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

• 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
• 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

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.

项目摘要 许多罕见的遗传性疾病包括一系列神经系统表型。皮质畸形 发育（MCDs）包括一组共有缺陷性皮质层压表型的综合征。 然而，鲜为人知的是，在受影响的MCDs皮质神经元的轴突和树突形态。在 特别是，兴奋性投射神经元的轴突形态是显著的，因为这些神经元 将新皮层与整个大脑的多个区域连接起来，这需要精确调节组织间质， 轴突分支然而，调节间质轴突分支的分子机制很差， 明白我们假设，在许多编码未研究蛋白质的罕见疾病相关基因中， 与此资助机会公告（FOA）中列出的罕见疾病（UPARDs）相关， 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。我们以前已经开发了一种研究完整皮层的方法， 在单细胞水平上的神经元形态，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UPARDs的105个基因进行遗传筛选， 在小鼠大脑皮层中以高水平表达。我们鉴定了这些基因 通过分析我们从出生后第13天（P13）上层皮层兴奋性神经元产生的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 靶向这些基因的sgRNA，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）电穿孔靶向第2/3层胼胝体投射神经元（CPN）， 下调所选基因的表达，以研究这些神经元在出生后第14天的形态 （P14）。我们将重点关注个体皮质间质轴突分支密度和分布的变化 层和顶端和基底树突的取向和分支。这个屏幕的主要结果是 将鉴定调节皮质兴奋性神经元形态的新蛋白质。这些结果将 作为进一步分析这些未充分研究的蛋白质的基础， 信号通路，调节神经元形态和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。