Critical involvement of extracellular ATP acting on P2RX7 purinergic receptors in photoreceptor cell death

Abstract

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7(-/-) mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP.

Figure 1

P2RX7 agonists induced retinal cell death in primary retinal cell cultures. The viability of retinal cells was assessed by incubation with calcein AM or MitoTracker CMTMRos after 24 hours of culture with ATP or BzATP. Photoreceptors were labeled by immunocytochemistry for recoverin (in green). A: The frequency of calcein⁺ (top panels; in blue) or CMTMRos⁺ (bottom panels; in red) photoreceptors decreased after incubation with 200 μmol/L to 5 mmol/L ATP in a dose-dependent manner. B: BzATP also reduced calcein⁺ (top panels) or CMTMRos⁺ (bottom panels) photoreceptors in a dose-dependent fashion. These quantifications are shown in the right panels in A and B. *P < 0.05; **P < 0.01.

Figure 2

BBG attenuated photoreceptor death as a P2RX7 antagonist. A: The viability of photoreceptors after 24 hours of continuous incubation with 1 to 10 μmol/L BBG. B: Photoreceptor death (induced by 1 mmol/L BzATP) was partially reversed by 30 minutes of transient incubation (BBG) or 24 hours of continuous incubation (co-BBG) of 1 to 10 μmol/L BBG or 1 to 10 μmol/L KN-62. C: Photoreceptor viability was preserved in BzATP-treated (1 mmol/L, 24 hours) photoreceptors from P2RX7^−/− mice (KO) compared with wild-type (WT) controls. D and E: Neutralizing antibody against TNF-α (MP6-XT22) or CD95 ligand (MFL3) failed to prevent BzATP-induced photoreceptor death; n = 10 per group. Scale bar = 20 μm. **P < 0.01.

Figure 3

P2RX7 induced caspase-8 cleavage and calcium influx in vitro. A: Electron microscopy showed the relatively well-preserved organelle of photoreceptors in primary cultures without BzATP administration (left panel). Apoptotic cell death was observed with chromatin condensation (middle panel) after BzATP administration. Necrotic cell death could also be detected after BzATP (right panel). Scale bar = 2 μm. B: Quantified results of cleaved caspase-8⁺ or TUNEL⁺ cells after 1 mmol/L BzATP administration in the presence or absence of 10 μmol/L BBG in primary cultures; n = 10 per group. *P < 0.05. C: Representative images of immunocytochemistry for cleaved caspase-8 (left panels, cleaved caspase-8 in red; middle panels, TUNEL in green; right panes, merged with Hoechst 33342 in blue). Cytoplasmic caspase-8 cleavage was detected in TUNEL-positive cells after BzATP administration and decreased by BBG treatment. D: The fluorescent images of calcium indicator Fluo-4 in primary retinal cell cultures before and 20 minutes after BzATP administration. The phase contrast images are shown in the left panels. The BzATP-induced increase of Fluo-4 fluorescence (top panels) was attenuated by adding 1 mmol/L EGTA (middle panels) or 10 μmol/L BBG (bottom panels). Scale bar = 5 μm.

Figure 4

P2RX7 stimulation induced photoreceptor apoptosis/necrosis in vivo. A: Representative TUNEL (in green) and propidium iodide (in red) staining of mouse retina after 3 (top panel) and 14 (bottom panel) days of gas vitrectomy. B: Representative images of cleaved caspase-8 in the mouse retina at 24 hours after the intraocular injection of 20 mmol/L BzATP (top left panel, Hoechst 33342 in blue; top right panel, cleaved caspase-8 in red; bottom left panel, TUNEL in green; bottom right panel, merged). In wild-type mice, cleaved caspase-8-positive staining was observed in TUNEL-positive apoptotic cells in the inner nuclear layer (INL) and the outer nuclear layer (ONL) after the vitreous injection of BzATP (white arrows). GCL, ganglion cell layer; IPL, inner plexiform layer. C: The BzATP injections dose-dependently induced photoreceptor apoptosis in the ONL, as detected by TUNEL (left panels), and this effect was decreased by co-injection of 500 μmol/L BBG (middle panels). Only very few TUNEL-positive photoreceptors were detected on injection of BzATP into P2RX7^−/− mice (right panels). TUNEL stainings are shown in green and propidium iodide in red. The quantitations are shown in D; n = 6 per group. Scale bar = 20 μm. **P < 0.01. E: Transmission electron microscopy showed BzATP-treated photoreceptors with characteristics of apoptosis, cell shrinkage, and chromatin condensation (white arrow) in the ONL and degenerated pedicles and spherules in the outer plexiform layer (OPL; black arrows). Apoptotic photoreceptors were decreased, and rod spherules and cone pedicles were well preserved in mice receiving BzATP plus BBG. Scale bar = 2 μm.

Figure 5

Neuroprotective effects of BBG in primary retinal cell cultures. A and B: Representative images of immunofluorescence detection of AIF (AIF in red, TUNEL in green, Hoechst33342 in blue) and cleaved caspase-9 (cleaved caspase-9 in red, TUNEL in green, Hoechst33342 in blue) in control cultures and after starvation in the absence or presence of BBG. Scale bar = 5 μm. The quantitations are shown in C; n = 10 per group. *P < 0.05. D: The dose-dependent neuroprotective effect of BBG; n = 10 per group. **P < 0.01.

Figure 6

Nutrient starvation-induced ATP release from primary retinal cells in vitro. The ATP levels in primary culture were measured by luciferin–luciferase assay. Total cellular levels of ATP were measured after permeabilization of plasma membranes, whereas the inferior detection limit of ATP levels was determined by adding apyrase to the culture supernatant fluids. Starvation was induced as in Figure 4 in the presence or absence of BBG or the ecto-ATPase inhibitor, β, γ-methylene-ATP (β, γ-Me-ATP); n = 10 per group. *P < 0.05, **P < 0.01.

Figure 7

Time-lapse imaging of ATP release induced by starvation. For dynamic imaging of the ATP release, light production from the luciferin–luciferase reaction was captured by an electron multiplier charge-coupled camera for 3 hours. Representative images are shown (see Supplemental Videos S1-S3 at http://ajp.amjpathol.org for total time-lapse images). Circles in the middle and bottom panels indicate events of ATP release induced by starvation, and arrows indicate the same location during the observation periods. Scale bar = 10 μm.