Regulation of Neuroligins and Effects on Synapse Number and Function

神经连接蛋白(NLGNs)是脑特异性细胞黏附分子。它们表达在突触后膜上，并与跨越突触间隙的突触前神经尿苷(NRXN)结合。有趣的是，在自闭症谱系障碍(ASD)患者中已经发现了NLGNs和NRXN的突变。这导致研究人员开发了基因工程NLGN小鼠模型来研究ASD的病因学。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亚型高度保守，但表现出明显的突触定位。然而，调控异构体特异性靶向和定位的分子机制还不是很清楚。我们专注于蛋白质-蛋白质相互作用和翻译后修饰在决定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连接素异构体上鉴定了几个不同的磷酸化位点。我们一直在研究相关的激酶及其与突触形成的生理相关性。 自闭症是一组神经发育障碍，具有较高的遗传易感性，男性的发病率高于女性。在具有智能障碍和自闭症特征的患者中，已在X连锁的NLGN3和4X中发现了各种点突变。有趣的是，迄今为止报道的所有与自闭症相关的NLGN3和NLGN4X点突变都位于它们的胞外区，除了精氨酸(R)704的NLGN4X胞内区的一个点突变，它被修改为半胱氨酸(C)。我们发现，在人类胚胎神经元中，内源性NLGN4X被T707处的蛋白激酶C(PKC)强烈磷酸化。这种自闭症突变(R704C)消除了T707的磷酸化，这是NLGN4X介导的兴奋性增强的关键。有趣的是，与其他NLGN ASD相关突变不同，R704C不会破坏NLGN4X的稳定性或表面表达，但仍会导致突触功能障碍。自闭症的小鼠模型揭示了兴奋性/抑制性传递失衡的作用，通常导致抑制性传递的直接增加。我们的结果建立了基因突变、关键的翻译后修饰和强大的突触变化之间的潜在因果关系，并将为阐明ASD的病理生理学提供见解。 在人类中也有NLGN-4Y，它位于Y染色体上，与NLGN-4X几乎相同。事实上，NL-4X和NL-4Y在胞外区只有8个氨基酸差异，在胞内区有5个氨基酸差异。然而，目前还没有关于NLGN-4Y磷酸化的研究。我们现在用不同的激酶结合质谱学比较了4X和4Y的PKC磷酸化，发现NL-4X和NL-4Y在不同的残基上被磷酸化。重要的是，PKC T707位点的磷酸化水平存在差异。这些结果表明，NL-4X和NL-4Y以一种根本不同的方式受到调控，我们正在跟进这些发现。我们相信，通过对NLGNs性别相关亚型的更好研究，我们希望了解与自闭症相关的性别偏见。 NLGN-1和支架蛋白PSD-95均定位于兴奋性突触。此外，NLGN-1通过胞质尾部的PDZ配体与PSD-95结合。在体外和异种细胞中，我们已经在PDZ配体附近的NLGN-1上发现了蛋白激酶A(PKA)的磷酸化位点。当我们在NLGN-1PKA位点引入模拟磷酸化突变时，NLGN-1与PSD-95之间的相互作用在体外和原位都减少了。因此，我们发现磷酸化调节PSD-95与NLGN-1的结合，就像我们对NMDARs所显示的那样。 我们还证明了磷酸化调节NLGN-2，NLGN-2是定位于抑制性突触并具有特定功能的神经连接素异构体。特别是，NLGN-2是用质谱仪检测到的PKA的底物。我们已经产生了针对这个位点的磷酸特异性抗体，并证明了它的特异性。有了这个伟大的工具，我们现在正在研究NLGN-2在体内的磷酸化和调节。NLGN-2的磷酸化受突触活性的调节，我们正在研究这一过程的确切机制。 最后，神经连接素胞外域的裂解是对突触活动的反应。我们已经发现了这种裂解的异构体特异性调节，并正在研究其调节的潜在机制。特别是，我们正在研究参与神经连接蛋白切割的蛋白水解酶和神经连接蛋白中的分子决定因素。

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 autism-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 autism 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. Mouse models of autism have uncovered a role for an imbalance of excitatory/inhibitory transmission, often resulting in direct increases in inhibitory transmission. 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 NLGN-4Y, which is located on the Y chromosome and is almost identical to NLGN-4X. In fact, NL-4X and NL-4Y have only eight amino acid differences in the extracellular domain and five in the intracellular domain. However, there are no studies on phosphorylation of NLGN-4Y. We have now compared the PKC phosphorylation of 4X vs 4Y using different kinases in conjunction with mass spectrometry, and find that NL-4X and NL-4Y are phosphorylated at different residues. Importantly, there is a difference in the levels of the phosphorylation of the PKC T707 site. These results suggest that NL-4X and NL-4Y are regulated in a fundamentally different manner, and we are following up on these findings. We believe through a better investigation of the sex-linked isoforms of NLGNs, we hope to understand the sex bias associated with ASDs. NLGN-1 and the scaffolding protein PSD-95 are both localized at excitatory synapses. In addition, NLGN-1 binds to PSD-95 through the PDZ ligand in the cytoplasmic tail. We have identified a protein kinase A (PKA) phosphorylation site on NLGN-1, 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 NLGN-1 and PSD-95 is decreased in vitro and in situ. Therefore, we find that phosphorylation regulates PSD-95 binding to NLGN-1, just as we have shown for NMDARs. We also have demonstrated that phosphorylation regulates NLGN-2, the neuroligin isoform that is located and specifically functions at inhibitory synapses. In particular, NLGN-2 is a substrate for PKA as detected using mass spectrometry. We have generated a phospho-specific antibody against this site, and demonstrate its specificity. With this great tool, we are now characterizing NLGN-2 phosphorylation and regulation in vivo. NLGN-2 phosphorylation is regulated by synaptic activity, and we are investigating the precise mechanisms by which this occurs. Finally, neuroligins undergo cleavage of their extracellular domain in response to synaptic activity. We have uncovered an isoform-specific regulation of this cleavage and are studying the mechanisms underlying its regulation. In particular, we are studying the proteases involved in neuroligin cleavage and the molecular determinants within the neuroligins.