Nogo-A/NgR信号通路在皮质发育障碍发生中的作用与机制研究

Heterogeneous neurons are important causes of malformations of cortical development(MCDs) causing refractory epilepsy.Abnormalities of Neural precursor cells (NPCs) migration may be one cause of Heterogeneous neurons. However, the specific mechanisms is still not clear.Our previous studies showed the expression of Nogo-A and its receptors NgR were upregulated in MCDs cortical lesions at both the mRNA and protein levels. In additon, we created an MCD mice model according to previous reports and found that the number of NPCs, migrating from SVZ, at cortical lesions of Nogo-A Knock out mice is larger than those of WT cotrol group. Thus, we suppose that the overexpression of Nogo-A may regulate the migration of misshapen cells by inhibiting the migration activity of NPCs and regulating the direction of cell growth cone and be related to the development or progression of MCD diseases.In this study, through comprehensive utilization of technologies such as morphology, molecular biology, electrophysiology, we analyzed the protein and messenger RNA(mRNA) levels of Nogo-A,NgR and its downstream targets in animal model and surgically resected cortical lesions of TSC tubers and FCDIIb.Using Nogo-A knockout animals,we investigate the possible downstream signaling pathway of Nogo-A which may be regulate the NPCs migration.Furthermore,we also investigate the influence of Nogo-A on synaptic remodeling, synaptic transmission and neural electrophysiological properties of MCDs cortical lesions. Clarifying above issues will be valuably to elucidate the pathological mechanism of MCDs, which will support us a new theoretical basis on the prevention and treatment of clinical MCDs and the resulting epilpsy.

异构神经元是皮质发育障碍（MCDs）发展为难治性癫痫的重要诱因，而神经前体细胞（NPCs）异常迁移是异构神经元形成的关键。本项目组率先发现Nogo-A及其受体在MCDs病灶中表达升高，并且伴随异常迁移的NPCs细胞数量增加，这提示Nogo-A可能参与了NPCs迁移的调控过程，继而诱发MCDs病灶产生。据此本研究拟进一步综合运用形态学、分子生物学、膜片钳等技术，研究Nogo-A/NgR及下游信号在MCDs 临床标本和动物模型中的表达和组织学定位；利用敲基因动物观察Nogo-A/NgR通路对NPCs向MCDs致痫灶迁移的影响并筛选参与迁移调节的Nogo-A下游信号分子，阐明下游信号分子表达与NPCs迁移的关系；探明Nogo-A对MCDs突触重构、突触传递及神经电生理特性的影响，进一步明确其调控NPCs参与MCDs发生的机制。阐明以上问题将为临床MCDs及所致癫痫的预防与诊治，提供新的理论依据。

本项目综合运用形态学、分子生物学、神经电生理、细胞培养、RNAi等技术研究：①Nogo-A/NgR及下游信号信号分子在MCDs临床标本和动物模型中的表达和组织学定位；②利用GFP荧光探针/分子对FCD冰冻损伤动物模型SVZ-NPCs进行标记，验证SVZ-NPCs向皮质损伤灶迁移分化过程；③通过外源性Nogo-A蛋白及外源性拮抗剂NEP1-40干扰，证实Nogo-A/NgR信号通路参与皮质损伤后SVZ-NPCs的迁移。④ 通过动物模型标本进一步观察Nogo-A/NgR及下游信号分子的表达变化进一步阐明影响SVZ-NPCs迁移的Nogo-A信号通路。⑤ 通过培养SVZ-NPCs细胞,采用RNAi等技术，证明Nogo-A的上调参与调节SVZ-NPCs的增值与活性并促进干细胞向胶质细胞分化。⑥利用膜片钳技术，探讨NgR修饰后的NPC细胞的电生理的变化以及对细胞内游离钙离子浓度的影响。⑦初步探讨Nogo-A蛋白通过调节Nav1.3的表达与定位参与MCDs的癫痫发生的可能机制。研究结果表明，Nogo-A及受体NgR的mRNA、蛋白表达水平在MCDs皮质致痫灶表达上调，并特异性强表达于与癫痫发作程度密切相关的“异构神经元”。进一步实验证明上调的Nogo-A可激活下游的NgR/LINGO-1/TORY-RhoA信号通路，该信号通路可以抑制NPCs的迁移活性，下调细胞的自我更新及增值能力，并促进干细胞向胶质细胞分化，以上机制可能是NPCs迁移后形成异构神经元参与MCD病理发生的重要原因之一。最后，通过电生理实验，初步证实Nogo-A信号的变化可以影响病灶区电生理特性。通过上述研究，初步为“Nogo-A通路参与MCDs的发生与癫痫点燃过程”的推测提供了形态学和功能学的实验证据。研究成果以论著形式发表SCI论文3篇，形成专利2项。参与国内、国际学术会议交流研究成果，作会议发言1次。协助培养研究生3名。