有丝分裂检验点与中枢神经系统非整倍体神经细胞的起源

In the central nervous system, 10-30% of neural stem cells(NSC), neural progenitor cells(NPC), neurons and glial cells were found to be aneuploidy, which is significantly higher than that of any other normal tissue cells. These aneuploid neural cells may contribute to diversity of neural cells, the complexity of brain and neural disease development. Why and how are these aneuploid neural cells generated? The causes and chromosomal constitution for the aneuploid neural cells are not defined so far. We have been working for years on the mitotic spindle assembly checkpoint (SAC), which is the major mechanism preventing the generation of aneuploidy, and found that the expression of multiple key genes of SAC were downregulated simultaneously in the neural differentiation of human embryonic stem cells. We hypothesize that neural differentiation inhibits SAC function, improves the frequency of the aneuploidy and enables the potential diversity of neural cells. In this proposal we will define the chromosomal alteration feature in aneuploid neural cells and characterize whether there are alterations of SAC components in terms of gene expression, protein modification, cellular distribution, centromeric localization and protein interactions in neural differention, when compared to that of SAC in other normal tissue cells. We will investigate whether and how the alterations in SAC contribute to the generation of aneuploid neural cells, with the gene knockdown or expression of mutants of SAC genes. We will further investigate what key neural differention molecules regulate the SAC system and what components in the SAC system are regulated. The study will reveal the chromosomal constitution feature of aneuploid neural cells, the alterations of SAC signaling in neurogenesis, the role of the interplay of SAC and neural differentiation mechanisms in the genesis of aneuploid neural cells. The study will generate valuable data critical for understanding the biological and clinic relevance of the aneuploid neural cells.

在正常中枢神经系统中发现10-30%的神经细胞是非整倍体，远高于身体其它组织。非整倍体神经细胞可能贡献神经细胞多样性或导致神经疾病。中枢神经为什么有高比例的非整倍体神经细胞？我们长期研究消除非整倍体产生的机制有丝分裂检验点(SAC)，发现SAC基因在干细胞神经分化中系统性地下调，推测神经分化抑制SAC以增加非整倍体发生和神经细胞多样性。项目首先研究体内中枢神经非整倍体细胞的染色体改变是否具有染色体倾向性及SAC组分的表达、定位、修饰、相互作用等工作机制的变化；然后在体外神经分化中用SAC基因干扰或缺陷突变体诱导产生非整倍体，研究非整倍体神经细胞是否由SAC变化导致，分析体外分化中非整倍体神经细胞的染色体改变特征及SAC工作机制和中枢神经系统是否一致；最后研究哪些神经分子调控哪些SAC组分及如何调控。项目从非整倍体神经细胞的染色体改变特征及发生机制二个新视角揭示非整倍体神经细胞的生物学意义。

在中枢神经系统中发现高达30%的神经细胞是非整倍体细胞，其非整倍体细胞占比远超身体其它组织。有研究证实非整倍体神经细胞能有效地整合进由二倍体神经元组成的神经网络并执行神经传导功能，因此非整倍体神经细胞提供了二倍体神经细胞之外的神经细胞多样性。本项目研究中枢神经系统为什么有高比例的非整倍体细胞。有丝分裂检测点是抑制和消除非整倍体产生的机制，我们研究发现神经分化过程中细胞分裂虽受有丝分裂检测点控制，但是有丝分裂检测点基因BUB1，BUBR1，BUB3，MAD1和MAD2的表达均被显著下调，有丝分裂检测点功能受到神经分化机制的抑制，从而提高了非整倍体细胞产生的几率。我们在神经分化过程中表达功能有一定缺陷的有丝分裂检测点蛋白（BUB3和MAD2的突变体）和着丝粒蛋白（CENP-A着丝粒定位缺陷突变体）以提高神经分化时非整倍体细胞的产生，以验证非整倍体神经细胞产生的机制和发现非整倍体神经细胞的染色体改变特征（获得或丢失某个染色体的倾向性）。项目提出有丝分裂检测点机制被神经分化机制抑制是中枢神经系统中非整倍体神经细胞大幅增加的原因，提示神经分化过程中细胞染色体均等分配保障机制服从神经细胞多样性需求而发生变化。项目增加了我们对非整倍体神经细胞发生机制及其生物学意义的认识。