Post-transcriptional regulation of Snap25 by Pumilio proteins in synaptogenesis

Pumilio 蛋白在突触发生中对 Snap25 的转录后调控

• 批准号: 10239034
• 负责人: Lisa Kathryn Randolph
• 金额: $ 4.6万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10239034
• 关键词: 3' Untranslated Regions; Address; Adult; Attention deficit hyperactivity disorder; Axon; Binding; Brain; Breathing; Cells; Complex; Cues; Data; Development; Developmental Process; Disease; Down-Regulation; Drosophila genus; Electrophysiology (science); Elements; Environment; Epilepsy; Genetic Transcription; Goals; Image; Immunofluorescence Immunologic; Immunohistochemistry; Immunoprecipitation; In Vitro; Lead; Measures; Mediating; Messenger RNA; Morphology; Mus; Neurodevelopmental Disorder; Neuromuscular Junction; Neurons; Neuropathogenesis; PHluorin; Play; Post-Transcriptional Regulation; Presynaptic Terminals; Process; Protein Biosynthesis; Proteins; RNA; RNA-Binding Proteins; Rattus; Regulation; Repression; Role; Schizophrenia; Signal Transduction; Single Nucleotide Polymorphism; Site; Slice; Synapses; Translations; Western Blotting; Work; experimental study; genetic manipulation; in vivo; insight; knock-down; neuronal cell body; neuronal circuitry; neuropsychiatric disorder; novel; prenatal; presynaptic; prevent; ranpirnase; response; spatiotemporal; synaptic function; synaptogenesis; synaptosomal-associated protein 25; transcriptome; two-photon; vesicular release

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.

项目摘要/摘要 神经元收发信号的能力是大脑最基本的功能的基础， 从调节呼吸到复杂的思维。为了让大脑正常运作，大脑的形成和 新突触的功能必须受到严格的调控。可以促进这一监管的一个机制是 局部蛋白质合成，允许对神经元的变化做出微调和空间精确的反应 环境。最近发现，突触体相关蛋白25的局部合成 (SNAP25)是突触前终末的形成和功能所必需的。然而， 控制SNAP25基因表达、定位、稳定性和翻译的机制在很大程度上还不清楚。 SNAP25此前曾被认为与几种神经精神障碍有关，特别是注意 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，阻断Snap25在轴突中的翻译可以防止集聚 突触前末端的突触前蛋白并减缓囊泡的释放，表明后- Snap25的转录调控对突触发育至关重要。核糖核酸结合蛋白Pumilio (PUM)1和2通过与Snap25的3‘非编码区结合来调节Snap25的mRNA。传统上被称为翻译抑制因子， Pum1和Pum2最近也被鉴定为轴突转录组的负调控因子。 因此，我假设Snap25的去抑制和突触前定位是通过发育 Pumilio蛋白的下调是适当控制突触前终末形成和功能所必需的， 而这些调节机制的破坏将导致突触前终末的异常形成和 突触释放。我将通过以下具体目标来解决这一假设：目标1将决定 Pumilio介导的调控在Snap25整体水平以及在Snap25定位和局部中的作用 突触前部位的翻译。目的2将研究Pumilio介导的Snap25调控在 突触前终末的形成和功能。我将使用全细胞和 原代大鼠神经元分区培养和器官型切片培养结合遗传学 操纵Pumilio和SNAP25。我将确定Pumilio击倒对Snap25本地化的影响 以及随后这一调节过程的中断如何影响突触前终末 形成和突触释放。总之，拟议中的实验将提供对后 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、突触功能和相关的神经发育障碍中的作用。