Regulation of Neuroligins and Effects on Synapse Number and Function

神经素（nlgn）是脑特异性细胞粘附分子。它们在突触后膜上表达，并结合跨越突触间隙的突触前神经素（NRXNs）。有趣的是，在自闭症谱系障碍（ASD）患者中发现了nlgn和nrxn的突变。这使得研究人员开发了基因工程的NLGN小鼠模型来研究自闭症的病因。这些研究表明，NLGN功能障碍可以改变大脑中抑制和兴奋的平衡。NLGN亚型是高度保守的，但显示出不同的突触定位。然而，调控异构体特异性靶向和定位的分子机制尚不清楚。我们专注于蛋白质-蛋白质相互作用和翻译后修饰在NLGN运输和功能调节中的作用。在过去的几年里，我们已经在不同的神经素异构体上发现了几个不同的磷酸化位点。我们一直在描述所涉及的激酶及其与突触形成的生理相关性。

Neuroligins (NLGNs) are brain-specific cell adhesion molecules. They are expressed on the postsynaptic membrane and bind to presynaptic neurexins (NRXNs) spanning the synaptic cleft. Interestingly, mutations in both NLGNs and NRXNs have been identified in Autism Spectrum Disorder (ASD) patients. This has led researchers to develop genetically engineered NLGN mouse models to study the etiology of ASDs. These studies have shown that NLGN dysfunction can shift the balance of inhibition and excitation in the brain. NLGN isoforms are highly conserved, yet display distinct synaptic localizations. However, the molecular mechanisms that regulate isoform-specific targeting and localization are not well understood. We focus on the role of protein-protein interactions and post-translational modifications in dictating NLGN trafficking and functional regulation. Over the last few years we have identified several different phosphorylation sites on the different neuroligin isoforms. We have been characterizing the kinases involved and the physiological relevance to synapse formation. ASDs are a group of neurodevelopmental disorders that have a high genetic predisposition and higher occurrence rates in males than females. A variety of point mutations have been identified in X-linked NLGN3 and 4X in patients with intellectual disability and symptoms characteristic of ASDs. Interestingly, all of the ASD-associated point mutations in NLGN3 and NLGN4X reported thus far reside in their extracellular domains except for a single point mutation in the intracellular domain of NLGN4X at arginine (R) 704, which is modified to a cysteine (C). We discovered that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707 in human embryonic neurons. This ASD-associated mutation (R704C) eliminates T707 phosphorylation, which is critical for NLGN4X-mediated excitatory enhancement. Interestingly, unlike other NLGN ASD-associated mutations, R704C, did not disrupt the stability or surface expression of NLGN4X, yet still led to synaptic dysfunction. Our results establish a potential causality between a genetic mutation, a key posttranslational modification, and robust synaptic changes and will provide insights in elucidating the pathophysiology of ASDs. In human there is also NLGN4Y, which is located on the Y chromosome and is almost identical to NLGN4X. In fact, NLGN4X and NLGN4Y have only eight amino acid differences in the extracellular domain and five in the intracellular domain. We just published our recent findings showing that NLGN4Y has a trafficking defect. Specifically, we used biochemistry, electrophysiology, and imaging analyses to study NLGN4Y and identified severe deficits in maturation, surface expression, and synaptogenesis compared to NLGN4X. Strikingly, the functional differences were primarily regulated by one amino acid difference with NLGN4X (P93 in NLGN4X, but S93 in NLGN4Y). Furthermore, we analyzed ASD-associated mutations in NLGN4X and identified a cluster in the region surrounding S93 in NLGN4X. Importantly, these de novo mutations identified in patients phenocopied NLGN4Y. Because NLGN4Y cannot compensate for the trafficking and functional deficits observed in ASD-associated NLGN4X mutations, our data reveal a potential pathogenic mechanism for male bias in NLGN4X-associated ASD. We continue to study NLGN4X and 4Y and believe that a better investigation of the sex-linked isoforms of NLGNs will lead to better insight regarding the sex bias associated with some cases of ASD. In another study, we characterized a functional interplay between NLGN1 and PSD-95. NLGN1 and the scaffolding protein PSD-95 are both localized at excitatory synapses. In addition, NLGN1 binds to PSD-95 through the PDZ ligand in the cytoplasmic tail. We identified a protein kinase A (PKA) phosphorylation site on NLGN1, near the PDZ ligand, in vitro and in heterologous cells. When we introduce a phospho-mimetic mutation at the NLGN-1 PKA site, the interaction between NLGN1 and PSD-95 is decreased in vitro and in situ. Furthermore, a phosphomimetic mutant displays reduced surface expression. Therefore, we find that phosphorylation regulates PSD-95 binding to NLGN1 and trafficking, just as we have shown for NMDARs. In addition to phosphorylation, neuroligins are regulated by other posttranslational modifications. Specifically, neuroligins undergo cleavage of their extracellular domain in response to synaptic activity. We have uncovered an isoform-specific regulation of this cleavage in which PKC activation dramatically increases NLGN3 cleavage. This is timely since the ectodomain of NLGN3 has been identified as a mitogen that regulates glioma proliferation. We are studying the mechanisms underlying the regulation of NLGN3 cleavage. In particular, we are studying the proteases involved in neuroligin cleavage and the molecular determinants within the neuroligins. We have generated an HA-tagged NLGN3 knock-in mouse so that we can track the endogenous cleavage of NLGN3 in vivo. We are currently using this mouse line to study NLGN3 cleavage and also the NLGN3 interactome. We have also studied other proteins that interact with neuroligins. The RhoGEF kalirin-7 is a brain-specific kalirin isoform thought to be an important signaling hub at the postsynaptic density. The mechanisms by which kalirin-7 regulates synaptic transmission, particularly which protein-protein interactions are important, remain largely unknown. To study kalirin-7 interactors, we have developed a kalirin-7 specific antibody and have used it to immunoprecipitate endogenous protein to screen for potential interactions using LC MS/MS. Of potential hits, members of the neuroligin family of cell adhesion molecules were of particular interest given that their phenotype and subcellular localization closely resembles that of kalirin-7. Using both in vitro and in vivo techniques we have validated this interaction, showing that kalirin-7 can interact with all members of the neuroligin family, but not all isoforms of kalirin can interact with neuroligins. We find that NLGN-dependent potentiation of synapses and spine growth are mediated, at least in part, by kalirin-7. Thus we identified the first downstream effector of NLGN1.