Neuroligin-2 as a central organizer of inhibitory synapses in health and disease (original) (raw)
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Genes, Brain and Behavior, 2009
Neuroligins (NL) are postsynaptic cell adhesion molecules that are thought to specify synapse properties. Previous studies showed that mutant mice carrying an autismassociated point mutation in NL3 exhibit social interaction deficits, enhanced inhibitory synaptic function and increased staining of inhibitory synaptic puncta without changes in overall inhibitory synapse numbers. In contrast, mutant mice lacking NL2 displayed decreased inhibitory synaptic function. These studies raised two relevant questions. First, does NL2 deletion impair inhibitory synaptic function by altering the number of inhibitory synapses, or by changing their efficacy? Second, does this effect of NL2 deletion on inhibition produce behavioral changes? We now show that although NL2-deficient mice exhibit an apparent decrease in number of inhibitory synaptic puncta, the number of symmetric synapses as determined by electron microscopy is unaltered, suggesting that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers. This decrease in inhibitory synaptic function in NL2-deficient mice correlates with a discrete behavioral phenotype that includes a marked increase in anxiety-like behavior, a decrease in pain sensitivity and a slight decrease in motor co-ordination. This work confirms that NL2 modulates inhibitory synaptic function and is the first demonstration that global deletion of NL2 can lead to a selective behavioral phenotype.
Proceedings of the National Academy of Sciences, 2013
The MAM domain-containing GPI anchor proteins MDGA1 and MDGA2 are Ig superfamily adhesion molecules composed of six IG domains, a fibronectin III domain, a MAM domain, and a GPI anchor. MDGAs contribute to the radial migration and positioning of a subset of cortical neurons during early neural development. However, MDGAs continue to be expressed in postnatal brain, and their functions during postnatal neural development remain unknown. Here, we demonstrate that MDGAs specifically and with a nanomolar affinity bind to neuroligin-2, a cell-adhesion molecule of inhibitory synapses, but do not bind detectably to neuroligin-1 or neuroligin-3. We observed no cell adhesion between cells expressing neuroligin-2 and MDGA1, suggesting a cis interaction. Importantly, RNAi-mediated knockdown of MDGAs increased the abundance of inhibitory but not excitatory synapses in a neuroligin-2dependent manner. Conversely, overexpression of MDGA1 decreased the numbers of functional inhibitory synapses. Likewise, coexpression of both MDGA1 and neuroligin-2 reduced the synaptogenic capacity of neuroligin-2 in an artificial synapse-formation assay by abolishing the ability of neuroligin-2 to form an adhesion complex with neurexins. Taken together, our data suggest that MDGAs inhibit the activity of neuroligin-2 in controlling the function of inhibitory synapses and that MDGAs do so by binding to neuroligin-2.
Neuron, 2017
Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) bind directly to neuroligin-1 (NL1) and neuroligin-2 (NL2), thereby respectively regulating excitatory and inhibitory synapse development. However, the mechanisms by which MDGAs modulate NL activity to specify development of the two synapse types remain unclear. Here, we determined the crystal structures of human NL2/MDGA1 Ig1-3 complex, revealing their stable 2:2 arrangement with three interaction interfaces. Cell-based assays using structure-guided, site-directed MDGA1 mutants showed that all three contact patches were required for the MDGA's negative regulation of NL2-mediated synaptogenic activity. Furthermore, MDGA1 competed with neurexins for NL2 via its Ig1 domain. The binding affinities of both MDGA1 and MDGA2 for NL1 and NL2 were similar, consistent with the structural prediction of similar binding interfaces. However, MDGA1 selectively associated with NL2, but not NL1, in v...
Neuroligin-1 Loss Is Associated with Reduced Tenacity of Excitatory Synapses
Neuroligins (Nlgns) are postsynaptic, integral membrane cell adhesion molecules that play important roles in the formation, validation, and maturation of synapses in the mammalian central nervous system. Given their prominent roles in the life cycle of synapses, it might be expected that the loss of neuroligin family members would affect the stability of synaptic organization, and ultimately, affect the tenacity and persistence of individual synaptic junctions. Here we examined whether and to what extent the loss of Nlgn-1 affects the dynamics of several key synaptic molecules and the constancy of their contents at individual synapses over time. Fluorescently tagged versions of the postsynaptic scaffold molecule PSD-95, the AMPA-type glutamate receptor subunit GluA2 and the presynaptic vesicle molecule SV2A were expressed in primary cortical cultures from Nlgn-1 KO mice and wild-type (WT) littermates, and live imaging was used to follow the constancy of their contents at individual synapses over periods of 8-12 hours. We found that the loss of Nlgn-1 was associated with larger fluctuations in the synaptic contents of these molecules and a poorer preservation of their contents at individual synapses. Furthermore, rates of synaptic turnover were somewhat greater in neurons from Nlgn-1 knockout mice. Finally, the increased GluA2 redistribution rates observed in neurons from Nlgn-1 knockout mice were negated by suppressing spontaneous network activity. These findings suggest that the loss of Nlgn-1 is associated with some use-dependent destabilization of excitatory synapse organization, and indicate that in the absence of Nlgn-1, the tenacity of excitatory synapses might be somewhat impaired.
Journal of Neuroscience, 28(24): 6055-6067; doi: 10.1523/JNEUROSCI.0032-08.2008, 2008
The level of excitation in the brain is kept under control through inhibitory signals mainly exerted by GABA neurons. However, the molecular machinery that regulates the balance between excitation and inhibition (E/I) remains unclear. Candidate molecules implicated in this process are neuroligin (NL) adhesion molecules, which are differentially enriched at either excitatory or inhibitory contacts. In this study, we use transgenic mouse models expressing NL1 or NL2 to examine whether enhanced expression of specific NLs results in synaptic imbalance and altered neuronal excitability and animal behavior. Our analysis reveals several abnormalities selectively manifested in transgenic mice with enhanced expression of NL2 but not NL1. A small change in NL2 expression results in enlarged synaptic contact size and vesicle reserve pool in frontal cortex synapses and an overall reduction in the E/I ratio. The frequency of miniature inhibitory synaptic currents was also found to be increased in the frontal cortex of transgenic NL2 mice. These animals also manifested stereotyped jumping behavior, anxiety, impaired social interactions, and enhanced incidence of spike-wave discharges, as depicted by EEG analysis in freely moving animals. These findings may provide the neural basis for E/I imbalance and altered behavior associated with neurodevelopmental disorders.
A Combined Transgenic Proteomic Analysis and Regulated Trafficking of Neuroligin-2
Journal of Biological Chemistry, 2014
Background: Brain inhibitory synaptic connections present challenges for molecular identification and imaging. Results: Transgenic mice expressing tagged neuroligin-2 were used to identify associated complexes and image inhibitory synapses in multiple brain regions. Conclusion: Neuroligin-2-associated complexes are enriched in synaptic components, and neuroligin-2 undergoes regulated dynamin-dependent endocytosis and retromer association. Significance: New data and approaches are presented for brain inhibitory synapse proteomics and imaging. Synapses, the basic units of communication in the brain, require complex molecular machinery for neurotransmitter release and reception. Whereas numerous components of excitatory postsynaptic sites have been identified, relatively few proteins are known that function at inhibitory postsynaptic sites. One such component is neuroligin-2 (NL2), an inhibitory synapse-specific cell surface protein that functions in cell adhesion and synaptic organization via binding to neurexins. In this study, we used a transgenic tandem affinity purification and mass spectrometry strategy to isolate and characterize NL2-associated complexes. Complexes purified from brains of transgenic His 6-FLAG-YFP-NL2 mice showed enrichment in the Gene Ontology terms cell-cell signaling and synaptic transmission relative to complexes purified from wild type mice as a negative control. In addition to expected components including GABA receptor subunits and gephyrin, several novel proteins were isolated in association with NL2. Based on the presence of multiple components involved in trafficking and endocytosis, we showed that NL2 undergoes dynamin-dependent endocytosis in response to soluble ligand and colocalizes with VPS35 retromer in endosomes. Inhibitory synapses in brain also present a particular challenge for imaging. Whereas excitatory synapses on spines can be imaged with a fluorescent cell fill, inhibitory synapses require a molecular tag. We find the His 6-FLAG-YFP-NL2 to be a suitable tag, with the unamplified YFP signal localizing appropriately to inhibitory synapses in multiple brain regions including cortex, hippocampus, thalamus, and basal ganglia. Altogether, we characterize NL2-associated complexes, demonstrate regulated trafficking of NL2, and provide tools for further proteomic and imaging studies of inhibitory synapses.
Neurexins, Neuroligins and LRRTMs: synaptic adhesion getting fishy
Journal of Neurochemistry, 2011
Recent studies have identified the leucine rich repeat protein LRRTM2 as a postsynaptic ligand of Neurexins. Neurexins also bind the postsynaptic adhesion molecules, Neuroligins. All three families of genes have been implicated in the etiologies of neurodevelopmental disorders, specifically autism spectrum disorders (ASDs) and schizophrenia. Does the binding promiscuity of Neurexins now suggest complex cooperativity or redundancy at the synapse? While recent studies in primary neuronal cultures and also systematic extracellular protein interaction screens suggest summative effects of these systems, we propose that studying these interactions in the developing zebrafish embryo or larvae may shed more light on their functions during synaptogenesis in vivo. These gene families have recently been extensively characterized in zebrafish, demonstrating high sequence conservation with the human genes. The simpler circuitry of the zebrafish, together with the characterization of the expression patterns down to single, identifiable neurons and the ability to knock-down or overexpress multiple genes in a rapid way lend themselves to dissecting complex interaction pathways. Furthermore, the capability of performing high-throughput drug screens suggests that these small vertebrates may prove extremely useful in identifying pharmacological approaches to treating ASDs.
The function of GABAergic synapses is critically shaped by cell adhesion proteins that recruit GABAARs to synapses and mediate transsynaptic signalling, but the synapse-type-specific function of such synaptic adhesion proteins and their mutual interaction remain incompletely understood. A ubiquitous cell adhesion protein at GABAergic synapses is Neuroligin-2 (Nlgn2), which recruits synaptic GABAARs by promoting the assembly of the postsynaptic gephyrin scaffold. While Nlgn2 is present at virtually all GABAergic synapses throughout the forebrain, its loss affects different GABAergic synapse subtypes with different severity, indicating that synapse-specific interactors and synapse-organizer-redundancies define the function of Nlgn2 for a given synapse type. Here we investigated how Nlgn2 function at GABAergic synapses in mouse hippocampal area CA1 is modulated by two recently identified interaction partners, MDGA1 and MDGA2. We show that Nlgn2 and MDGA1 colocalize most prominently in ...