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

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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.