The role of Poorly Characterized Disease-related Proteins in Cortical Development

特征不明的疾病相关蛋白在皮质发育中的作用

基本信息

批准号：
10725259
负责人：
ALEX L KOLODKIN
金额：
$ 16.38万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10725259
关键词：
Affect Apical Attention Axon Binding Binding Sites Bioinformatics Brain Brain Diseases Brain region CRISPR/Cas technology Cell Compartmentation Cells Cerebral cortex Cloning Cloning Vectors Cortical Malformation Data Set Databases Defect Dendrites Development Developmental Delay Disorders Disease Down-Regulation Electroporation Embryo Embryonic Development Enzymes Epigenetic Process Foundations Functional disorder Funding Opportunities Gene Targeting Generations Genes Genetic Genetic Screening Goals Human Image Impaired cognition In Situ Hybridization Individual Knock-in Label Libraries Light Mediating Molecular Morphology Motor Skills Mus Mutate Mutation Neocortex Neurologic Neurons Outcome Pathogenesis Patients Phenotype Phosphorylation Site Play Process Proteins Rare Diseases Regulation Rest Role Seizures Signal Pathway Somatosensory Cortex Surveys Symptoms Syndrome Tamoxifen Time density excitatory neuron experimental study gain of function in utero insight interstitial knock-down loss of function migration neocortical novel postnatal promoter protein function rare genetic disorder small hairpin RNA transcription factor transcriptome sequencing vector

项目摘要

PROJECT SUMMARY Many rare genetic diseases include a range of neurological phenotypes. Malformations of Cortical Development (MCDs) includes a group of syndromes that share defective cortical lamination phenotypes. However, little is known regarding axonal and dendritic morphology of neurons in affected MCDs cortices. In particular, axonal morphologies of excitatory projection neurons are remarkable because these neurons connect neocortex with multiple regions throughout the brain, and this requires precise regulation of interstitial axon branching. However, molecular mechanisms that regulate interstitial axon branching are poorly understood. We hypothesize that among many rare disease-related genes encoding Understudied Proteins Associated with Rare Diseases (UPARDs) listed in this funding opportunity announcement (FOA), there are a significant number of genes encoding UPARDs that are directly involved in the regulation of cortical neuron axon and dendrite morphology. We have previously developed an approach for studying complete cortical neuronal morphology at the single cell level which at the same time can be combined with targeted genetic or epigenetic perturbations. In this project, we will perform a genetic screen of 105 genes encoding UPARDs listed in this FOA which are expressed at high levels in the mouse cerebral cortex. We identified these genes by analysis of our scRNAseq dataset generated from postnatal day 13 (P13) upper layer cortical excitatory neurons and other publicly available RNAseq or in situ hybridization databases. We will generate a library of sgRNAs targeting these genes and clone them into vectors carrying the Cas9 enzyme. We will use in utero electroporation at embryonic day 15.5 (E15.5) to target layer 2/3 callosal projection neurons (CPNs) and downregulate expression of selected genes to study the morphology of these neurons at postnatal day 14 (P14). We will focus on changes in the density and distribution of interstitial axon branches in individual cortical layers and on the orientation and branching of apical and basal dendrites. The major outcome of this screen will be the identification of novel proteins that regulate cortical excitatory neuron morphology. These results will serve as a foundation for further analyses of these understudied proteins, allowing for the identification of novel signaling pathways that modulate neuronal morphology and for a better understanding of the pathogenesis of rare diseases connected with dysfunction of these proteins.

项目概要许多罕见的遗传疾病包括一系列神经表型。皮质畸形发育（MCD）包括一组具有缺陷的皮质层压表型的综合征。然而，对于受影响的 MCD 皮层神经元的轴突和树突形态知之甚少。在特别是，兴奋性投射神经元的轴突形态非常引人注目，因为这些神经元将新皮质与整个大脑的多个区域连接起来，这需要间质的精确调节轴突分支。然而，调节间质轴突分支的分子机制尚不明确。明白了。我们假设在许多编码待研究蛋白质的罕见疾病相关基因中与本资助机会公告 (FOA) 中列出的罕见疾病 (UPARD) 相关，有大量编码 UPARD 的基因直接参与皮质神经元的调节轴突和树突形态。我们之前开发了一种研究完整皮质的方法单细胞水平的神经元形态，同时可以与靶向遗传或表观遗传扰动。在这个项目中，我们将对编码 UPARD 的 105 个基因进行遗传筛选本 FOA 中列出的这些药物在小鼠大脑皮层中高水平表达。我们鉴定了这些基因通过分析出生后第 13 天 (P13) 上层皮质兴奋性生成的 scRNAseq 数据集神经元和其他公开可用的 RNAseq 或原位杂交数据库。我们将生成一个库 sgRNA 靶向这些基因并将它们克隆到携带 Cas9 酶的载体中。我们将在子宫内使用在胚胎第 15.5 天 (E15.5) 电穿孔至目标层 2/3 胼胝体投射神经元 (CPN) 和下调选定基因的表达，以研究出生后第 14 天这些神经元的形态（第 14 页）。我们将重点关注个体皮质间质轴突分支的密度和分布的变化层以及顶端和基底树突的方向和分支。此屏幕的主要成果将鉴定调节皮质兴奋性神经元形态的新型蛋白质。这些结果将为进一步分析这些待研究的蛋白质奠定基础，从而识别新的蛋白质调节神经元形态的信号通路，并更好地了解发病机制与这些蛋白质功能障碍有关的罕见疾病。