STRUCTURAL BASIS OF PRION INFECTION: Tracking fluorescent prions to define their pathogenic pathways by cutting-edge light and electron microscopy

朊病毒是引起人类疯牛病和克雅氏病的传染因子，在医学研究中是独一无二的。与所有其他传染因子（细菌和病毒）不同，这种传染性颗粒不包含遗传信息，而是由一种叫做朊病毒蛋白（PrP）的人体自身蛋白质的畸形、流氓形式的团块组成。一旦在体内形成，流氓PrP颗粒（朊病毒）就像种子一样，将正常的PrP转化为与自己相似的PrP，引发连锁反应，导致朊病毒在整个大脑中逐渐积累。这种积累会导致神经细胞死亡，导致严重的脑损伤、痴呆，最终导致感染者死亡。了解朊病毒结构的特殊之处以及它们如何生长和杀死神经细胞变得越来越重要，因为人们认为，随着畸形蛋白质种子的扩散，类似的过程也与阿尔茨海默氏症和帕金森病等更常见的脑部疾病有关。目前，由于缺乏有效的治疗方法，对痴呆症患者的机构护理每年要花费英国国家医疗服务体系数百亿英镑。尽管经过几十年的研究，人们仍然不清楚朊病毒是如何生长的，也不清楚它们是如何杀死神经细胞的。造成这些知识空白的一个主要原因是，从未在分离的活神经细胞中充分详细地观察到朊病毒感染。然而，随着光学和电子显微镜技术的进步，我们现在可以直接看到朊病毒感染细胞的过程。本课题的研究目的是：1)发现在感染的初始阶段，朊病毒会与神经细胞的哪个部分结合；2)确定朊病毒会移动到神经细胞的哪个部分，从而产生毒性作用；3)确定参与每个部分过程的神经细胞的特定结构成分。为了实现这些目标，我将使用经过基因改造的细胞来生产带有内置荧光化学标签的PrP，这种标签会发出光，可以用新的、强大的光学显微镜看到。这将使在活细胞感染过程中实时跟踪“发光”的朊病毒成为可能。一旦确定了感染的具体步骤，并且我们确切地知道在细胞内的哪里可以找到朊病毒，我将使用强大的电子显微镜放大这些位置。来自电子显微镜的高分辨率图像将使我们能够确定细胞的哪些结构成分直接与朊病毒相互作用。通过这项工作，我希望能够首次详细了解朊病毒如何与细胞相互作用，从而导致它们的致命影响。重要的是，了解朊病毒与细胞相互作用的结构成分将确定药物的关键靶点，这些药物可能能够阻止大脑内的朊病毒感染。

Prions, the infectious agents causing mad cow disease (BSE) and CJD in humans are unique in medical research. Unlike all other infectious agents (bacteria and viruses) the infectious particle does not contain genetic information but instead consists of clumps of misshapen, rogue forms of one of the body's own proteins called the prion protein (PrP). Once formed in the body, rogue PrP particles (prions) act as seeds to convert normal PrP into a likeness of themselves setting off a chain reaction leading to progressive accumulation of prions throughout the brain. This accumulation causes nerve cells to die leading to severe brain damage, dementia and ultimately death of the infected individual. Understanding what is special about the structure of prions and how they grow and kill nerve cells is increasingly important as it is thought that similar processes, with the spread of growing misshapen protein seeds, are also involved in more common forms of brain disease such as Alzheimer's and Parkinson's diseases. Currently, in the absence of effective treatments, institutional care for patients with dementia costs the UK NHS tens of billions of pounds each year.Despite decades of research, it is still not clear how prions grow or how they kill nerve cells. A major reason for these gaps in our knowledge is that prion infection has never been observed in sufficient detail in isolated living nerve cells. However recent advances in technology with light and electron microscopes will now allow us to directly see prions as they infect cells. The research aims of this proposal are 1) to find out which parts of the nerve cell prions bind to during initial phases of infection, 2) to identify which part of the nerve cell prions move to in order to cause their toxic effects, 3) to identify specific structural components of the nerve cell that are involved in each part of the process. To achieve these aims, I will use cells that have been genetically modified to produce PrP with a built-in fluorescent chemical tag which will produce light that can be seen using new, powerful, light microscopes. This will enable tracking of "glowing" prions in real time as infection proceeds in living cells. Once the precise steps of infection are worked out and we know exactly where to find prions within the cell, I will zoom-in on these locations using powerful electron microscopes. High-resolution images from electron microscopes will enable us to pinpoint which structural components of the cell are directly interacting with prions. Through this work I hope to provide the first detailed understanding of the how prions interact with cells to cause their lethal effects. Importantly, knowing the structural components of the cell that prions interact with will identify key targets for drugs that may be able to stop prion infection within the brain.