Ephrin-B2通过调控GABA抑制性中间神经元发育影响小鼠精神分裂症样行为的机制研究

Schizophrenia is a devastating mental disorder with a complex etiology. Its cause and progression, though incompletely understood, are recently found to be linked with the developmental disorder of GABAergic interneuron. It is suggested that normal brain development and function is impaired long before the onset of the first full-blown clinical symptoms. Biological understanding of genetic risk factors may be able to link neurodevelopmental etiopathogenesis to adult onset and pathophysiology of schizophrenia. Eph receptor tyrosine kinases and their membrane anchored Ephrin ligands have critical roles in neuronal maturation and synapse formation upon axon-dendrite contact. It is worth noting that clinical evidence revealed an association between abnormal Ephrin-B2 gene expression and schizophrenia. Consistently, our preliminary results demonstrated schizophrenia-like behavioral deficits in Ephrin-B2lacZ/+ heterozygous mice with C-terminal truncated Ephrin-B2-β-galactosidase fusion protein. In this study, we aim to clarify the precise role of Ephrin-B2 in mediating schizophrenia-like behavioral deficits in mice. We intend to use Ephrin-B2lacZ/+ mice, Ephrin-B2Cre/loxp mice as well as mice with stereotaxic injections of adeno-associated viral over-expressing Ephrin-B2. In combination with morphological study and biochemical study, we would like to focus on how Ephrin-B2 mediated signaling pathways affect the GABAergic interneuron development and synaptic connection. Furthermore, we would like to figure out the relationship in-between spatial-temporal expression of Ephrin-B2, abnormal GABAergic interneuron phenotypes and behavioral deficits in mice. In conclusion, we hope to provide the scientific evidence for demonstrating the effects of Ephrin-B2 on the progression of schizophrenia, which would help to find novel therapeutic mechanism for treating schizophrenia.

精神分裂症是神经发育密切相关的疾病，因此与发育相关的基因表达及功能的异常对精神分裂症发生发展的影响受到普遍关注。Eph-Ephrin家族分子是一类介导神经元发育、突触间信号传递的重要分子。证据显示Ephrin-B2表达异常与精神分裂症可能存在相关性。我们前期研究发现其在中枢神经系统表达降低会导致成年小鼠表现精神分裂症样行为异常。本课题将继续运用Ephrin-B2lacZ/+转基因小鼠、Ephrin-B2条件性敲除小鼠及构建病毒特异性过表达Ephrin-B2技术，探讨Ephrin-B2在大脑GABA能中间神经元的定位、对其发育表型的影响等。继而结合组织细胞形态学、动物行为学等手段，探讨Ephrin-B2分子时空表达与中间神经元结构功能变化、小鼠精神分裂症样行为的关系及相关信号转导机制。本研究预期结果将有助于阐释Ephrin-B2参与调控的GABA能中间神经元发育影响精神分裂症发生发展的机制。

本研究率先证明突触发育调节分子Ephrin-B2与精神分裂症相关阴性症状及认知症状等异常行为的发生发展密切相关。我们通过在神经系统发育的关键时期（新生期）、特定抑制性神经元中敲除Ephrin-B2基因及病毒定位过表达系统，分别在青春期、成年期检测小鼠行为学，发现Ephrin-B2基因时空特异敲除小鼠表现出部分精神分裂症样行为学特点。因此，我们首次证实Ephrin-B2基因变异可以诱导出小鼠精神分裂症样行为表型，尤其是以社交受损为代表的阴性核心症状。我们首次通过蛋白组学技术筛选确认Ephrin-B2介导行为异常的关键下游分子HDAC4，这从分子机制水平上更为清晰的阐释突触发育分子Ephrin-B2介导的关键反向信号通路。为进一步模拟精神分裂症病人临床表现的行为异常，我们采用条件性敲除Ephrin-B2依次联合使用幼鼠生长发育过程各期的应激性生活事件（即母婴分离、隔离饲养及足部电击诱发的回避反应）、青春期谷氨酸系统的损伤破坏小鼠感觉运动门控功能，模拟精神分裂症的自然发病过程，造成小鼠成年后出现与精神分裂症相类似的行为学表现及大脑功能异常，以此构建突触发育调节分子Ephrin-B2与环境“多重打击”交互作用精神分裂症动物模型，发现该类模型小鼠表现出更为显著的精神分裂症样行为。这说明我们以前脉冲抑制和小鼠行为学改变作为评价指标体系，衡量多重打击法联合调控神经突触发育制备精神分裂症动物模型具有可行性并可以形成标准化操作流程。继而我们以该模型对精神分裂症治疗药物的反应为模型预测效度评价指标，采用奥氮平、利培酮、阿立哌唑对模型进行评估，发现奥氮平及阿立哌唑可以缓解模型小鼠的精神分裂症样行为。综上所述，本项目阐释了精神分裂症核心症状--社交受损等异常行为发生过程中的突触发育作用机制。通过外界不良环境刺激的交互造模与评估，我们建立了精神分裂症小鼠模型的构建标准体系和评价标准体系。通过本项目，我们发现了突触发育分子Ephrin-B2所关联的组蛋白去乙酰化酶的逆转社交受损行为异常的潜在靶点，我们同时也成功建立了新一代的外部环境刺激--神经发育相结合的精神分裂症动物模型。该靶点及模型稳定可重复性强，可推广用于今后精神分裂症阴性症状的病因机制研究及药物筛选。