New chemical and microscopy tools targeting the complement system in neuroinflammation

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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.