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

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.

MicroRNAs (miRNAs) are small noncoding endogenous RNA molecules that repress target mRNAs through complementary binding in the message 3'-UTR. They underpin a powerful mechanism for fine-tuning protein expression in multiple physiological and pathological processes. A large proportion of miRNAs are expressed in the brain, and most of these are found in neuronal dendrites associated with synapses. Many have been assigned roles in modulating the local translation of proteins that are essential to dendritic spine morphogenesis, synaptic function and memory formation. MiRNAs silence target mRNAs via the RNA-induced silencing complex (RISC), of which Argonaute proteins (Ago) are the major subunit. The subcellular localisation of translational repression via miRNA and RISC in neurons is unknown, and the mechanisms for activity-dependent modulation are also unclear. Our preliminary data show that PICK1, which regulates AMPAR trafficking and dendritic spine morphology during synaptic plasticity, interacts with Ago2 in neurons. PICK1 increases the association of Ago2 with endosomes and inhibits translational repression via the 3'UTR for Limk1, which is a target for the dendritically-localised mir-134. Furthermore, the Ago2-PICK1 interaction is regulated by the induction of synaptic plasticity. Our hypothesis is that PICK1 is involved in fine-tuning translational repression in dendrites in response to specific types of synaptic stimulation by regulating Ago2 subcellular localisation and function.
We will use biochemical and cell imaging approaches to investigate the role of PICK1 in regulating the localisation and function of Ago2 in neurons under basal conditions and following the induction of synaptic plasticity, and determine whether such a mechanism is involved in regulating dendritic spine morphology. This work will fill critical gaps in our understanding about how the repression of target mRNA translation by miRNAs is regulated by neuronal activity.

Who will benefit from the research?

In addition to the specific academic beneficiaries described in the appropriate section of this proposal, the pharmaceutical industry 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. 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.

The public:
The public will benefit from the increase in knowledge about brain function. The brain is a very important organ, commanding special interest from the public, because it holds our memories, governs our behaviour, and processes our senses and perceptions. 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. Other neuroscience activities led by Bristol researchers - eg during Brain Awareness Week (a biennial hands-on research festival with a total audience of 4,700) - are equally popular with public audiences, as are public talks on neuroscience topics held regularly by Bristol Neuroscience (BN) http://www.bristol.ac.uk/neuroscience/society/public-past.

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.