P2X7 receptor function in microglia in relation to glaucoma

Felgate, Matthew (2019) P2X7 receptor function in microglia in relation to glaucoma. Doctoral thesis, University of East Anglia.

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Purpose: The purpose of this research was to investigate the role of P2X7 in microglial function, in relation to glaucoma. Specifically, a P2X7 knockout (K/O) microglial cell line was generated and used to explore the responses of purinergic stimulation, including [Ca2+]i signalling, cytotoxicity, and IL-1β production and release. Purinergic mediated consequences of oxygen glucose deprivation were also briefly explored.

Methods: P2X7 K/O microglial cells were generated using CRISPR gene editing technology on BV-2 microglia, and tested for P2X7 expression using flow cytometry, gDNA PCR, qRT-PCR and Western blotting. [Ca2+] responses to purinergic agonist stimulation were measured in Fura-2 loaded BV-2 and P2X7 K/O cells. Viability and cytotoxicity of purinergic stimulated BV-2 cells were assessed with MTS, LDH and caspase-3/7 staining assays. IL-1β mRNA and protein expression was measured with qRT-PCR and ELISAs respectively. BV-2 cells were subject to OGD using a custom incubator

Results: CRISPR modification generated a cell line, which upon analysis demonstrated knockout of P2X7 protein expression. Purinergic agonists demonstrated a multifaceted [Ca2+]i response mediated by multiple receptors including P2X7, P2X4, P2Y2 and P2Y6, with P2X7 giving a sustained rise in [Ca2+]i . Microglia were sensitive to ATP-induced cytotoxicity mediated by P2X7. An initial lower level of ATP-induced toxicity was P2X7-independent and mediated by ADP. ATP treatment was a sufficient priming stimulus for IL-1β mRNA and protein and P2X7 was responsible for IL-1β release upon subsequent, in a P2X7-dependent manner.

Conclusions: The generation of a P2X7 K/O microglial cell line has provided a wealth of information that can contribute to the understanding of the role of microglia in the pathophysiological mechanisms in glaucoma.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Chris White
Date Deposited: 14 Apr 2021 08:33
Last Modified: 14 Apr 2021 08:33
URI: https://ueaeprints.uea.ac.uk/id/eprint/79733


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