The P2X7 receptor drives microglial activation and proliferation: a trophic role for P2X7R pore

Abstract

Microglial activation is an integral part of neuroinflammation associated with many neurodegenerative conditions. Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X(7) receptor (P2X(7)R) expression in the neuroinflammatory foci where activated microglia are a coexisting feature. Whether P2X(7)R overexpression is driving microglial activation or, conversely, P2X(7)R overexpression is a consequence of microglial activation is not known. We report that overexpression alone of a purinergic P2X(7)R, in the absence of pathological insults, is sufficient to drive the activation and proliferation of microglia in rat primary hippocampal cultures. The trophic responses observed in microglia were found to be P2X(7)R specific as the P2X(7)R antagonist, oxidized ATP (oxATP), was effective in markedly attenuating microgliosis. oxATP treatment of primary hippocampal cultures expressing exogenous P2X(7)Rs resulted in a significant decrease in the number of activated microglia. P2X(7)R is unusual in exhibiting two conductance states, a cation channel and a plasma membrane pore, and there are no pharmacological agents capable of cleanly discriminating between these two states. We used a point mutant of P2X(7)R (P2X7RG345Y) with intact channel function but ablated pore-forming capacity to establish that the trophic effects of increased P2X(7)R expression are exclusively mediated by the pore conductance. Collectively, and contrary to previous reports describing P2X(7)R as a "death receptor," we provide evidence for a novel trophic role for P2X(7)R pore in microglia.

Figure 1.

Comparative morphology of glia expressing P2X₇R-EGFP and EGFP. A, B, Individual glia from hippocampal neuronal–glia mixed cultures expressing P2X₇R-EGFP. Cells have the characteristic morphology of activated microglia, with large soma and lamellipodia (arrowheads). Arrow on image B highlights P2X₇R-EGFP hotspots from which lamellipodia appear to originate. C, D, Representative fields of glia from hippocampal mixed cultures expressing P2X₇R-EGFP (C) or EGFP (D). P2X₇R-EGFP-expressing glia (C) often occurred in discrete “islands.” Inset in image D (scale bar, 10 μm) shows ramified morphology of microglia expressing EGFP. E, Individual microglia expressing P2X₇R-EGFP in microglia-enriched culture displayed similar morphology to glia from the neuronal–glia mixed cultures. Arrow highlights lamellipodia. F, Microglia expressing EGFP (enriched culture) displayed ramified morphology with 6–7 primary processes and small cell body. Images are representative of at least 20 fields, each from separate experiments. Scale bars, 20 μm.

Figure 2.

Imunohistochemical confirmation of morphological microglial identification. A, Fluorescence in glia expressing P2X₇R-EGFP is not colocalized with that of astrocytes stained with astrocytic marker GFAP. Green, P2X₇R-EGFP; red, GFAP. B, P2X₇R-EGFP fluorescence colocalizes with that of the microglial marker isolectin GS-IB₄. Green, P2X₇R-EGFP; red, isolectin GS-IB₄. Scale bars, 20 μm. C, The majority of glia expressing P2X₇R-EGFP in mixed cultures, and morphologically characterized to be activated, were isolectin GS-IB₄ positive (90%; n = 192) and GFAP negative (94%; n = 176; χ² test, p < 0.001). D, P2X₇R overexpression in primary hippocampal cultures drives microglial activation. There was a significantly larger number of activated microglia in mixed cultures transfected with P2X₇R-EGFP than with EGFP alone. Cell numbers were determined from 291 and 249 randomly selected fields (x, y dimensions: 230 × 230 μm), respectively, from at least five independent experiments, 72 h post-transfection. Data are mean ± SEM. ***p < 0.001 (Student's t test).

Figure 3.

Microglial activation was attenuated with oxATP. A, The number of P2X₇R-EGFP-expressing activated microglia was reduced at 72 h post-transfection in mixed cultures treated with oxATP (250 μm, 3 h) at 21 h post-transfection. A total of 100 and 99 randomly selected fields (from multiple experiments), with and without oxATP pretreatment, respectively, were examined and the number of activated microglia was quantified. Data are mean ± SEM. **p < 0.01 (Student's t test). B, Process retraction and microglial activation viewed dynamically with time-lapse microscopy. After BzATP stimulation, process retraction was significantly faster (μm/s) for microglia expressing the wild-type (wt) construct P2X₇R-EGFP (n = 5) than for those expressing the wt construct but pretreated with oxATP (n = 5) or expressing EGFP alone (n = 6). C, Images show a P2X₇R-EGFP-expressing microglia (enriched culture) 72 h post-transfection. Application of 100 μm BzATP caused the cell to retract its primary process (arrow), assuming a more activated phenotype. D, EGFP-expressing microglia (enriched culture) 72 h post-transfection. Application of 100 μm BzATP caused few morphological changes (arrows). Each panel is representative of at least five independent experiments. Time from the onset of experiment is indicated on the top right corner of each image. Scale bar, 20 μm.

Figure 4.

P2X7RG345-EGFP, a receptor with unaltered channel, but abolished pore function. A, Microglia from mixed cultures expressing P2X7RG345Y-EGFP had a nonactivated morphology. Scale bar, 20 μm. B, Representative graphs showing Ca²⁺ channel characteristics of P2X₇R-EGFP (wt; top) and P2X7RG345Y-EGFP (mutant; bottom). Microglia expressing the exogenous constructs were loaded with fluo-4 and treated with either BzATP (100 μm) or ATP (1 mm), with or without pretreatment with oxATP (500 μm, 3 h before experiment). Ca²⁺ signals were sampled every 15 s for 25 min. Data are absolute fluorescence intensity as a function of time (in minutes). Arrows indicate time of application of 1 mm ATP or 100 μm BzATP. Stars (--★--) indicate the nominal region of pore conductance (see Materials and Methods) evident for the wt construct but absent for response of mutant P2X₇R. C, Collated RFI data following BzATP (100 μm) application in multiple experiments for wild-type (wt) (n = 76) and mutant (n = 42) expressing microglia. Data are represented as mean ± SEM (p > 0.05, no significant difference; Student's t test). D, Collated RFI data, comparing the channel property of wt (n = 17) and mutant (n = 13) receptors following ATP (1 mm) application (p > 0.05, no significant difference; Student's t test). The ATP-induced Ca²⁺ channel response of both mutant (n = 11) and wt (n = 11) was significantly reduced (**p < 0.01, Student's t test) when cells were pretreated with oxATP (500 μm, 3 h before experiment). Data are mean ± SEM. E, An assay for pore formation using ethidium⁺ uptake into activated microglia expressing the wild-type receptor, P2X₇R-EGFP (n = 5 cells), or the mutant receptor, P2X7RG345Y-EGFP (n = 7 cells). The increase in ethidium⁺ fluorescence intensity was measured for 80 min after application of 12.5 μm ethidium⁺ bromide and 100 μm BzATP simultaneously (arrow indicates time of application). F, Representative images of nuclear ethidium⁺ incorporation by microglia expressing P2X₇R-EGFP (wt; top) or P2X7RG345Y-EGFP (mutant; bottom) after application of 25 μm ethidium⁺ bromide and 1 mm ATP. Graph shows collated data (n = 3) of nuclear ethidium⁺ intensity after exposure (49 min) to 25 μm ethidium bromide and 1 mm ATP. Data are mean ± SEM, *p <0.05, Student's t test.

Figure 5.

Microglia expressing wild-type receptor release more TNF-α and show a larger degree of intrinsic pore capacity than microglia expressing mutant receptors. A, Microglia expressing P2X₇R-EGFP (n = 99 cells examined from multiple experiments) and P2X7RG345Y-GFP (n = 95), at 72 h after transfection, or left untransfected (n = 71) were exposed to 12.5 μm ethidium bromide for 30 min, to get an indication of the degree of P2X₇R pores inherent in the culture environment. Ethidium⁺ intensity in the nucleus of activated microglia was measured as an indicator pore capacity in these cells. Results are shown as mean ± SEM. ***p < 0.001 (one-way ANOVA followed by Tukey's HSD post hoc test). B, A representative fluorescent image of a field of glia expressing P2X₇R-EGFP and stained with ethidium bromide. Ethidium⁺ intensity in the nucleus (arrowheads) was quantified with a confocal fluorescence microscope. Scale bar, 10 μm. C, An ELISA for TNF-α showing significantly higher levels of the proinflammatory cytokine release in microglia-enriched cultures expressing P2X₇R-EGFP (n = 8 independent experiments, each being measured in duplicate) than in those expressing P2X7RG345Y-EGFP (n = 8) or left untransfected (n = 6). Results show mean ± SEM. *p < 0.05 (Student's t test).

Figure 6.

LPS-activated untransfected microglia show an enhanced level of P2X₇R pore activity. A, LPS-stimulated untransfected microglia identified with isolectin GS IB₄ (red) show nuclear incorporation of YOPRO1 (green). Scale bar, 10 μm. B, Untransfected microglia exposed to LPS (200 ng/ml, n = 69) showed an enhanced level of P2X₇R pore activity as measured by YOPRO1 nuclear intensity, compared with cultures not treated with LPS (n = 65) or those treated with LPS (200 ng/ml) but subsequently exposed to oxATP (250 μm; n = 55) before YOPRO1 staining. Results are shown as mean ± SEM of YOPRO1 nuclear intensity. **p < 0.01 (one-way ANOVA followed by Tukey's HSD post hoc test).