Wnt Regulation of Cell Cycle in Cortical Neurogenesis

描述（由申请人提供） Wnts是细胞外信号，已知其调节与发育性疾病（如自闭症和精神分裂症）相关区域的神经发生，但介导这些作用的细胞周期机制仍不明确。我们的目的是研究Wnt-3a在培养物和体内前脑神经发生和G1细胞周期机制调节中的作用，如下：1）用Wnt-3a构建体转染的成纤维细胞的条件培养基处理胚胎大鼠皮层细胞培养物。将通过[3 H]胸苷掺入评估S期进入，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进行测量，并通过激酶试验评估细胞周期蛋白/CDK复合物活性。3)Wnt-3a对体内增殖和细胞周期调节的急性作用将使用经子宫内膜脑室内（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作用。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广泛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.