基于iPS技术的AUTS2基因缺陷细胞模型的建立及机制研究

Autism, a complex neurodevelopmental disorder with high genetic heterogeneity, has become a serious common disorder to children’s health. As the etiology and pathogenesis are unclear, no effective treatments available. In our previous study, an exonic deletion of autism susceptibility candidate 2 (AUTS2) was identified in a patient with autism. His urinary cells (UCs) were cultured and reprogrammed to non-integrating induced pluripotent stem cells (iPSCs), and then differentiated to neural progenitor cells (NPCs). Compared with UCs-derived iPSCs/NPCs obtained from a healthy child, the expression of AUTS2 from the patient’s iPSCs/NPCs was decreased and Rac1 was also showed down-regulated. We tried to induce NPCs to mature neurons and found that electrophysiological function of the neuron from the patient was impaired. Given that Auts2 deletion could result in abnormal neuronal migration and neurite formation via repressing Rac1 activity in mice, we speculate that AUTS2 may affect electrophysiological function of neuron by binding Rac1. To confirm this hypothesis, the patient’s UCs-derived iPSCs/NPCs/neurocytes were established to explore the role of AUTS2 and pathogenesis of autism in order to provide a scientific and experimental basis for discovery of new pharmacological targets and individualized treatment.

孤独症是具有高度遗传异质性的神经发育障碍性疾病，已成为严重影响儿童健康的常见病。因病因和发病机制不明，迄今仍缺乏有效治疗措施。我们前期在1例孤独症患儿中检测到孤独症易感候选基因2（AUTS2）缺失突变，收集患儿尿液，将尿液细胞（UCs）重编程为iPSCs，并进一步诱导为神经前体细胞（NPCs），发现与健康儿童的iPSCs/NPCs相比，AUTS2表达降低，Rac1表达也下调；我们尝试将NPCs分化为神经元，发现其电生理功能异常。动物实验显示Auts2缺陷可抑制Rac1活性导致神经元迁移及神经突形成缺陷，因此我们推测人AUTS2可以通过调控Rac1的表达影响神经细胞电生理功能。为证实本假说，本研究拟建立AUTS2缺陷患儿iPSCs/NPCs模型，以此探讨AUTS2和Rac1对神经细胞表型及电生理功能的影响，揭示AUTS2缺陷所致孤独症的分子机制，为寻找孤独症个体化治疗药物靶点提供依据。

孤独症易感候选基因2（AUTS2）与儿童孤独症的发病密切相关，但机制并不清楚。我们前期收集了1例AUTS2缺失突变的孤独症患儿的尿液细胞并重编程为iPSCs。在本课题中，我们将其进一步诱导为神经前体细胞（NPCs），发现与健康儿童的NPCs相比，患儿NPCs中AUTS2表达降低，Rac1表达也下调。进一步将NPCs分化为神经元，发现患儿来源的神经元细胞表型和电生理功能均异常。随后将AUTS2在神经细胞中过表达后，神经元的神经突显著变长变粗，与正常神经元也有差别。而双荧光素酶报告载体实验和蛋白质免疫共沉淀实验发现AUTS2能够直接调控RAC1的启动子促进其表达，而与RAC1蛋白无直接相互作用。最后，转录组测序发现AUTS2缺陷的神经细胞中DCX等下游基因的表达也发生了变化，且DCX的启动子受到AUTS2的调控。我们的研究结果初步揭示了AUTS2缺陷导致孤独症的分子机制，为孤独症的临床诊断和治疗提供了理论依据，对寻找孤独症个体化的药物治疗靶点具有重要意义。