Purinergic Signalling in Müller Cells in the Context of Glaucomatous Neuroinflammation and Neurodegeneration

Habib, Sofia (2023) Purinergic Signalling in Müller Cells in the Context of Glaucomatous Neuroinflammation and Neurodegeneration. Doctoral thesis, University of East Anglia.

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Abstract

Purpose: Glaucoma describes progressive optic neuropathies characterised by retinal ganglion cell (RGC) death with corresponding loss of visual field. Our group demonstrated P2X7 receptor (P2X7R)-mediated RGC death, increase in interleukin- 1β (IL-1β) and interleukin-10 (IL-10) expression in the human retina. Müller cells may have a role in glaucomatous neuroinflammation and neurodegeneration. The purpose of this research was to: (i) identify the purinergic (P2) receptors; (ii) determine if stimulation of the P2X7R mediates cell-death and (iii) determine if stimulation of the P2X7R mediates IL-1β and IL-10 cytokine expression in human Müller cells.

Methods: P2 receptor mRNA expression in MIO-M1 cells (immortalised human Müller cell line) and retinal tissue was evaluated using real time quantitative polymerase chain reaction (RT-qPCR). Purinergic (P2) evoked intracellular Ca2+ responses were measured using calcium microfluorimetry. Change in cell viability or cell cytotoxicity was determined with MTS and LDH assays respectively. IL-1R1, IL-α, IL-1β and IL-10 mRNA and protein were measured with RT-qPCR and enzyme-linked immunosorbent assay (ELISA) respectively. Inhibitors of IL-1β downstream signalling pathways were used to investigate IL-1β induced IL-10 release.

Results: MIO-M1 cells expressed mRNA for P2X4-7, P2Y1, P2Y2, P2Y4, and P2Y12-14 with enriched expression of P2X7R and P2Y4R compared to human retinal tissue. Intracellular Ca2+ responses provided functional evidence for P2X7R, P2Y1R, P2Y12R and P2Y14R. Prolonged stimulation of the P2X7R showed no significant change in viability or death of MIO-M1 cells. Stimulation of the P2X7R did not cause an increase in IL-1α, IL-1β or IL-10 mRNA expression. IL-1R1 was expressed in MIO-M1 cells. IL- 1β stimulation of IL-1R1 produced a 6.2-fold rise in IL-10 mRNA expression (p=0.03) and 1.37-fold rise in intracellular protein (p<0.0001) at 24 hours.

Conclusions: Evidence demonstrated functioning P2X7R, P2Y1R, P2Y14R and suggested functioning P2Y4R, P2Y12R in human Müller cells. Other groups established high concentrations of ATP stimulate P2X7R mediated IL-1β release from microglial cells. Here it is suggested retinal IL-1β activates IL-1R1 in Müller cells causing IL-10 protein production. Thereby, high concentrations of ATP in the human retina may cause activation of an IL-1β-IL-10 axis mediated by the Müller cell. There was no evidence that the P2X7R in the human Müller cell contributed to the proposed IL-1β- 1L-10 axis.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Chris White
Date Deposited:	21 Jun 2023 12:17
Last Modified:	21 Jun 2023 12:17
URI:	https://ueaeprints.uea.ac.uk/id/eprint/92457
DOI:

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