SSA: Phosphoregulation of argonaute function in synaptic plasticity and memory

MicroRNAs(MiRNAs)通过与RNA诱导沉默复合体(RISC)中的ArgAerte(AGO)蛋白结合，抑制靶mRNAs的翻译，对蛋白质合成的微调起着至关重要的作用。长期的突触可塑性是通过改变神经回路来实现学习和记忆的基础，神经回路的一个主要组成部分是树突棘的形态变化，树突棘包含突触后机制。受调控的miRNA活性通过调节众多突触蛋白的翻译在这一过程中发挥关键作用，miRNA调节失调与涉及突触功能障碍的神经疾病有关，如阿尔茨海默病。突触后NMDAR的刺激和随之而来的细胞内钙依赖信号是大脑突触可塑性的主要上游组成部分。我们最近的体外工作发现，Ser387上Ago2的磷酸化是调控特定RISC蛋白质-蛋白质相互作用和随后的miRNA依赖的基因沉默以控制NMDAR依赖的树突状突起可塑性的重要机制(Rajgor等人，EMBO J.，2018)。在这个项目中，我们的目标是在体内研究这些机制；我们假设，特定类型的突触可塑性，因此记忆过程，需要依赖于Ser387磷酸化的RISC蛋白-蛋白质相互作用和miRNA活性的变化。为了研究Ago2磷酸化在突触可塑性和记忆中的功能作用，该项目将在一系列行为测试和脑片突触电生理分析中比较S387A(磷酸缺失)Ago2基因敲入小鼠和野生型小鼠。这些将被选择来评估一系列不同的学习和记忆过程，其中涉及不同类型的突触可塑性。例如，物体识别记忆需要大脑皮层长时程增强(LTD)，或者空间记忆需要海马长时程增强(LTP)。为了确定哪些miRNAs参与其中，我们将从相关脑区制备的裂解物中进行Ago2免疫沉淀，通过微阵列分析结合的miRNAs，并通过Western blotting分析RISC组装。此外，还将进行蛋白质组学筛查，以公正的方式分析RISC蛋白质之间的相互作用。通过Ago2磷酸化调节RISC在学习和记忆中的作用尚不清楚，因此该项目将在miRNA依赖的脑功能机制中定义新的概念。它还将在生物化学、电生理学和行为神经科学方面提供出色的培训。我们正在寻找一名有热情和创新精神的学生，拥有神经科学、生物或医学学位。该项目将由乔纳森·汉利教授(生物化学)、克里亚·沃伯顿教授(行为神经科学)和乔恩·布朗博士(突触生理学)监督。欲了解更多信息，请联系乔纳森·汉利(jon.hanley@bristol.ac.uk)。

MicroRNAs (miRNAs) are fundamentally important for fine-tuning protein synthesis by associating with Argonaute (Ago) proteins in the RNA-induced silencing complex (RISC) to repress the translation of target mRNAs. Long-term synaptic plasticity underlies learning and memory by modifying neural circuitry, a major component of which is morphological changes of dendritic spines, which contain the postsynaptic machinery. Regulated miRNA activity plays a key role in this process by modulating the translation of numerous synaptic proteins, and miRNA dysregulation is implicated in neurological disorders that involve synaptic dysfunction, such as Alzheimer's disease. Stimulation of postsynaptic NMDARs and consequent calcium-dependent intracellular signalling is the major upstream component of synaptic plasticity in the brain. Our recent in vitro work identified phosphorylation of Ago2 at Ser387 as an essential mechanism for regulating specific RISC protein-protein interactions and consequent miRNA-dependent gene silencing to control NMDAR-dependent dendritic spine plasticity (Rajgor et al., EMBO J., 2018). In this project, we aim to investigate these mechanisms in vivo; we hypothesise that specific types of synaptic plasticity, and hence memory processes, require Ser387 phosphorylation-dependent changes in RISC protein-protein interactions and miRNA activity. To examine functional roles of Ago2 phosphorylation in synaptic plasticity and memory, this project will compare S387A (phospho-null) Ago2 knock-in mice with wild-type litter-mates in a battery of behavioural tests and brain slice synaptic electrophysiology assays. These will be chosen to assess a diverse range of learning and memory processes, which involve different types of synaptic plasticity. E.g. object recognition memory requires cortical long-term depression (LTD), or spatial memory involves hippocampal long-term potentiation (LTP). To define which miRNAs are involved, we will carry out Ago2 immunoprecipitations from lysates prepared from relevant brain regions, to analyse bound miRNAs by microarrays and RISC assembly by Western blotting. In addition, a proteomics screen will be carried out to analyse RISC protein-protein interactions in an unbiased manner. A role for RISC regulation by Ago2 phosphorylation in learning and memory is as yet unexplored, therefore this project will define novel concepts in miRNA-dependent mechanisms of brain function. It will also provide an excellent training in biochemistry, electrophysiology and behavioural neuroscience. We are looking for an enthusiastic and innovative student with a degree in neuroscience, biological or medical science. The project will be supervised by Prof. Jonathan Hanley (biochemistry), Prof Clea Warburton (behavioural neuroscience) and Dr. Jon Brown (synaptic physiology). For further information, please contact Jonathan Hanley (jon.hanley@bristol.ac.uk).