FMRP-Mediated Gene Regulation in Human Brain Development

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and a leading genetic cause of autism. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein forming a messenger ribonucleoprotein complex with polyribosomes in the regulation of protein synthesis at synapses. Three-dimensional (3D) aggregate culture of human-induced pluripotent stem cells (iPSCs) has evolved from embryoid body cultures, quite faithfully following human organogenesis, and provides a new platform to investigate human brain development in a dish, otherwise inaccessible to experimentation. To determine whether the loss of FMRP could alter the development of human brain organoids, we have generated forebrain organoids from three FXS male patients and three healthy male controls. We observed reduced proliferation of neural progenitor cells and premature neural differentiation as well as perturbed cell cycle progression in FXS forebrain organoids. There is also a deficit in the production of GABAergic neurons as well as an altered balance between the number of excitatory and inhibitory neurons in FXS organoids. Interestingly these deficits were not observed with FXS mouse model. To compare the differential gene expression caused by the loss of FMRP between human and mouse, we then performed RNA-seq to identify the differentially expressed genes using both mouse embryonic brain cortex and human forebrain organoids at the comparable developmental stages. We detected very few genes differentially expressed in the absence of Fmrp in mouse. However, we identified 200 genes downregulated and 126 genes up-regulated in human FXS organoids, indicating human-specific impact caused by the loss of FMRP. Our bulk RNA-seq analyses revealed a more pervasive gene expression alteration in human organoids. Thus, it is plausible to speculate that there could be important human-specific genes that might be related with FXS pathogenesis. To test this idea, we used human forebrain organoids and the mouse embryonic brains in parallel to perform enhanced crosslinking and FMRP immunoprecipitation followed by high-throughput sequencing and bioinformatics analysis (CLIP-seq). Our analyses identified more than 3,700 mRNA bound by FMRP in human organoids. Of the 3,700 mRNAs, ~1,600 overlapped with mouse mRNAs. Further, ~80% of the identified mouse mRNA species overlap with previously published results in mouse. To better understand the developmental, cellular and molecular changes and to gain insight beyond bulk gene expression analysis in forebrain organoids from FXS patients compared to control, we next performed single cell RNA-seq (scRNA-seq) to characterize distinct cell populations, specific cell types and cell states. We found that the population of specific neuron type were changed upon the loss of FMRP. Next, investigated if there were developmental differences in the transitioning from the early, primitive state to one of the more committed end states between control and FXS organoids, with many cells distributed along a “trajectory” between them. we found several bifurcation points of FXS cells from the general trajectory, and the cells of these subpopulations with altered neuronal states are strongly enriched for FXS. These results together suggest that the loss of FMRP could cause neurodevelopmental deficits specifically in human, and fragile X organoids could provide a unique platform to study the molecular pathogenesis of FXS and identify human-specific druggable targets for FXS and autism in general. Overall design: RNA-seq of human organoids, human fetal brain and mouse fetal brain (control and FXS), eCLIP-seq of human organoids and mouse fetal brain (control and FXS), single-cell RNA-seq of human organoids (control and FXS).

脆性X综合征（Fragile X Syndrome, FXS）是最常见的遗传性智力障碍类型，也是自闭症的主要遗传诱因。FXS由功能性脆性X智力低下蛋白（fragile X mental retardation protein, FMRP）缺失导致。FMRP是一种RNA结合蛋白，可与多核糖体形成信使核糖核蛋白复合物，调控突触处的蛋白质合成。人类诱导多能干细胞（human-induced pluripotent stem cells, iPSCs）的三维（3D）聚集培养体系源自胚状体培养技术，能够高度忠实模拟人类器官发生过程，为在体外研究人类脑发育提供了全新平台——这是传统实验手段难以实现的。为明确FMRP缺失是否会改变人类脑类器官的发育，我们从3名FXS男性患者与3名健康男性对照个体中诱导生成了前脑类器官。研究观察到，FXS前脑类器官中存在神经祖细胞增殖能力下降、神经分化提前，以及细胞周期进程紊乱的现象；同时，FXS类器官中γ-氨基丁酸能神经元（GABAergic neurons）生成存在缺陷，兴奋性与抑制性神经元的数量平衡也出现异常。值得注意的是，此类缺陷并未在FXS小鼠模型中被观测到。为比较人类与小鼠中FMRP缺失引发的差异基因表达差异，我们分别选取发育阶段匹配的小鼠胚胎大脑皮层与人类前脑类器官，通过RNA测序（RNA-seq）鉴定差异表达基因。结果显示，小鼠中因Fmrp缺失导致的差异表达基因极少；而人类FXS类器官中，共有200个基因下调、126个基因上调，表明FMRP缺失对人类存在特异性影响。我们的批量RNA测序（bulk RNA-seq）分析显示，人类类器官中的基因表达改变更为广泛。据此我们推测，可能存在一批关键的人类特异性基因，与FXS的发病机制密切相关。为验证这一假说，我们并行使用人类前脑类器官与小鼠胚胎大脑，开展了增强型交联免疫沉淀结合高通量测序（enhanced crosslinking and FMRP immunoprecipitation followed by high-throughput sequencing and bioinformatics analysis, CLIP-seq）分析。结果在人类类器官中鉴定出超过3700种与FMRP结合的mRNA；其中约1600种与小鼠mRNA存在重叠。进一步分析显示，鉴定出的小鼠mRNA中约80%与已发表的小鼠相关研究结果重合。为更深入解析FXS患者前脑类器官的发育、细胞及分子变化，并挖掘批量基因表达分析之外的深层机制，我们随后开展了单细胞RNA测序（single cell RNA-seq, scRNA-seq），以表征不同细胞群、特定细胞类型及细胞状态。我们发现，特定神经元类型的群体在FMRP缺失后发生了改变。接下来，我们探究了对照与FXS类器官在从早期原始状态向更成熟终末状态转变过程中的发育差异——两类器官的大量细胞沿一条“轨迹”分布。我们发现，FXS细胞在该总体轨迹上存在多个分叉点，且这些神经元状态异常的亚群细胞显著富集于FXS样本中。综上，上述结果表明，FMRP缺失仅会在人类中引发神经发育缺陷，而脆性X类器官可为研究FXS的分子发病机制提供独特平台，并有望鉴定出FXS及广义自闭症的人类特异性可药用靶点。本研究整体实验设计如下：人类类器官、人类胎儿脑组织及小鼠胎儿脑组织（对照与FXS组）的RNA测序；人类类器官与小鼠胎儿脑组织（对照与FXS组）的增强型交联免疫沉淀测序（eCLIP-seq）；以及人类类器官（对照与FXS组）的单细胞RNA测序。