Analysis of epigenetic and neuronal circuit changes in autism on the single-cell level

单细胞水平分析自闭症的表观遗传和神经元回路变化

基本信息

批准号：
10696952
负责人：
Dmitry Velmeshev
金额：
$ 24.75万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-02 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10696952
关键词：
ASD patient ATAC-seq Affect Alleles Area Autopsy Award Bar Codes Brain Candidate Disease Gene Cell Nucleus Cells Child Chromatin Chromatin Remodeling Factor Cre lox recombination system DNA-Binding Proteins Data Data Set Development Engineering Enterobacteria phage P1 Cre recombinase Epigenetic Process Exhibits Gene Expression Gene Expression Profile Generations Genes Genetic Engineering Genetic Heterogeneity Genetic Transcription Genome Genomic approach Genomics Goals Heterogeneity Human Joints Label Lead Libraries Maps Modeling Molecular Mus Mutate Neocortex Neurodevelopmental Disorder Neuroglia Neurons Oligonucleotides Pathogenesis Pathway interactions Patients Phase Phenotype Poly A Polyadenylation Postdoctoral Fellow Process Protocols documentation Publishing RNA Rabies Rabies virus Recurrence Regulatory Element Reporter Research Research Personnel Research Project Grants Risk Risk Factors Signal Transduction Synapses Synaptic Transmission System Systems Biology Techniques Testing Tissues Training United States Viral Virion Virus Wild Type Mouse Work XCL1 gene autism spectrum disorder autistic brain tissue cell cortex cell type clinical heterogeneity cohort comparison control effective therapy epigenetic profiling epigenetic regulation genetic risk factor global health helicase in vivo insight loss of function loss of function mutation molecular pathology mouse model neocortical neural circuit neuronal circuitry novel precision medicine sequencing platform single nucleus RNA-sequencing single-cell RNA sequencing skills targeted treatment tool transcriptome sequencing transcriptomics vector viral RNA

项目摘要

Project Summary/Abstract Autism Spectrum Disorder (ASD) is a neurodevelopmental disease affecting almost 2% of children in the US alone. Despite its genetic and clinical heterogeneity, recent systems biology and genomics studies demonstrated that ASD converges on a specific set of cellular pathways. Epigenetic regulation and synaptic signaling emerged as the two most prominent pathways in ASD, with many high-confidence genetic risk factors and dysregulated genes involved in these processes. This observation prompted a hypothesis that epigenetic dysregulation leads to improper neuronal circuit development and function, which has been demonstrated in mouse models of epigenetic regulators recurrently mutated in ASD, such as CHD8 (Chromodomain Helicase DNA Binding Protein 8). However, the exact epigenetic changes, cell types they affect and the neuronal circuitry changes resulting from epigenetic dysregulation in ASD are unknown. Recently, single-cell genomics approaches, including single- cell RNA sequencing and single-cell ATAC sequinning, offered unprecedented new level of detail of cellular and molecular composition of the brain, as well as processes underlying its development. In my postdoc, I applied single-nucleus RNA sequencing to human post-mortem cortical tissue from ASD patients to gain insight into the molecular changes associated with ASD in specific neuronal and glial subtypes. One of the most important insights from this work is the implication of upper-layer cortical neurons as the cell type most affected by ASD- associated transcriptional changes. This observation raises questions about the origin and functional effects of such changes on specific neuronal circuits. As part of the Aim 1 of my K99 proposal, I will test the hypothesis that gene expression changes in ASD are driven by changes in epigenetic states of specific cell types. To that end, I will perform a joint RNA-seq and ATAC-seq profiling of neocortical tissue of ASD patients and controls to identify cell type-specific epigenetic changes. Then, I will develop and test a high-throughput synaptic tracing technique by combining barcoded rabies virus library with single-nucleus RNA sequencing (Aim 2 of K99 phase). Finally, using the training, tools and preliminary data from the K99 phase of my proposal, I will launch an independent research project that focuses on investigating cell-type specific epigenetic and neuronal circuitry changes in the Chd8+/ mouse model during development (R00 phase). I will first apply the joint RNA-seq/ATAC- seq protocol to study epigenetic changes in specific cell types during development caused by the loss of one of Chd8 alleles. By crossing the Chd8+/ mouse with reporter lines expressing Cre recombinase in specific neuronal subtypes, such as upper-layer cortical neurons (Cux2-Cre), I will be able to use the barcoded rabies virus library and single-nucleus RNA-seq to identify changes in specific components of cortical circuitry as the result of Chd8 haploinsufficiency. I believe that the K99-R00 award will allow me to form a unique research direction and establish myself as a successful independent investigator in the area of autism and single-cell genomics.

项目概要/摘要自闭症谱系障碍 (ASD) 是一种神经发育疾病，影响美国近 2% 的儿童独自的。尽管存在遗传和临床异质性，但最近的系统生物学和基因组学研究表明自闭症谱系障碍（ASD）集中在一组特定的细胞通路上。表观遗传调控和突触信号传导出现作为自闭症谱系障碍 (ASD) 中两个最重要的途径，具有许多高置信度遗传风险因素和失调参与这些过程的基因。这一观察结果提出了一个假设，即表观遗传失调会导致神经元回路发育和功能不当，这已在小鼠模型中得到证实表观遗传调节因子在 ASD 中反复突变，例如 CHD8（染色质解旋酶 DNA 结合蛋白） 8).然而，确切的表观遗传变化、它们影响的细胞类型以及导致的神经元回路变化自闭症谱系障碍 (ASD) 中的表观遗传失调引起的影响尚不清楚。最近，单细胞基因组学方法，包括单细胞细胞 RNA 测序和单细胞 ATAC 亮片加工，提供了前所未有的细胞和细胞细节的新水平大脑的分子组成，以及其发育的过程。在我的博士后期间，我申请了对 ASD 患者死后皮质组织进行单核 RNA 测序，以深入了解特定神经元和神经胶质亚型中与 ASD 相关的分子变化。最重要的之一这项工作的见解表明，上层皮质神经元是受自闭症谱系障碍 (ASD) 影响最严重的细胞类型。相关的转录变化。这一观察结果提出了关于起源和功能效应的问题特定神经元回路的这种变化。作为 K99 提案目标 1 的一部分，我将检验假设 ASD 中基因表达的变化是由特定细胞类型的表观遗传状态的变化驱动的。对此最后，我将对 ASD 患者和对照的新皮质组织进行联合 RNA-seq 和 ATAC-seq 分析，以识别细胞类型特异性的表观遗传变化。然后，我将开发并测试高通量突触追踪通过将带有条形码的狂犬病病毒库与单核RNA测序相结合的技术（K99阶段的目标2）。最后，利用我提案 K99 阶段的培训、工具和初步数据，我将启动一个专注于研究细胞类型特异性表观遗传和神经元回路的独立研究项目 Chd8+/小鼠模型在开发过程中（R00阶段）的变化。我将首先应用联合RNA-seq/ATAC- seq 协议，用于研究特定细胞类型在发育过程中由于其中一种缺失而引起的表观遗传变化 Chd8 等位基因。通过将 Chd8+/ 小鼠与在特定神经元中表达 Cre 重组酶的报告系杂交亚型，例如上层皮质神经元 (Cux2-Cre)，我将能够使用带条形码的狂犬病病毒库和单核 RNA-seq 来识别 Chd8 导致的皮质电路特定成分的变化单倍体不足。我相信K99-R00奖将使我形成独特的研究方向并使自己成为自闭症和单细胞基因组学领域成功的独立研究者。