Neuronal vulnerability to ischaemia: the role of AMPA receptor trafficking.

The aim of this research is to define mechanisms that contribute to the dysfunction or death of nerve cells in the brain following brain injury, such as a stroke, or other neurological disorders.

Brain ischaemia occurs when the blood supply to a part of the brain is interrupted, for example by blockage of an artery (a stroke) or by heart failure. Brain damage caused by stroke has huge personal, social and economic impact. An estimated 150,000 people per year suffer from a stroke, making it the third most common cause of death in the UK. It is also one of the most common causes of severe disability, and more than 250,000 people live with disabilities caused by this condition.

Nerve cells (neurons) in the brain need a constant blood supply to provide essential factors for life, especially oxygen and glucose, and without these factors, neurons malfunction and die. An intriguing aspect of ischaemia is that neurons in certain brain regions are more likely to die as a result of an ischaemic injury than others. The aim of this proposal is to investigate the mechanisms that underlie these differences, with the hope that the knowledge we gain from our research could be used to make vulnerable neurons less likely to die following brain ischaemia.

Neurons communicate with each other via chemicals known as neurotransmitters, which are released from one neuron to be received by another via special receptors across a gap called a synapse. During the oxygen and glucose deprivation (OGD) associated with ischaemia, the properties of a very important type of neurotransmitter receptor (called the AMPA receptor), change in such a way that it allows more calcium than usual to enter neurons. It is this abnormal calcium entry that leads to neuronal death hours or days after brain ischaemia.

We have shown that less vulnerable neurons respond to OGD with different alterations of AMPA receptor properties, which are likely to result in reduced calcium entry into the cell. We propose to study the molecular mechanisms that underlie how AMPA receptors change during OGD in the different types of neurons, and investigate the idea that subtle differences in the way AMPA receptors change at synapses has a profound influence on the likelihood that the neurons will die.

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. We will also use slices of rat brain that keep some of the important connections between neurons. Using these cells and slices we will be able to understand more about the mechanisms that occur in response to OGD in neurons. We will then modify these mechanisms using drugs or genetic techniques to see if we can stop neurons from dying.

It has been shown that other disorders, including motor neuron disease and traumatic brain injury (physical impact injury), involve similar changes in AMPA receptors at synapses. This work will therefore provide crucial information about how neurons respond to a range of neurological disorders.

Transient global brain ischaemia induces delayed neuronal death in hippocampal CA1 region via GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs). This occurs via two mechanisms: a delayed downregulation of GluA2 mRNA expression, and a rapid trafficking event during the insult whereby a fraction of the normal complement of GluA2-containing Ca2+-impermeable AMPA receptors are replaced by CP-AMPARs. Certain populations of pyramidal neurons show a greater vulnerability to ischaemia than others. Typically, in the mature brain, hippocampal CA1 neurons are considerably more vulnerable than their counterparts in CA3 or cortical regions.
Our data suggest that oxygen/glucose deprivation (OGD) causes distinct AMPAR trafficking events in CA1, CA3 and cortical neurons. We propose that these differences contribute to the differential vulnerability to ischaemia, and that manipulating these processes could influence the vulnerability of neurons in response to OGD.
We will use electrophysiology, biochemical and cell imaging techniques to define OGD-induced AMPAR trafficking events in neuronal populations that exhibit different vulnerabilities to ischaemia. We will focus on the differences between cortical and hippocampal CA1 neurons, because OGD induces a rapid expression of CP-AMPARs in both neuronal populations, but the precise mechanisms are different. In particular, we will investigate the role of subunit-specific endocytosis, recycling and degradation, and the function of subunit interacting proteins in controlling the expression of CP-AMPARs at the synapse. In addition, we will apply genetic or peptide-based tools that prevent or reduce synaptic CP-AMPAR expression to cell death assays, with the aim of improving neuronal viability in response to OGD.

Who will benefit from the research?

1) Clinical researchers and clinicians involved in treating patients with brain ischaemia and diseases with related mechanisms. Included in this is the National Health Service who bear the current costs of such diseases.
2) The pharmaceutical industry.
3) People at risk from brain ischaemia and diseases with related mechanisms.
4) The general public.

How will they benefit?

Clinicians/clinical researchers and the NHS:
An estimated 150,000 people per year suffer from a stroke, which is the most prevalent form of brain ischaemia. It is one of the most common causes of severe disability, affecting more than 250,000 people, placing a massive financial burden on the healthcare systems in the UK and globally. An increase in knowledge about the molecular mechanisms that determine neuronal vulnerability will lead to the development of therapies, which will have a significant impact on the work of clinicians treating such patients. Any successful treatments would have a massive financial benefit.
Since the trafficking mechanisms that regulate the expression of Ca2+-permeable AMPARs during brain ischaemia are likely to be similar to those in other disease states such as traumatic brain injury and motor neuron disease/ALS, our work may also impact on the development of therapies for these conditions, and hence on the work of the clinicians involved.

The pharmaceutical industry:
Brain ischaemia is currently not a major focus for pharmaceutical companies. This is likely to be because of the lack of targets that are accessible for therapeutic intervention during a clinically relevant time frame. A primary objective of our proposed research is to identify mechanisms that could be targets for such intervention, and if such mechanisms were found to be useful drug targets, the pharmaceutical industry may initiate a programme to develop effective stroke therapies. This represents a very large potential for impact.

People at risk from brain ischaemia and diseases with related mechanisms:
Such people will benefit from the potential improvements to therapeutic interventions that may arise from this work. As described above, clinicians will use the increased knowledge about the mechanisms of ischemic injury in the brain to treat people who suffer brain ischaemia. This benefit may take several years before it is realised.

The public:
The public will benefit from the increase in knowledge about important disease processes in the brain. 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.

The social impact and economic costs of brain ischaemia are enormous, and growing with the ageing population. Therefore our work will benefit society from the advances we make in investigating mechanisms that 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.