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

在医学研究中，疯牛病(BSE)和CJD的致病因子--普恩病毒是独一无二的。与所有其他感染性物质(细菌和病毒)不同，感染性颗粒不包含遗传信息，而是由一团畸形的、流氓形式的人体自身蛋白质组成，称为普恩蛋白(PrP)。一旦在体内形成，流氓PrP粒子(PrP)就像种子一样，将正常的PrP转化为与自身相似的东西，引发连锁反应，导致PrP在大脑中逐渐积累。这种积累会导致神经细胞死亡，导致严重的脑损伤、痴呆症，并最终导致感染者死亡。了解Pron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，随着不断生长的畸形蛋白质种子的传播，类似的过程也涉及到更常见的大脑疾病，如阿尔茨海默氏症和帕金森氏症。目前，在缺乏有效治疗的情况下，机构对痴呆症患者的护理每年花费英国NHS数百亿英镑。尽管进行了数十年的研究，但仍不清楚普里恩是如何生长的，或者它们是如何杀死神经细胞的。我们认识这些空白的一个主要原因是，在分离的活着的神经细胞中从来没有足够详细地观察到Pron感染。然而，光学和电子显微镜技术的最新进展现在可以让我们直接看到普恩病毒感染细胞的情况。这项建议的研究目的是1)找出在感染的初始阶段神经细胞的Pron与哪些部分结合，2)确定神经细胞的Prion移动到哪些部分以引起它们的毒性作用，3)确定参与这一过程每个部分的神经细胞的特定结构成分。为了实现这些目标，我将使用经过转基因的细胞来生产带有内置荧光化学标签的PrP，这种标签将产生使用新的、强大的光学显微镜可以看到的光。当感染在活细胞中进行时，这将使实时跟踪“发光的”普恩病毒成为可能。一旦确定了感染的确切步骤，并且我们确切地知道了在细胞内的哪里可以找到普恩病毒，我就会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准确地确定细胞的哪些结构组件直接与普里恩相互作用。通过这项工作，我希望提供第一个详细的了解如何与细胞相互作用，导致他们的致命影响。重要的是，了解与Prion相互作用的细胞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Prion在大脑中的感染。

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.