Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Claire Mitchell

Abstract

The P2X7 receptor (P2X7R) is an ionotropic receptor that responds to high concentrations of extracellular adenosine triphosphate (ATP) leading to NF-κB-mediated inflammatory responses. P2X7 receptors are important regulators of microglial cells, the innate immune effector cells of the central nervous system (CNS). P2X7R stimulation and microglial morphological and molecular changes have been implicated in loss of neurons accompanying elevation of intraocular pressure (IOP) and associated ATP release. However, the mechanisms linking these processes remain unclear. Recent developments implicating astrocyte inflammation as the intermediaries between microglia inflammation and neural loss further complicate the relationship. We sought to interrogate the consequences of P2X7R stimulation to microglia morphology, the molecular alterations to microglia and astrocytes, in the retina, and the P2X7R role in loss of neurons from transient elevation of IOP. We administered P2X7R agonist BzATP to mice retinae to examine microglia morphological differences and inflammatory gene expression differences. This work was supported with in vitro exposure of cultured microglia to P2X7R stimulation. Finally, we used a model of elevated IOP to determine the effects on microglia and astrocytes. We found that retinal exposure to BzATP led to process retraction and elevation of Iba1 in microglia, as well as elevation of both pro-inflammatory (Tnfa, Nos2) and anti-inflammatory (Arg1, Chil3) markers. Transient elevation of IOP induced similar microglial process retraction and retinal gene expression, and these morphological and molecular differences were dampened in retinae derived from P2X7-/- mice. BzATP-injected retinae upregulated genes associated with induction of astrocyte neurotoxic inflammation. Finally, loss of neural populations in retinae subjected to elevated IOP was reduced in retinae from P2X7-/- mice. Furthermore, as P2X7R affects multiple metabolic processes, we sought to review literature to examine the intersection between microglial P2X7R stimulation and microglia phagocytosis or autophagy. We outlined P2X7R-mediated reduction of phagocytosis, transient induction of autophagy, and disruption of microglia lysosomes with longer exposure, and demonstrated a role for lysosomal leakage in cytokine release. These studies highlight the diverse consequences of microglial P2X7R stimulation, and indicate that stimulation plays a key role in retinal degeneration accompanying elevated IOP.

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