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

神经元的基本功能是接收、整合和传输信号。为此，大多数神经元通过形成单个轴突和多个树突来发展极性。然而，很少有人知道的神经元极化的分子基础。为了解决这个问题，我们进行了蛋白质组分析，培养海马神经元，使用高灵敏度的大凝胶双向电泳（2-DE）。本研究共鉴定了94个在神经元极化初期表达上调的蛋白和92个在轴突中富集的蛋白，并对其中的两个新蛋白shootin 1和single进行了功能分析和分子网络分析。最终shootin 1在单个神经突中不对称地积累，这导致轴突诱导极化。shootin 1顺行运输到生长锥和扩散回索马;抑制这种运输防止其在神经元中的不对称积累。通过过量的shootin 1扰乱shootin 1的不对称组织破坏极化，而抑制shootin 1表达延迟极化。功能分析表明，shootin 1通过PI 3-激酶途径调节神经元极性。这些结果表明shootin 1参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，Singar表达上调，并在极化后保持在较高水平。通过RNA干扰敲低信号表达导致携带多余轴突的神经元数量增加。相反，过表达的信号抑制过剩轴突的形成诱导的过量水平shootin 1。这些结果表明，Singar通过维持单个轴突来确保神经元极性的鲁棒性

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