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Proteomic identification of molecules involved in neuronal polarity formation and analysis of their intracellular molecular networks

参与神经元极性形成的分子的蛋白质组学鉴定及其细胞内分子网络的分析

基本信息

批准号：
15310140
负责人：
INAGAKI Naoyuki
金额：
$ 9.79万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

The basic function of neurons is to receive, integrate and transmit signals. To do so, most neurons develop polarity by forming a single axon and multiple dendrites. However, little is known about the molecular basis of neuronal polarization. To approach this problem, we performed proteome analyses of cultured hippocampal neurons using highly sensitive large-gel two-dimensional electrophoresis (2-DE). In this project, we identified 94 proteins up-regulated during the initial step of neuronal polarization and 92 proteins enriched in axons, and analyzed the functions and molecular networks of novel proteins, shootin1 and singar.Shootin1 became up-regulated during polarization of hippocampal neurons and began fluctuating accumulation among multiple neurites. Eventually shootin1 accumulated asymmetrically in a single neurite, which led to axon induction for polarization. Shootin1 was transported anterogradely to the growth cones and diffused back to the soma ; inhibiting this transport prevented its asymmetric accumulation in neurons. Disturbing the asymmetric organization of shootin1 by excess shootin1 disrupted polarization, while repressing shootin1 expression delayed polarization. Functional analysis suggested that shootin1 regulates neuronal polarity through the PI 3-kinase pathway. These results suggest that shootin1 is involved in the generation of internal asymmetric signals required for neuronal polarization.Singar becomes up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Knockdown of singar expression by RNA interference led to an increase in the population of neurons bearing surplus axons. Conversely, overexpression of singar suppressed the formation of surplus axons induced by excess level of shootin1. These results suggest that singar ensures the robustness of neuronal polarity by maintaining a single axon

神经元的基本功能是接收、整合和传递信号。为了做到这一点，大多数神经元通过形成单个轴突和多个树突来形成极性。然而，人们对神经元极化的分子基础知之甚少。为了探讨这个问题，我们使用高灵敏的大分子双向凝胶电泳法(2-DE)对培养的海马神经元进行了蛋白质组分析。在本项目中，我们鉴定了94个在神经元极化初期上调的蛋白质和92个富含在轴突中的蛋白质，并分析了新的蛋白质Shootin1和Single的功能和分子网络。Shootin1在海马神经元极化过程中上调，并开始在多个神经元之间波动积累。最终，Shootin1在单个轴突中不对称地积累，导致轴突诱导极化。Shootin1被顺向运输到生长锥，然后扩散回胞体；抑制这种运输可以阻止其在神经元中的不对称积累。过量的Shootin1干扰了Shootin1的不对称组织，破坏了极化，同时抑制了Shootin1的表达，延迟了极化。功能分析表明，Shootin1通过PI3K途径调节神经元的极性。这些结果表明，Shootin1参与了神经元极化所需的内部不对称信号的产生。在培养的海马神经元极化过程中，Shootin1上调并保持在高水平。通过RNA干扰抑制信号的表达，导致承载多余轴突的神经元数量增加。相反，SINAR的过表达抑制了由过量的Shootin1诱导的多余轴突的形成。这些结果表明，SIGAR通过维持单一轴突来确保神经元极性的稳健性