Regulation of Neuroligins and Effects on Synapse Number and Function

神经胶质素（Neuroligins，NLGNs）是脑特异性细胞粘附分子。它们在突触后膜上表达，并与跨越突触间隙的突触前神经毒素（NRXN）结合。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NLGN小鼠模型来研究ASD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，但显示不同的突触定位。然而，调节异构体特异性靶向和定位的分子机制还不清楚。我们专注于蛋白质-蛋白质相互作用和翻译后修饰在决定NLGN运输和功能调节中的作用。在过去的几年中，我们已经确定了不同的神经配素亚型上的几个不同的磷酸化位点。我们一直在研究与突触形成有关的激酶及其生理相关性。 ASD是一组神经发育障碍，具有较高的遗传易感性，男性的发生率高于女性。在患有智力残疾和ASD特征性症状的患者中，已在X连锁NLGN 3和4X中鉴定出多种点突变。有趣的是，迄今为止报道的NLGN 3和NLGN 4X中的所有自闭症相关点突变都存在于它们的胞外结构域中，除了NLGN 4X的胞内结构域中精氨酸（R）704处的单个点突变，其被修饰为半胱氨酸（C）。我们发现，内源性NLGN 4X是强大的磷酸化蛋白激酶C（PKC）在T707人胚胎神经元。这种自闭症突变（R704 C）消除了T707磷酸化，这对NLGN 4X介导的兴奋性增强至关重要。有趣的是，与其他NLGN ASD相关突变不同，R704 C没有破坏NLGN 4X的稳定性或表面表达，但仍然导致突触功能障碍。自闭症的小鼠模型已经揭示了兴奋性/抑制性传递失衡的作用，通常导致抑制性传递的直接增加。我们的研究结果建立了一个潜在的因果关系之间的基因突变，一个关键的翻译后修饰，和强大的突触变化，并将提供见解，阐明ASD的病理生理学。 在人类中也有NLGN-4 Y，它位于Y染色体上，与NLGN-4X几乎相同。事实上，NL-4X和NL-4 Y在细胞外结构域中只有8个氨基酸差异，在细胞内结构域中只有5个氨基酸差异。然而，没有关于NLGN-4 Y磷酸化的研究。我们现在已经使用不同的激酶结合质谱法比较了4X与4 Y的PKC磷酸化，发现NL-4X和NL-4 Y在不同的残基处磷酸化。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-4 Y的调节方式根本不同，我们正在跟进这些发现。我们相信，通过对NLGN性连锁亚型的更好研究，我们希望了解与ASD相关的性别偏见。 NLGN-1和支架蛋白PSD-95都位于兴奋性突触。此外，NLGN-1通过胞质尾中的PDZ配体与PSD-95结合。我们已经确定了一个蛋白激酶A（PKA）的磷酸化位点NLGN-1，附近的PDZ配体，在体外和异源细胞。当我们在NLGN-1 PKA位点引入磷酸模拟突变时，NLGN-1和PSD-95之间的相互作用在体外和原位都降低了。此外，拟磷酸化突变体显示减少的表面表达。因此，我们发现磷酸化调节PSD-95与NLGN-1的结合和运输，就像我们对NMDAR所显示的那样。 除了磷酸化，神经配素还受到其他翻译后修饰的调节。具体地，神经配蛋白响应于突触活性而经历其细胞外结构域的裂解。我们已经发现了这种裂解的亚型特异性调节，其中PKC活化显著增加NLGN 3裂解。这是及时的，因为NLGN 3的胞外域已被鉴定为调节胶质瘤增殖的有丝分裂原。我们正在研究NLGN 3切割的调节机制。特别是，我们正在研究的蛋白酶参与神经连接蛋白的切割和内的神经连接蛋白的分子决定簇。 我们还开始研究与神经配素相互作用的其他蛋白质。RhoGEF kalirin-7是脑特异性kalirin同种型，被认为是突触后密度的重要信号传导枢纽。kalirin-7调节突触传递的机制，特别是哪些蛋白质-蛋白质相互作用是重要的，在很大程度上仍然未知。为了研究kalirin-7相互作用，我们已经开发了一种kalirin-7特异性抗体，并使用它来免疫沉淀内源性蛋白质，以筛选潜在的相互作用，使用LC MS/MS。潜在的命中，细胞粘附分子的神经配蛋白家族的成员是特别感兴趣的，因为它们的表型和亚细胞定位非常相似的kalirin-7。使用体外和体内技术，我们已经验证了这种相互作用，表明kalirin-7可以与神经配蛋白家族的所有成员相互作用，但不是所有kalirin的亚型都可以与神经配蛋白相互作用。我们目前正在更精确地定义交互域。这些数据已经开始建立一个新的异构体之间的特异性相互作用，这两个主要的蛋白质的突触后密度。

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. Furthermore, a phosphomimetic mutant displays reduced surface expression. Therefore, we find that phosphorylation regulates PSD-95 binding to NLGN-1 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 also embarked on a study of 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 are currently defining the interactions domains with greater precision. These data have begun to establish a novel isoform-specific interaction between these two major proteins of the postsynaptic density.