The molecular roles of RFX3 in neurodevelopment and Autism Spectrum disorder

项目摘要 自闭症谱系障碍(ASD)是最常见的神经发育障碍，但 ASD发病的神经生物学机制在很大程度上仍然未知。大规模 对自闭症患者的外显子组测序研究已经显著识别了100多个基因 与ASD风险相关。ASD风险基因的功能特征可以为深入了解 ASD的发病机制。我们和其他人最近发现了新的功能丧失变异体 转录因子RFX3是ASD相对常见的单基因病因，意味着 RFX3在人类神经发育中的重要作用。我们已经发现证据表明RFX3可能 作为II/III层神经元发育和功能的关键转录调节因子：ITS 在皮质II/III层兴奋性神经元中表达显著丰富，并且RFX3结合 Motif特异地富含在人类胎儿生发区的可及染色质区域 和II/III层兴奋性神经元。在这项拟议的研究中，我将解决以下假设 RFX3调节II/III层兴奋性神经元的关键神经发育过程 影响神经元形成和功能的其他ASD危险基因的表达。在《目标1》中，我将 通过对人皮质神经元中RFX3调控的基因和信号转导通路的研究 RFX3全基因组结合位点及其缺失引起的转录变化 RFX3单倍体不足的人IPSC来源神经元的占有率。在目标2中，我将评估 RFX3单倍体缺陷对人脑皮质神经元形成和突触功能的影响 IPSC衍生的前脑有机化合物。我将使用单细胞RNA测序来识别细胞的变化 RFX3缺陷型有机体的类型组成和发育轨迹的变化， 和多电极阵列评估RFX3缺陷类器官的突触可塑性平衡 与同基因对照相比。综上所述，这项建议将对 人类神经元中受RFX3调控的转录程序，以及RFX3单倍体不足的原因 扰乱神经元的发育和功能。这将有助于更好地理解ASD 神经生物学，以及针对自闭症的新靶向疗法的开发。

Project Abstract Autism Spectrum Disorder (ASD) is the most common neurodevelopmental disorder, yet the neurobiological mechanisms underlying ASD pathogenesis remain largely unknown. Large-scale exome sequencing studies of individuals with ASD have identified over 100 genes significantly associated with ASD risk. Functional characterization of ASD risk genes can provide insight to ASD pathogenesis. We and others have recently identified de novo loss-of-function variants in the transcription factor RFX3 as a relatively common monogenic cause of ASD, implying an important role for RFX3 in human neurodevelopment. We have found evidence that RFX3 may be a critical transcriptional regulator of the development and function of layer II/III neurons: its expression is significantly enriched in cortical layer II/III excitatory neurons, and the RFX3 binding motif is specifically enriched in accessible chromatin regions of the human fetal germinal zone and layer II/III excitatory neurons. In this proposed research, I will address the hypothesis that RFX3 regulates key neurodevelopmental processes in layer II/III excitatory neurons and the expression of other ASD risk genes that affect neuronal formation and function. In Aim 1, I will identify the genes and pathways regulated by RFX3 in human cortical neurons by profiling the genome-wide binding sites of RFX3 and the transcriptional changes induced by loss of RFX3 occupancy in RFX3 haploinsufficient human iPSC-derived neurons. In Aim 2, I will evaluate the effect of RFX3 haploinsufficiency on cortical neuron formation and synaptic function in human iPSC-derived forebrain organoids. I will use single-cell RNA-sequencing to identify changes in cell type composition and infer alterations in developmental trajectories in RFX3 deficient organoids, and multielectrode array to assess synaptic plasticity balance in RFX3 deficient organoids compared to isogenic controls. Taken together, this proposal will yield insight on the transcriptional programs regulated by RFX3 in human neurons, and how RFX3 haploinsufficiency disrupts neuronal development and function. This will allow for improved understanding of ASD neurobiology, and the development of novel targeted therapies for ASD.