TPH2基因突变对血清素神经元的影响及其在创伤后应激障碍中作用机制的研究

In the brain, tryptophan hydroxylase-2 (TPH2) gene is uniquely expressed in serotonin neurons. It is reported that TPH2 gene mutation is associated with the pathogenesis of post-traumatic stress disorder (PTSD). However, the effects of TPH-2 gene mutation on the serotonin neurons of PTSD patients and the associated mechanism of PTSD pathogenesis are largely unknown. Recently, we transplanted the iPSC-derived serotonergic progenitors into the brain of the newborn mice, they turned into serotonergic neurons which showed the migration and projection to the host’s cerebellum, hindbrain and spinal cord. The research wrote above is the foundation for us to explore the effects of TPH2 gene mutation on PTSD-SN. In this study, iPSC generated from PTSD patients with TPH2 mutations will be differentiated into serotonin neurons. Detecting celluar morphology, formation of synapse, electrophysiology, neurotransmitter releasing and transplantation of PTSD iPSC-derived serotonin neurons into PTSD animal model will be applied to determine the pathological phenotype. Then, TPH2 gene in healthy serotonin neurons will be mutated and mutated TPH2 gene in PTSD-serotonin neurons will be corrected by gene-editing via CRISPR/Cas9. Phenotypical alterations between before and after gene-editing will help us to expose and confirm the effects of TPH2 mutations on PTSD-serotonin neurons, which will help us to reveal the underlying mechanism by which TPH2 mutations promotes PTSD, and provide a new method to diagnose and treat PTSD.

脑内色胺酸羟化酶-2(TPH2)基因仅在血清素神经元(SN)中表达，研究发现该基因突变与创伤后应激障碍(PTSD)发病密切相关。但尚无研究报道该基因突变对PTSD患者SN的影响及其在PTSD发病中的作用机制。申请人近期研究发现诱导性多能干细胞(iPSC)来源的SN前体移植入小鼠脑内后分化成熟为SN，并迁移入宿主小脑、后脑及脊髓，这为从分子、细胞和动物水平研究TPH2基因突变对PTSD-SN的影响奠定了基础。在此研究基础上，本项目将定向分化PTSD病人iPSC获得SN，通过检测细胞形态、突触形成、电生理、血清素的合成分泌及体内移植等方法发现疾病表型；再通过基因编辑，分别突变或纠正健康SN或PTSD-SN的TPH2基因，观察基因编辑前后SN的表型变化，明确TPH2基因突变是否是引起PTSD-SN异常表型的主因，进而阐明TPH2基因突变在PTSD发病中的作用机制，为PTSD诊疗提供新策略。

脑内色胺酸羟化酶-2基因仅在血清素神经元中表达，临床关联性研究发现该基因突变与创伤后应激障碍(PTSD)发病密切相关。但尚无研究报道该基因突变对血清素神经元的影响及其在PTSD发病中的作用机制。申请人前期研究发现诱导性多能干细胞(iPSC)来源的血清素神经前体移植入小鼠脑内后分化成熟为血清素神经元，并迁移入宿主小脑、后脑及脊髓，这为从分子、细胞和动物水平研究TPH2基因突变对PTSD的影响奠定了基础。在此基础上，本项目应用CRISPR/Cas9基因编辑技术，将野生型人类胚胎干细胞的TPH2基因精准点突变，并将此突变细胞构建成TPH2-EGFP报告细胞，向血清素神经元定向分化。研究发现，（1）TPH2-EGFP报告细胞表达的EGFP能够忠实显示血清素神经元，这为血清素神经元的纯化和活细胞示踪提供了基础工具；（2）点突变的细胞定向分化为血清素神经前体、神经元的过程以及血清素神经元的形态无明显异常，提示点突变对血清素神经元的发育过程和细胞形态影响不明显；（3）点突变导致血清素神经元电生理功能异常，可能是导致PTSD发病的功能性原因。