Post-transcriptional regulation of Snap25 by Pumilio proteins in synaptogenesis

项目总结/摘要 神经元发送和接收信号的能力是大脑最基本功能的基础， 从调节呼吸到复杂的思考。为了使大脑正常运作， 新突触的功能必须受到严格的调控。一种有助于这种调节的机制是 局部蛋白质合成，这使得微调和空间精确的反应，神经元的变化， 环境最近发现突触体相关蛋白25的局部合成 突触前位点的SNAP 25是突触前末梢形成和发挥功能所必需的。然而，在这方面， 控制SNAP 25 mRNA、定位、稳定性和翻译的机制在很大程度上是未知的。 SNAP 25先前已涉及几种神经精神疾病，特别是关注 缺陷/多动障碍（ADHD）。SNAP25的减少也与精神分裂症有关 和癫痫。在体外，特异性地阻断轴突中Snap25的翻译可以防止细胞聚集。 突触前蛋白在突触前末端，并减缓囊泡释放，表明后 Snap25的转录调节对于突触发育至关重要。RNA结合蛋白Pumilio (Pum)1和2通过与Snap25的3 ′ UTR结合来调节Snap25 mRNA的表达。传统上被称为翻译阻遏物， Pum1和Pum2最近也被确定为轴突转录组的负调节因子。 因此，我推测发育过程中Snap25的去阻遏和突触前定位可能是一个重要的机制。 Pumilio蛋白的下调是正确控制突触前末端形成和功能所必需的， 并且这些调节机制的破坏将导致异常的突触前末端形成， 突触释放我将通过以下具体目标来解决这个假设：目标1将决定 Pumilio介导的调节在整体Snap25水平以及Snap25定位和局部中的作用 在突触前位点的翻译。目的2将研究Pumilio介导的Snap25调控在 突触前末梢的形成和功能。我将使用全细胞和 区室化原代大鼠神经元培养和器官型切片培养结合遗传学 Pumilio和SNAP25的操作。我将确定Pumilio击倒如何影响Snap25本地化 和翻译，以及随后这种调节过程的中断如何影响突触前末梢 形成和突触释放。总之，拟议的实验将提供新的见解后， Snap25在发育过程中的转录调控机制，以及这些机制如何发挥作用。 在突触发生、突触功能和相关神经发育障碍中的作用。

Project Summary/Abstract The ability of neurons to send and receive signals underlies the most essential functions of the brain, from regulating breathing to complex thought. In order for the brain to function properly, the formation and function of new synapses must be tightly regulated. One mechanism that can contribute to this regulation is local protein synthesis, which allows for finely tuned and spatially precise responses to changes in the neuron’s environment. It has recently been discovered that the local synthesis of synaptosomal-associated protein 25 (SNAP25) at the presynaptic site is required for the formation and function of presynaptic terminals. However, the mechanisms controlling SNAP25 mRNA, localization, stability, and translation are largely unknown. SNAP25 has previously been implicated in several neuropsychiatric disorders, in particular attention deficit/hyperactivity disorder (ADHD). Reductions in SNAP25 have also been associated with schizophrenia and epilepsy. In vitro, blocking the translation of Snap25 specifically in axons prevents the clustering of presynaptic proteins at the presynaptic terminal and slows vesicular release, indicating that the post- transcriptional regulation of Snap25 is crucial for synapse development. The RNA-binding proteins Pumilio (Pum) 1 and 2 regulate Snap25 mRNA by binding to its 3′UTR. Traditionally known as translational repressors, Pum1 and Pum2 have also recently been identified as negative regulators of the axonal transcriptome. Therefore, I hypothesize that de-repression and presynaptic localization of Snap25 by developmental downregulation of Pumilio proteins is required for proper control of presynaptic terminal formation and function, and that disruption of these regulatory mechanisms will lead to aberrant presynaptic terminal formation and synaptic release. I will address this hypothesis through the following Specific Aims: Aim 1 will determine the role of Pumilio-mediated regulation in overall Snap25 levels as well as in Snap25 localization and local translation at the presynaptic site. Aim 2 will investigate the role of Pumilio-mediated Snap25 regulation in presynapstic terminal formation and function. I will investigate these questions using both whole-cell and compartmentalized primary rat neuronal culture and organotypic slice culture in combination with genetic manipulation of Pumilio and SNAP25. I will determine how Pumilio knockdown impacts Snap25 localization and translation, and subsequently how disruption of this regulatory process impacts presynaptic terminal formation and synaptic release. Together, the proposed experiments will provide novel insight into post- transcriptional mechanisms of Snap25 regulation during development, and how these mechanisms may play a role in synaptogenesis, synapse function, and associated neurodevelopmental disorders.