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.

Rapid morphologic changes to microglial cells and upregulation of mixed microglial activation state markers induced by P2X7 receptor stimulation and increased intraocular pressure

“…S 3 a, b). Similar analysis confirmed identity of brain microglia as shown in more detail recently [ 51 ]. To support the microglial identity of the cells, the ability of lipopolysaccharides (LPS) and interleukin-4 (IL-4) to induce expression of activation state markers was determined using qPCR, as these two agonists are traditionally associated with classical and alternative activation states, respectively [ 28 , 52 ].…”

“…A 1-min addition of BzATP raised cytoplasmic Ca 2+ in the microglial cells (Fig. 3 b, c); the response was rapid, with most cells showing a response within 20 s. The response was also reversible upon wash-out of BzATP, and repeatable upon reapplication; these characteristics are consistent with an ionotropic channel with little inactivation like the P2X7 receptor as observed previously [ 53 , 54 ], and resembled the response reported recently in isolated brain microglia [ 51 ]. The P2X7 receptor-specific inhibitor A839977 [ 55 ] significantly reduced the Ca 2+ rise triggered by BzATP, with the robust response to BzATP after removal of the A839977 confirming this decrease (Fig.…”

“…S 3 c). These responses resemble those recently found for isolated brain microglia [ 51 ] and suggest cells cultured under these conditions responded as predicted for microglial cells.…”

Crosstalk Between Dysfunctional Mitochondria and Inflammation in Glaucomatous Neurodegeneration

“…While expanding evidence for both inflammation and mitochondrial dysfunction supports crosstalk, the degree of interaction may be influenced by several key factors, and suggests several key targets for intervention ( Table 1 ). For example, the microglial activation state is expected to have a considerable impact on waste accumulation and impaired mitophagy ( Campagno and Mitchell, 2021 ). Investigations into compartmentalized interaction between ASC, NLRP3 and oxidative stress in soma, axon, and synapse has particular relevance for glaucoma given the ganglion cell architecture.…”

“…Whether P2Y12 receptors play a direct role in microglial surveillance, or potentiate the activity of THIK-1 potassium channels as recently suggested ( Madry et al, 2018 ), P2Y12 receptor stimulation by ATP clearly contributes to surveillance. Stimulation of the P2X7 receptor has also been implicated in microglial phagocytosis and degradation ( Campagno and Mitchell, 2021 ), an effect which may have particular impact in aging cells. Inhibition of the P2X7 receptor was shown to reduce microglia activation in D2 mice ( Romano et al, 2020 ), suggesting a key role for the receptor in the inflammatory response in glaucoma.…”