利用大脑皮质类器官模型探索奥氮平调控Nodal对早期胚胎大脑发育的影响

Olanzapine is the most frequently used antipsychotic, and concerns have been raised about a potential association with abnormal fetal brain development. The cerebral organoids—derived from human pluripotent stem cells (hPSCs)—undergoes early stage neural tube formation, followed by neuroepithelia differentiation, eventually developed into self-organized, brain-specific epithelial tissue. It offers a promising approach for investigating the mystery of human brain, which can mimic the endogenous development of human brain. We have previously generated human cerebral organoids using human-induced pluripotent stem cells and treated with different concentrations of olanzapine to study the influence. We found that the neural epithelium tissues formed in olanzapine group have more buds compared with control group. Further analysis demonstrated that Nodal is highly expressed in olanzapine-treated group. Therefore, we suppose that Nodal is closely participated in the promotion of cerebral organoids development by olanzapine treatment. We determine to establish the cerebral organoid model to investigate the effect of long treatment of olanzapine on early brain development. Then, we plan to explore the role of Nodal in regulating the development promotion effect of olanzapine, identify potential related targets, explicit molecular mechanisms and signaling pathways. Therefore, the implement of our project is helpful to provide new evidence for olanzapine use with a vital clinical significance.

奥氮平是临床处方量排第一的抗精神病药物，其在孕期使用可能导致胚胎大脑发育异常。大脑皮质类器官是由干细胞定向诱导分化形成的大脑皮质结构，可一定程度上在体外模拟人类胚胎早期大脑的发育过程和结构特点。申请人前期建立了大脑皮质类器官模型，发现奥氮平对类器官分化过程中的神经上皮形成有重要影响。相比于对照组，奥氮平处理组的神经上皮组织形成的神经芽突明显增多。进一步RNA测序发现奥氮平组Nodal生长分化因子显著升高。由此我们提出了“Nodal生长分化因子可能通过相应的通路调节，参与奥氮平对大脑皮质类器官分化的促进作用”的科学假说。本课题拟在此基础上进一步探讨：奥氮平长时间处理对大脑皮质类器官分化、脑区形成和神经细胞的影响；奥氮平调控Nodal来促进大脑类器官分化的相关靶点和具体机制。本项目的完成将阐明奥氮平促进大脑皮质类器官分化的分子机制，为奥氮平用药提供新的实验依据和数据支持，具有重要的临床意义。

精神分裂症是一种重型的精神疾病，奥氮平是临床处方量排第一的抗精神病药物，既往研究提示其在孕期使用可能导致胚胎大脑发育异常。不断积累的证据显示，妊娠期使用SGAs导致新生儿严重畸形的绝对风险很低，孕早期暴露于SGAs不显著升高严重畸形风险。然而，既往研究缺乏对脑区影响的直接证据。课题组利用大脑皮质类器官模型观察奥氮平对早期大脑胚胎发育的影响，发现奥氮平对类器官分化过程中的神经上皮形成有重要影响。对处理后不同时间点的奥氮平组和对照组分别进行了RNA测序，发现奥氮平促进大脑类器官的前额发育。与已有的大脑发育图谱（the Human Developing Brain atlas）对比，24天的奥氮平处理组与更高胎龄的发育图谱一致，提示奥氮平处理相对于对照组有促进胎脑发育的效果，主要促进的脑区为前额皮质。免疫荧光染色也观察到了奥氮平组神经干细胞SOX2和前额标志物FoxG1的高表达。对12天的两组RNA seq测序发现， MXL1/PCAT14/Nodal等基因在奥氮平组显著高表达。进一步通路分析提示，Nodal相关通路在奥氮平组中起重要作用，可能与神经上皮促进有关。为了进一步明确奥氮平是否通过激活Nodal 生长分化因子来促进大脑皮质类器官的分化，我们在奥氮平处理组中加入Nodal 抑制剂SB431542，观察奥氮平导致的大脑皮质类器官分化促进作用能否被抑制，并与奥氮平处理组、SB431542组，溶媒对照组比较。结果发现，Nodal抑制剂加入后类器官的芽突生成减少，同时伴有Sox2和Pax6的表达减弱。课题组随后采用基因编辑技术敲低Nodal的表达进行了进一步验证。上述研究阐明了奥氮平促进大脑皮质类器官分化的分子机制，为奥氮平孕期用药提供新的实验依据和数据支持，具有重要的临床意义。

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