Regulation of microRNA-mediated local translation in neurons by Argonaute phosphorylation

这项研究的目的是研究大脑中的神经细胞如何控制其结构和功能的长期变化，以响应来自其他神经细胞的通信。大脑中的神经细胞(神经元)通过称为突触的连接相互通信，突触位于神经元表面的小突起中，称为树突。一种化学物质(神经递质)从神经元释放出来，穿过突触激活邻近神经元上的受体。突触可以通过改变树突棘表面突触上发现的受体的数量，以及通过改变容纳突触的脊椎的大小和形状来改变其强度。这一过程(称为突触可塑性)被认为是学习和记忆的基础，因为记忆存储在由突触连接的神经元电路中，当记忆形成或丧失时，突触会被修改。为了保留长期记忆，神经元需要改变与决定突触上的脊椎结构或受体数量有关的蛋白质机制。蛋白质是通过翻译DNA序列(基因)中编码的遗传信息而形成的。DNA和蛋白质之间的中间产物被称为信使RNA，神经元可以将信使RNA运送到靠近突触的神经元部分，并在特定时间局部控制特定蛋白质的合成，这些蛋白质对这些突触非常重要。另一种类型的分子，称为微型RNA(MiRNA)，可以与mRNA结合，阻止mRNA向蛋白质的转化。这一过程受到非常精确的调控，以控制局部蛋白质的合成，但在突触可塑性过程中如何调控这一过程尚不清楚。一种名为ArgAerte2的蛋白质是细胞机械的重要组成部分，它促进miRNA阻止蛋白质合成。我们的初步实验结果表明，ArgAerte 2在突触可塑性的诱导下发生了化学修饰。这影响了它与miRNA活性所需的其他重要蛋白质的结合能力。我们的主要假设是，导致可塑性的突触刺激触发了ArgAerte 2的这种修饰，增强了它与其他蛋白质的结合，从而增加了miRNA介导的对蛋白质合成的抑制，以控制重要突触蛋白质的水平。我们还提出，这一机制发生在局部水平，因此蛋白质合成的抑制发生在受刺激的突触附近。我们的目标是通过一些实验方法来检验这些假设。我们的实验将在从大鼠大脑中获得的神经元上进行，并在体外保持“活”。我们将使用显微镜观察神经递质受体的位置和树突棘的结构，并使用电生理记录来研究突触功能。我们将在神经元ArgAerte2中引入基因突变，这种突变将模仿或阻止可塑性刺激引起的化学修饰。这将使我们能够研究所提出的机制是否参与调节突触结构、受体补体和/或突触功能。我们还将进行生化实验，以准确地研究可塑性刺激如何导致ArgAerte 2的修饰，以及这一机制是否特异性地调节突触蛋白的合成。我们的实验将使我们能够更多地了解调控神经元蛋白质合成的局部控制机制，以响应突触活动，从而进一步了解构成长期记忆的机制。

The aim of this research is to investigate a mechanism for how nerve cells in the brain control long-term changes in their structure and function in response to communication from other nerve cells.Nerve cells (neurons) in the brain communicate with one another at connections called synapses, which are located in small protrusions on the neuronal surface called dendritic spines. A chemical (neurotransmitter) is released from a neuron and travels across the synapse to activate receptors on the adjacent neuron. Synapses can change their strength by altering the number of receptors found at the synapse on the surface of the dendritic spine, and also by changing the size and shape of the spine that houses the synapse. This process (known as synaptic plasticity) is thought to underlie learning and memory, because memories are stored in circuits of neurons connected by synapses, which are modified when a memory is formed or lost.In order to retain long-term memories, neurons need to change the protein machinery involved in determining spine structure or receptor number at the synapse. Proteins are made by translating genetic information encoded in DNA sequences (genes). An intermediate between DNA and protein is called messenger RNA (mRNA), and neurons can transport mRNA to the parts of the neuron close to synapses and locally control the synthesis of specific proteins that are important for those synapses at a particular time. Another type of molecule, called micro RNA (miRNA) can bind to mRNA and stop the translation of mRNA into protein. This process is very precisely regulated to control local protein synthesis, but how this is regulated during synaptic plasticity is unknown.A protein called Argonaute 2 is an important component of the cell machinery that promotes the block of protein synthesis by miRNA. Results from our preliminary experiments indicate that Argonaute 2 is chemically modified in response to the induction of synaptic plasticity. This affects its ability to bind to other important proteins required for miRNA activity. Our main hypothesis is that the synaptic stimuli that lead to plasticity trigger this modification of Argonaute 2, enhancing its binding to other proteins, and consequently increasing the miRNA-mediated repression of protein synthesis to control the levels of important synaptic proteins. We also propose that this mechanism occurs on a local level, so that the repression of protein synthesis occurs in the vicinity of the stimulated synapse. We aim to test these hypotheses by a number of experimental approaches. Our experiments will be carried out on neurons obtained from rat brains and kept 'alive' in vitro. We will use microscopy to visualise the location of neurotransmitter receptors and the structure of dendritic spines, and electrophysiological recordings to investigate synaptic function. We will introduce genetic mutations in neuronal Argonaute 2 that will mimic or prevent the chemical modification caused by the plasticity stimulus. This will allow us to investigate whether the proposed mechanism is involved in regulating synaptic structure, receptor complement and/or synaptic function. We will also carry out biochemical experiments to investigate precisely how the plasticity stimulus causes the modification of Argonaute 2, and whether this mechanism specifically regulates the synthesis of synaptic proteins. Our experiments will enable us to understand more about the mechanisms that regulate the local control of protein synthesis in neurons in response to synaptic activity, and hence further our knowledge of the mechanisms that underlie long-term memory.