Cellular Mechanisms of Neuroligin-4 Gene in Human Neurons

修改后的项目摘要/摘要部分 X染色体连锁神经连接蛋白-4(NLGN4)是一种在人脑皮质中大量表达的突触后细胞黏附分子，但其细胞功能和分子特性尚不清楚。人类NLGN4由一个独特的氨基酸序列组成，在传统的啮齿动物模型中不是进化上保守的，这限制了我们研究这个人类特有的基因如何影响突触组织。不同的NLGN4同源基因之间的这种固有的物种差异突显了迫切需要产生一个人体模型系统来揭示其人类特有的机制。基因工程和多能干细胞表观遗传重编程领域的最新技术进步为我们提供了一个独特的机会来研究NLGN4的机制属性，同时保持人类细胞环境的保真度。在这项提案中，我们的目标是利用来自人类干细胞的神经元亚型来评估我们的中心假设，即NLGN4在定义兴奋性突触与抑制性突触的输入输出参数方面发挥指导作用。我们预计NLGN4通过其胞内和胞外结构域与突触蛋白的子集建立分子相互作用，这些结构域共同调节其适当的成熟、运输和功能。不同NLGN4基序的氨基酸序列以及某些关键残基的翻译后修饰可能在决定其功能特异性方面发挥重要作用。目的1：探讨NLGN4是如何调节突触网络活动的，我们将通过共聚焦成像和电生理记录观察NLGN4对突触形态和传递的不利影响。在目标2：我们将通过进行系统的结构-功能和生化分析，确定NLGN4的不同氨基酸残基如何差异调节其特征。在目标3：我们将使用严格的免疫共沉淀、细胞聚集和邻近依赖的生物素化实验来研究NLGN4‘S与其他突触蛋白结合如何定义其功能特性。这个项目基本上将提供关于NLGN4功能的全面知识，以及它与其他NLGNs的异同。使用NLGN4作为模型，这种广泛的互补方法也将使我们能够获得关于人类突触环境的基本信息，以及突触前或突触后的CAM如何调节其组成和活动。

Modified Project Summary/Abstract Section The X-chromosome linked Neuroligin-4 (NLGN4) is a postsynaptic cell-adhesion molecule (CAM) abundantly expressed in human cerebral cortex, however, its cellular function and molecular properties remain relatively unclear. Human NLGN4 consists of a unique amino-acid sequence that is not evolutionarily well-conserved in conventional rodent models, limiting our ability to investigate how this human-specific gene impacts synapse organization. This inherent species differences between diverse NLGN4 orthologs underscore the immediate need to generate a human model system to uncover its human-specific mechanisms. Recent technological advances in the fields of genetic engineering and epigenetic reprogramming of pluripotent stem cells provide us with a unique opportunity to examine the mechanistic properties of NLGN4, while maintaining the fidelity of human cellular context. In this proposal, we aim to utilize neuronal subtypes derived from human stem cells to assess our central hypotheses that NLGN4 plays an instructive role in defining the input-output parameters of excitatory vs. inhibitory synapses. We anticipate that NLGN4 establishes molecular interactions with a subset of synaptic proteins via its intra- and extracellular domains, which collectively regulate its proper maturation, trafficking, and function. Both the amino-acid sequence of different NLGN4 motifs as well as post-translational modifications at some those critical residues might play significant roles in determining its functional specificity. In aim 1: To inquire how NLGN4 can modulate synaptic network activity, we will either completely eliminate its endogenous expression in human neurons or introduce loss-of-function mutation, and inspect adverse effects on synaptic morphology and transmission using confocal imaging and electrophysiological recording. In Aim 2: We will determine how distinct amino-acid residues of NLGN4 can differentially regulate its characteristics, by performing systematic structure-function and biochemical analyses. In Aim 3: We will investigate how NLGN4’s binding to other synaptic proteins may define its functional identity, using rigorous co-immunoprecipitation, cell-aggregation, and proximity-dependent biotinylation assay. This project will essentially provide a comprehensive knowledge about NLGN4 function, its similarities and differences with other NLGNs. Using NLGN4 as a model, this extensive set of complementary approaches would also allow us to acquire fundamental information about human synaptic environment and how pre- or postsynaptic CAMs modulate its composition and activity.