Temporal Single Cell RNAseq to Identify Genes and Pathways Affected by 15q11.2 Duplication in Autism iPSC-Derived Differentiating Cortical Neurons

时间单细胞 RNAseq 识别自闭症 iPSC 衍生的分化皮质神经元中受 15q11.2 重复影响的基因和通路

• 批准号: 9200627
• 负责人: Wei Zhang
• 金额: $ 22.45万
• 依托单位: JUVOBIO PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-16 至 2018-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9200627
• 关键词: Academic support; Address; Affect; Autistic Disorder; Bioinformatics; Biological Markers; Brain; Cell Count; Cell Line; Cell Proliferation; Cells; Child; Clinical; Companions; Computer Simulation; Copy Number Polymorphism; DNA Sequence Alteration; Data; Data Quality; Data Set; Databases; Deposition; Development; Developmental Disabilities; Diagnosis; Dimensions; Disease; Disease Progression; Disease model; Drug Targeting; Environmental Risk Factor; Epilepsy; Exposure to; Frequencies; Gene Expression; Genes; Genetic Predisposition to Disease; Glutamates; Health Priorities; Heterogeneity; In Vitro; Lateral; Lead; Maps; Marketing; Mendelian disorder; Molecular; Molecular Profiling; Morphology; Mutation; Nature; Nervous System Physiology; Neuronal Differentiation; Neurons; Pathogenesis; Pathology; Pathway interactions; Patients; Pharmacologic Substance; Phase; Phenotype; Play; Population; Price; Process; Protocols documentation; Proxy; Publishing; Reagent; Research; Research Personnel; Research Support; Resources; Risk Factors; Role; Sampling; Schizophrenia; Scientist; Sorting - Cell Movement; Synapses; Synaptic plasticity; System; Therapeutic; Time; Tissue Sample; United States; Up-Regulation; autism spectrum disorder; cell type; database structure; diagnostic biomarker; disease mechanisms study; disease phenotype; disorder control; drug discovery; genetic risk factor; induced pluripotent stem cell; innovation; insight; interest; microdeletion; molecular dynamics; nerve stem cell; nervous system disorder; neuron development; neuronal circuitry; novel therapeutics; peer; public health priorities; reconstitution; relating to nervous system; research study; tool; transcriptome; transcriptome sequencing

PROJECT SUMMARY We aim to establish a single-cell RNAseq database of differentiating cortical neuronal progenitors (NPCs) and neurons derived from patient-specific induced pluripotent stem cells (iPSCs) for neuro- developmental diseases (NDDs). The database will also include data on differentiation, morphology, formation and functionality of synapses, annotations of analytical findings, and will be supported by the most advanced single-cell RNAseq bioinformatics tools. We will provide all this, together with corresponding NPCs and neurons for research reagents, as tool to support academic research and drug discovery for NDDs in the field. One in 68 children born in 2002 are diagnosed with Autism Spectrum Disorder (ASD), a public health priority in the US. Genetic predispositions in ASD are thought to contribute to the primary pathology by altering neuronal development, evidenced partly by altered gene expressions. Patient-specific induced pluripotent stem cells (iPSCs) have been shown to recapitulate specific disease phenotypes through the neurogenic process and can serve as effective disease models. Hence, single-cell RNAseq along the accessible and controlled process of differentiating ASD-specific iPSCs into neural progenitors and subtypes of neurons can provide insights into the temporal and multi-lineage dimensions of ASD pathogenesis and biomarkers for diagnostics, progression, and therapeutics discovery. Copy-number variants (CNVS) at 15q11.2 is a prominent risk factor for neurological disorders including ASD, epilepsy, and schizophrenia. 15q11-q13 duplication/triplication represent the most common CNVs in patients with ASD (up to 3%). Duplications and microdeletions can both lead to the same disorders, suggesting the importance of this region in normal neurological functions and the necessity to study the impact of both duplications and deletions for a full understanding of the mechanisms. Phase I utilizes four 15q11.2 duplication iPSC lines from ASD patients and two control iPSC lines to: Aim 1: Characterize neural differentiation of ASD and control iPSC-derived neurons. Differentiate iPSCs into cortical NPCs, glutamatergic, and GABAergic subtypes. Identify deficits in ASD lines through morphological studies and structural analyses of synapses. Aim 2: Generate single-cell RNAseq datasets of differentiating NPCs and neuronal subtypes at nine time points during differentiation. Aim 3: Perform bioinformatics analyses. Reconstitute the molecular dynamics underlying neuronal differentiation. Validate experimental conditions including sampling frequency and number of cells. Identify, validate molecular signatures underlying 15q11.2 duplication’s impact on neuronal differentiation and functions. Phase II: utilize the experimental conditions established here to generate single-cell RNAseq datasets from multiple ASD iPSC lines that harbor different genetic mutations to be included in the database; build database structure and user interface, and seek to identify aberrant differentiation and functional development caused by ASD mutations as well as genes and pathways that are commonly and differentially affected across multiple ASD mutations. We will devote resources to annotate the database with our own findings and those that are published by peers to enhance its utility to subscribers.

项目摘要 我们旨在建立一个分化皮质神经祖细胞的单细胞RNASEQ数据库 （NPC）和来自患者特异性诱导的多能干细胞（IPSC）的神经元用于神经 - 发育疾病（NDDS）。该数据库还将包括有关差异化，形态，形成的数据 突触的功能，分析结果的注释，将由最先进的 单细胞RNASEQ生物信息学工具。我们将提供所有这些，以及相应的NPC和 研究试剂的神经元，作为支持该领域NDD的学术研究和药物发现的工具。 2002年出生的68名儿童被诊断出患有自闭症谱系障碍（ASD），这是公共卫生 在美国的优先事项。据认为，ASD中的遗传易感性通过改变而导致主要病理 神经元发育，部分通过基因表达改变。患者特异性诱导的多能 已显示干细胞（IPSC）通过神经源概括了特定的疾病表型 过程并可以充当有效的疾病模型。因此，沿可访问的单细胞RNASEQ和 将ASD特异性IPSC分化为神经元祖细胞和神经元亚型的受控过程可以 提供有关ASD发病机理和生物标志物的临时和多条尺寸的见解 诊断，进展和治疗发现。 15q11.2处的拷贝数变体（CNV）是神经系统疾病的重要危险因素 ASD，癫痫和精神分裂症。 15Q11-Q13重复/三次列式代表最常见的CNV ASD患者（最多3％）。重复和微缺失都可以导致相同的疾病， 提出该区域在正常神经功能方面的重要性，并需要研究影响 重复和删除都可以充分理解机制。 第一阶段利用了来自ASD患者的四个15q11.2重复IPSC线，并使用两条对照IPSC线。 AIM 1：表征ASD和控制IPSC衍生神经元的神经元分化。区分IPSC 进入皮质NPC，谷氨酸能和GABA能亚型。识别通过ASD线中的定义 突触的形态学研究和结构分析。 AIM 2：生成九个分化NPC和神经元亚型的单细胞RNASEQ数据集 分化过程中的时间点。 AIM 3：执行生物信息学分析。重建神经元基础的分子动力学 分化。验证实验条件，包括采样频率和细胞数量。确认， 验证15q11.2重复对神经元分化和功能的影响。 第二阶段：利用此处建立的实验条件来生成从 多个ASD IPSC系列具有不同的基因突变，该突变包含在数据库中；构建数据库 结构和用户界面，并寻求确定由异常差异化和功能开发 ASD突变以及通常影响多个的基因和途径 ASD突变。我们将使用我们自己的发现和那些的资源注释数据库 由同龄人发行，以增强其对订户的效用。