MicroRNA control of local synaptic protein synthesis in neuronal dendrites

神经元是一种独特的细胞类型，因为它们是极端极化的，具有从细胞体中的细胞核延伸几毫米的长突起。这提出了一个细胞生物学挑战：远端突触的蛋白质表达是如何调节的？一种解决方案是蛋白质合成的调节是分散的，在树突中局部控制翻译以根据特定突触的需要提供蛋白质。内源性RNA分子，通过与RNA诱导沉默复合物（RISC）中的Argonaute（Ago）蛋白结合来抑制靶mRNA的翻译，并且对精细生物学至关重要。在许多细胞过程中调节蛋白质合成。长期突触可塑性是学习和记忆的基础，通过修改神经回路，其中一个主要组成部分是树突棘的形态变化，其中包含突触后机制。调节的miRNA活性通过调节决定棘形态的细胞骨架蛋白的翻译在该过程中起关键作用。miRNA失调与包括阿尔茨海默病在内的几种涉及突触功能障碍的神经系统疾病有关。一个关键问题是这种翻译控制是如何“局部”的，即，基因沉默是否会沿着树突扩散到邻近未受刺激的突触，如果是的话，这是如何调节的？这一点很重要，因为赫布学习的主流理论假设可塑性是突触特异性的，而新出现的证据表明并非如此。我们最近定义了响应于NMDA受体刺激导致树突棘收缩而快速增加miRNA介导的基因沉默的机制。我们的假设是，miRNA的活性在靠近刺激的棘突处被调制，以局部调节翻译，从而仅影响少数相邻棘突的形态。主要的实验方法将是通过谷氨酸释放的单突触刺激，其次是Live（例如，使用多聚化Sun-Tags的新生肽的单分子成像）和固定细胞成像（例如，使用多聚化Sun-Tags的新生肽的单分子成像）。（例如嘌呤霉素邻近连接测定）成像技术来分析特定蛋白质的翻译和树突区域中棘形态的动态成像。该项目将研究通过诱变基本RISC蛋白质所涉及的机制。此外，分子水平的计算机模拟模型将被开发来测试我们的假设和解剖系统中实验上不可分离的部分。例如，分别比较1）miRNA和信号因子的扩散，2）mRNA、miRNA和信号因子的相对数量，3）树突和棘的几何形状对限制蛋白质翻译到局部空间区室的作用。这个联合实验-计算项目将定义突触局部翻译的关键机制。

Neurons are a unique cell type because they are extremely polarised, with long processes extending millimetres from the nucleus in the cell body. This poses a cell biological challenge: how is protein expression at distal synapses regulated? One solution is that regulation of protein synthesis is decentralised, with local control of translation in dendrites to supply proteins according to the requirements of specific synapses.MicroRNAs (miRNAs) are small, endogenous RNA molecules that repress the translation of target mRNAs by associating with Argonaute (Ago) proteins in the RNA-induced silencing complex (RISC) and are fundamentally important for fine-tuning protein synthesis in numerous cellular processes. 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 cytoskeletal proteins that determine spine morphology. miRNA dysregulation is implicated in several neurological disorders that involve synaptic dysfunction, including Alzheimer's disease.A key question is how "local" is this control of translation, i.e., does gene silencing spread along the dendrite to neighbouring unstimulated synapses, and if so, how is this regulated? This is important because dominant theories of Hebbian learning assume that plasticity is synapse-specific, while emerging evidence suggests otherwise. We have recently defined mechanisms for rapidly increasing miRNA-mediated gene silencing in response to NMDA receptor stimulation to cause dendritic spine shrinkage. Our hypothesis is that miRNA activity is modulated close to the stimulated spine to locally regulate translation and hence influence the morphology of only a small number of neighbouring spines.The main experimental approach will be single-synapse stimulation by glutamate uncaging, followed by live (e.g. single-molecule imaging of nascent peptides using multimerized Sun-Tags) and fixed-cell (e.g. puromycin-proximity ligation assays) imaging techniques to analyse the translation of specific proteins and dynamic imaging of spine morphology in that region of dendrite. The project will investigate the mechanisms involved via mutagenesis of essential RISC proteins.In addition, molecular-level computer simulation models will be developed to test our hypotheses and dissect parts of the system that are not experimentally dissociable. For example, separately comparing the roles of 1) diffusion of miRNA and signalling factors, 2) relative numbers of mRNAs, miRNAs and signalling factors, 3) dendrite and spine geometry for restricting protein translation to localised spatial compartments.This joint experimental-computational project will define key mechanisms of local translation at synapses.