The activity-dependent regulation of Argonaute 2 function in neurons by PICK1.

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)从神经元中释放出来，穿过突触激活相邻神经元中的受体。突触可以通过改变突触中神经元表面发现的受体的数量，以及通过改变容纳突触的树突的大小和形状来改变它们的强度(称为突触可塑性)。这一过程被认为是学习和记忆的基础，因为记忆很可能存储在相互连接的神经元电路中。为了保持长期记忆，神经元需要合成额外的蛋白质成分，这些蛋白质成分对于维持脊椎结构的变化或突触上受体数量的变化非常重要。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(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对这些突触重要的特定蛋白质的合成。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。我们已经发现，一种已知参与突触可塑性的蛋白质(称为PICK1)与另一种蛋白质(称为ArgAerte2)相互作用，后者是细胞机制的重要组成部分，促进miRNA与mRNA的关联，从而阻止蛋白质的合成。初步实验表明，PICK1通过与ArgAerte2相互作用，可以解除这种蛋白质合成的障碍，并增加特定蛋白质的产生。我们的主要假设是PICK1通过与Argavite2的相互作用在调节接近活性突触的蛋白质合成中发挥重要作用。我们旨在通过分析PICK1如何调控ArgAerte2功能的确切机制来检验这一假说。我们将使用既定的方法来分析蛋白质合成，同时在不同的突触活动条件下操纵PICK1-ArgAerte2的相互作用。我们的初步结果表明，PICK1将Argavite2锚定到神经元内称为内小体的膜结合结构上，内小体在突触可塑性过程中参与将重要的受体运输到突触。我们的假设之一是，这些贩运事件与PICK1对ArgAerte2的调控有关。MiRNAs的一个重要功能是控制蛋白质的局部合成，这些蛋白质决定树突棘的大小或形状，这是与突触连接的强度相关的一个重要因素。我们将研究PICK1是否参与树突局部蛋白质的合成，并通过其与Argavite2的相互作用调节树突棘的大小。我们的大部分实验将使用从大鼠大脑获得的神经元进行。这些神经元可以从大脑中分离出来，然后在培养皿中保持“活着”。使用这些细胞，我们将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 in the adjacent neuron. Synapses can change their strength (known as "synaptic plasticity") by altering the number of receptors found on the surface of the neuron in the synapse, and also by changing the size and shape of the dendritic spine that houses the synapse. This process is thought to underlie learning and memory, because the memory is likely to be stored in a circuit of interconnected neurons.In order to retain long-term memories, neurons need to synthesize additional protein components that are important for maintaining the changes in spine structure, or the changes in 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 a particular protein that is 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.We have found that a protein (called PICK1), which is known to be involved in synaptic plasticity over the timescale of hours, interacts with another protein (called Argonaute2), which is an important component of the cell machinery that promotes the association of miRNA with mRNA to block protein synthesis. Preliminary experiments suggest that by interacting with Argonaute2, PICK1 can relieve this block of protein synthesis and increase the production of specific proteins. Our main hypothesis is that PICK1 plays an important role in regulating protein synthesis close to active synapses via its interaction with Argonaute2.We aim to test this hypothesis by analysing the precise mechanism for how PICK1 regulates the function of Argonaute2. We will use established methods for analysing protein synthesis while manipulating the PICK1-Argonaute2 interaction under different conditions of synaptic activity. Our preliminary results suggest that PICK1 anchors Argonaute2 to membrane-bound structures inside the neuron called endosomes, which are involved in trafficking important receptors to the synapse during synaptic plasticity. One of our hypotheses is that these trafficking events are linked to the regulation of Argonaute2 by PICK1. One important function of miRNAs is controlling the local synthesis of proteins that determine the size or shape of dendritic spines, which is an important factor that correlates with the strength of a synaptic connection. We will investigate whether PICK1 is involved in local protein synthesis in dendrites and consequent regulation of dendritic spine size via its interaction with Argonaute2.Most of our experiments will be carried out using neurons obtained from the rat brain. These neurons can be isolated from the brain and then kept 'alive' in a petri dish. Using these cells we will be able 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.