The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells

Abstract

Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1β secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca2+-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space.

Inflammasome activation by danger signals: extracellular ATP and pH

“…As for the ATP/P2X7R pathway, it has also been reported that ATP induced a reduction in intracellular pH in rat pancreatic ducts via P2X7R activation [22]. We further observed that the activation of the P2X7R by ATP induced an increase in lysosomal pH in microglial cells [23], which might have caused a reduction in cytosolic pH by impairing the intracellular proton balance. These studies imply that P2X7R-mediated intracellular acidification is implicated in the ATP-induced activation of inflammasomes (Fig.…”

Purinergic receptor X7 is a key modulator of metabolic oxidative stress-mediated autophagy and inflammation in experimental nonalcoholic steatohepatitis

“…This may be due to a different mechanism of a dysfunctional lysosomal function compared with our model that primarily evidenced LC3B depletion. Recent studies also link P2X7 receptor to a dysfunctional lysosome and autophagy protein LC3B (25,33). In both cases, the fate of the cell and its link to immune activation remain a dysregulated lysosomal compartment and are regulated by ATP-binding P2X7 receptors in the latter study (25).…”

“…Metabolic oxidative stress also, mainly characterized by oxidatively modified proteins, has been known to induce chaperone-mediated autophagy, increased substrate translocation by Hsc 70 toward the lysosomal membrane, and increased LAMP2A levels (17). Furthermore, P2X7 receptors have been associated with autophagy, disruption of normal lysosomal functions, and release of autophagolysosomes to the extracellular matrix in the microglial cells, causing an increase in inflammation (33). Our present study shows that there was a significant downregulation of both early and late autophagy proteins, including LAMP2A in P2X7 receptor gene-deleted mice.…”

“…3, A and B, and 4E contrasting result might point to a translational level regulation and can be explored further. These events can be crucial mediators of chaperone-mediated autophagy in the hepatic lobe, thus increasing inflammation, in a manner perhaps similar to P2X7 receptor-mediated release of autophagolysosomes/ phagolysosomes into the extracellular matrix, causing increase in inflammation (33). The above mechanism of release of phagolysosomes to the extracellular matrix might be speculative for NASH at this point, but this study certainly proves the dependence of depleted LC3B and increased levels of LAMP2A and Hsc 70 on P2X7 receptor in both models of experimental NASH.…”

Physiology of Microglia

“…Prominent microglial cell death was also observed in organotypic rat hippocampal slices treated with a combination of LPS and ATP (1 mM) or BzATP (100 M); this cell death was prevented by pharmacological blockade of P2X 7 receptors (49). The P2X 7 receptors are also involved in regulation of microglial autophagy through the release of autolysosomes (893,895).…”