Wnt Regulation of Cell Cycle in Cortical Neurogenesis

描述(由申请人提供) WNTs是已知的细胞外信号，调节与自闭症和精神分裂症等发育性疾病相关的区域的神经发生，但调节这些影响的细胞周期机制尚不清楚。本研究旨在研究WNT-3a在体外培养的大鼠前脑神经发生和G1期细胞周期机制调控中的作用，主要内容如下：(1)将WNT-3a基因导入大鼠胚脑皮质细胞培养的条件培养液中。用[~3H]胸腺嘧啶核苷掺入法检测细胞周期进入S时相，用溴脱氧尿嘧啶核苷标记法检测细胞周期。2)用蛋白印迹法检测WNT-3a对细胞周期调节蛋白水平的影响，包括D和E细胞周期蛋白、细胞周期蛋白依赖性激酶(CDKs)和CIP/KIP CDK抑制物，用蛋白印迹法检测细胞周期蛋白/CDK复合体的活性。3)采用侧脑室注射法检测WNT-3a对体内细胞增殖和细胞周期调控的急性影响。通过细胞电穿孔GFP：WNT-3a共转染对神经发生的持续影响将被评估。明确这些在大脑发育中介导Wnt信号的机制对于理解和治疗弥漫性神经疾病至关重要。

DESCRIPTION (provided by applicant) Wnts are extracellular signals known to regulate neurogenesis in regions associated with developmental diseases such as autism and schizophrenia, yet cell cycle mechanisms mediating these effects remain undefined. We aim to study Wnt-3a's role in forebrain neurogenesis and G1 cell cycle machinery regulation in culture, and in vivo, as follows: l) Embryonic rat cortical cell cultures will be treated with media conditioned by fibroblasts transfected with Wnt-3a constructs. S-phase entry will be assessed by [3H]Thymidine incorporation, and characterized cellularly by bromodeoxyuridine labeling. 2) Wnt-3a's effects on cell cycle regulatory protein levels, including D and E cyclins, cyclin dependent kinases (CDKs), and CIP/KIP CDK inhibitors, will be measured by western blotting, and cyclin/CDK complex activity will be assessed by kinase assays. 3) Acute effects of Wnt-3a on proliferation and cell cycle regulation in vivo, will be determined using transuterine intracerebroventricular (ICV) injection. Sustained effects on neurogenesis will be assessed using GFP:Wnt-3a co-transfection by cell electroporation. Defining these mechanisms mediating Wnt signaling in brain development is critical to understanding and treating pervasive neurological diseases.