P2RX7 plays a critical role in extracellular vesicle-mediated secretion of pathogenic molecules from microglia and astrocytes

Abstract

Extracellular vesicle (EV) secretion is mediated by purinergic receptor P2X₇ (P2RX7), an ATP-gated cation channel highly expressed in microglia. We have previously shown that administration of GSK1482160, a P2RX7 selective inhibitor, suppresses EV secretion from murine microglia and prevents tauopathy development, leading to the recovery of the hippocampal function in PS19 mice, expressing P301S tau mutant. It is yet unknown, however, whether the effect of GSK1482160 on EV secretion from glial cells is specifically regulated through P2RX7. Here we tested GSK1482160 on primary microglia and astrocytes isolated from C57BL/6 (WT) and P2rx7^-/- mice and evaluated their EV secretion and phagocytotic activity of aggregated human tau (hTau) under ATP stimulation. GSK1482160 treatment and deletion of P2rx7 significantly reduced secretion of small and large EVs in microglia and astrocytes in both ATP stimulated or unstimulated condition as determined by nanoparticle tracking analysis, CD9 ELISA and immunoblotting of Tsg101 and Flotilin 1 using isolated EVs. GSK1482160 treatment had no effect on EV secretion from P2rx7 ^-/- microglia while we observed significant reduction in the secretion of small EVs from P2rx7 ^-/- astrocytes, suggesting its specific targeting of P2RX7 in EV secretion except small EV secretion from astrocytes. Finally, deletion of P2rx7 suppressed IL-1β secretion and phagocytosed misfolded tau from both microglia and astrocytes. Together, these findings show that GSK1482160 suppresses EV secretion from microglia and astrocytes in P2RX7-dependment manner, and P2RX7 critically regulates secretion of IL-1β and misfolded hTau, demonstrating as the viable target of suppressing EV-mediated neuroinflammation and tau propagation.

Keywords: ATP; CD9; GSK1482160; P2RX7; Tsg101; astrocytes; extracellular vesicle; flotillin‐1; microglia.

FIGURE 1

Primary culture of microglia and EV isolation. (a) Schematic diagram of the experimental design. (b) Immunocytochemistry of primary cultured microglia by Iba1 (green) and GFAP (red). (c) Western blotting of the WT microglial cell lysate and isolated EVs from and P2rx7 ^–/– microglia for CD11B, TMEM119, CD81, Cytochrome C and GM130.

FIGURE 2

Quantification of microglia derived EVs by NTA, immunoblotting and ELISA. (a) NTA plot of mode size and concentration. (b and c) Quantification of EV particle numbers by NTA for small EVs (b) and large EVs (c). (d–f) Immunoblotting and semi quantification of Tsg101 and Flotilin 1 in microglia‐derived EVs. (g, h) CD9 ELISA (g) and IL‐1β ELISA of microglia‐derived EVs (h). (b–h) ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 between WT and KO within the same treatment condition, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 between control and treated condition of the same genotype group, ^┼ p < 0.05, ^┼┼ p < 0.01, ^┼┼┼ p < 0.001, ^┼┼┼┼ p < 0.0001 between ATP and ATP+GSK of the same genotype group as determined by two‐way ANOVA followed by Tukey's multiple comparison test (N = 3 per group). Data are representative of at least three independent experiments. Graphs indicate mean ± s.e.m. NTA, nanoparticle tracking analysis.

FIGURE 3

Primary culture of astrocytes and EV isolation. (a) Immunocytochemistry of primary cultured astrocytes by GFAP (green) and S100B (red). (b) Western blotting of the WT astrocyte cell lysate and isolated EVs from WT and P2rx7 ^–/– astrocytes for EAAT1, ITGA6, CD81, Cytochrome C and GM130.

FIGURE 4

Quantification of astrocyte derived EVs by NTA, immunoblotting and ELISA. (a) NTA plot of mode size and concentration (b–c) Quantification of EV particle numbers by NTA for small EVs (b) and large EVs (c). (d–f), Immunoblotting and semi quantification of Tsg101 and Flotilin 1 in astrocyte‐derived EVs. (g) CD9 ELISA and (h) IL‐1β ELISA of astrocyte‐derived EVs. (b–h) *p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 between WT and KO within the same treatment condition, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 between control and treated condition of the same genotype group, ^┼ p < 0.05, ^┼┼ p < 0.01, ^┼┼┼ p < 0.001, ^┼┼┼┼ p < 0.0001 between ATP and ATP+GSK of the same genotype group as determined by two‐way ANOVA followed by Tukey's multiple comparison test (N = 5 per group for b–c, N = 3 per group for e–h). Data are representative of at least three independent experiments. Graphs indicate mean ± s.e.m. NTA, nanoparticle tracking analysis.

FIGURE 5

Evaluation of soluble and EV‐containing hTau aggregates secreted from microglia and astrocytes. EVs were isolated from the culture media of WT or P2rx7 ^–/– microglia (a, b) and astrocytes (c, d) after phagocytosis of 10 µg/mL aggregated hTau, followed by ATP stimulation. (a, c) Soluble hTau ELISA, (b, d) hTau ELISA of isolated EVs after proteinase K (PK) treatment. p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 between WT and KO within the same treatment condition, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 between control and treated condition of the same genotype group as determined by two‐way ANOVA followed by Tukey's multiple comparison test (N = 3 per group). Data are representative of at least three independent experiments. Graphs indicate mean ± s.e.m.