Cellular Mechanisms of Neuroligin-4 Gene in Human Neurons

Neuroligin-4 基因在人类神经元中的细胞机制

基本信息

批准号：
10367707
负责人：
Soham Chanda
金额：
$ 37.08万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-19 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10367707
关键词：
Adverse effects Affect Affinity Amino Acid Sequence Amino Acids Arginine Binding Biochemical Biological Assay Biological Models Biological Process Biotinylation Birth Brain Brain Stem Cause of Death Cell Adhesion Molecules Cell Aggregation Cell physiology Cerebral cortex Characteristics Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Data Defect Electrophysiology (science)Embryo Environment Epigenetic Process Epitopes Excitatory Synapse Exhibits Extracellular Domain Family member Gene Targeting Genes Genetic Engineering Glutamine Human Inhibitory Synapse Institutes Instruction Knockout Mice Knowledge Laboratories Link Mass Spectrum Analysis Mediating Methods Modeling Molecular Morphology Mus Mutagenesis Mutate N-Glycosylation Site Neurons Neurotransmitter Receptor Orthologous Gene Output Play Pluripotent Stem Cells Post-Translational Protein Processing Process Property Protein Isoforms Proteins Recombinants Research Respiratory Center Rodent Rodent Model Role Series Single Nucleotide Polymorphism Specificity Structure Synapses System Techniques Variant X Chromosome base confocal imaging density differentiation protocol experimental study glycosylation human interactome human model human stem cells imaging genetics induced pluripotent stem cell insight loss of function loss of function mutation mutant neural circuit novel overexpression postsynaptic presynaptic public health relevance relating to nervous system scaffold small molecule species difference stem cells trafficking transcription factor transmission process

项目摘要

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.

修改后的项目摘要/摘要部分 X 染色体连接的 Neuroligin-4 (NLGN4) 是一种在人大脑皮层中大量表达的突触后细胞粘附分子 (CAM)，然而，其细胞功能和分子特性仍相对不清楚。在传统的啮齿动物模型中，这种基因在进化上并没有得到很好的保守，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NLGN4直向同源物之间的这种固有的物种差异强调了迫切需要。生成人类模型系统以揭示其人类特异性机制。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检查 NLGN4 的机制特性，同时保持人类的保真度。在本提案中，我们的目标是利用源自人类干细胞的神经亚型来评估我们的中心假设，即 NLGN4 在定义兴奋性与抑制性的输入输出参数方面发挥着指导作用。我们预计 NLGN4 通过其胞内和胞外结构域与突触蛋白子集建立分子相互作用，共同调节不同 NLGN4 基序的氨基酸序列以及突触后的功能。一些关键残基的翻译修饰可能在确定其功能特异性方面发挥重要作用。在目标 1 中：为了探究 NLGN4 如何调节突触网络活动，我们将完全消除其内源表达。目标 2：我们将通过以下方式确定 NLGN4 的不同氨基酸残基如何差异调节其特征。目标 3：我们将使用严格的免疫共沉淀研究 NLGN4 与其他突触蛋白的结合如何定义其功能特性。该项目本质上将提供有关 NLGN4 功能、其与其他 NLGN 的相似性和差异的全面分析知识，使用 NLGN4 作为模型，这套广泛的补充方法也将使我们能够获得基本的知识。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信息。