Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7 Receptor Expression and Retinal Ganglion Cell Death

P2X7 receptor (P2X7R), an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC) apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I) agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis), as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL), which was reversed by coinjection of brilliant blue G (BBG), a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death.

ATP, released predominantly from glial cells, plays important roles in modulating a variety of physiological and pathological processes by activating Gq-coupled purinergic receptors [13][14][15][16][17][18][19][20]. P2X 7 receptor (P2X 7 R), one of purinergic (P2) receptors, is an ATP-sensitive ligand-gated cation channel. Activation of P2X 7 Rs leads to ion channel open, which is permeable for small cations (Na + , Ca 2+ , and K + ). Repeated or prolonged activation of P2X 7 Rs under some pathological conditions may result in the formation of a nonselective channel/pore, through which large molecules up to 600-800 Da can pass, thus ultimately leading to cell death [21][22][23]. It was reported that P2X 7 Rs were expressed in retinal ganglion cells (RGCs), and lots of evidence has demonstrated that activation of P2X 7 Rs may contribute to glaucomatous RGC death [24][25][26]. In addition, activation of P2X 7 Rs in cultured rat brain astrocytes enhanced metabotropic purinergic receptor P2Y 2 mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signaling pathways [27]. In the present study, we aimed to explore whether and how activated Müller cells may modulate P2X 7 R expression in RGCs through ATP release.
(NIH) guidelines for the Care and Use of Laboratory Animals and the guidelines of Nantong University on the ethical use of animals. For intravitreal injection experiments, Sprague-Dawley rats (100-300 g) were obtained from Nantong Laboratory Animal Company and maintained under conditions of a 12 h light/dark cycle. Postnatal 1 d Sprague-Dawley rats were used for primary retinal neuronal cell culture. During this study, all possible efforts were made to minimize the number of animals used and their suffering.

Intravitreal Injection.
Rats were anesthetized with an intramuscular injection of a mixture of ketamine (25 mg/kg) and xylazine (10 mg/kg). The pupil was dilated with tropicamide drops, and 10 M (S)-3,5-dihydroxyphenylglycine (DHPG), 10 M ATP, or 10 M BBG dispersed in 2 L of 0.9% saline was injected into the vitreous space through a postlimbus spot using Hamilton microinjector (Hamilton) under a stereoscopic microscope. The needle was inserted 2 mm behind the temporal limbus and directed to the optic nerve. Eyes that received only an injection of saline in the same manner served as a control group. The eyes with cataract, endophthalmia, or other damage after injection were excluded.

Primary Retinal Neuronal Cell
Culture. Primary retinal neuronal cell culture was prepared as described previously [28] with minor modification. Briefly, retinas of newborn Sprague-Dawley rats (1 d) were digested with trypsin (0.25% for 15 min at 37 ∘ C) and retinal neurons were dissociated mechanically by using a fire-polished Pasteur pipette. The cell suspension was plated onto 35 mm poly-D-lysine-coated dishes at a density of 1.2 × 10 6 and cultured in a neurobasal medium (Gibco BRL, Life Technologies, Rockville, MD, USA), supplemented with 2% B27 and 2 mM glutamine, in a humidified 5% CO 2 incubator at 37 ∘ C. RGCs were identified by using antibody against Brn3a, a RGC marker. Experiments were performed on the 8th day of neurons in culture.

Retrograde Labeling and Counting of RGCs.
To assess the effect of DHPG on RGCs in vivo, retrograde labeling of RGCs was performed. After anesthetization, the rats were placed in the stereotactic apparatus (Stoelting, Wood Dale, IL, USA) and the brain surface was exposed by perforating the parietal bone to facilitate dye injection. 2 L of 2% fluorogold (FG; Biotium, Hayward, CA, USA) was injected into both superior colliculi and dorsal lateral geniculate nuclei. After seven days, rats from different groups were sacrificed and eyeballs were enucleated and placed in 4% paraformaldehyde for 4 h. The whole retina was then carefully dissected, flattened, and mounted with the vitreous side up on slides. Photographs were captured using a fluorescent microscope (Leica, Germany) and FG-labeled RGCs were counted in a masked fashion by the same investigator using automated particle counting software in ImagePro Version 6.0 (Media Cybernetics, Bethesda, USA). The number of labeled cells in 12 photographs of each retina (three photographs per retinal quadrant) at 1/6, 3/6, and 5/6 of the retinal radius was summed together and expressed as mean RGC densities/mm 2 for each group.

Western Blot.
Western blot analysis was conducted as previously described with some modifications [3]. After washed with PBS solution, rat retinas or cells were lysed in lysis buffer (containing 1 M Tris-HCl at pH 7.5, 1% Triton X-100, 1% Nonidet p-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 g/mL leupeptin, 10 g/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined by the BCA method (Pierce). Protein samples (1.0 g/L, 15 L) were subjected to 10% SDS-PAGE using a Mini-Protean 3 electrophoresis system (Bio-Rad) and electrotransferred to polyvinylidene fluoride membranes using a Mini TransBlot electrophoretic transfer system (Bio-Rad). The membranes were blocked with 5% skimmed milk at room temperature for 1 h and then incubated with rabbit antibody against-P2X 7 R (1 : 1000, Abcam). The blots were washed with TBST and incubated with HRP-conjugated goat antimouse or anti-rabbit IgG (1 : 4000, Jackson ImmunoResearch Laboratories) for 1 h at room temperature and visualized with enhanced chemifluorescent reagent ECL (Thermo Scientific, Rockford, IL, USA) and exposed to X-ray film in the dark. The experiments were performed in triplicate, and the protein bands were quantitatively analyzed with Image J Analysis software.
2.6. Immunofluorescence Staining. Immunohistochemistry was performed following the procedure described in detail previously [3]. After rats were anesthetized and perfused with 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4), the left eyeballs were removed and postfixed in 4% paraformaldehyde for 2-4 h, followed by dehydration with graded sucrose solutions at 4 ∘ C (4 h in 20% and overnight in 30%). The retinas were vertically sectioned at 14 m thickness on a freezing microtome (Leica, Nussloch, Germany). The slices were collected and mounted on chrome-alum-gelatincoated slides. After washing with 0.01 M PBS (pH 7.4), the sections were blocked in 6% bovine serum albumin (Sigma, St. Louis, MO, USA) in PBS plus 0.1% Triton X-100 at room temperature for 2 h and then incubated with a rabbit antibody against-P2X7 receptor antibody (1 : 400) or mouse monoclonal against-GFAP (1 : 500, Sigma-Aldrich) antibody at 4 ∘ C for 48 h. Immunoreactive proteins were visualized by incubating with FITC-conjugated goat anti-mouse IgG (1 : 200 dilution; Jackson, Immunoresearch Laboratories, Wes Grove, PA, USA). The samples were mounted with antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and the immunofluorescence images were visualized with a Leica confocal laser scanning microscope (Leica, Germany).
For immunocytochemistry, cells were washed in PBS and fixed with 4% fresh PFA solution for 40 min at room temperature. Cells were rinsed in PBS, permeabilized with 0.1% Triton X-100, and incubated in PBS containing 1% BSA for 2 h to block the nonspecific binding sites. Triplicate wells were incubated with monoclonal antibodies against Brn3a (1 : 200, Sigma-Aldrich), or a rabbit antibody against-P2X 7 receptor antibody (1 : 400) at 4 ∘ C overnight. On the following day, the appropriate second antibodies and 4,6-diaminodiphenyl-2phenylindole (DAPI; Sigma-Aldrich) were added in a dark room and incubated for 2-3 h. After washing, preparations were mounted and detected by a fluorescent microscope (Leica, Germany).

Statistical Analysis.
All data were expressed as means ± SE. Differences among groups were analyzed by one-way ANOVA, followed by multiple comparison tests (LSD). All statistical analyses were carried out by the aid of SPSS 17.0 software package and significance level was set at < 0.05.

Results
Our previous study has demonstrated that DHPG, an mGluR I agonist, may induce Müller cell gliosis by inhibiting inward rectifying K + channels [3]. We examined whether Müller cell gliosis by activation of mGluR I may induce changes in P2X 7 R expression in RGCs by using immunohistochemistry and Western blotting. Firstly, we confirmed DHPG-induced Müller cell gliosis as our previous report [3]. Figure 1(a) shows that intravitreal injection of DHPG (10 M, 2 L) indeed induced an increase in GFAP expression in the retinal section obtained from 2 weeks after the injection (a2), as compared with that in the normal physiological solution-(NS-) injected retinal section (a1). We then examined changes in P2X 7 R protein expression after the DHPG injection. As shown in Figures 1(b)-1(d), in the NS-injected retinal section (Control, Ctr), P2X 7 R proteins were expressed in cells of the ganglion cell layer (GCL) and the inner nuclear layer (INL), as well as in the outer plexiform layer (OPL) ((b1) and (d1)). P2X 7 R expression showed a remarkable increase in retinal section obtained from the rat at 2 weeks after the DHPG injection, especially in the GCL (Figures 1(b)-1(d), (b2), and (d2)). Since mGluR I expresses extensively in retinal cells, the DHPG-induced upregulation of P2X 7 R expression in the cells of GCL may be mediated by bioactive substances released from activated Müller cells and/or by direct action of DHPG on the cells of GCL. ATP, a P2X 7 R ligand, is one of extracellular signaling molecules for glia-neuron crosstalk, which may be abundantly released from activated Müller cells. We examined whether ATP is involved in the DHPG effect on P2X 7 R expression by acting on P2X 7 Rs. Brilliant blue G (BBG, 10 M, 2 L), a specific P2X 7 R antagonist, was intravitreally coinjected with DHPG (10 M). Retinal slice was made 2 weeks after the injection for immunohistochemistry. As shown in Figures 1(b)-1(d), the DHPG-induced increase in P2X 7 R expression in the cells of GCL in retinal section was significantly reduced by BBG injection ((b3) and (d3)). Similar observations were obtained in other five eyes. Western blotting revealed that total P2X 7 R protein in the DHPG-injected retinas was profoundly increased to 185.0 ± 6.0% of the control, and the protein level was reduced to 121.0 ± 8.0% of the control by coinjection of BBG ( = 6, < 0.001) (Figures 1(e) and 1(f)). These results suggest that activated Müller cells induced by DHPG may release ATP, in turn acting on the cells of GCL to upregulate P2X 7 R expression.
We then tested whether ATP treatment may induce changes in P2X 7 R expression. ATP (10 M, 2 L) was intravitreally injected every 7 days, and retinal sections or whole retinal extracts were made at different times after the injection for immunohistochemistry and Western blotting, respectively. As shown in Figure 2(a), weak positive signals of P2X 7 R proteins were seen in the retinal section obtained from the NS-injected rat (control) ((a1) and (c1)), especially in the GCL and the INL. P2X 7 R expression was significantly increased in the sections obtained from ATP-injected rats started from 1 week ((a2) and (c2)) and through 6 weeks after the injection ((a3)-(a5) and (c3)-(c5)), as compared with the control (a1). Consistently, Western blot experiments showed that P2X 7 R protein levels were significantly increased after ATP treatment (Figure 2(d)). The average density of P2X 7 R proteins slightly increased (104.1 ± 18.0% of control, = 9, = 0.71) at 1 week after the ATP-injection, while it considerably increased at 2 weeks after the injection (122.7 ± 13.3% of the control, = 9, < 0.01) and through 6 weeks (128.2 ± 8.1% of the control, = 9, < 0.01 at 4 weeks; 156.8 ± 8.8% of the control, = 9, < 0.001 at 6 weeks) (Figure 2(e)). These results suggest that ATP indeed induces upregulation of P2X 7 R protein expression in the cells of GCL.

Discussion
In the present study, we found that retinal Müller cell activation induced by the mGluR I agonist DHPG upregulated P2X 7 R expression in the cells of GCL, which was mediated by ATP released from Müller cells, thus contributing to RGC death.
Our previous study has demonstrated that activation of mGluR I induced Müller cell gliosis by inhibiting Kir K + (a1) (a2) (a3) currents, especially Kir4.1 mediated currents, in a rat chronic ocular hypertension model [3]. The mGluR I agonist DHPG treatment of cultured Müller cells or intravitreal injection modulated Kir4.1 proteins and Kir4.1 mRNAs, leading to increase of GFAP expression [3,6], suggesting that DHPGinduced GFAP expression may be used as a Müller cell gliosis model. In the present study, we confirmed that DHPG injection indeed induced upregulation of GFAP expression in Müller cells. One of our major findings is that DHPG injection significantly upregulated P2X 7 R expression in the cells of GCL in addition to Müller cell activation. The DHPGinduced effect on P2X 7 R expression in the cells of GCL was mediated by ATP released by activated Müller cells. Even though previous study reported that activation of P2X 7 Rs in cultured astrocytes enhanced P2Y 2 mRNA expression [27], for our knowledge, the present study is the first report, showing the relationship between the activated Müller cells and P2X 7 R expression in RGCs, in which ATP plays a vital role. This is supported by the experimental evidence. Firstly, DHPG injection-induced upregulation of P2X 7 R expression in the cells of GCL was reversed by the P2X 7 R blocker BBG ( Figure 1). Secondly, intravitreal injection of ATP may directly induce upregulation of P2X 7 R expression in the cells of GCL ( Figure 2). Thirdly, ATP treatment of cultured retinal neurons resulted in upregulation of P2X 7 R expression in RGCs (Figure 3). It should be noted that RGCs also expressed mGluR I; therefore, we cannot exclude a possibility that DHPG may directly activate mGluR I in RGCs and then increase P2X 7 R expression in these cells. However, considering the facts that the DHPG-induced upregulation of P2X 7 R expression was reversed by coinjection of the P2X 7 R blocker BBG ( Figure 1) and DHPG-induced reduction in the number of RGCs was partially rescued by BBG (Figure 4), we speculate that the DHPG effects on P2X 7 R expression were mainly mediated by ATP that released from activated Müller cells. Indeed, we have observed that concentrations of extracellular ATP were increased in cultured Müller cells exposed to DHPG (unpublished data). Regarding the mechanisms underlying ATP-induced changes in P2X 7 R expression, one possibility is that under pathological conditions prolonged stimulation of P2X 7 Rs by ATP may increase intracellular Ca 2+ concentration and then trigger intracellular Ca 2+ -dependent signaling pathways, thus increasing P2X 7 R expression [27,30]. It is noteworthy that in the present study we did not identify the cell types in the GCL. There was evidence showing that P2X 7 Rs were also expressed in amacrine cells and glial cells [31][32][33][34][35][36]. Therefore, the increase in P2X 7 R protein expression in the retinal sections, as shown in Figures 1 and 2, may be in displaced amacrine cells, and/or in glial cells in the GCL. However, it should be noted that ATP-induced increase in P2X 7 R proteins was mainly in the GCL and inner nuclear layer (INL) (Figure 2), and ATP could induce an increase in P2X 7 R protein expression in cultured rat RGCs (Figure 3). We speculated that the increased expression of P2X 7 R proteins in the GCL indeed occurred in RGCs, even though we cannot exclude a possibility that these events maybe also occurred in displayed amacrine cells and glial cells.
RGC apoptotic death is a common feature in retinal neurodegenerative diseases, such as glaucoma. It is noteworthy that overactivation of P2X7Rs induced upregulation of P2X7R expression in the cells of GCL (Figure 2), in addition to directly leading to RGC apoptosis. In turn, upregulation of P2X 7 R expression aggravated RGC apoptosis. Therefore, the present study provides evidence that under pathological conditions ATP, released by activated Müller cells, may induce RGC apoptotic death through two pathways, directly acting on P2X 7 Rs and upregulating P2X 7 R expression.
Another important finding in the present study is that both DHPG and ATP intravitreal injection resulted in a reduction in the number of FG-positive RGCs, and the DHPG effect was partially rescued by coinjecting the P2X 7 R blocker BBG. These results provide direct evidence that activated Müller cells in some retinal diseases and injuries contribute to RGC death via elevated ATP/P2X 7 R activity [37][38][39]. On the other hand, our present results indicate that blocking Müller cell gliosis and inhibiting ATP/P2X 7 R activity may be potentially useful for the therapeutic management of RGC death in retinal diseases and injuries, such as glaucoma.