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

项目摘要 Rebecca穆勒博士是一位执业儿童精神病学家，也是一位早期研究员，有着良好的记录 开发分子遗传工具来阐明神经发育障碍的机制。她的长- 长期目标是确定细胞类型，电路和调控网络的影响，损失的染色体结构域解旋酶 DNA结合蛋白8（CHD 8），一个高置信度的自闭症谱系障碍（ASD）风险基因。穆勒博士发起了 她在与James Noonan博士合作期间对CHD 8的研究。由马修州立大学共同指导 医学博士，博士她成功地获得了国家心理健康研究所和 西蒙斯自闭症研究基金会，以确定CHD 8的影响及其对监管网络的损失 她发现发育中的大脑皮层中的CHD 8调节其他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.和穆罕默德·奥斯曼·乔汉医学博士神经行为障碍转化动物模型专家 以及单胺能神经传递的研究。总之，他们发现减少刻板印象， 抑制多巴胺后，雄性Chd 8 +/-小鼠的过度运动活动减弱，但雌性Chd 8 +/-小鼠的过度运动活动未减弱 转运蛋白，对多巴胺受体激动剂有完整的反应，表明CHD 8在 中脑和纹状体之间多巴胺能回路的发展。本R 01的总体目标 这项研究的目的是阐明CHD 8基因突变影响细胞的机制和对细胞凋亡至关重要的途径。 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细胞 转录组学和细胞类型特异性功能基因组学与电生理学和行为探测 CHD 8表达缺失的动物模型。 以下三个独立的具体目标将解决CHD 8调节基因的中心假设 这对基底神经节和多巴胺能系统的功能至关重要，CHD 8的缺失将 这些基因和其他ASD风险基因失调。该项目将1）识别细胞，转录， 用单个核RNA研究发育中Chd 8 +/-小鼠腹侧中脑和纹状体的表观遗传变化 测序（snRNAseq）和染色质可及性（snATACseq），2）阐明CHD 8缺失如何导致缺陷 在基底神经节中使用纤维光度法和体内DA刺激的分子传递中， 使用组成性和条件性CHD 8单倍不足小鼠模型的细胞外记录，和3） 使用染色质在多巴胺能系统的特定细胞类型中定位CHD 8结合活性 免疫沉淀和有条件生物素化的Chd 8-标签转基因的测序。通过研究CHD 8， 针对其他ASD风险基因的关键调节分子，穆勒博士将填补在 CHD 8在ASD相关行为中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.