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

修订项目总结/摘要： 复杂的神经回路在发育过程中精确地组装在人类大脑中大约800亿个神经元之间。哺乳动物成簇的原钙粘蛋白（Pcdh）基因，编码一个家族的高度多样化的细胞表面的嗜同性结合蛋白，提供了一个独特的细胞表面的身份，或正常的神经回路组装所需的条形码个别神经元。以前对成簇的Pcdh基因和蛋白质的结构、功能和基因表达研究揭示了Pcdh条形码产生和发挥功能的复杂机制，以及Pcdh突变功能丧失对小鼠神经回路组装和行为的影响。Pcdh条形码的一个显著特征是它由相互作用的神经元膜上的细胞表面蛋白质“晶格”组成。Pcdh条形码已显示在小鼠的神经突自我回避、神经元平铺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-细胞相互作用的显性功能丧失Pcdh突变。 虽然未能准确地组装神经回路与神经发育和神经精神障碍有关，但PCDH基因簇中单个PCDH基因中单核苷酸变异（SNV）的出现不符合自闭症谱系障碍（ASD）队列的大规模DNA测序研究中遗传关联的全基因组统计关联标准。然而，在理解PCDH基因簇的调控和功能方面的进展以及Simons Simplex队列中ASD先证者的淋巴母细胞系（LCL）的最近可用性，这些先证者在PCDH基因中具有破坏性的从头SNV，这为进一步探索PCDH基因在源自人诱导多能干细胞的人神经元中的功能提供了独特的机会。 目的是对ASD病例中鉴定的PCDH基因簇中的从头SNV进行系统分析。在目的1中，我们将确定PCDH亚型中的SNV如何影响PCDH蛋白在基于细胞的体外嗜同性结合测定中的嗜同性相互作用和细胞内定位。在目标2中，我们将产生具有功能丧失PCDH突变的CRISPR-Cas9编辑的hiPSC，并将它们分化成神经元以评估hiPSC衍生的神经元中的自我回避缺陷。在目标3中，我们将研究PCDH亚型中SNV对小鼠神经回路和功能连接的影响，使用多巴胺能神经元作为模型。这些拟议的研究应显着推进我们的理解PCDH基因座在神经回路组装在大脑发育过程中的功能作用。

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.