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This study asked whether the P2X7 receptor was necessary and sufficient to trigger astrocyte polarization into neuroinflammatory activation states. Intravitreal injection of agonist BzATP increased gene expression of pan-astrocyte activation markers Gfap , Steap4 , and Vim and A1-type astrocyte activation markers C3 , Serping1 , and H2T23 , but also the Cd14 and Ptx3 genes usually associated with the A2-type astrocyte activation state and Tnfa , IL1a , and C1qa , assumed to be upstream of astrocyte activation in microglia. Correlation analysis of gene expression suggested the P2X7 receptor induced a mixed A1/A2-astrocyte activation state, although A1-state genes like C3 increased the most. A similar pattern of mixed glial activation genes occurred one day after intraocular pressure (IOP) was elevated in wild-type mice, but not in P2X7 -/- mice, suggesting the P2X7 receptor is necessary for the glial activation that accompanies IOP elevation. In summary, this study suggests stimulation of the P2X7R is necessary and sufficient to trigger the astrocyte activation in the retina following IOP elevation, with a rise in markers for pan-, A1-, and A2-type astrocyte activation. The P2X7 receptor is expressed on microglia, optic nerve head astrocytes, and retinal ganglion cells (RGCs) in the retina, and can be stimulated by the mechanosensitive release of ATP that accompanies IOP elevation. Whether the P2X7 receptor connects this mechanosensitive ATP release to microglial and astrocyte polarization in glaucoma remains to be determined.

Cytokine IL-1β is an early component of inflammatory cascades, with both priming and activation steps required before IL-1β release. Here, the P2X7 receptor (P2X7R) for ATP was shown to both prime and release IL-1β 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-1β protein colocalizing with CX3CR1-GFP in retinal wholemounts consistent with microglial cells. ATP also triggered release of IL-1β 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-1β 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-1β 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-1β.

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 Ca 2+ 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., (© 2021. The Author(s).)

Microglial cells regulate neural homeostasis by coordinating both immune responses and clearance of debris, and the P2X 7 receptor for extracellular ATP plays a central role in both functions. The P2X 7 receptor is primarily known in microglial cells for its immune signaling and NLRP3 inflammasome activation. However, the receptor also affects the clearance of extracellular and intracellular debris through modifications of lysosomal function, phagocytosis, and autophagy. In the absence of an agonist, the P2X 7 receptor acts as a scavenger receptor to phagocytose material. Transient receptor stimulation induces autophagy and increases LC3-II levels, likely through calcium-dependent phosphorylation of AMPK, and activates microglia to an M1 or mixed M1/M2 state. We show an increased expression of Nos2 and Tnfa and a decreased expression of Chil3 ( YM1 ) from primary cultures of brain microglia exposed to high levels of ATP. Sustained stimulation can reduce lysosomal function in microglia by increasing lysosomal pH and slowing autophagosome-lysosome fusion. P2X 7 receptor stimulation can also cause lysosomal leakage, and the subsequent rise in cytoplasmic cathepsin B activates the NLRP3 inflammasome leading to caspase-1 cleavage and IL-1β maturation and release. Support for P2X 7 receptor activation of the inflammasome following lysosomal leakage comes from data on primary microglia showing IL-1β release following receptor stimulation is inhibited by cathepsin B blocker CA-074. This pathway bridges endolysosomal and inflammatory roles and may provide a key mechanism for the increased inflammation found in age-dependent neurodegenerations characterized by excessive lysosomal accumulations. Regardless of whether the inflammasome is activated via this lysosomal leakage or the better-known K + -efflux pathway, the inflammatory impact of P2X 7 receptor stimulation is balanced between the autophagic reduction of inflammasome components and their increase following P2X 7 -mediated priming. In summary, the P2X 7 receptor modulates clearance of extracellular debris by microglial cells and mediates lysosomal damage that can activate the NLRP3 inflammasome. A better understanding of how the P2X 7 receptor alters phagocytosis, lysosomal health, inflammation, and autophagy can lead to therapies that balance the inflammatory and clearance roles of microglial cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Campagno and Mitchell.)

Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active the NLRP3 inflammasome and release IL-1β, but stimulation also leads to release of other cytokines. As cytokine signaling by retinal pigmented epithelial (RPE) cells is implicated in retinal neurodegeneration, the role of P2X7R in release of cytokine IL-6 from RPE cells was investigated. P2X7R stimulation triggered IL-6 release from primary mouse RPE, human iPS-RPE and human ARPE-19 cells. IL-6 release was polarized, with predominant rise across apical membranes. IL-6 release was inhibited by P2X7R antagonists A438079, A839977, and AZ10606120, but not the NRTI lamivudine (3TC), P2X1R antagonist NF279, or P2Y1R antagonist MRS2179. P2X7R-mediated IL-6 release required extracellular Ca 2+ and was blocked by Ca 2+ chelator BAPTA. IL-6 release and Ca 2+ elevation occurred rapidly, consistent with vesicular IL-6 staining in unstimulated cells. P2X7R stimulation did not trigger IL-1β release in these unprimed cells. P2X7R-mediated IL-6 release was enhanced in RPE cells from the ABCA4 -/- mouse model of retinal degeneration. In summary, P2X7R stimulation triggers rapid Ca 2+ -dependent IL-6 release across the apical membrane of RPE cells.

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.

The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4 -/- mouse model of Stargardt's disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro , but identification of an appropriate receptor is crucial for manipulating this pathway in vivo . As the P2Y 12 receptor for ADP is coupled to the inhibitory G i protein, we asked whether blocking the P2Y 12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4 -/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4 -/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y 12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y 12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and "non-targeting" scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1 -/- cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca 2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.

The transient receptor potential cation channel mucolipin 1 (TRPML1) channel is a conduit for lysosomal calcium efflux, and channel activity may be affected by lysosomal contents. The lysosomes of retinal pigmented epithelial (RPE) cells are particularly susceptible to build-up of lysosomal waste products because they must degrade the outer segments phagocytosed daily from adjacent photoreceptors; incomplete degradation leads to accumulation of lipid waste in lysosomes. This study asks whether stimulation of TRPML1 can release lysosomal calcium in RPE cells and whether such release is affected by lysosomal accumulations. The TRPML agonist ML-SA1 raised cytoplasmic calcium levels in mouse RPE cells, hesRPE cells, and ARPE-19 cells; this increase was rapid, robust, reversible, and reproducible. The increase was not altered by extracellular calcium removal or by thapsigargin but was eliminated by lysosomal rupture with glycyl-l-phenylalanine-β-naphthylamide. Treatment with desipramine to inhibit acid sphingomyelinase or YM201636 to inhibit PIKfyve also reduced the cytoplasmic calcium increase triggered by ML-SA1, whereas RPE cells from TRPML1 -/- mice showed no response to ML-SA1. Cotreatment with chloroquine and U18666A induced formation of neutral, autofluorescent lipid in RPE lysosomes and decreased lysosomal Ca 2+ release. Lysosomal Ca 2+ release was also impaired in RPE cells from the ATP-binding cassette, subfamily A, member 4 -/- mouse model of Stargardt's retinal dystrophy. Neither TRPML1 mRNA nor total lysosomal calcium levels were altered in these models, suggesting a more direct effect on the channel. In summary, stimulation of TRPML1 elevates cytoplasmic calcium levels in RPE cells, but this response is reduced by lysosomal accumulation.-Gómez, N. M., Lu, W. Lim, J. C., Kiselyov, K., Campagno, K. E., Grishchuk, Y., Slaugenhaupt, S. A., Pfeffer, B., Fliesler, S. J., Mitchell, C. H. Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation.