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

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.

MicroRNAs (miRNAs) are small noncoding endogenous RNA molecules that repress translation of target mRNAs via binding to the 3'UTR. MiRNAs silence target mRNAs by associating with Argonaute (Ago) proteins to form the RNA-induced silencing complex (RISC), underpinning a powerful mechanism for fine-tuning protein expression in multiple cellular processes. A large proportion of miRNAs are expressed in the brain, and many are found in neuronal dendrites close to synapses. Recent reports show that a number of specific miRNAs are required for synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic function. However, it is unknown how the induction of synaptic plasticity stimulates RISC activity to rapidly and locally repress translation of synaptic proteins. Our preliminary data show that NMDAR stimulation (cLTD) causes an increase in Ago2 phosphorylation at serine 387, which enhances its binding to the essential RISC binding partner GW182. This in turn promotes gene silencing via miR-134, which is a dendritically targeted miRNA. These observations lead to our hypothesis that NMDA-stimulated Ago2 phosphorylation transiently regulates its interaction with specific protein partners to rapidly repress translation of synaptic proteins, and that this is an essential mechanistic component of synaptic and/ or structural plasticity. We further propose that this mechanism controls RISC activity at a local level, such that stimulation of a single synapse affects translational repression close to the stimulated spine. We will test these hypotheses using biochemical, cell imaging and electrophysiological approaches. We have validated serine 387 phospho-null and phospho-mimic Ago2 mutants, which are key tools for the functional experiments. This work will fill critical gaps in our understanding about the mechanisms of miRNA-mediated gene silencing in synaptic plasticity.

Who will benefit from the research?
In addition to the specific academic beneficiaries described in the appropriate section of this proposal, sectors of the pharmaceutical industry working to develop effective drug therapies for neurological diseases and the general public will benefit from this work. Indirectly, and in the long term, the ageing population and people suffering from neurological diseases will also benefit. In addition, undergraduate and postgraduate students will benefit from our work. Therefore, there is the potential for beneficial impact on both the health and wealth of the UK.

How will they benefit?
The pharmaceutical industry:
Our work will identify mechanisms involved in modulating translational repression in neuronal dendrites. Since such regulation by microRNAs has been implicated in ageing and in numerous diseases such as schizophrenia, Alzheimer's, Huntington's, Parkinson's and autism spectrum disorders, these pathways represent promising targets for therapeutic intervention, and a number of pharmaceutical companies are now engaged in research programmes with this objective. Therefore our work will impact on the development of therapies for conditions associated with age and cognitive decline and these are of major interest to pharmaceutical companies. JRM and MCA have ongoing collaborations with Eli Lilly & co (through the Centre for Cognitive Neuroscience) and co-supervise CASE award studentships at Bristol University.

People suffering from neurological disease:
The social impact and economic costs of neurological diseases are enormous, and growing with the ageing population. Therefore our work will benefit society from the advances we make in investigating mechanisms that underlie the diseases outlined above, and will benefit the economy both in terms of costs saved in care for patients suffering from these conditions, and also in profits from pharmaceuticals developed and sold. We acknowledge that these indirect benefits may take several years before they are realised.

The public:
Our work will impact several public audiences, including school pupils, teachers and the general public. Understanding more about the functioning of the brain, including fundamental processes like learning and memory, is of significant interest to many groups. At a recent public engagement event for schools and families (Changing Perspectives) neuroscience activities were one of the most popular of the range of hands-on science stalls on offer. School pupils were engrossed by testing their memories, and these activities could easily be expanded to include demonstrations of the molecular mechanisms of protein translation in neurons and how this influences memory and learning. Other neuroscience activities led by Bristol researchers - for example during Brain Awareness Week and Discover - are equally popular with public audiences, as are public talks on neuroscience topics held regularly by Bristol Neuroscience.

Students:
This research will provide new opportunities for postgraduate research students who will carry out their projects in our labs, especially since the role of miRNA-mediated translational regulation in neurons is a relatively new area of research for my lab. It will also contribute to attracting talented students to the UK, therefore bringing highly educated and talented people into this country. Undergraduate students at Bristol will benefit from learning about our research in lecture courses before it is published.