ADAM10通路在小脑神经细胞发育中的作用和机制研究

The cerebellum is a morphologically unique brain structure made up of an elaborate set of folia separated by fissures. Recent evidence suggests that the cerebellum participates in higher order functions, including cognition, emotion and language processing, in addition to its well-documented role in coordinating proprioceptive-motor processing. A variety of clinical diseases are related to its developmental abnormalities. Adam10 (a disintegrin and metalloprotease 10) is a member of the ADAMs family, and plays important roles in the development of the cerebral cortex. Currently, the role of adam10 gene in the development of cerebellum is unclear. In previous study, we utilized the Cre /loxp conditional gene knockout technology to obtain the neuranal adam10 gene knockout mice, and showed that neuronal adam 10 gene knockout mice display developmental abnormalities, mainly including decreased size, disorganized foliation and lamination of the cerebellar cortex, abnormal migration and distribution of the cells. In this study, based on the previous adam10 gene knockout mouse model,by use of the immunofluorescence,immunohistochemistry, western blot, immunoprecipitation, in situ hybridization, in vivo electroporation, we aim to investigate the abnormal development of the cerebellum, especially the changes of neuroglial proliferation,apoptosis, differentiation and migration, and protein expression levels of the related signal molecules, to reveal the roles and the molecular mechanism of adam10 mediating cerebellar development, to expand the field of research on cerebellum development, and to provide new theoretical bases for the clinical cerebellar diseases prevention and gene therapy.

小脑是运动协调的重要器官，其发育异常与临床多种疾病相关。Adam10（A Disintegrin And Metalloprotease 10）基因是Adams家族的重要成员，研究表明其在哺乳动物大脑皮层发育过程中发挥重要作用。目前，adam10基因在小脑发育过程的作用尚不清楚。我们前期采用Cre/loxP条件性基因敲除技术获得了神经细胞特异性adam10基因敲除小鼠，发现该小鼠小脑分叶和皮层结构缺陷、细胞发育及分布异常。本项目拟在前期已获得的adam10基因敲除鼠模型基础上，采用免疫组织化学、免疫印迹及共沉淀、原位杂交和在体电转染等技术，研究adam10基因敲除对小脑星形胶质细胞、颗粒细胞和浦肯野细胞的增生、凋亡、分化、迁移等的影响，并分析相关蛋白分子及其信号通路的改变，另通过挽救实验进一步验证adam10基因在小脑发育过程中的作用。该课题的完成将为临床神经系统相关疾病的防治提供新思路。

Adam10基因在小脑的广泛表达提示其在小脑发育过程中可能发挥重要的调控作用。胚胎发育早期敲除神经系统adam10基因导致小鼠在出生前死亡，未能深入研究adam10基因在小脑发育过程中的作用。我们通过利用CRE-LOXP系统实现胶质细胞 （包括放射状胶质细胞）adam10基因特异性敲除，由于选择性地敲除胶质细胞的adam10基因，adam10基因敲除小鼠可以存活到出生后3周左右，这为我们研究adam10基因在小脑发育过程中的作用提供了前提。我们研究结果发现adam10基因敲除小鼠小脑最早在胚胎18.5天出现发育异常，形态学上主要表现为小脑分叶和皮层分层缺陷。敲除鼠小脑星形胶质细胞最早在胚胎18.5天出现异常，主要表现为相邻脑叶基底部细胞胞体排列紊乱。随着发育进行，Bergmann胶质细胞形态分化障碍，失去放射状突起，不能与脑膜接触形成终足结构。同时，胶质细胞BLBP蛋白表达水平下降，胶质细胞凋亡增加。颗粒细胞最早在出生后4天左右出现迁移定位异常，表现为颗粒细胞异常聚集在相邻脑叶融合处而不能内迁；到出生后21天左右，部分颗粒细胞不能内迁进入内颗粒细胞层，而是异常聚集在脑膜表面或者相邻脑叶融合处。另外，颗粒细胞前体细胞的增生下降，成熟的颗粒细胞凋亡增加。浦肯野细胞发育异常主要是成熟、定位障碍，并且这种异常是非细胞自主性的继发性改变。出生后特异性敲除胶质细胞或颗粒细胞adam10基因都不引起小脑发育异常。Adam10基因敲除导致Notch I蛋白表达水平下调，但不影响β-catenin、N-cadherin和L1CAM蛋白表达水平。我们的研究提示胶质细胞adam10基因对于小脑的分叶形成和皮层结构的建立起到关键性的作用，同时对胶质细胞的发育和颗粒细胞的迁移也起到非常重要的作用，其机制可能涉及adam10-Notch-BLBP信号通路。