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，长突起从细胞体中的细胞核延伸几毫米。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控是分散的，通过局部控制树突中的翻译来根据特定突触的要求提供蛋白质。MicroRNAs(MiRNAs)是一种小的内源RNA分子，通过与RNA诱导沉默复合体(RISC)中的ArgAerte(AGO)蛋白质结合来抑制目标mRNAs的翻译，在许多细胞过程中对微调蛋白质合成至关重要。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活性通过调节细胞骨架蛋白的翻译在这一过程中发挥关键作用，细胞骨架蛋白决定了脊柱的形态。MiRNA调节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的“局部”控制，即基因沉默是否沿着树突传播到邻近的未受刺激的突触，如果是，这是如何调节的？这一点很重要，因为Hebbian学习的主导理论假设可塑性是突触特有的，而新出现的证据表明并非如此。我们最近确定了快速增加miRNA介导的基因沉默的机制，以响应NMDA受体的刺激而导致树突棘收缩。我们的假设是，miRNA的活性在受刺激的脊椎附近被调节，以局部调节翻译，因此只影响少量相邻脊椎的形态。主要的实验方法将是通过谷氨酸去掉的单突触刺激，然后是活的(例如，使用多聚体太阳标签的新生多肽的单分子成像)和固定细胞成像技术(例如，嘌呤霉素邻近连接试验)，以分析特定蛋白质的翻译和树突该区域脊柱形态的动态成像。该项目将研究通过突变基本RISC蛋白所涉及的机制。此外，还将开发分子级别的计算机模拟模型来测试我们的假设，并剖析系统中实验上无法分离的部分。例如，分别比较1)miRNA和信号因子的扩散，2)mRNAs、miRNAs和信号因子的相对数量，3)树突和棘突几何形状限制蛋白质翻译到局部空间间隔的作用。这一联合实验和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.