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

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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.