Use of PrP transgenic Drosophila to measure mammalian prion infectivity

使用 PrP 转基因果蝇测量哺乳动物朊病毒感染性

• 批准号: NC/K000462/1
• 负责人: Raymond Bujdoso
• 金额: $ 33.35万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FK000462%2F1
• 关键词: Use; PrP; transgenic; Drosophila; measure

Prion diseases are invariably fatal neurodegenerative diseases of the brain that affect humans and other vertebrate species. These diseases include CJD of humans, BSE of cattle and scrapie of sheep. Prion diseases occur when a protein termed PrP undergoes a change in shape, and aggregates in the brain where it damages neurons. While other neurodegenerative diseases also occur through their own specific protein misfolding event, prion diseases are unique because they are transmissible, within individuals of the same species and also between different species. During prion disease, the transmissible infectious agent associated with these conditions, which is thought to be the misfolded form of PrP, accumulates in the brain of affected individuals. This infectious agent is termed a prion. Prions are a significant risk to public health through their potential to spread from animals to humans, seen by the appearance of BSE in UK cattle and subsequent emergence of variant CJD in humans. The most common route of transmission of prion diseases is by oral inoculation with prion-infected material i.e. by inadvertently eating prions. Individuals that become infected in such a manner accumulate prion infectivity in lymphoid tissue and blood. It is important to determine how much prion infectivity is present in the brain and different tissues, and blood from prion-infected individuals. This is necessary to understand the biology of infectious prions, to ensure the safety of animal products destined for human consumption, and to help to establish a blood test for these diseases. The prion is an unconventional infectious agent and the only reliable method to detect its presence is to inoculate experimental animals with prion-infected samples and see if the recipients develop prion disease. Inevitably, this has led to a large number of experimental animals, in particular mice and sheep, being used to assess mammalian prion infectivity, and as a consequence, dying from terminal prion disease. This proposal aims to reduce and replace, where possible, the use of mice and sheep to measure prion infectivity with an invertebrate host, Drosophila melanogaster. This in turn, will help to refine prion infectivity experiments by using less sentient animals that are not known to have a pain system or a comparable level of awareness as mammalian hosts. We will model scrapie of sheep in Drosophila. We have already generated Drosophila transgenic for sheep PrP and have shown these flies are susceptible to the toxic effects of sheep prions. Adult flies, fed prion infectivity as larvae, show a decreased ability to move and show the presence of the misfolded form of PrP in their brains. In the experiments of this proposal, we intend to validate the sensitivity of our new prion fly model to determine the extent to which our new animal model can replace mice and sheep in measuring prion infectivity. Our use of PrP transgenic Drosophila will produce humane, high-quality, reproducible prion-based science whilst assisting the 3Rs strategy. Good quality, statistically significant data will be generated using PrP transgenic Drosophila. Large numbers of flies can easily be produced and housed, and relevant numbers of replicates can be tested at the same time using a financially viable model. Our previous experience using mammalian hosts to study infectious prion disease will enable us to directly compare and highlight the benefits gained by using PrP transgenic Drosophila so we can sensibly inform other scientists and encourage less dependency on mouse and sheep prion bioassays. We will use previously generated prion-infected samples to test in our Drosophila model so that no other experimental animals need to be injected with prion material in order to obtain samples for use within this study. We have preliminary test results from many of these samples so they can be directly compared with the results we generate with our PrP transgenic Drosophila.

Pron疾病总是致命的大脑神经退行性疾病，影响人类和其他脊椎动物物种。这些疾病包括人类的CJD、牛的BSE和绵羊的瘙痒病。当一种名为PrP的蛋白质发生形状变化并聚集在大脑中，损害神经元时，Prion疾病就会发生。虽然其他神经退行性疾病也通过它们自己特定的蛋白质错误折叠事件发生，但Pron疾病是独特的，因为它们可以在同一物种的个体内传播，也可以在不同物种之间传播。在PrP疾病期间，与这些疾病相关的可传播的感染源，被认为是PrP的错误折叠形式，在受影响的人的大脑中积累。这种感染性病原体被称为普恩病毒。从英国牛的疯牛病和随后在人类中出现的变异CJD可以看出，普恩病毒有可能从动物传播到人类，从而对公共卫生构成重大风险。普恩病毒病最常见的传播途径是口服感染普恩病毒的物质，即无意中吃了普恩病毒。以这种方式感染的个体在淋巴组织和血液中积累了普恩病毒的传染性。重要的是要确定普恩病毒感染者的大脑和不同组织以及血液中存在多少普恩病毒感染性。这对于了解传染性普恩的生物学，确保供人类食用的动物产品的安全，以及帮助建立针对这些疾病的血液检测是必要的。Prion是一种非常规的感染性物质，检测其存在的唯一可靠方法是用感染了Prion的样本接种实验动物，看看接受者是否会患上Prion病。不可避免地，这导致了大量的实验动物，特别是小鼠和绵羊，被用来评估哺乳动物普恩病毒的传染性，结果是死于终末期普恩病毒病。这项提议旨在尽可能减少和取代使用老鼠和绵羊来测量无脊椎动物宿主--黑腹果蝇--的普恩传染性。反过来，这将有助于通过使用感知程度较低的动物来改进普恩病毒的感染性实验，这些动物并不像哺乳动物宿主那样具有疼痛系统或类似的意识水平。我们将在果蝇中制作绵羊瘙痒的模型。我们已经为绵羊PrP培育了转基因果蝇，并表明这些果蝇对绵羊PrP的毒性作用很敏感。成年果蝇，在幼虫时期喂食普恩病毒后，行动能力下降，大脑中出现错误折叠形式的PrP。在这项提议的实验中，我们打算验证我们新的Pron Fly模型的敏感性，以确定我们的新动物模型在测量Pron感染性方面可以在多大程度上取代老鼠和绵羊。我们使用PrP转基因果蝇将产生人性化的、高质量的、可重现的基于PrP的科学，同时帮助3Rs战略。使用PrP转基因果蝇将产生高质量、有统计学意义的数据。可以很容易地生产和安置大量的苍蝇，并且可以使用经济上可行的模型同时测试相关数量的重复。我们以前使用哺乳动物宿主研究传染性Pron疾病的经验将使我们能够直接比较和突出使用PrP转基因果蝇所获得的好处，这样我们就可以明智地告知其他科学家，并鼓励减少对小鼠和绵羊Prion生物检测的依赖。我们将使用之前产生的普恩病毒感染的样本在我们的果蝇模型中进行测试，这样就不需要为其他实验动物注射普恩病毒材料来获得在本研究中使用的样本。我们有许多样本的初步测试结果，因此可以直接与我们用PrP转基因果蝇产生的结果进行比较。