Regulation of Arp2/3-mediated actin polymerisation by PICK1 in neuronal function

Nerve cells (neurons) in the brain communicate with one another at connections called synapses. 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. This process is thought to underlie learning and memory, because the memory is likely to be stored in a circuit of interconnected neurons. An important mechanism for moving receptors around neurons involves a protein called actin, which forms filaments that shrink and grow to physically manoeuvre parts of the cell or its constituents. We have found that a protein (called PICK1) is involved in controlling the formation of these actin filaments, and in this way controls the movement of neurotransmitter receptors to or from the synapse. Individual neurons are constituents of neuronal circuits that control complex behaviour or memory systems, and these neurons receive various inputs that trigger biochemical reactions inside them. These biochemical reactions can influence countless different processes in cells, and we propose that PICK1's regulation of actin could be controlled in this way. Therefore, in the proposed research, we aim to investigate how PICK1 is 'switched on' or 'switched off' with respect to its control of actin filaments. We are going to investigate three types of biochemical reaction in this work, all of which are specific varieties of well-known cellular mechanisms. We will initially carry out experiments on protein molecules in test-tubes to study the biochemical processes, and then do experiments in living neurons cultured in vitro to investigate how manipulating these biochemical reactions affects synaptic plasticity. We will use two approaches to study synaptic plasticity in neurons. First, we will visualise the movement of neurotransmitter receptors using microscopy, and second, we will analyse the activity of neurons by recording their electrical activity. This work is important because it will lead to a wealth of new information about synaptic plasticity, and hence learning and memory mechanisms. The movement of neurotransmitter receptors to and from the synapse is thought to underlie the altered neuronal activity in several brain diseases, such as stroke, Alzheimer's, and also in drug addiction. Therefore, the mechanisms that we will study in this research will add to our knowledge about these debilitating diseases, and may contribute to developing therapies. In addition, the control of actin is absolutely essential to numerous processes in all of the cells in our bodies, not just neurons. Therefore, this work will provide important information that will enhance the study of many other cellular processes and disease mechanisms.

The dynamic actin cytoskeleton is crucial for regulating cell morphology and vesicle trafficking by exerting mechanical forces that alter the shape of membranes. The Arp2/3 complex stimulates the formation of actin networks that mediate such membrane changes, and is highly regulated so that changes in cell morphology or vesicle trafficking occur at appropriate times and subcellular locations. We recently identified PICK1 as an Arp2/3 inhibitor, and demonstrated that this activity is required for NMDA-induced AMPAR internalisation and also for the development of appropriate dendritic morphology. Arp2/3 activators such as N-WASP and WAVE are subject to extremely tight regulation by a number of intracellular signalling pathways, consistent with a profound importance for controlling actin polymerisation. Since receptor trafficking and dendritic development are so crucial for the modification and function of neuronal circuits, we propose that the inhibition of Arp2/3 activity by PICK1 is under a similarly high level of control via multiple signalling pathways. Our hypothesis is that Arp2/3-mediated actin polymerisation is regulated by Arf1-dependent signalling via PICK1, and also that certain kinase pathways regulate the phosphorylation state of PICK1 to modulate its inhibition of Arp2/3 activity. In addition, we propose that calcium ions may also regulate actin assembly via PICK1. We aim to study these phenomena using biochemical/ molecular approaches followed by cell biological and electrophysiological analyses with AMPAR trafficking, Long Term Depression and dendritic morphology as assays for PICK1-Arp2/3 regulation. This work will not only contribute to our knowledge of the function and formation of neural circuits that underlie learning and memory, but will also identify novel pathways for the functional control of actin polymerisation in neurons.

Who will benefit from the research? As well as the specific academic beneficiaries listed in the 'Academic Beneficiaries' section, the public and wider academic community will benefit from the increase in knowledge about basic cell biology and brain function. Sectors of the pharmaceutical industry working to develop effective drug therapies for neurological diseases will also benefit from the proposed work. Indirectly, and in the long term, people suffering from such diseases or drug addiction may also benefit. Therefore, there is the potential for beneficial impact on both the health and wealth of the UK. How will they benefit from this research? AMPA receptor (AMPAR) trafficking is now thought to be involved in several neurological diseases, such as stroke, amyotrophic lateral sclerosis (motor neuron disease), bipolar disorder and Alzheimer's disease. In addition, PICK1 has been identified as a potential candidate for a schizophrenia susceptibility gene. Therefore, the mechanisms that we will study in our research will be incorporated into the body of knowledge about these debilitating diseases, for example, informing the development of animal models of disease, which amongst other things, pharmaceutical companies extensively utilise as part of the drug development process. Furthermore, since AMPAR trafficking has been shown to be a crucial step in the specialised form of synaptic plasticity that leads to drug addiction in rodent models, our work will benefit researchers who are working directly on the mechanisms that underlie drug addiction. The social impact and economic costs of the diseases mentioned above are enormous. Therefore our work will benefit society from the advances we make in investigating mechanisms that may underlie such diseases, 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 by UK-based companies. We acknowledge that these indirect benefits may take several years before they are realised. What will be done to ensure that they have the opportunity to benefit from this research? We aim to publish our work in high profile journals, so that our findings are easily accessed by pharmaceutical researchers. JGH and JRM are involved in collaborations with two different pharmaceutical companies, which will allow us to present our research to the industry, and pursue and potential for drug development. We will engage with the public, by taking part in activities such as 'junior science cafes' in schools. In addition, we actively encourage lab members to take part in other public engagement activities within schools such as the 'researchers in residence' scheme. The applicants have already participated in events aimed at explaining the basics of neuroscience to children at '@Bristol', which is a local hands-on interactive science centre, and they are committed to participating in further events in the future. In addition, the public can access neuroscience research via the MRC centre for Synaptic Plasticity and Bristol Neuroscience (BN) webpages. BN is an organisation that brings together all aspects of neuroscience research in Bristol, specifically with the aim of advancing understanding of the basic principles of the nervous system and development of new treatments for neurological disorders and disease. BN has close links to the Institute for Advanced Studies, which has a commitment to public engagement in science as well as involvement in establishing the National Co-ordinating Centre for Public Engagement (hosted jointly by Bristol University and The University of The West of England). Through this a number of activities are arranged such as talks at local schools, involvement in summer school programmes, interaction with the media and events to increase public understanding of science.