Cellular and Molecular Mechanisms of Behavioral Dysfunction in a Zebrafish Model of CHARGE Syndrome

电荷综合征斑马鱼模型行为障碍的细胞和分子机制

• 批准号: 10372659
• 负责人: Kurt C. Marsden
• 金额: $ 41.12万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372659
• 关键词: Acoustic Stimulation; Acoustics; Affect; Anxiety; Area; Atlases; Attention deficit hyperactivity disorder; Auditory; Behavior; Behavior Therapy; Behavioral; Behavioral Mechanisms; Behavioral Symptoms; Brain; Brain region; CHARGE syndrome; CHD7 gene; CRISPR/Cas technology; Cells; Choanal Atresia; Chromatin Remodeling Factor; Clutch Size; Coloboma; Computer software; DNA Binding; Data; Defect; Development; Disease; Ear; Elements; Eye; Fertilization; Functional disorder; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genital; Genitalia; Genitourinary system; Genotype; Glutamates; Goals; Heart; Heart Abnormalities; Heterozygote; Human Genetics; Image; Impairment; Informal Social Control; Intellectual functioning disability; Label; Larva; Lighting; Link; Live Birth; Location; Measures; Mediating; Methods; Microinjections; Microscopy; Modeling; Molecular; Molecular Target; Morphology; Mutate; Nervous system structure; Neurodevelopmental Disorder; Neurons; Obsessive-Compulsive Disorder; Optics; Pain; Pathway interactions; Patients; Pattern; Pharmacology; Photic Stimulation; Population; Proteins; Proteome; Proteomics; Regulation; Research; Resources; Role; Sampling; Sensory; Shapes; Site; Sleep Disorders; Startle Reaction; Stereotyping; Stimulus; Structure; System; Techniques; Therapeutic; Tissues; Transgenic Organisms; Visual; Work; Zebrafish; auditory processing; autism spectrum disorder; base; behavioral phenotyping; brain morphology; cell type; chromatin remodeling; clinically relevant; common symptom; driving behavior; human disease; in vivo; in vivo calcium imaging; in vivo imaging; loss of function mutation; malformation; molecular dynamics; morphometry; motor impairment; mutant; neural circuit; neurobehavioral; neurodevelopment; neuronal circuitry; novel; novel diagnostics; novel therapeutic intervention; relating to nervous system; response; transcriptome; transcriptome sequencing; transcriptomics; visual information

Project Summary CHARGE syndrome is a rare, multi-system disorder that affects approximately 1 in 10,000 live births. The most common symptoms include ocular coloboma, choanal atresia, heart defects, genital abnormalities, ear malformations, and an array of neuro-behavioral difficulties. Sensory under- or over-load, motor impairments, enhanced pain, sleep disorders, attention deficit hyperactivity disorder (ADHD), obsessive compulsive disorder (OCD), intellectual disability, anxiety, and autism are all frequently observed in CHARGE. Treatments for these behavioral symptoms are limited, highlighting an area of critical need in understanding the mechanisms underlying these defects in order to develop new therapeutic approaches. Two-thirds of CHARGE cases are caused by loss-of-function mutations in chd7, which encodes a DNA-binding, ATP-dependent chromatin remodeling protein. chd7 is highly expressed in the developing vertebrate brain and regulates transcription of several key neurodevelopmental genes, but direct cellular and molecular links between chd7 function and behavioral regulation have not been established. This project leverages the larval zebrafish model, which has rapidly emerged as a powerful system for investigating the development and function of behavioral circuits as well as human genetic disease. Using CRISPR/Cas9 we have established a chd7-null line and have characterized several morphological and behavioral phenotypes reflective of CHARGE. In response to acoustic stimuli, chd7 mutants perform normal short-latency startle responses (SLCs) but have impaired long-latency escape responses (LLCs). Similarly, chd7 mutants respond normally to increases in illumination but are deficient in responding to decreases in illumination. These deficits are independent of morphological defects in the eyes and ears, indicating that chd7 likely regulates specific behavioral circuits in the brain. In Aim 1 we will systematically interrogate the known circuit elements driving these behaviors using in vivo calcium imaging and cell-specific rescue to locate the sites of chd7 action. We will then comprehensively define the brain regions that are dependent on chd7 using whole-brain morphometry and activity analyses. In Aim 2 we will apply state-of- the-art proteomic and transcriptomic approaches to identify molecular pathways that link chd7 with these changes in brain structure and function. By analyzing samples from three developmental timepoints, we will also define the temporal dynamics of these changes. Finally, we will use a systematic CRISPR/Cas9 approach to validate the top proteomics- and transcriptomics-based chd7 targets in vivo by measuring brain development and behavior in mutant larvae. Overall, the results of this work will establish direct links between chd7, its molecular targets, and behavioral circuits. Furthermore, these aims will generate a powerful set of broadly useful resources for interrogating the cellular and molecular bases of chd7-dependent neural development.

项目概要 CHARGE 综合征是一种罕见的多系统疾病，影响大约万分之一的活产儿。最 常见症状包括眼部缺损、后鼻孔闭锁、心脏缺陷、生殖器异常、耳部畸形 畸形和一系列神经行为困难。感觉负荷不足或过载、运动障碍、 疼痛加剧、睡眠障碍、注意力缺陷多动障碍 (ADHD)、强迫症 （强迫症）、智力障碍、焦虑和自闭症都是 CHARGE 中常见的现象。针对这些情况的治疗 行为症状是有限的，突出了理解其机制的关键需要领域 找出这些缺陷的根源，以开发新的治疗方法。三分之二的指控案件是 由 chd7 的功能缺失突变引起，chd7 编码 DNA 结合、ATP 依赖性染色质 重塑蛋白。 chd7 在发育中的脊椎动物大脑中高度表达并调节 几个关键的神经发育基因，但 chd7 功能和功能之间有直接的细胞和分子联系 行为监管尚未建立。该项目利用幼虫斑马鱼模型，该模型具有 迅速成为研究行为回路的发展和功能的强大系统 以及人类遗传病。使用 CRISPR/Cas9，我们建立了 chd7-null 系并已 表征了反映电荷的几种形态和行为表型。响应声学 刺激下，chd7突变体执行正常的短潜伏期惊吓反应（SLC），但长潜伏期受损 逃避反应（LLC）。同样，chd7 突变体对光照的增加反应正常，但缺乏 响应照明的减少。这些缺陷与眼睛的形态缺陷无关 和耳朵，表明 chd7 可能调节大脑中的特定行为回路。在目标 1 中，我们将 使用体内钙成像系统地询问驱动这些行为的已知电路元件， 细胞特异性救援以定位 cdh7 作用位点。然后我们将全面定义大脑区域 使用全脑形态测量和活动分析依赖于chd7。在目标 2 中，我们将应用状态- 最先进的蛋白质组学和转录组学方法来鉴定将 chd7 与这些物质联系起来的分子途径 大脑结构和功能的变化。通过分析三个发育时间点的样本，我们还将 定义这些变化的时间动态。最后，我们将使用系统的 CRISPR/Cas9 方法 通过测量大脑发育来验证体内基于蛋白质组学和转录组学的顶级 chd7 靶点 和突变幼虫的行为。总体而言，这项工作的结果将在 chd7、其 分子目标和行为回路。此外，这些目标将产生一套强大的、广泛有用的 用于探究 CHD7 依赖性神经发育的细胞和分子基础的资源。

项目成果

Morphological and sensorimotor phenotypes in a zebrafish CHARGE syndrome model are domain-dependent.

斑马鱼 CHARGE 综合征模型中的形态和感觉运动表型是领域依赖性的。

• 发表时间: 2023-06
• 作者: Hodorovich, Dana R;Lindsley, Patrick M;Berry, Austen A;Burton, Derek F;Marsden, Kurt C
• 通讯作者: Marsden, Kurt C