Cellular Mechanisms of Neuroligin-4 Gene in Human Neurons

修改项目摘要/摘要部分 X染色体连锁的神经连接素4（Neuroligin-4，NLGN-4）是一种在人类大脑皮层中大量表达的突触后细胞粘附分子（postsynaptic cell-adhesion molecule，CAM），但其细胞功能和分子特性尚不清楚。人类NLGN 4由一个独特的氨基酸序列组成，在传统的啮齿动物模型中进化上并不保守，限制了我们研究这种人类特异性基因如何影响突触组织的能力。不同NLGN 4直系同源物之间的这种固有物种差异强调了产生人类模型系统以揭示其人类特异性机制的迫切需要。在多能干细胞的遗传工程和表观遗传重编程领域的最新技术进展为我们提供了一个独特的机会来研究NLGN 4的机械特性，同时保持人类细胞环境的保真度。在这个提议中，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NLGN 4在定义兴奋性与抑制性突触的输入-输出参数方面起着指导性作用。我们预计NLGN 4通过其胞内和胞外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。NLGN 4不同基序的氨基酸序列以及一些关键残基的翻译后修饰可能在决定其功能特异性方面起重要作用。目标1：为了探究NLGN 4如何调节突触网络活动，我们将完全消除其在人类神经元中的内源性表达或引入功能丧失突变，并使用共聚焦成像和电生理记录来检查对突触形态和传递的不利影响。目标2：我们将确定如何不同的氨基酸残基NLGN 4可以差异调节其特性，通过进行系统的结构功能和生化分析。目标3：我们将研究如何NLGN 4的结合到其他突触蛋白可能定义其功能的身份，使用严格的免疫共沉淀，细胞聚集，和邻近依赖性生物素化测定。该项目将基本上提供有关NLGN 4功能的全面知识，其与其他NLGN的相似性和差异。使用NLGN 4作为模型，这种广泛的互补方法也将使我们能够获得有关人类突触环境以及突触前或突触后CAM如何调节其组成和活动的基本信息。

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.