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

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Description We have published a paper in the Journal of Biological Chemistry. This demonstrates that the interaction between the two key proteins under study (PICK1 and Argonaute2) interact in a Ca2+ dependent manner. This is a mechanism for transducing NMDA receptor activation during synaptic plasticity into changes in protein translation via microRNA activity.
Another paper is published in EMBO Journal. This defines a specific phosphorylation site on Argonaute2 that is up-regulated in response to NMDAR stimulation, causes an increase in binding to RISC protein partners, and consequently an increase in specific silencing events. This mechanism is essential for dendritic spine shrinkage.
Exploitation Route Mechanisms for the regulation of microRNA activity by neuronal activity are likely to play a critical role in processes of neuronal function and neuronal development. They are also likely to be involved in numerous neurological disorders. Indeed, we have pilot data indicating that the mechanism we describe in the EMBO paper is involved in Alzheimer's disease. We recently secured funding from ARUK to pursue this.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Alzheimer's Research UK, Interdisciplinary Research Grant
Amount £249,000 (GBP)
Funding ID ARUK-IRG2018C-001 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 03/2023
 
Description Regulation of microRNA-mediated local translation in neurons by Argonaute phosphorylation
Amount £490,661 (GBP)
Funding ID BB/R006938/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 07/2021
 
Description Grant application to ARUK, collaboration with Exeter University 
Organisation University of Exeter
Country United Kingdom 
Sector Academic/University 
PI Contribution Successful grant application to Alzheimer's Research UK. We contributed the ideas for the application, and will contribute the biochemistry/ cell imaging aspects of the work.
Collaborator Contribution Electrophysiology, in vivo surgeries, Alzheimer's mouse models.
Impact Alzheimer's Research UK Interdisciplinary Research Grant.
Start Year 2017