Microglial mechanisms of cell death and the role of the P2X7 receptor in relation to glaucoma

Newman, Jack (2023) Microglial mechanisms of cell death and the role of the P2X7 receptor in relation to glaucoma. Doctoral thesis, University of East Anglia.

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Purpose: The purpose of this research was to investigate mechanisms of cell death in microglia in the context of neurodegenerative disease including glaucoma. The effects of ATP stimulation on cell membrane disruption and cell death in microglia was investigated and inhibitors used to characterise the pathways involved. The mechanisms and pathways between microglia and macrophages were compared and finally the effects of pathophysiological insults on cell membrane disruption and cell death in microglia were explored using inhibitors again to characterise the pathways involved.

Methods: BV2, P2X7K/O BV2 microglia and J774 macrophages were the cell lines used. Cell membrane disruption was measured using YO-PRO and propidium iodide (PI) dye, while cell death was investigated using cell death assays (LDH and MTS). Cell morphological changes were observed by phase contrast microscopy. Inhibitors for cell death associated proteins were utilised to investigate cell death pathways. Other methods used include SDS-PAGE and Western blot to detect the pyroptotic protein gasdermin D protein and qPCR for measuring mRNA gene expression levels of proteins associated with cell death.

Results: ATP stimulation caused membrane disruption and cell death in BV2 microglia that was P2X7 dependent. However, inhibitors of apoptotic (Z-DEVD-FMK), pyroptotic (MCC950, Ac-YVAD-cmk and Necrosulfonamide) and necroptotic proteins (necrostatin), as well as, other caspase inhibitors (Z-VAD-FMK and Z-IETD-FMK) and calpain inhibitors (PD150606 and CAT811) caused no difference in these changes. The J774 macrophages underwent a pyroptotic cell death, in both LPS and non-LPS primed cells after ATP stimulation, that could be blocked with inhibitors of pyroptosis. LPS priming had no effect on parameters measured in BV2 microglia and cleaved gasdermin D was present in J774 macrophages and not the BV2 microglia after ATP stimulation. The pathophysiological stressors, amyloid-β, oxidative stress, ischaemia and pH, all caused cell death, but the inhibitors predominantly had no effect. Levels of mRNA of activation/pyroptosis-associated proteins levels showed a similar profile between LPS and most of the stressors.

Conclusions: ATP and the pathophysiological stressors all caused damage to the microglia but the pathway this is occurring through is a non-pyroptotic mechanism. This is different to that seen in macrophages. The pathophysiological stressors also caused expression changes indicative of activation. Further elucidation could help in the understanding and development of novel therapies for neurodegenerative disease including Alzheimer’s disease and glaucoma.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Nicola Veasy
Date Deposited: 08 Jul 2024 14:20
Last Modified: 08 Jul 2024 14:20
URI: https://ueaeprints.uea.ac.uk/id/eprint/95828


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