New chemical and microscopy tools targeting the complement system in neuroinflammation

阿尔茨海默病是导致老年残疾和死亡的主要原因，影响着全世界数百万人。该疾病的一个特征是淀粉样斑块与大脑炎症相结合。越来越多的证据表明，先天免疫系统的一部分，即补体系统，可能是减少包括阿尔茨海默病在内的神经退行性疾病的神经炎症的关键。具体而言，补体蛋白C5水平在ALS患者中升高（DOI:10.1016/j.jneuroim.2014.09.005），慢性应激上调神经退行性大鼠的C5水平（DOI:10.1007/s10571-017-0491-3）。该项目旨在通过开发尖端的化学、计算和成像工具，更好地了解C5在神经炎症中的作用。在补体级联中，C5被C5转化酶裂解为C5a和C5b。C5a是一种过敏毒素，调节其他免疫细胞的反应，C5b与C6复合物开始终末补体途径，导致膜攻击复合物的形成和细胞凋亡。抑制C5可以减少神经炎症，同时允许对感染的正常免疫反应，因此可能成为神经退行性疾病药物的新靶点。我们寻求开发新的工具来成像，调节和观察C5切割的神经炎症效应：1。开发一种单分子显微镜法，在分子水平上实时成像C5裂解。(图1)。B)我们将利用商业的和重组的C5和C6生成C5裂解的荧光报告基因，通过单分子TIRF显微镜直接成像裂解或结合C6，实时量化这一过程的分子动力学。开发新的C5裂解小分子抑制剂。与Supervisor 3一起，我们开发了一个计算管道来筛选C5裂解的新小分子调节剂（图1C）。这些将在导师2的实验室中设计和合成，并使用单分子测定法以及标准PAGE和SPR测定法进行测试。验证C5切割抑制减少神经炎症的假设。我们已经证明，用现有的C5裂解抑制剂（H1H）治疗小鼠小胶质细胞，在用炎症细胞因子干扰素- α治疗后，有益于细胞健康（图1D）。我们将用H1H和新开发的抑制剂在人类细胞系上进一步验证这一假设。我们还将开发先进的细胞健康检测方法，使用机器学习来表征细胞形态，并使用pH敏感的荧光染料来测量淀粉样蛋白吞噬作用。成果：C5的分子特征，神经炎症细胞生物学的新工具和理解，药物发现的新工具，新的潜在药物。拟议的项目符合BBSRC的生物科学促进健康的战略研究重点，特别是终身健康和生物技术促进健康方面的挑战。它也符合BBSRC对生物科学促进健康研究优先事项的关键愿景之一，即开发多学科方法和利用新兴技术来支持人类和动物健康的改善。这个项目包含了多学科基础研究的一个重要部分，但它也有潜在的额外维度，即基于C5信号干扰物及其在减少大脑炎症效应方面的可能作用，可能导致新的疗法和治疗方法。

Alzheimer's disease is a leading cause of late-life disability and death, affecting millions worldwide. A hallmark of the disease are amyloid plaques combined with inflammation in the brain. There is growing evidence that part of the innate immune system, the complement system, may be key to reducing neuroinflammation in neurodegenerative diseases, including Alzheimer's. Specifically, levels of the complement protein C5 are elevated in ALS patients (DOI:10.1016/j.jneuroim.2014.09.005) and chronic stress upregulates C5 in neurodegenerative rats (DOI:10.1007/s10571-017-0491-3). This project seeks to better understand the role of C5 in neuroinflammation by developing cutting-edge chemical, computational and imaging tools.In the complement cascade, C5 is cleaved by C5 convertase to C5a and C5b. C5a is an anaphylatoxin, regulating the response of other immune cells, and C5b complexes with C6 to commence the terminal complement pathway which leads to formation of the membrane attack complex and apoptosis. Inhibiting C5 may reduce neuroinflammation, while allowing for normal immune responses to infection, and so may be a new target for drugs for neurodegenerative disease. We seek to develop new tools to image, modulate and observe the neuroinflammation effects of C5 cleavage by:1. Developing a single-molecule microscopy assay to image C5 cleavage in real time at the molecular level. (Fig 1A.B) We will generate a fluorescent reporter for C5 cleavage using commercial and recombinant C5 and C6, to either image cleavage directly or binding of C6 using single-molecule TIRF microscopy to quantify the molecular dynamics of this process in real time.2. Developing new small molecule inhibitors of C5 cleavage. With Supervisor 3, we have developed a computational pipeline to screen for new small molecule modulators of C5 cleavage (Fig. 1C). These will be designed and synthesised in Supervisor 2's lab and tested using the single-molecule assay but also standard PAGE and SPR assays.3. Testing the hypothesis that C5 cleavage inhibition reduces neuroinflammation. We have shown that treatment of mouse microglial cells with an existing C5 cleavage inhibitor (H1H) benefits cell health following treatment with the inflammatory cytokine interferon-alpha (Fig 1D). We will further test this hypothesis on human cell lines with H1H and newly developed inhibitors. We will also develop advanced assays for cell health, using machine learning to characterise cell morphology and measure amyloid beta phagocytosis using pH sensitive fluorescence dyes.Outcomes: molecular characterisation of C5, new tools and understanding of the cell biology of neuroinflammation, new tools for drug discovery, new potential drugs.The proposed project maps to the BBSRC's strategic research priority of Biosciences for Health, specifically the challenges in lifelong health and biotechnology for health. It also fits with one of BBSRC's key visions for the Biosciences for Health research priority, of developing multidisciplinary approaches and exploiting emerging technologies to underpin improvements in both human and animal health. This project contains a major piece of multidisciplinary fundamental research, but it also has the added dimension of potentially leading to new therapies and treatments based upon C5 signalling disruptors and their possible role in reducing inflammatory effects within the brain.