Pumilio2-mediated control of local protein expression in neurons

突触处的局部蛋白质合成的调节在初级神经元中特别重要。在这里，一组特定的mRNA在RNA结合蛋白（RBP）的帮助下被包装成核糖核蛋白颗粒（RNP或RNA颗粒），并沿沿着转运到突触附近的树突中。人们普遍认为，翻译在运输过程中受到抑制。然而，涉及翻译调控的潜在机制和分子仍然知之甚少。神经元特异性RBP Pumilio2（Pum2）是许多生物体和细胞类型中众所周知的保守翻译调节因子。在此，Pum2参与了eIF4E mRNA以及编码几种钠通道（如Nav1.1和Nav1.6）的mRNA的翻译调节。Pum2缺陷的原代海马神经元在树突棘形态发生以及神经元的兴奋性方面表现出缺陷。重要的是要注意，小鼠中Pum2敲低导致癫痫发作。Pum2是含有Staufen2（Stau2）的RNP的组分，其中Stau2是另一种众所周知的参与树突状mRNA定位的RBP。因此，推测Pum2参与神经元中局部转录物的局部表达的控制是诱人的。为了系统地了解Pum2在这一过程中的作用，我们计划将联合收割机从小鼠脑中分离RNA颗粒与野生型和Pum2缺陷小鼠脑的多核糖体梯度分离相结合，以鉴定生理学相关的Pum2靶mRNA。在与Medenbach（项目9）的合作方法中，我们将建立一个来自小鼠神经元组织的体外翻译系统，以重现调控。与Kiebler实验室中复杂的小鼠模型相结合，将使我们能够阐明Pum2介导的蛋白质合成控制的潜在机制，特别强调成熟突触的局部翻译，并从分子上深入了解失调如何导致疾病。

Regulation of local protein synthesis at the synapse is particularly important in primary neurons. Here, a set of specific mRNAs is packaged with the help of RNA-binding proteins (RBPs) into ribonucleoprotein particles (RNPs or RNA granules) and transported along microtubules into dendrites near synapses. It is widely believed that translation is repressed during transport. However, the underlying mechanisms and molecules involved in translational regulation remain poorly understood. The neuron-specific RBP Pumilio2 (Pum2) is a well-known, conserved translational regulator in many organisms and cell types. Here, Pum2 has been implicated in the translational regulation of eIF4E mRNA as well as mRNAs coding for several sodium channels such as Nav1.1 and Nav1.6. Pum2-deficient primary hippocampal neurons display deficits in dendritic spine morphogenesis as well as in the excitability of neurons. It is important to note that Pum2 knock-down in mice causes epileptic seizures. Pum2 is a component of Staufen2 (Stau2)-containing RNPs, with Stau2 being another well-known RBP involved in dendritic mRNA localization. Therefore, it is tempting to speculate that Pum2 is involved in the control of local expression of localized transcripts in neurons. To gain systematic insight into the role of Pum2 in this process, we plan to combine RNA granule isolation from mouse brain with polysome gradient fractionation of wild type and Pum2-deficient mouse brains to identify physiologically relevant Pum2 target mRNAs. In a collaborative approach with Medenbach (Project 9), we will establish an in vitro translation system from mouse neuronal tissue to recapitulate regulation. The combination with sophisticated mouse models in the Kiebler lab will then allow us to elucidate the underlying mechanisms of Pum2 mediated control of protein synthesis with a special emphasis on local translation at mature synapses and to gain molecular insight into how misregulation results in disease.