PICK1 and cortactin as antagonistic regulators of Arp2/3-mediated actin polymerisation in GluA2-dependent AMPA receptor trafficking.

The aim of this research is to study specific aspects of how nerve cells in the brain communicate with each other, and how this communication can change during learning and also as a result of certain kinds of brain disorder.

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.

AMPA receptors are the neurotransmitter receptors involved in most synaptic excitation in the brain, and they are made up of distinct protein components, different combinations of which govern how the receptors work. The presence of a component called GluA2 is important for two reasons. First, a number of other proteins physically interact with GluA2 in order to move AMPA receptors to or from the synapse. Second, it influences the entry of calcium ions into the neuron. AMPA receptors lacking GluA2 are permeable to calcium, so they allow calcium to flow into the cell, but those that contain GluA2 are calcium impermeable. Calcium is very important for triggering a wide range of biochemical processes inside neurons that can lead to short or long-term changes to the function of the neuron.

Previous work has suggested that some kinds of learning involve changing the total number of AMPA receptors localised to synapses, and others involve reducing the GluA2 content of the receptors at synapses for short periods of time. Both of these processes require the movement of receptors via GluA2, so it is crucial to understand how this happens. In certain disease states such as stroke, traumatic brain injury and motor neuron disease, longer-lasting removal of GluA2-containing AMPA receptors can lead to too much calcium entry, resulting in the dysfunction or death of the neurons affected.

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. A protein called PICK1 interacts with GluA2 and regulates the removal of AMPA receptors from synapses by inhibiting the formation of these actin filaments. Another protein, called cortactin, stimulates the formation of actin filaments, and we have found that it also binds directly to GluA2. Our preliminary experiments suggest that cortactin might function in an opposing manner to PICK1, and stabilise synaptic GluA2. We propose to study precise molecular mechanisms that control the GluA2-dependent movement of AMPA receptors at synapses, focussing on PICK1 and cortactin.

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 AMPA receptors at synapses in normal and disease states. We will use advanced forms of light microscopy as well as electron microscopy to visualise the precise location of these proteins relative to each other under conditions that lead to changes in synaptic levels of GluA2. We will use genetic techniques to manipulate interactions between relevant proteins and determine what effect this has on GluA2 movement under these conditions.

This work will provide crucial mechanistic information about how neurons regulate the function of AMPA receptors, which are the most important neurotransmitter receptors in the brain. This will have wide-reaching implications for our understanding of learning and memory processes as well as a range of neurological diseases.

The precise regulation of AMPA receptor (AMPAR) trafficking is crucial to excitatory neurotransmission, synaptic plasticity and the consequent formation and modification of neural circuits. AMPARs are assemblies of subunits GluA1-4, and the vast majority contain GluA2, which is particularly important because it renders AMPARs Ca2+-impermeable, and it binds accessory proteins involved in trafficking AMPARs to and from the synapse. The small proportion of AMPARs that do not contain GluA2 are Ca2+-permeable (CP-AMPARs). Therefore GluA2-dependent trafficking can lead to changes in the total number of receptors at the synapse, or to changes in their Ca2+-permeability, which can have profound implications for signaling during specific forms of synaptic plasticity and for neuronal viability under pathological conditions. PICK1 binds GluA2 and inhibits Arp2/3-mediated actin polymerisation. During Long-Term Depression, PICK1 recruitment leads to a reduction in total synaptic AMPARs, whereas during certain pathological insults (eg oxygen/glucose deprivation), GluA2-containing AMPARs are replaced by a small number of GluA2-lacking CP-AMPARs. Cortactin activates the Arp2/3 complex, and we show here that it also binds directly to GluA2. Our hypothesis is that cortactin and PICK1 antagonistically modulate actin dynamics to precisely control GluA2-dependent trafficking. We further propose that PICK1 and cortactin function as an antagonistic pair at endocytic sites as well as at recycling endosomes, and that this is a crucial factor in determining whether CP-AMPARs are expressed at synapses. We will use a range of biochemical, cell imaging and electrophysiological techniques to analyse the function of cortactin relative to PICK1 in the control of GluA2-dependent AMPAR endocytosis and recycling. This work will contribute to our knowledge about AMPAR trafficking mechanisms that are central to learning and memory and a number of neurological diseases.

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, people suffering from neurological diseases may 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 that regulate the trafficking of the GluA2 subunit of AMPARs. GluA2-specific AMPAR trafficking, specifically resulting in the synaptic expression of GluA2-lacking, Ca2+-permeable AMPARs, is thought to be a key event in a number of neurological disorders such as ischaemia, traumatic brain injury, chronic pain, drug addiction and neurodegenerative diseases. Therefore our work will impact on the development of therapies for these conditions by 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.