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。 NLGN1的定位和突触效应是兴奋性突触所特有的，NLGN1在兴奋性突触上增强突触蛋白的募集并增强兴奋性突触传递。这些NLGN1效应依赖于突触活性和CaMKII。我们最近发现CaMKII强效和特异性地磷酸化NLGN1的胞内结构域。我们发现NLGN1有一个单一的显性CaMKII位点T739，它在培养的神经元和体内对视觉刺激的突触活动中被磷酸化。此外，一个缺磷突变体(T739A)降低了NLGN1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减少。我们的发现首次显示了CaMKII和NLGN1之间的直接功能相互作用，这是兴奋性突触的两个主要成分。 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。

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.