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Matthew Felgate, Leanne Stokes, David C Broadway, Julie Sanderson; The role of the P2X7 receptor in microglia in relation to glaucoma. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5302.
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We previously found that death of retinal ganglion cells (RGCs) and expression of the neuroinflammatory marker interleukin-1β (IL-1β) can be caused by activation of the purinergic P2X7 receptor (P2X7R) in the human retina. Microglia are key regulators of neuroinflammation and the P2X7R is proposed to play a key role in this process. The aim of this research was to use CRISPR technology to investigate P2X7R function in microglial cells.
A P2X7R knockout (KO) BV2 mouse microglial cell line was developed using CRISPR/Cas9 gene editing. Absence of the P2X7R was assessed by Western blot, flow cytometry and P2X7-dependent Ca2+ signalling (Fura-2 fluorimetric assay). Cell viability and death were quantified by MTS and LDH assays respectively. Induction of IL-1β mRNA was evaluated using qRT-PCR.
Western blots and flow cytometry confirmed the knockout of P2X7R in BV2 cells (n=3). ATP and BzATP (50µM-1mM & 5µM-100µM respectively) significantly increased intracellular Ca2+ in BV2 cells (n=3;p<0.05). ATP caused a biphasic response, showing an initial peak followed by a lower sustained phase of raised intracellular Ca2+. BzATP caused only the sustained phase. The P2X7R antagonist AZ10606120 (10µM) inhibited the sustained phase of the Ca2+ response. In P2X7 KO BV2 cells, no sustained phase was seen with ATP and BzATP caused no response (n=3).BV2 cells exhibited a dose-dependent decrease in cell viability and increase in cell death in response to ATP (50μM–3mM; n=4;p<0.05) and BzATP (5µM–300μM; n=4;p<0.05). AZ10606120 (10µM) protected against ATP- and BzATP-induced cell death (n=4;p<0.05). In P2X7 KO BV2 cells, no significant decrease in cell viability was observed at any of the ATP concentrations tested (10µM-5mM) (n=4).ATP (300μM) increased IL-1β mRNA expression in BV2 cells by 5-fold at 24h (n=4;p<0.05). A similar effect was demonstrated in P2X7 KO BV2 cells (n=4). BzATP (30μM) caused no significant changes in IL-1β expression (n=4).
We successfully developed a stable P2X7R deficient BV2 cell line and demonstrated that P2X7 activation caused death of BV2 cells. ATP-mediated IL-1β mRNA induction was not mediated by the P2X7R. Microglia play an important role in retinal homeostasis and cytokine production. Understanding the role of the P2X7R in microglial activation and neuroinflammation may provide insight into the role of ATP signalling in neurodegeneration in glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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