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

本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信息。大脑中的神经细胞（神经元）通过被称为突触的连接相互交流，突触位于神经元表面被称为树突棘的小突起中。一种化学物质（神经递质）从一个神经元释放出来，并通过突触激活相邻神经元上的受体。突触可以通过改变树突棘表面突触上的受体数量，以及改变容纳突触的棘的大小和形状来改变它们的强度。这个过程（被称为突触可塑性）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回路中，当记忆形成或丢失时，突触会被修改。为了保持长期记忆，神经元需要改变参与决定脊柱结构或突触受体数量的蛋白质机制。蛋白质是通过翻译编码在DNA序列（基因）中的遗传信息而产生的。DNA和蛋白质之间的一种中间物质被称为信使RNA (mRNA)，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对这些突触重要的特定蛋白质的合成。另一种称为微RNA （miRNA）的分子可以与mRNA结合并阻止mRNA转化为蛋白质。这一过程被非常精确地调控以控制局部蛋白质合成，但在突触可塑性过程中是如何调控的尚不清楚。一种叫做Argonaute 2的蛋白质是细胞机制的重要组成部分，它促进了miRNA对蛋白质合成的阻断。我们的初步实验结果表明，Argonaute 2的化学修饰是为了响应突触可塑性的诱导。这影响了它与miRNA活性所需的其他重要蛋白质结合的能力。我们的主要假设是，导致可塑性的突触刺激触发Argonaute 2的这种修饰，增强其与其他蛋白质的结合，从而增加mirna介导的蛋白质合成抑制，以控制重要突触蛋白的水平。我们还提出这种机制发生在局部水平上，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脑中获得的神经元上进行，并在体外保持“活”状态。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Argonaute 2中引入基因突变，以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。我们还将开展生化实验，精确研究可塑性刺激如何引起Argonaute 2的修饰，以及该机制是否特异性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突触活动对神经元中蛋白质合成的局部控制的调节机制，从而进一步了解长期记忆的机制。

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.