrane geometry, and reveal mechanisms underlying variations in NL-NRX affinities modulation. We further show that, despite lower affinity, NL2-NRX1β interaction alone is capable of tethering different lipid membranes in total reconstitution, and that NL2 and NRX1β cluster at inter-cellular junctions without the need of other synaptic components. In addition, NL2 combines with the master post-synaptic scaffolding protein gephyrin and clusters neurotransmitter receptors at cellular membrane. These findings suggest dual roles of NL2 - NRX1β interaction - both as mechanical tether, and as signaling receptors, to ensure correct spatial and chemical coordination between two cells to generate function synapses.

Astrocytic neuroligin 3 regulates social memory and synaptic plasticity through adenosine signaling in male mice

Social memory impairment is a key symptom of many brain disorders, but its underlying mechanisms remain unclear. Neuroligins (NLGs) are a family of cell adhesion molecules essential for synapse development and function and their dysfunctions are linked to neurodevelopmental and neuropsychiatric disorders, including autism and schizophrenia. Although NLGs are extensively studied in neurons, their role in glial cells is poorly understood. Here we show that astrocytic deletion of NLG3 in the ventral hippocampus of adult male mice impairs social memory, attenuates astrocytic Ca2+ signals, enhances the expression of EAAT2 and prevents long-term potentiation, and these impairments are rescued by increasing astrocyte activity, reducing EAAT2 function or enhancing adenosine/A2a receptor signaling. This study has revealed an important role of NLG3 in astrocyte function, glutamate homeostasis and social memory and identified the glutamate transporter and adenosine signaling pathway as potential therapeutic strategies to treat brain disorders.

Effects of Human Neural Stem Cells Overexpressing Neuroligin and Neurexin in a Spinal Cord Injury Model.

Recent studies have highlighted the therapeutic potential of stem cells for various diseases. However, unlike other tissues, brain tissue has a specific structure, consisting of synapses. These synapses not only transmit but also process and refine information. Therefore, synaptic regeneration plays a key role in therapy of neurodegenerative disorders. Neurexins (NRXNs) and neuroligins (NLGNs) are synaptic cell adhesion molecules that connect pre- and postsynaptic neurons at synapses, mediate trans-synaptic signaling, and shape neural network properties by specifying synaptic functions. In this study, we investigated the synaptic regeneration effect of human neural stem cells (NSCs) overexpressing NRXNs (F3.NRXN) and NLGNs (F3.NLGN) in a spinal cord injury model. Overexpression of NRXNs and NLGNs in the neural stem cells upregulated the expression of synaptophysin, PSD95, VAMP2, and synapsin, which are synaptic markers. The BMS scores indicated that the transplantation of F3.NRXN and F3.NLGN enhanced the recovery of locomotor function in adult rodents following spinal cord injury. Transplanted F3.NRXN and F3.NLGN differentiated into neurons and formed a synapse with the host cells in the spinal cord injury mouse model. In addition, F3.NRXN and F3.NLGN cells restored growth factors (GFs) and neurotrophic factors (NFs) and induced the proliferation of host cells. This study suggested that NSCs overexpressing NRXNs and NLGNs could be candidates for cell therapy in spinal cord injuries by facilitating synaptic regeneration.

Systematic Review of the Literature on Single-Nucleotide Polymorphisms within the Neuroligin (NLGN) Gene in the Development of Autism Spectrum Disorder

Systematic Review of the Literature on Single-Nucleotide Polymorphisms within the Neuroligin (NLGN) Gene in the Development of Autism Spectrum Disorder

Hippocampal neurogenesis modulated by Quinic acid: A therapeutic strategy for the neurodegenerative disorders.

Neural progenitor cells (NPCs) reside in the brain and participate in the mechanism of neurogenesis that permits the brain to generate the building blocks for enhancement of cognitive abilities and acquisition of new skills. The existence of NPCs in brain has opened a novel dimension of research to explore their potential for treatment of various neurodegenerative disorders. The present study provides novel insights into the intracellular mechanisms in neuronal cells proliferation, maturation and differentiation regulated by Quinic acid (QA). Furthermore, this study might help in discovery and development of lead molecule that can overcome the challenges in the treatment of neurodegenerative diseases. The growth supporting effect of QA was studied using MTT assay. For that purpose, hippocampal cell cultures of neonatal rats were treated with different concentrations of QA and incubated for 24, 48 and 72 h. Gene and protein expressions of the selected molecular markers nestin, neuron-specific class III beta-tubulin (Tuj-1), neuronal nuclear protein (NeuN), neuronal differentiation 1 (NeuroD1), glial fibrillary acidic protein (GFAP), neuroligin (NLGN) and vimentin were analyzed. QA-induced cell proliferation and differentiation of hippocampal progenitor cells was also accompanied by significantly increased expression of progenitor and immature neuronal marker, mature neuronal marker and differentiating factor, that is, nestin, Tuj-1, NeuN and NeuroD1, respectively. On the other hand, vimentin downregulation and constant GFAP expression were observed following QA treatment. Additionally, the effects of QA on the recovery of stressed cells was studied using in vitro model of oxygen glucose deprivation (OGD). It was observed that hippocampal cells were able to recover from OGD following the treatment with QA. These findings suggest that QA treatment promotes hippocampal neurogenesis by proliferating and differentiating of NPCs and recovers neurons from stress caused by OGD. Thus, the neurogenic potential of QA can be explored for the treatment of neurodegenerative disorders.

Autism-associated neuroligin 3 deficiency in medial septum causes social deficits and sleep loss in mice.

Patients with autism spectrum disorder (ASD) frequently experience sleep disturbance. Genetic mutations in the neuroligin 3 (NLG3) gene are highly correlative with ASD and sleep disturbance. However, the cellular and neural circuit bases of this correlation remain elusive. Here, we found that the conditional knockout of Nlg3 (Nlg3-CKO) in the medial septum (MS) impairs social memory and reduces sleep. Nlg3 CKO in the MS caused hyperactivity of MSGABA neurons during social avoidance and wakefulness. Activation of MSGABA neurons induced social memory deficits and sleep loss in C57BL/6J mice. In contrast, inactivation of these neurons ameliorated social memory deficits and sleep loss in Nlg3-CKO mice. Sleep deprivation led to social memory deficits, while social isolation caused sleep loss, both resulting in a reduction in NLG3 expression and an increase in activity of GABAergic neurons in the MS from C57BL/6J mice. Furthermore, MSGABA-innervated CA2 neurons specifically regulated social memory without impacting sleep, whereas MSGABA-innervating neurons in the preoptic area selectively controlled sleep without affecting social behavior. Together, these findings demonstrate that the hyperactive MSGABA neurons impair social memory and disrupt sleep resulting from Nlg3 CKO in the MS, and achieve the modality specificity through their divergent downstream targets.

Reversing anxiety by targeting a stress-responsive signaling pathway

Current treatments of anxiety and depressive disorders are plagued by considerable side effects and limited efficacies, underscoring the need for additional molecular targets that can be leveraged to improve medications. Here, we have identified a molecular cascade triggered by chronic stress that exacerbates anxiety- and depressive-like behaviors. Specifically, chronic stress enhances Src kinase activity and tyrosine phosphorylation of calmodulin, which diminishes MyosinVa (MyoVa) interaction with Neuroligin2 (NL2), resulting in decreased inhibitory transmission and heightened anxiety-like behaviors. Importantly, pharmacological inhibition of Src reinstates inhibitory synaptic deficits and effectively reverses heightened anxiety-like behaviors in chronically stressed mice, a process requiring the MyoVa-NL2 interaction. These data demonstrate the reversibility of anxiety- and depressive-like phenotypes at both molecular and behavioral levels and uncover a therapeutic target for anxiety and depressive disorders.

Repeated mild traumatic brain injury in the parietal cortex inhibits expressions of NLG-1 and PSD-95 in the medulla oblongata of mice

To assess the effects of repeated mild traumatic brain injury (rmTBI) in the parietal cortex on neuronal morphology and synaptic plasticity in the medulla oblongata of mice. Thirty-two male ICR mice were randomly divided into sham operation group (n=8) and rmTBI group (n=24). The mice in the latter group were subjected to repeated mild impact injury of the parietal cortex by a free-falling object. The mice surviving the injuries were evaluated for neurological deficits using neurological severity scores (NSS), righting reflex test and forced swimming test, and pathological changes of the neuronal cells in the medulla oblongata were observed with HE and Nissl staining. Western blotting and immunofluorescence staining were used to detect the expressions of neuroligin 1(NLG-1) and postsynaptic density protein 95(PSD-95) in the medulla oblongata of the mice that either survived rmTBI or not. None of the mice in the sham-operated group died, while the mortality rate was 41.67% in rmTBI group. The mice surviving rmTBI showed significantly reduced NSS, delayed recovery of righting reflex, increased immobility time in forced swimming test (P < 0.05), and loss of Nissl bodies; swelling and necrosis were observed in a large number of neurons in the medulla oblongata, where the expression levels of NLG-1 and PSD-95 were significantly downregulated (P < 0.05). The mice that did not survive rmTBI showed distorted and swelling nerve fibers and decreased density of neurons in the medulla oblongina with lowered expression levels of NLG-1 and PSD-95 compared with the mice surviving the injuries (P < 0.01). The structural and functional anomalies of the synapses in the medulla oblongata may contribute to death and neurological impairment following rmTBI in mice.

Rare heterozygous genetic variants of NRXN and NLGN gene families involved in synaptic function and their association with neurodevelopmental disorders.

The interaction of neurexins (NRXNs) in the presynaptic membrane with postsynaptic cell adhesion molecules called neuroligins (NLGNs) is critical for this synaptic function. Impaired synaptic functions are emphasized in neurodevelopmental disorders to uncover etiological factors. We evaluated variants in NRXN and NLGN genes encoding molecules located directly at the synapse in patients with neuropsychiatric disorders using clinical exome sequencing and chromosomal microarray. We presented detailed clinical findings of cases carrying heterozygous NRXN1 (c.190C>T, c.1679C>T and two copy number variations [CNVs]), NRXN2 (c.808dup, c.1901G>T), NRXN3 (c.3889C>T), and NLGN1 (c.269C>G, c.473T>A) gene variants. In addition, three novel variants were identified in the NRXN1 (c.1679C>T), NRXN3 [c.3889C>T (p.Pro1297Ser)], and NLGN1 [c.473T>A (p.Ile158Lys)] genes. We emphasize the clinical findings of CNVs of the NRXN1 gene causing a more severe clinical presentation than single nucleotide variants of the NRXN1 gene in this study. We detected an NRXN2 gene variant (c.808dup) with low allelic frequency in two unrelated cases with the same diagnosis. We emphasize the importance of this variant for future studies. We suggest that NRXN2, NRXN3, and NLGN1 genes, which are less frequently reported than NRXN1 gene variants, may also be associated with neurodevelopmental disorders.

Structural and functional reorganization of inhibitory synapses by activity-dependent cleavage of neuroligin-2

Recent evidence has demonstrated that the transsynaptic nanoscale organization of synaptic proteins plays a crucial role in regulating synaptic strength in excitatory synapses. However, the molecular mechanism underlying this transsynaptic nanostructure in inhibitory synapses still remains unclear and its impact on synapse function in physiological or pathological contexts has not been demonstrated. In this study, we utilized an engineered proteolysis technique to investigate the effects of acute cleavage of neuroligin-2 (NL2) on synaptic transmission. Our results show that the rapid cleavage of NL2 led to impaired synaptic transmission by reducing both neurotransmitter release probability and quantum size. These changes were attributed to the dispersion of RIM1/2 and GABAA receptors and a weakened spatial alignment between them at the subsynaptic scale, as observed through superresolution imaging and model simulations. Importantly, we found that endogenous NL2 undergoes rapid MMP9-dependent cleavage during epileptic activities, which further exacerbates the decrease in inhibitory transmission. Overall, our study demonstrates the significant impact of nanoscale structural reorganization on inhibitory transmission and unveils ongoing modulation of mature GABAergic synapses through active cleavage of NL2 in response to hyperactivity.

Neuroligin1 in excitatory synapses contributes to long-term cognitive impairments after repeated neonatal sevoflurane exposures

BackgroundRepeated sevoflurane exposures in neonatal rats may lead to neuronal apoptosis affecting long-term cognitive function, the mechanism is unknown. Neuroligin1 (NL1) is essential for normal excitatory transmission and long-term synaptic plasticity in the hippocampus of intact animals. Herein, we explore the role of NL1 in hippocampal excitatory synapses on long-term cognitive impairments induced by repeated sevoflurane exposures in neonatal rats. MethodsFrom postnatal day six (P6) to P8, neonatal rats were exposed to 30% oxygen or 3% sevoflurane +30% oxygen for 2 h daily. Rats from each litter were randomly assigned to five groups: control group (Con), native control adeno-associated virus (NC-AAV) group (Con + NC-AAV), sevoflurane group (Sev), sevoflurane + recombinant RNAi adeno-associated virus targeting NL1 downregulation (NL1−-AAV) group (Sev + NL1−-AAV) and control + recombinant RNAi adeno-associated virus targeting NL1 upregulation (NL1+-AAV) group (Con + NL1+-AAV). Animals were injected with NC-AAV or NL1-AAV into the bilateral hippocampal CA1 area and caged on P21. From P35 to P40, behavioral tests including open field (OF), novel object recognition (NOR), and fear conditioning (FC) tests were performed to assess cognitive function in adolescent rats. In another experiment, rat brains were harvested for immunofluorescence staining, western blotting, co-immunoprecipitation, and real-time polymerase chain reaction (PCR). ResultsWe found that the mRNA and protein levels of NL1 were substantially higher in the Sev group than in the Con group. Immunofluorescence showed that NL1 and PSD95 were highly colocalized in hippocampal CA1 area and vesicular GABA transporter (vGAT) around neurons decreased after repeated sevoflurane exposures. Co-immunoprecipitation showed that the amount of PSD95 with NL1 antibody was significantly increased in the Sev group compared to the Con group. These rats had a poorer performance in the NOR and FC tests than control rats when they were adolescents. These results were reversed by NL1−-AAV injection into the CA1 area. NL1+-AAV group was similar to the Sev group. ConclusionWe have demonstrated that repeated neonatal sevoflurane exposures decreased inhibitory synaptic inputs (labelled by vGAT) around neurons, which may influence the upregulation of NL1 in hippocampal excitatory synapses and enhanced NL1/PSD95 interaction, ultimately leading to long-term cognitive impairments in adolescent rats. Injecting NL1−-AAV reversed this damage. These results suggested that NL1 in excitatory synapses contributes to long-term cognitive impairments after repeated neonatal sevoflurane exposures.

Differential contribution of canonical and noncanonical NLGN3 pathways to early social development and memory performance

Neuroligin (NLGN) 3 is a postsynaptic cell adhesion protein organizing synapse formation through two different types of transsynaptic interactions, canonical interaction with neurexins (NRXNs) and a recently identified noncanonical interaction with protein tyrosine phosphatase (PTP) δ. Although, NLGN3 gene is known as a risk gene for neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID), the pathogenic contribution of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ pathways to these disorders remains elusive. In this study, we utilized Nlgn3 mutant mice selectively lacking the interaction with either NRXNs or PTPδ and investigated their social and memory performance. Neither Nlgn3 mutants showed any social cognitive deficiency in the social novelty recognition test. However, the Nlgn3 mutant mice lacking the PTPδ pathway exhibited significant decline in the social conditioned place preference (sCPP) at the juvenile stage, suggesting the involvement of the NLGN3-PTPδ pathway in the regulation of social motivation and reward. In terms of learning and memory, disrupting the canonical NRXN pathway attenuated contextual fear conditioning while disrupting the noncanonical NLGN3-PTPδ pathway enhanced it. Furthermore, disruption of the NLGN3-PTPδ pathway negatively affected the remote spatial reference memory in the Barnes maze test. These findings highlight the differential contributions of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ synaptogenic pathways to the regulation of higher order brain functions associated with ASD and ID.

Frontostriatal circuit dysfunction leads to cognitive inflexibility in neuroligin-3 R451C knockin mice.

Cognitive and behavioral rigidity are observed in various psychiatric diseases, including in autism spectrum disorder (ASD). However, the underlying mechanism remains to be elucidated. In this study, we found that neuroligin-3 (NL3) R451C knockin mouse model of autism (KI mice) exhibited deficits in behavioral flexibility in choice selection tasks. Single-unit recording of medium spiny neuron (MSN) activity in the nucleus accumbens (NAc) revealed altered encoding of decision-related cue and impaired updating of choice anticipation in KI mice. Additionally, fiber photometry demonstrated significant disruption in dynamic mesolimbic dopamine (DA) signaling for reward prediction errors (RPEs), along with reduced activity in medial prefrontal cortex (mPFC) neurons projecting to the NAc in KI mice. Interestingly, NL3 re-expression in the mPFC, but not in the NAc, rescued the deficit of flexible behaviors and simultaneously restored NAc-MSN encoding, DA dynamics, and mPFC-NAc output in KI mice. Taken together, this study reveals the frontostriatal circuit dysfunction underlying cognitive inflexibility and establishes a critical role of the mPFC NL3 deficiency in this deficit in KI mice. Therefore, these findings provide new insights into the mechanisms of cognitive and behavioral inflexibility and potential intervention strategies.

Establishment of transgenic fluorescent mice for labeling synapses and screening synaptogenic adhesion molecules.

Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (Syt1-tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1-tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.

On-Site Formation of Functional Dopaminergic Presynaptic Terminals on Neuroligin-2-Modified Gold-Coated Microspheres.

Advancements in neural interface technologies have enabled the direct connection of neurons and electronics, facilitating chemical communication between neural systems and external devices. One promising approach is a synaptogenesis-involving method, which offers an opportunity for synaptic signaling between these systems. Janus synapses, one type of synaptic interface utilizing synaptic cell adhesion molecules for interface construction, possess unique features that enable the determination of location, direction of signal flow, and types of neurotransmitters involved, promoting directional and multifaceted communication. This study presents the first successful establishment of a Janus synapse between dopaminergic (DA) neurons and abiotic substrates by using a neuroligin-2 (NLG2)-mediated synapse-inducing method. NLG2 immobilized on gold-coated microspheres can induce synaptogenesis upon contact with spatially isolated DA axons. The induced DA Janus synapses exhibit stable synaptic activities comparable to that of native synapses over time, suggesting their suitability for application in neural interfaces. By calling for DA presynaptic organizations, the NLG2-immobilized abiotic substrate is a promising tool for the on-site detection of synaptic dopamine release.

Mice with an autism-associated R451C mutation in neuroligin-3 show intact attention orienting but atypical responses to methylphenidate and atomoxetine in the mouse-Posner task.

Atypical attention orienting has been associated with some autistic symptoms, but the neural mechanisms remain unclear. The human Posner task, a classic attention orienting paradigm, was recently adapted for use with mice, supporting the investigation of the neurobiological underpinnings of atypical attention orienting in preclinical mouse models. The current study tested mice expressing the autism-associated R451C gene mutation in neuroligin-3 (NL3) on the mouse-Posner (mPosner) task. NL3R451C and wild-type (WT) mice were trained to respond to a validly or invalidly cued target on a touchscreen. The cue was a peripheral non-predictive flash in the exogenous task and a central spatially predictive image in the endogenous task. The effects of dopaminergic- and noradrenergic-modulating drugs, methylphenidate and atomoxetine, on task performance were assessed. In both tasks, mice were quicker and more accurate in the validly versus invalidly cued trials, consistent with results in the human Posner task. NL3R451C and WT mice showed similar response times and accuracy but responded differently when treated with methylphenidate and atomoxetine. Methylphenidate impaired exogenous attention disengagement in NL3R451C mice but did not significantly affect WT mice. Atomoxetine impaired endogenous orienting in WT mice but did not significantly affect NL3R451C mice. NL3R451C mice demonstrated intact attention orienting but altered responses to the pharmacological manipulation of the dopaminergic and noradrenergic networks. These findings expand our understanding of the NL3R451C mutation by suggesting that this mutation may lead to selective alterations in attentional processes.

GABAA receptors and neuroligin 2 synergize to promote synaptic adhesion and inhibitory synaptogenesis.

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), β(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and post-synaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2β2γ2-GABAARs versus extrasynaptic type α4β3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2β2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2β2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4β3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2β2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit.

Developmental synapse pathology triggered by maternal exposure to the herbicide glufosinate ammonium

Environmental and genetic factors influence synapse formation. Numerous animal experiments have revealed that pesticides, including herbicides, can disturb normal intracellular signals, gene expression, and individual animal behaviors. However, the mechanism underlying the adverse outcomes of pesticide exposure remains elusive. Herein, we investigated the effect of maternal exposure to the herbicide glufosinate ammonium (GLA) on offspring neuronal synapse formation in vitro. Cultured cerebral cortical neurons prepared from mouse embryos with maternal GLA exposure demonstrated impaired synapse formation induced by synaptic organizer neuroligin 1 (NLGN1)–coated beads. Conversely, the direct administration of GLA to the neuronal cultures exhibited negligible effect on the NLGN1-induced synapse formation. The comparison of the transcriptomes of cultured neurons from embryos treated with maternal GLA or vehicle and a subsequent bioinformatics analysis of differentially expressed genes (DEGs) identified “nervous system development,” including “synapse,” as the top-ranking process for downregulated DEGs in the GLA group. In addition, we detected lower densities of parvalbumin (Pvalb)-positive neurons at the postnatal developmental stage in the medial prefrontal cortex (mPFC) of offspring born to GLA–exposed dams. These results suggest that maternal GLA exposure induces synapse pathology, with alterations in the expression of genes that regulate synaptic development via an indirect pathway distinct from the effect of direct GLA action on neurons.

Multiple N-linked glycosylation sites critically modulate the synaptic abundance of neuroligin isoforms

In recent years, elegant glycomic and glycoproteomic approaches have revealed an intricate glycosylation profile of mammalian brain with enormous spatial and temporal diversities. Nevertheless, at a cellular level, it is unclear how these post-translational modifications affect various proteins to influence crucial neuronal properties. Here, we have investigated the impact of N-linked glycosylation on neuroligins (NLGNs), a class of cell-adhesion molecules that play instructive roles in synapse organization. We found that endogenous NLGN proteins are differentially glycosylated across several regions of murine brain in a sex-independent but isoform-dependent manner. In both rodent primary neurons derived from brain sections and human neurons differentiated from stem cells, all NLGN variants were highly enriched with multiple N-glycan subtypes, which cumulatively ensured their efficient trafficking to the cell surface. Removal of these N-glycosylation residues only had a moderate effect on NLGNs' stability or expression levels but particularly enhanced their retention at the endoplasmic reticulum. As a result, the glycosylation-deficient NLGNs exhibited considerable impairments in their dendritic distribution and postsynaptic accumulation, which in turn, virtually eliminated their ability to recruit presynaptic terminals and significantly reduced NLGN overexpression-induced assemblies of both glutamatergic and GABAergic synapse structures. Therefore, our results highlight an essential mechanistic contribution of N-linked glycosylations in facilitating the appropriate secretory transport of a major synaptic cell-adhesion molecule and promoting its cellular function in neurons.