Physiological role of protein synthesis in nerve growth cone

本研究的目的是阐明局部蛋白质合成在细胞外刺激引起的神经生长锥运动性和形态变化中的生理作用。2002年，我们利用培养在层粘连蛋白上的鸡背根神经节细胞，研究了局部蛋白质合成在神经生长因子诱导的神经突起生长中的作用。我们首先研究了钙调节蛋白翻译因子-2(EEF2)的表达和分布模式。我们发现磷酸化的eEF2分布在细胞体和生长锥体中，但表达水平较低。高KCl2刺激引起细胞生长锥体塌陷，突起生长受到抑制，磷酸化eEF2表达水平随细胞内钙瞬变而显著增加，提示生长锥内eEF2呈钙依赖性磷酸化，参与了调控突起生长的信号级联反应。2003年，我们研究了局部蛋白质合成在信号3A(Sema3A)诱导的生长锥崩溃中的作用。Sema3A诱导eIF4E的磷酸化，并用茴香霉素阻断蛋白质合成，抑制生长锥体的塌陷。经Fyn激酶抑制剂Lavemdustin-A处理后，Sema3A诱导的eIF4E磷酸化水平降低。由于Sema3A信号被认为不依赖于钙信号，因此推测由局部蛋白质合成介导的轴突突起和生长锥塌陷是独立调节的。

The aim of this study is to clarity the physiological roles of local protein synthesis in change of motility and morphology of nerve growth cone in response to extracellular stimuli. In 2002, we examined the roles of local protein synthesis in neurite outgrowth induced by nerve growth factor using chick dorsal root ganglion cells cultured on laminin. We first investigated the expression and distribution patterns of calcium-regulated protein translational factor, eukaryotic elongation factor-2 (eEF2). We found that phosphorylated eEF2 was distributed in the cell body and the growth cones, but the expression level was low. The cells exposed to high KCl stimulus showed growth cone collapse and inhibition of neurite outgrowth, and the expression level of phosphorylated eEF2 was significantly increased with intracellular calcium transient induced by high KCl stimulus, suggesting that eEF2 within growth cones was phosphorylated in a calcium-dependent manner and was involved in the signal cascade regulating neurite outgrowth. In 2003, we examined the roles of local protein synthesis in growth cone collapse induced by semaphoring 3A (Sema3A). Sema3A induced phosphorylation of eIF4E and blockade of protein synthesis with anisomycin inhibited the growth cone collapse. The cells to expose to Fyn kinase inhibitor, lavemdustin-A showed decrease of eIF4E phosphorylation of eIF4E induced by Sema3A. Since Sema3A signaling is considered to be independent of calcium signaling, it is suggested that neurite outgrowth and growth cone collapse mediated by local protein synthesis are independently regulated.