The P2X7 Receptor in Microglial Cells Modulates the Endolysosomal Axis, Autophagy, and Phagocytosis

Abstract

Microglial cells regulate neural homeostasis by coordinating both immune responses and clearance of debris, and the P2X₇ receptor for extracellular ATP plays a central role in both functions. The P2X₇ 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 P2X₇ 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. P2X₇ 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 P2X₇ 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 P2X₇ receptor stimulation is balanced between the autophagic reduction of inflammasome components and their increase following P2X₇-mediated priming. In summary, the P2X₇ 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 P2X₇ receptor alters phagocytosis, lysosomal health, inflammation, and autophagy can lead to therapies that balance the inflammatory and clearance roles of microglial cells.

Keywords: NLRP3; P2X7; autophagy; cathepsin B; lysosomes; microglia; neuroinflammation; phagocytosis.

Figure 1

P2X₇ receptor modulation of clearance pathways in microglial cells. (A) Under control conditions when extracellular adenosine triphosphate (ATP) levels (ATP_o) are too low to activate the P2X₇ receptor, it acts as a scavenger receptor to aid in the phagocytosis of extracellular waste. The luminal pH of lysosomes in microglial cells is sufficiently acidic (green) to enable efficient degradative enzyme activity. (B) Following transient stimulation with high concentrations of ATP_o, the P2X₇ receptor no longer acts as a scavenger receptor. Autophagy is stimulated, shown by a transient rise in levels of autophagosomal lipid marker microtubule-associated protein 1A/1B-light chain 3-II (LC3-II). Material is degraded and microglia express genes and proteins associated with a mixed M1/M2 activation state. (C) A more prolonged stimulation of the P2X₇ receptor leads to an elevated lysosome pH (red), with autophagosomal contents being delivered extracellularly instead of to the lysosome. Markers of accumulation, including LC3-II (green) and sequestosome-1 (SQSTM1/p62; blue) are elevated. Microglia express genes and proteins associated with an M1 activation state.

Figure 2

Inflammasome activation by the P2X₇ receptor and the balance between priming and autophagic degradation of inflammasome components. Stimulation of the P2X₇ receptor triggers the assembly and activation (step #2) of the NLRP3 inflammasome following efflux of K⁺ through the open channel (blue arrow), eventually leading to cleavage of pro-IL-1β (red star) into the mature form and release through the gasdermin D pore (blue). The P2X₇ receptor can also activate the NLRP3 inflammasome and subsequent IL-1β release by raising cytoplasmic cathepsin B (CatB), following its initiation of lysosomal leakage through an unknown mechanism (purple arrows). P2X₇ receptor stimulation can prime inflammasome components via NF-κB (step #1, red arrow) and increase available inflammasome components. Component levels can also be decreased following autophagic degradation (green arrow), limiting the inflammatory potential of P2X₇ receptor stimulation. The balance between priming and autophagic degradation will influence the inflammatory impact of P2X₇ receptor stimulation.

Figure 3

ATP-dependent IL-1β release from primary microglial cells involves cathepsin B. (A) Purity of primary mouse brain microglia cultures shown with high staining for marker Iba1 (red) and low staining for astrocytic marker GFAP (green). (B) Primary microglial cultures increased expression of Nos2 with 4 h exposure of LPS but not IL-4, as expected for microglial cells polarized into the M1-state (****p < 0.001). Exposure to 1 mM ATP for 4 h increased expression of Nos2 (****p < 0.001) and Tnfa (**p = 0.0042) and decreased expression of Chil3 (*p = 0.014) as compared to control (Ctrl) using quantitative PCR. (C) Evidence of functional P2X₇ receptors in primary murine microglial cells indicated by the robust, reversible and repeatable elevation of cytoplasmic calcium when briefly exposed to 100 μM BzATP (line); cells were loaded with Ca²⁺ indicator fura-2. (D) Release of cytokine IL-1β triggered by exposure to 1 mM ATP for 1 h was reduced by cathepsin B inhibitor CA-074 (10 μM). Microglial cells were primed with 500 ng/ml LPS for 4 h and IL-1β was measured with an ELISA. Approaches as described previously (Lim et al., ; Lu et al., ; Shao et al., 2020). All work was approved by the University of Pennsylvania IACUC.