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

• 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
• 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

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.

项目摘要/摘要 自闭症谱系障碍(ASD)是一种神经发育疾病，在美国几乎有2%的儿童受到影响 独自一人。尽管它具有遗传和临床异质性，但最近的系统生物学和基因组学研究表明 ASD会聚在一组特定的细胞通路上。表观遗传调控和突触信号的出现 作为ASD中最突出的两条途径，具有许多高置信度的遗传危险因素和调控失调 参与这些过程的基因。这一观察结果引发了一种假说，即表观遗传失调导致 与不正常的神经元电路发育和功能有关，这已在小鼠模型中得到证明 表观遗传调节因子在ASD中反复突变，如CHD8(Chromodomain Helicase DNA Binding Protein) 8)。然而，确切的表观遗传学变化、它们影响的细胞类型以及由此导致的神经元电路变化 ASD的表观遗传失调所致的发病机制尚不清楚。最近，单细胞基因组学方法，包括单细胞基因组学方法， 细胞RNA测序和单细胞ATAC片断，提供了前所未有的细胞和 大脑的分子组成，以及大脑发育的基本过程。在我的博士后生涯中，我申请 对ASD患者死后皮质组织进行单核RNA测序以了解 与ASD相关的特定神经元和神经胶质亚型的分子变化。其中最重要的一个 这项工作的见解是，上层皮质神经元是受ASD影响最大的细胞类型。 相关的转录变化。这一观察结果提出了关于脑脊液的起源和功能影响的问题。 特定神经元回路上的这种变化。作为我的K99提案的目标1的一部分，我将测试这个假设 ASD的基因表达变化是由特定细胞类型的表观遗传状态的变化驱动的。到那时候 最后，我将对ASD患者和对照组的新皮质组织进行联合RNA-SEQ和ATAC-SEQ分析，以 识别特定细胞类型的表观遗传学变化。然后，我将开发和测试一个高通量的突触跟踪 将条形码狂犬病病毒文库与单核RNA测序(K99期目标2)相结合的技术。 最后，使用我提案的K99阶段的培训、工具和初步数据，我将启动 专注于研究细胞类型特定的表观遗传和神经元回路的独立研究项目 CHD8+/小鼠模型在发育期间(R00阶段)的变化。我将首先应用联合RNA-SEQ/ATAC- SEQ方案，研究特定细胞类型在发育过程中因丢失其中一种而引起的表观遗传变化 CHD8等位基因。CHD8+/小鼠与表达Cre重组酶的报告基因在神经元中的杂交 亚型，如上层皮质神经元(Cux2-Cre)，我将能够使用条形码狂犬病病毒库 和单核RNA-seq，以确定CHD8导致的皮质回路特定成分的变化 单倍体功能不全。我相信K99-R00奖将让我形成独特的研究方向和 使自己在自闭症和单细胞基因组学领域成为一名成功的独立研究员。