Regulation of Neuroligins and Effects on Synapse Number and Function

Neuroligins 的调节及其对突触数量和功能的影响

• 批准号: 10263050
• 负责人: Katherine Roche
• 金额: $ 258.46万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10263050
• 关键词: Amino Acids; Antibodies; Arginine; Binding; Binding Proteins; Biochemistry; Biological; Brain; Cell Adhesion Molecules; Cells; Characteristics; Chemosensitization; Cyclic AMP-Dependent Protein Kinases; Cysteine; Cytoplasmic Tail; DNA Sequence Alteration; Data; Defect; Development; Electrophysiology (science); Embryo; Equilibrium; Etiology; Excitatory Synapse; Extracellular Domain; Family; Female; Functional disorder; Gene Family; Genetic Engineering; Genetic Predisposition to Disease; Glioma; Growth; Human; Image; In Situ; In Vitro; Inhibitory Synapse; Intellectual functioning disability; Investigation; Knock-in Mouse; Ligands; Link; Maintenance; Mediating; Mediator of activation protein; Mitogens; Molecular; Mus; Mutation; NRCAM gene; Neurodevelopmental Disorder; Neurons; Pathogenicity; Patients; Peptide Hydrolases; Phenocopy; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Point Mutation; Post-Translational Protein Processing; Postsynaptic Membrane; Protein Family; Protein Isoforms; Protein Kinase; Protein Kinase C; Proteins; Publishing; Regulation; Reporting; Research Personnel; Rodent; Role; Scaffolding Protein; Sex Bias; Signal Transduction; Site; Surface; Symptoms; Synapses; Synaptic Cleft; Synaptic Transmission; Techniques; Time; Vertebral column; Y Chromosome; autism spectrum disorder; autistic; de novo mutation; density; in vivo; insight; interest; male; member; mimetics; mouse model; mutant; neuroligin 1; postsynaptic; presynaptic; protein protein interaction; response; sex; synaptogenesis; trafficking

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.

Neuroligins (NLGN) 是大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，自闭症谱系障碍 (ASD) 患者中已发现 NLGN 和 NRXN 突变。这促使研究人员开发出基因工程 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抑制和兴奋的平衡。 NLGN 亚型高度保守，但显示出不同的突触定位。然而，调节异构体特异性靶向和定位的分子机制尚不清楚。我们重点研究蛋白质-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，我们在不同的神经肽亚型上鉴定了几个不同的磷酸化位点。我们一直在表征所涉及的激酶以及与突触形成的生理相关性。 自闭症谱系障碍（ASD）是一组具有高度遗传倾向的神经发育障碍，男性发病率高于女性。在患有智力障碍和 ASD 症状特征的患者中，X 连锁 NLGN3 和 4X 中发现了多种点突变。有趣的是，迄今为止报道的 NLGN3 和 NLGN4X 中所有与 ASD 相关的点突变都位于其细胞外结构域，除了 NLGN4X 细胞内结构域精氨酸 (R) 704 处的单点突变（该突变被修饰为半胱氨酸 (C)）。我们发现内源性 NLGN4X 在人胚胎神经元的 T707 处被蛋白激酶 C (PKC) 强烈磷酸化。这种 ASD 相关突变 (R704C) 消除了 T707 磷酸化，这对于 NLGN4X 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，R704C 不会破坏 NLGN4X 的稳定性或表面表达，但仍然导致突触功能障碍。我们的结果建立了基因突变、关键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。 人类中也存在NLGN4Y，它位于Y染色体上，与NLGN4X几乎相同。事实上，NLGN4X 和 NLGN4Y 在胞外结构域中只有 8 个氨基酸差异，在胞内结构域中只有 5 个氨基酸差异。我们刚刚发布了最新的研究结果，表明 NLGN4Y 存在贩运缺陷。具体来说，我们使用生物化学、电生理学和成像分析来研究 NLGN4Y，并发现与 NLGN4X 相比，其在成熟、表面表达和突触发生方面存在严重缺陷。引人注目的是，功能差异主要由与 NLGN4X 的一个氨基酸差异（NLGN4X 中的 P93，但 NLGN4Y 中的 S93）调节。此外，我们分析了 NLGN4X 中与 ASD 相关的突变，并在 NLGN4X 中 S93 周围区域鉴定了一个簇。重要的是，这些从头突变是在 NLGN4Y 表型复制的患者中发现的。由于 NLGN4Y 无法补偿 ASD 相关 NLGN4X 突变中观察到的运输和功能缺陷，因此我们的数据揭示了 NLGN4X 相关 ASD 中男性偏见的潜在致病机制。我们继续研究 NLGN4X 和 4Y，并相信对 NLGN 的性别相关异构体进行更好的研究将有助于更好地了解与某些 ASD 病例相关的性别偏见。 在另一项研究中，我们描述了 NLGN1 和 PSD-95 之间的功能相互作用。 NLGN1 和支架蛋白 PSD-95 均位于兴奋性突触。此外，NLGN1 通过细胞质尾部的 PDZ 配体与 PSD-95 结合。我们在体外和异源细胞中鉴定了 NLGN1 上 PDZ 配体附近的蛋白激酶 A (PKA) 磷酸化位点。当我们在 NLGN-1 PKA 位点引入磷酸模拟突变时，NLGN1 和 PSD-95 之间的相互作用在体外和原位都会减少。此外，拟磷突变体表现出表面表达减少。因此，我们发现磷酸化调节 PSD-95 与 NLGN1 的结合和运输，正如我们对 NMDAR 的研究一样。 除了磷酸化之外，neuroligin 还受到其他翻译后修饰的调节。具体而言，神经肽响应突触活动而对其胞外结构域进行裂解。我们发现了这种裂解的异构体特异性调节，其中 PKC 激活显着增加了 NLGN3 裂解。这是及时的，因为 NLGN3 的胞外域已被确定为调节神经胶质瘤增殖的有丝分裂原。我们正在研究 NLGN3 裂解调控的机制。特别是，我们正在研究参与神经连接蛋白裂解的蛋白酶以及神经连接蛋白内的分子决定因素。我们已经生成了 HA 标记的 NLGN3 敲入小鼠，以便我们可以在体内追踪 NLGN3 的内源性裂解。我们目前正在使用该小鼠系来研究 NLGN3 裂解以及 NLGN3 相互作用组。 我们还研究了与神经连接蛋白相互作用的其他蛋白质。 RhoGEF kalirin-7 是一种大脑特异性的 kalirin 亚型，被认为是突触后密度的重要信号中枢。 Kalirin-7 调节突触传递的机制，特别是哪些蛋白质-蛋白质相互作用很重要，目前仍然很大程度上未知。为了研究 kalirin-7 相互作用蛋白，我们开发了一种 kalirin-7 特异性抗体，并用它免疫沉淀内源蛋白，以使用 LC MS/MS 筛选潜在的相互作用。在潜在的热门产品中，细胞粘附分子的 Neuroligin 家族成员特别令人感兴趣，因为它们的表型和亚细胞定位与 kalirin-7 非常相似。使用体外和体内技术，我们验证了这种相互作用，表明 Kalirin-7 可以与 Neuroligin 家族的所有成员相互作用，但并非所有 Kalirin 亚型都可以与 Neuroligin 相互作用。我们发现 NLGN 依赖性的突触增强和棘生长至少部分是由 kalirin-7 介导的。因此，我们确定了 NLGN1 的第一个下游效应子。