IMPACT OF LOSS OF THE AUTISM RISK GENE CHD8 ON EXPRESSION, EPIGENETICS AND CELLULAR FUNCTIONING OF THE DOPAMINERGIC SYSTEM

项目总结 丽贝卡·穆勒博士是一名执业的儿童精神病学家，也是一名早期研究员，有着良好的记录 开发分子遗传学工具来阐明神经发育障碍的机制。她长长的- 术语目标是确定受染色体结构域解旋酶丢失影响的细胞类型、回路和调节网络 DNA结合蛋白8(CHD8)是一种高度可信的自闭症谱系障碍(ASD)危险基因。穆勒博士发起 她在与詹姆斯·努南(James Noonan)在马修州立大学(Matthew State)共同指导下获得博士学位期间对CHD8的研究， 医学博士，博士。她成功地从国家精神卫生研究所和 西蒙斯自闭症研究基金会确定CHD8及其损失对监管网络的影响 她还表明，发育中的皮质中的CHD8调节着其他ASD风险基因。 她正在哥伦比亚大学与Jeremy Veenstra-VanderWeele合作扩展这项研究， M.D.和Muhammed Osman Chohan，医学博士，神经行为障碍的翻译动物模型专家 以及单胺能神经传递的研究。在一起，他们发现减少了刻板印象， 抑制多巴胺后，雄性CHD8+/-小鼠的过度运动活动减弱，但雌性CHD8+/-小鼠的运动过度活动减弱 转运蛋白，对多巴胺受体激动剂有完整的反应，表明CHD8在 中脑和纹状体之间多巴胺能回路的发育。本次R01的总体目标 建议阐明CHD8基因突变影响细胞和关键途径的机制 ASD和其他神经发育障碍的核心症状，通过整合最先进的单核 转录学和细胞类型特异性功能基因组学与电生理和行为探测 CHD8表达缺失的动物模型。 以下三个独立的具体目标将解决CHD8调节基因的中心假设 对基底节和多巴胺能系统的功能至关重要，CHD8的丢失将 这些基因和其他自闭症风险基因的失调。该项目将1)确定细胞、转录和 用单核RNA研究CHD8+/-小鼠腹侧中脑和纹状体发育中的表观遗传学变化 测序(SnRNAseq)和染色质可及性(SnATACseq)，2)阐明CHD8缺失是如何导致缺陷的 纤维光度法和活体实验研究DA刺激在基底神经节的分子传递 使用结构性和条件性CHD8单倍体功能不全小鼠模型的细胞外记录，以及3) 用染色质定位多巴胺能系统中特定细胞类型的CHD8结合活性 条件性生物素化CHD8标签转基因的免疫沉淀和测序。通过研究CHD8，一个 针对其他ASD风险基因的关键调控分子，Muhle博士将填补 CHD8在ASD相关行为所涉及的电路中的作用。

PROJECT SUMMARY Dr. Rebecca Muhle is a practicing child psychiatrist and an Early Stage Investigator with a strong track record of developing molecular genetic tools to elucidate the mechanism of neurodevelopmental disorders. Her long- term goal is to identify cell types, circuits and regulatory networks affected by loss of Chromodomain Helicase DNA-binding Protein 8 (CHD8), a high confidence autism spectrum disorder (ASD) risk gene. Dr. Muhle initiated her studies of CHD8 during her fellowship with James Noonan, Ph.D. with co-mentorship by Matthew State, M.D., Ph.D. She successfully obtained research funding from the National Institute for Mental Health and the Simons Foundation for Autism Research to determine the impact of CHD8 and its loss on regulatory networks of the developing cortex, and she showed that CHD8 in developing cortex regulates other ASD risk genes. She is extending this research at Columbia University in collaboration with Jeremy Veenstra-VanderWeele, M.D. and Muhammed Osman Chohan, M.D., experts in translational animal models of neurobehavioral disorders and the study of monoaminergic neurotransmission. Together, they have found reduced stereotypy and attenuated hyperlocomotor activity in male, but not female Chd8+/- mice after inhibition of the dopamine transporter, with an intact response to dopamine receptor agonism, suggesting that CHD8 plays a critical role in the development of the dopaminergic circuit between the midbrain and striatum. The overall objective of this R01 proposal is to elucidate the mechanisms whereby CHD8 genetic mutations affect cells and pathways critical for core symptoms of ASD and other neurodevelopmental disorders, by integrating state-of-the-art single nuclear transcriptomics and cell type specific functional genomics with electrophysiologic and behavioral probing of animal models with a loss of CHD8 expression. The following three independent specific aims will address the central hypothesis that CHD8 regulates genes that are critical for the function of the basal ganglia and dopaminergic system, and that loss of CHD8 will dysregulate these genes and other ASD risk genes. This project will 1) identify the cellular, transcriptional, and epigenetic changes in the developing Chd8+/- mouse ventral midbrain and striatum using single nuclear RNA sequencing (snRNAseq) and chromatin accessibility (snATACseq), 2) elucidate how CHD8 loss causes a defect in the molecular transmission of the DA stimulus in the basal ganglia using fiber photometry and in vivo extracellular recordings using constitutive and conditional CHD8 haploinsufficiency mouse models, and 3) localize CHD8 binding activity in specific cell types of the dopaminergic system using chromatin immunoprecipitation and sequencing of a conditionally biotinylated Chd8-tag transgene. By studying CHD8, a key regulatory molecule that targets other ASD risk genes, Dr. Muhle will close a critical knowledge gap in the role of CHD8 in the circuitry implicated in ASD-related behaviors.