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

大脑由血液提供氧气和葡萄糖，大脑内的细胞将氧气和葡萄糖转化为能量以维持大脑功能。因此，持续提供血液供应至关重要，因为当流向大脑的血液受阻（例如中风）时，可能会发生严重的脑损伤。中风是第三大死亡原因和残疾的主要原因，每年花费英国经济37亿英镑。中风只有一种被批准的治疗方法，可以分解血栓，只有不到15%的患者有资格接受这种治疗。因此，发现新的中风治疗策略是必要的。重要的是，中风的一个主要后果是血脑屏障（BBB）的破坏，血脑屏障是一种阻止物质从血液进入大脑的物理屏障。血脑屏障的破坏会引起肿胀并损害脑细胞。许多类型的细胞形成并维持BBB。这组细胞统称为神经血管单位（NVU），在控制大脑内的血液流动方面也很重要。人们正在尝试寻找治疗中风的药物靶点，以保护神经元（大脑的主要细胞类型）和保护NVU功能（保持BBB关闭和维持血液流动）。我们认为其中一个靶点是细胞中的生化信号通路，称为错构蛋白/哺乳动物雷帕霉素靶点（mTOR）通路。Hamartin是一种蛋白质，可以阻断另一种称为mTOR的蛋白质的活性，从而阻止细胞生长和其他细胞功能。我们最近发现，增加错构蛋白可以保护中风后的神经元。我们也有初步的数据表明，增加错构蛋白可以保护内皮细胞（一种重要的细胞类型的血流和血脑屏障功能）。mTOR通路与衰老、痴呆和血流量减少有关，因此阻断mTOR可能是中风后保护大脑的有用策略。该提案希望回答5个问题：1）大鼠中风后大脑中hamartin/mTOR通路的活性是否发生变化？2)增加hamartin或阻断mTOR可以保护中风后的大脑吗？3)在正常情况下和中风后，阻断mTOR是否会改变大脑中的血流量？4)中风后阻断mTOR时，哪些细胞类型负责保护大脑？5)人类中风后大脑中hamartin/mTOR通路的活性是否发生变化？为了回答问题1，将评估患有实验性中风的大鼠的大脑中hamartin/mTOR通路的蛋白质的变化。为了回答问题2，实验性中风的大鼠将接受一种增加大脑中hamartin水平的病毒，而其他大鼠将接受阻断mTOR的药物。我们希望增加hamartin或阻断mTOR可以减少中风后对大脑的损害。为了回答问题3，我们将给普通大鼠注射mTOR阻断剂，看看这是否能在正常情况下以及大脑受到刺激时改变血流。然后我们将观察mTOR阻断剂是否也会改变中风后的血流。问题4将回答mTOR在哪些细胞中具有保护作用。不同的细胞将从大鼠大脑中单独分离出来，并暴露在缺氧和缺糖的环境中，模拟中风。病毒会增加Hamartin，药物会阻断mTOR，这将告诉我们哪些脑细胞受到保护。为了回答问题5，将评估已故人类卒中患者的大脑中错构蛋白/mTOR通路蛋白的变化。这将表明，这一途径受到人类和大鼠中风的影响。我们希望这些研究将揭示阻断mTOR通路是否会对大脑提供保护，经过进一步的开发，可以为中风患者提供一种新的治疗选择。

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.