Role of actin cytoskeleton in the axonal growthcone during brain development

在神经网络形成过程中，轴突生长锥的形态发生及其功能受到抑制，使脑功能无法正常发育。为了阐明直接参与轴突生长体形态发生和功能的肌动蛋白细胞骨架的调控机制，本研究以原代培养的海马神经元为研究对象，以肌动蛋白丝为研究对象，分析了调节其理化生化特性的肌动蛋白结合蛋白蛋白网络的变化。我们首先用免疫细胞化学方法分析了不同发育阶段海马神经元轴突生长锥体中drebrin的分布。在第2期神经元中，drebrin定位于生长锥过渡区。免疫细胞化学还显示，神经滨素1在生长素中也显示出与drebrin相似的定位。我们进一步分析了2期生长锥中各种肌动蛋白相关蛋白的定位。微管未侵入过渡区。为了区分drebrin E和drebrin A，我们研制了drebrin A特异性抗体。该抗体明确表明生长锥只含有drebrin E.该抗体进一步表明成人脑迁移神经元只表达drebrin E.我们建立了成人脑迁移神经元的新鉴定方法。利用这种方法，我们在成年大鼠梨状皮质中发现了迁移神经元。我们最终发现，RNAi敲低drebrin导致轴突生长受到抑制。

During the neuronal network formation, the inhibition of axonal growthcone morphogenesis and its function make it impossible for the brain function to develop normally. In this study, in order to elucidate the regulatory mechanism of actin cytoskeleton, which is directly involved in the morphogenesis and function of axonal growthcones, we focused in the actin filament and analyzed the change in the protein network of actin-binding proteins which regulate the physicochemical and biochemical characters using primary cultured hippocampal neurons. We first analyzed the distribution of drebrin in the axonal growthcone of cultured hippocampal neurons at various developmental stages using immunocytochemistry. In the neuron of stage 2, drebrin was localized at transitional area of growthcones. The immunocytochemistry also showed that Neurabin 1 showed similar localization of in the growthcone to drebrin. We further analyzed localization of various actin-related proteins in the growthcones at stage 2. Microtubules did not invade into the transitional area. In order to discriminate drebrin E from drebrin A, we developed drebrin A-specific antibody. This antibody clearly showed that growthcone only has drebrin E. This antibody further showed that migrating neurons in the adult brain only express drebrin E. We developed the new identification method of migrating neurons in the adult brain. Using this method, we discovered migrating neurons in the piriform cortex in adult rats. We finally showed that drebrin knockdown by RNAi resulted the inhibition of axonal growth.