Neurovascular protection of the brain following ischaemic stroke and the role of the hamartin/mTOR pathway

The brain is supplied with oxygen and glucose delivered by the blood, and cells within the brain convert that oxygen and glucose into energy to maintain brain function. Therefore, it is critical that blood supply is continuously available, because when blood flow to the brain is blocked such as in stroke, severe brain damage can occur. Stroke is the third leading cause of death and leading cause of disability costing the UK economy £3.7 billion per year. Stroke has only one approved treatment which breaks down blood clots and less than 15% of patients are eligible for this treatment. Therefore, discovering novel strategies for stroke treatment is warranted. Importantly, a major consequence of stroke is the breakdown of the blood-brain barrier (BBB), a physical barrier that prevents passage of material from the blood to the brain. Breakdown of this BBB causes swelling and damages brain cells. Many types of cells form and maintain the BBB. Collectively this grouping of cells is called the neurovascular unit (NVU), and is also important in controlling blood flow within the brain. Attempts are being made to find drug targets for stroke treatment that protect neurons (the brain's major cell type) and conserve NVU function (keeping the BBB closed and maintaining blood flow). We believe one such target to be a biochemical signalling pathway in cells called the hamartin/mammalian target of rapamycin (mTOR) pathway. Hamartin is a protein that acts to block the activity of another protein called mTOR, resulting in the prevention of cell growth and other cell functions. We recently showed that increasing hamartin can protect neurons after stroke. We also have preliminary data that shows that increasing hamartin can protect endothelial cells (an important cell type for blood flow and BBB function). The mTOR pathway has been linked with ageing, dementia and decreased blood flow, so blockade of mTOR may be a useful strategy for protection of the brain following stroke. This proposal hopes to answer 5 questions:

1) Does the activity of the hamartin/mTOR pathway change in the brain after stroke in rats?
2) Can increasing hamartin or blocking mTOR protect the brain after stroke?
3) Does blocking mTOR change blood flow in the brain under normal conditions and following stroke?
4) Which individual cell types are responsible for protection of the brain when blocking mTOR after stroke?
5) Does the activity of the hamartin/mTOR pathway change in the brain after human stroke?

To answer question 1, brains from rats that have had an experimental stroke will be assessed for changes in proteins of the hamartin/mTOR pathway. To answer question 2, rats that had an experimental stroke will receive a virus that increases hamartin levels in the brain, while other rats will receive drugs that block mTOR. We hope that increasing hamartin or blocking mTOR will reduce damage to the brain after stroke. To answer question 3, we will give ordinary rats mTOR blockers to see if this can alter blood flow under normal conditions and when the brain is stimulated. We will then see if mTOR blockers also change blood flow after stroke. Question 4 will then answer in what cells is mTOR being protective. Different cells will be individually isolated from rat brains and exposed to an environment that lacks oxygen and glucose, mimicking a stroke. Hamartin will be increased with a virus or mTOR will be blocked with drugs, and this will tell us which brain cells are protected. To answer question 5, brains from deceased human stroke patients will be assessed for changes in proteins of the hamartin/mTOR pathway. This will show that this pathway is affected by stroke in humans as well as rats. We hope that these studies will reveal whether blocking the mTOR pathway will provide protection to the brain, which after further development, could provide a novel treatment option for stroke patients.

Ischaemic stroke, caused by a thrombus in a major cerebral artery, leads to depletion of the supply of oxygen and glucose to the brain causing neuronal death. A major consequence of this is blood-brain barrier (BBB) breakdown producing extravasation of harmful plasma components. BBB integrity is regulated by cells of the neurovascular unit (NVU) such as brain endothelial cells, pericytes, and astrocytes, which are also cell types involved in cerebral blood flow (CBF) regulation. Drug targets that protect neurons and maintain NVU function by restricting BBB breakdown and preserving CBF are desired as a therapeutic strategy for stroke. One such target could be the hamartin/mammalian target of rapamycin (mTOR) pathway. We have shown that hamartin, a protein that forms part of the tuberous sclerosis complex and inhibits mTOR, protects both neuronal cells and brain endothelial cells following oxygen-glucose deprivation (OGD) in vitro. The rat middle cerebral artery occlusion (MCAO) model will then be used to assess how the hamartin/mTOR pathway is affected under ischaemic conditions, and to test whether hamartin upregulation (with lentiviral particles) or mTOR inhibition (with drugs) can provide protection to the brain and maintain BBB integrity. We will then assess the effects of mTOR inhibitors on basal and evoked CBF using the whisker stimulation model in naïve and MCAO rats, with a focus on capillary flow and pericyte function. The cell types and mechanisms responsible for neurovascular function preservation will be determined using individual NVU cell cultures exposed to OGD and treated with mTOR inhibitors. To provide clinical relevance of this pathway, we will explore changes in the hamartin/mTOR pathway following human stroke. Overall, these results will hopefully provide the basis for which further pre-clinical development of mTOR inhibitors for stroke can begin, with the ultimate aim of improving stroke patient outcome in the clinic.

Stroke victims, carers and the NHS: The largest beneficiary of this research will be stroke patients, who we aim to improve treatment of in the long term. In addition, carers of stroke patients and the NHS will benefit due to the potential to improve outcome of patients. Stroke is a devastating disease with up to 33% mortality. There is currently only one treatment for ischaemic stroke available, the clot buster recombinant tissue plasminogen activator (rtPA), and this only works in a minority of patients. Stroke patients' dependence on others means the economic and societal cost of stroke is enormous. Our research has the potential of developing a novel treatment option that could be employed instead of, or in combination with rtPA, with the hope of improving outcome for stroke patients. Victims, carers and health service providers will be kept informed through public engagement seminars, media or online reports, and presentations/discussions with stroke clinicians.

Industry: Pharmaceutical and biotechnology companies will also benefit from our research. We are using a novel approach (focussing on the neurovascular unit) and a putative target for stroke (the hamartin/mTOR pathway), which may encourage interest within these companies to promote the development of novel modulators (inhibitors or activators; small molecules or peptides) of the hamartin/mTOR pathway. Importantly, these approaches may reinvigorate private sector interest in stroke research. We already have a number of links with industry, and through Genentech who formulate rtPA, we will be able to acquire the use of novel mTOR inhibitors (see Case for Support). Through public and private dissemination, industrial colleagues will be made aware of our research at the outset of the project, and future discussions may foster new collaborations for drug development for stroke.

Academic researchers: Other beneficiaries from our research include academic researchers, not only in the stroke field, but in neuroscience in general. The function of the hamartin/mTOR in the brain is not well established and further insight into this pathway under physiological conditions as well as in disease is urgently required, which we will attempt to elucidate with this project. Stroke researchers will also specifically benefit from our approach of focussing on the maintenance of the neurovascular unit as well as trying to protect neurons. Publication of this research with open access alongside presentations at neuroscience conferences will create exposure of our research to a wider academic audience.

Early career researchers: This research will provide an opportunity for an early stage post-doctoral research fellow to spend 3 years on a specific project and to learn a wide variety of techniques. They will benefit by fostering collaborations, the timely publication of results, presentations at conferences, and interaction with other junior and senior scientists within the stroke field and beyond.

Policy makers: This project may influence policy makers on the treatment of stroke and animal research. With only one approved treatment for ischaemic stroke, discovering important pathways with therapeutic potential may lead to changes in stroke treatment policy and how stroke will be treated in the future. Pre-clinical studies (such as this proposal) necessitate the use of animals. We are always eager to discuss with policy makers (and the public) about our research and promote the use of animals for scientific research where there is sufficient justification.

General public: The public engagement of science is extremely important to keep the general public informed, and to understand the need for certain research to be conducted. Throughout the duration of this project, we will participate in a number of public engagement events including Brain Awareness Week.