Retinal microglial cells increase expression and release of IL-1ß when exposed to ATP.

Cytokine IL-1ß is an early component of inflammatory cascades, with both priming and activation steps required before IL-1ß release. Here, the P2X7 receptor (P2X7R) for ATP was shown to both prime and release IL-1ß from retinal microglial cells. Isolated retinal microglial cells increased expression of Il1b when stimulated with endogenous receptor agonist extracellular ATP; ATP also rapidly downregulated expression of microglial markers Tmem119 and Cd206. Changes to all three genes were reduced by specific P2X7R antagonist A839977, implicating the P2X7R. Microglial cells expressed the P2X7R on ramifications and responded to receptor agonist BzATP with robust and rapid rises in intracellular Ca 2+ . BzATP increased expression of IL-1ß protein colocalizing with CX3CR1-GFP in retinal wholemounts consistent with microglial cells. ATP also triggered release of IL-1ß from isolated retinal microglia into the bath; release was inhibited by A839977 and induced by BzATP, supporting a role for the P2X7R in release as well as priming. The IL-1ß release triggered by ATP was substantially greater from microglial cells compared to astrocytes from the optic nerve head region. Il1b expression was increased by a transient rise in intraocular pressure and Il1b levels remained elevated 10 days after a single IOP elevation. In summary, this study suggests the P2X7 receptor can both prime IL-1ß levels in microglial cells and trigger its release. The P2Y12R was previously identified as a chemoattractant for retinal microglia, suggesting the recruitment of the cells towards the source of released extracellular ATP could position microglia for P2X7R receptor, enabling both priming and release of IL-1ß.

Rapid morphologic changes to microglial cells and upregulation of mixed microglial activation state markers induced by P2X7 receptor stimulation and increased intraocular pressure.

BACKGROUND: The identification of endogenous signals that lead to microglial activation is a key step in understanding neuroinflammatory cascades. As ATP release accompanies mechanical strain to neural tissue, and as the P2X7 receptor for ATP is expressed on microglial cells, we examined the morphological and molecular consequences of P2X7 receptor stimulation in vivo and in vitro and investigated the contribution of the P2X7 receptor in a model of increased intraocular pressure (IOP). METHODS: In vivo experiments involved intravitreal injections and both transient and sustained elevation of IOP. In vitro experiments were performed on isolated mouse retinal and brain microglial cells. Morphological changes were quantified in vivo using Sholl analysis. Expression of mRNA for M1- and M2-like genes was determined with qPCR. The luciferin/luciferase assay quantified retinal ATP release while fura-2 indicated cytoplasmic calcium. Microglial migration was monitored with a Boyden chamber. RESULTS: Sholl analysis of Iba1-stained cells showed retraction of microglial ramifications 1 day after injection of P2X7 receptor agonist BzATP into mouse retinae. Mean branch length of ramifications also decreased, while cell body size and expression of Nos2, Tnfa, Arg1, and Chil3 mRNA increased. BzATP induced similar morphological changes in ex vivo tissue isolated from Cx3CR1+/GFP mice, suggesting recruitment of external cells was unnecessary. Immunohistochemistry suggested primary microglial cultures expressed the P2X7 receptor, while functional expression was demonstrated with Ca2+ elevation by BzATP and block by specific antagonist A839977. BzATP induced process retraction and cell body enlargement within minutes in isolated microglial cells and increased Nos2 and Arg1. While ATP increased microglial migration, this required the P2Y12 receptor and not P2X7 receptor. Transient elevation of IOP led to microglial process retraction, cell body enlargement, and gene upregulation paralleling changes observed with BzATP injection, in addition to retinal ATP release. Pressure-dependent changes were reduced in P2X7-/- mice. Death of retinal ganglion cells accompanied increased IOP in C57Bl/6J, but not P2X7-/- mice, and neuronal loss showed some association with microglial activation. CONCLUSIONS: P2X7 receptor stimulation induced rapid morphological activation of microglial cells, including process retraction and cell body enlargement, and upregulation of markers linked to both M1- and M2-type activation. Parallel responses accompanied IOP elevation, suggesting ATP release and P2X7 receptor stimulation influence the early microglial response to increased pressure.

Oral Delivery of the P2Y12 Receptor Antagonist Ticagrelor Prevents Loss of Photoreceptors in an ABCA4-/- Mouse Model of Retinal Degeneration.

Purpose: Accumulation of lysosomal waste is linked to neurodegeneration in multiple diseases, and pharmacologic enhancement of lysosomal activity is hypothesized to reduce pathology. An excessive accumulation of lysosomal-associated lipofuscin waste and an elevated lysosomal pH occur in retinal pigment epithelial cells of the ABCA4-/- mouse model of Stargardt's retinal degeneration. As treatment with the P2Y12 receptor antagonist ticagrelor was previously shown to lower lysosomal pH and lipofuscin-like autofluorescence in these cells, we asked whether oral delivery of ticagrelor also prevented photoreceptor loss. Methods: Moderate light exposure was used to accelerate photoreceptor loss in albino ABCA4-/- mice as compared to BALB/c controls. Ticagrelor (0.1%-0.15%) was added to mouse chow for between 1 and 10 months. Photoreceptor function was determined with electroretinograms, while cell survival was determined using optical coherence tomography and histology. Results: Protection by ticagrelor was demonstrated functionally by using the electroretinogram, as ticagrelor-treated ABCA4-/- mice had increased a- and b-waves compared to untreated mice. Mice receiving ticagrelor treatment had a thicker outer nuclear layer, as measured with both optical coherence tomography and histologic sections. Ticagrelor decreased expression of LAMP1, implicating enhanced lysosomal function. No signs of retinal bleeding were observed after prolonged treatment with ticagrelor. Conclusions: Oral treatment with ticagrelor protected photoreceptors in the ABCA4-/- mouse, which is consistent with enhanced lysosomal function. As mouse ticagrelor exposure levels were clinically relevant, the drug may be of benefit in preventing the loss of photoreceptors in Stargardt's disease and other neurodegenerations associated with lysosomal dysfunction.