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。 NLGN 1的定位和突触效应对兴奋性突触是特异性的，在兴奋性突触处NLGN 1增强突触蛋白的募集并增强兴奋性突触传递。这些NLGN 1效应依赖于突触活性和CaMKII。我们最近发现，CaMKII稳健和特异性磷酸化NLGN 1的细胞内结构域。我们发现，NLGN 1有一个单一的优势CaMKII位点，T739，这是磷酸化的突触活动在培养的神经元和在体内响应视觉刺激。此外，磷酸缺陷突变体（T739 A）减少了NLGN 1的基础和活性驱动的表面表达，导致NLGN介导的兴奋性突触增强作用减少。我们的研究结果是第一次显示CaMKII和NLGN 1，兴奋性突触的两个主要组成部分之间的直接功能相互作用。 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。

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. The localization and synaptic effects of NLGN1 are specific to excitatory synapses at which NLGN1 enhances the recruitment of synaptic proteins and potentiates excitatory synaptic transmission. These NLGN1 effects are dependent on synaptic activity and CaMKII. We recently found that CaMKII robustly and specifically phosphorylates the intracellular domain of NLGN1. We showed that NLGN1 has a single dominant CaMKII site, T739, which is phosphorylated in response to synaptic activity in cultured neurons and in vivo in response to visual stimulation. Furthermore, a phospho-deficient mutant (T739A) reduces the basal and activity-driven surface expression of NLGN1, leading to a reduction in NLGN-mediated excitatory synaptic potentiation. Our findings are the first to show a direct functional interplay between CaMKII and NLGN1, two primary components of excitatory synapses. 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.