伴侣分子AnxA6调节孕晚期胎脑白质发育形成作用机制的研究

Inhibited proliferation and differentiation of neural progenitor cells (NPCs) has been implicated in the maldevelopment of white matter during the third trimester of pregnancy. Evidences have shown that the noncanonical Wnt/Ryk (receptor related to tyrosine kinase) signaling pathway participates in differentiation of NPCs, However, the mechanisms of Ryk regulates the differentiation of NPCs into oligodendrocytes (OLs) are still unclear. Our preliminary experiment results show that the nuclear membrane chaperone molecule annexin A6(AnxA6) interact with the Ryk protein. AnxA6 has a typical structure features of EF-hand motif (Ca2+-binding domain) by using Bioinformatics approaches prediction. The number of differentiated OLs from Ryk overexpression NPCs was found increase, and the expression of calcium sensing receptor also showed higher level than the normal control. Therefore, we hypothesize that AnxA6 may play a key role of molecular chaperone in cooperating with Ca2+ to regulate the nucleoplasm distribution of Ryk, as well as accelerate the transcription of the downstream target genes during NPCs differentiation into OLs. In this project, we will utilize Bioinformatics spatial analysis, RNAi knockdown and embryo electric transfection technology to determine whether AnxA6 synergizes with Ryk to regulate NPCs differentiation and promote the development of OLs in vitro and in vivo. This research results gained from the proposed studies will identify the function of the annexin in the development of the embryological nervous system and illuminate the molecular (or pathogenic) mechanisms of the AnxA6/ Ca2+ interacte with Wnt/Ryk signaling pathway during fetal neurogenesis, and provide the theoretical and experimental basis for preventing and treating the abnormal development of white matter.

孕晚期胎脑白质发育不良与神经祖细胞(NPCs)增殖分化能力受抑制有关。已明确非经典Wnt/Ryk通路参与NPCs分化调节，但如何调控实现向少突胶质细胞(OLs)分化及所涉及的具体分子机制知之甚少。本课题前期预实验观察到OLs分化中伴侣分子Annexin A6(AnxA6)是Ryk相互作用蛋白；生物信息学预测AnxA6具有EFhand结构域特征；高表达Ryk的NPCs分化OLs比例增多且伴钙敏感受体的表达增加。我们推测：AnxA6可能作为Ryk调控子与Ca2+协同影响Ryk活性和其核浆分布，启动下游基因参与NPCs向OLs分化。本项目将采用生物信息预测模拟技术、RNAi技术和胚胎电转染等技术，从离体和在体两方面，明确AnxA6的EFhand结合域对Ryk调控NPCs向少突分化中的协同作用和影响机制。阐明AnxA6/Ca2+与Wnt/Ryk信号通路间的调控机制，为防治白质发育不良提供实验依据。

孕晚期胎脑白质发育不良与神经祖细胞(NPCs)增殖分化能力受抑制有关。但如何调控实现向少突胶质细胞(OLs)分化及所涉及的具体分子机制知之甚少。本研究课题聚焦伴侣分子Annexin A6(AnxA6) 与Ryk ，构建过表达载体和干扰病毒载体，发现AnxA6表达能够上调早期OLs分化的转录因子Olig2的表达，参与促进OL突起扩展增大的能力，且受Ca2+调控而发挥作用。而Ryk表达受干扰后，能够影响AnxA6和Olig2的表达，使NPCs分化突起延长受抑制。表明AnxA6与Ryk相互作用共同影响NPCs向OL的分化能力。同时本研究还发现，孕晚期胎脑中AnxA6阳性细胞表达在白质区域与PDGFα阳性细胞分布近似。在高糖等因素显著活化ClC-2，导致孕晚期脑白质中ROS水平提升，抑制OLs的分化和髓鞘成熟。显著增加脑白质区TUNEL阳性细胞数，显著减少PDGFα阳性细胞数目，提示孕晚期高糖、缺氧等因素可明显导致发育早期脑白质中凋亡细胞的增多，影响了OPCs/PreOLs的分化能力。进一步证实孕晚期胎脑白质中髓鞘形成障碍可能与Caspase3蛋白表达增高和p-Akt蛋白表达减少相关联。上述相关机制的明确，为寻找孕晚期髓鞘发育的保护效应，有效预防和治疗髓鞘发育障碍性疾病提供实验证据。