Wnt Regulation of Cell Cycle in Cortical Neurogenesis

描述（由申请人提供） Wnt 是已知可调节与自闭症和精神分裂症等发育疾病相关区域的神经发生的细胞外信号，但介导这些作用的细胞周期机制仍不清楚。我们的目标是研究Wnt-3a在培养物和体内的前脑神经发生和G1细胞周期机制调节中的作用，如下：l)将用Wnt-3a构建体转染的成纤维细胞条件培养基处理胚胎大鼠皮质细胞培养物。 S期进入将通过[3H]胸苷掺入进行评估，并通过溴脱氧尿苷标记进行细胞表征。 2) 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 (CDK) 和 CIP/KIP CDK 抑制剂，将通过蛋白质印迹法测量，并通过激酶测定评估细胞周期蛋白/CDK 复合物活性。 3) Wnt-3a对体内增殖和细胞周期调节的急性影响将使用经子宫脑室内(ICV)注射来确定。将通过细胞电穿孔使用 GFP:Wnt-3a 共转染来评估对神经发生的持续影响。定义这些在大脑发育中介导 Wnt 信号传导的机制对于理解和治疗普遍性神经系统疾病至关重要。

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.