The impact of loss of function DNA sequence variants in the human protocadherin gene cluster on neural circuit assembly.

人类原钙粘蛋白基因簇中功能丧失 DNA 序列变异对神经回路组装的影响。

• 批准号: 10736632
• 负责人: THOMAS P MANIATIS
• 金额: $ 74.86万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736632
• 关键词: Affect; Alleles; B-Lymphocytes; Bar Codes; Behavior; Binding; Binding Proteins; Biological; Biological Assay; Bipolar Disorder; Brain; CRISPR/Cas technology; Cell Aggregation; Cell Communication; Cell Culture Techniques; Cell Line; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Cellular Assay; Chromatin; Collaborations; Complex; DNA Sequence; DNA sequencing; Data; Defect; Development; Disease; Dominant-Negative Mutation; Dorsal; Epilepsy; Etiology; Failure; Family; Future; Gene Cluster; Gene Expression; Genes; Genetic; Genetic Risk; Goals; Guide RNA; Human; Human Genetics; In Vitro; Individual; Intellectual functioning disability; Joints; K562 Cells; Knockout Mice; Knowledge; Link; Major Depressive Disorder; Mammals; Membrane; Methods; Microcephaly; Modeling; Molecular; Mus; Mutation; Nature; Neurites; Neurodevelopmental Disorder; Neurons; Pathogenesis; Pathway interactions; Pattern; Play; Privatization; Protein Isoforms; Proteins; Regulation; Role; Schizophrenia; Single Nucleotide Polymorphism; Structure; Surface; Synapses; Testing; Therapeutic; Therapeutic Intervention; Transfection; Variant; Work; anxiety-related behavior; autism spectrum disorder; causal variant; cell transformation; cohort; excitatory neuron; functional restoration; gain of function; gene function; genetic association; genome-wide; in vivo; induced pluripotent stem cell; inhibitory neuron; insight; knock-down; loss of function; lymphoblastoid cell line; mutant; neural; neural circuit; neural network; neuronal circuitry; neuropsychiatric disorder; novel; novel therapeutic intervention; prevent; proband; protein structure; targeted treatment; trafficking; transforming virus; variant detection

REVISED PROJECT SUMMARY/ABSTRACT: Complex neural circuits are accurately assembled between the approximately 80 billion neurons in the human brain during development. The mammalian clustered protocadherin (Pcdh) genes, which encode a family of highly diverse cell-surface homophilic binding proteins, provide individual neurons with a unique cell-surface identity, or barcode required for normal neural circuit assembly. Previous structural, functional, and gene expression studies of the clustered Pcdh genes and proteins have revealed complex mechanisms by which Pcdh barcodes are generated and function, as well as the impact of loss of function Pcdh mutations on neural circuit assembly and behavior in mice. A remarkable feature of the Pcdh barcode is that it consists of a cell surface protein “lattice” on the membranes of interacting neurons. The Pcdh barcode has been shown to play a critical role in neurite self-avoidance, neuronal tiling and normal behavior in mice. Dominant loss of function Pcdh mutations that disrupt cell-cell interactions have been demonstrated in cell culture studies. Although the failure to accurately assemble neural circuits has been linked to neurodevelopmental and neuropsychiatric disorders, the occurrence of single nucleotide variants (SNVs) in individual PCDH genes across the PCDH gene cluster do not meet a genome wide statistical association criteria for genetic association in large scale DNA sequencing studies of autism spectrum disorder (ASD) cohorts. However, advances in understanding the regulation and function of the PCDH gene cluster and the recent availability of lymphoblastoid cell lines (LCLs) from ASD probands in the Simons Simplex cohort bearing disruptive de novo SNVs in PCDH genes presents a unique opportunity to further explore PCDH gene function in human neurons derived from human induced pluripotent stem cells. The aims are to conduct a systematic analysis of de novo SNVs across the PCDH gene clusters identified in ASD cases. In Aim 1 we will determine how SNVs in PCDH isoforms impact homophilic interactions and intracellular localization of PCDH proteins in cell-based in-vitro homophilic binding assays. In Aim 2, we will generate CRISPR-Cas9 edited hiPSCs bearing loss of function PCDH mutations and differentiate them into neurons to assess deficits in self-avoidance in the hiPSC-derived neurons. In Aim 3, we will investigate the impact of SNVs in PCDH isoforms on neural circuits and functional connectivity in mice, using serotonergic neurons as a model. These proposed studies should significantly advance our understanding of the functional role of the PCDH locus in neural circuit assembly during brain development.

修订项目摘要/摘要： 在发育过程中，复杂的神经元准确地组装在人脑中约800亿个神经元之间。哺乳动物聚集蛋白（PCDH）基因编码高度多样的细胞表面均匀结合蛋白的家族，为单个神经元提供具有独特的细胞表面识别性或正常神经元组装所需的条形码。簇状的PCDH基因和蛋白质的先前结构，功能和基因表达研究揭示了生成PCDH条形码和功能的复杂机制，以及功能PCDH突变损失对小鼠神经元电路组装和行为的影响。 PCDH条形码的一个显着特征是它由相互作用神经元机理的细胞表面蛋白“晶格”组成。 PCDH条形码已被证明在小鼠的神经自我避免，神经元瓷砖和正常行为中起关键作用。在细胞培养研究中已经证明了破坏细胞 - 细胞相互作用的功能PCDH突变的主要丧失。 尽管无法准确组装神经元回路已与神经发育和神经精神疾病联系在一起，但是在PCDH基因群中，单个PCDH基因中单个核苷酸变化（SNVS）的发生不符合大型DNA Sequencs cohism sebism sebism semism symism sebism的基因组统计关联的基因组统计学标准。然而，在了解PCDH基因簇的调节和功能方面的进步以及最近在Simons Simplex COHORT中使用ASD问题的淋巴母细胞系（LCLS）的可用性在PCDH基因中具有颠覆性的DE NOVO SNVS带来了一个独特的机会，这是一个独特的机会，可以进一步探索人类神经元的PCDH基因，从而探索了人类诱导的多个细胞的PCDH基因。 目的是对ASD病例中确定的PCDH基因簇进行从头SNV的系统分析。在AIM 1中，我们将确定PCDH同工型中的SNV如何影响PCDH蛋白在基于细胞的视频内同质结合测定中的均质相互作用和细胞内定位。在AIM 2中，我们将生成CRISPR-CAS9编辑的HIPSC，具有功能PCDH突变的功能丧失，并将其区分为神经元，以评估HIPSC衍生神经元中自避免的缺陷。在AIM 3中，我们将使用Seratonagic神经元作为模型研究SNV对PCDH同工元对小鼠神经元和功能连通性的影响。这些提出的研究应大大提高我们对PCDH基因座在脑发育过程中神经元电路组装中功能作用的理解。