AMPK-dependent and independent actions of P2X7 in regulation of mitochondrial and lysosomal functions in microglia

Abstract

BackgroundP2X7 is ubiquitously expressed in myeloid cells and regulates the pathophysiology of inflammatory diseases. Since mitochondrial function in microglia is highly associated with microglial functions in controlling neuronal plasticity and brain homeostasis, we interested to explore the roles of P2X7 in mitochondrial and lysosomal functions as well as mitophagy in microglia.MethodsP2X7−/− bone marrow-derived macrophages (BMDM), primary microglia and BV-2 immortalized microglial cells were used to detect the particular protein expression by immunoblotting. Mitochondrial reactive oxygen species (mitoROS), intracellular calcium, mitochondrial mass and lysosomal integrity were examined by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics and mitophagy after P2X7 activation.ResultsIn primary microglia, BV-2 microglial cells and BMDM, P2X7 agonist BzATP triggered AMPK activation and LC3II accumulation through reactive oxygen species (ROS) and CaMKKII pathways, and these effects were abolished by P2X7 antagonist A438079 and P2X7 deficiency. Moreover, we detected the dramatic decreases of mitochondrial OCR and mass following P2X7 activation. AMPK inhibition by compound C or AMPK silencing reversed the P2X7 actions in reduction of mitochondrial mass, induction of mitochondrial fission and mitophagy, but not in uncoupling of mitochondrial respiration. Interestingly, we found that P2X7 activation induced nuclear translocation of TFEB via an AMPK-dependent pathway and led to lysosomal biogenesis. Mimicking the actions of BzATP, nigericin also induced ROS-dependent AMPK activation, mitophagy, mitochondrial fission and respiratory inhibition. Longer exposure of BzATP induced cell death, and this effect was accompanied by the lysosomal instability and was inhibited by autophagy and cathepsin B inhibitors.ConclusionAltogether ROS- and CaMKK-dependent AMPK activation is involved in P2X7-mediated mitophagy, mitochondrial dynamics and lysosomal biogenesis in microglial cells, which is followed by cytotoxicity partially resulting from mitophagy and cathepsin B activation.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0293-3) contains supplementary material, which is available to authorized users.

P2X7 Receptor is Involved in Mitochondrial Dysfunction Induced by Extracellular Alpha Synuclein in Neuroblastoma SH-SY5Y Cells

“…Previously, the involvement of P2X7R in modulation of AMPK activity and regulation of autophagic flux was highlighted. It was documented that AMPK is a key signalling modulator of P2X7R to induce mitophagy and mitochondrial fission in microglia [113] and that the deleterious effect of P2X7R predominantly include lysosomal impairment in microglial cells [113,114]. Conversely, in our study, we demonstrated that α-Syn-dependent stimulation of P2X7R in neuronal cells results in inhibition of AMPK activity, followed by the inhibition of Ulk-1, which might have the negative effect on autophagy initiation.…”

Targeting the Microglial NLRP3 Inflammasome and Its Role in Parkinson's Disease

“…Activation of the P2X7 receptor by its agonist, BzATP, led to ROS‐ and CaMKK‐dependent AMPK activation, disruption of mitochondrial dynamics, autophagy, and lysosomal biogenesis followed by partial cell death. The cytotoxicity was also a result of P2X7‐mediated mitophagy and cathepsin B activation in microglial cells . Moreover, the loss of activity of 2 mitophagy regulators, Parkin or PINK1, is directly linked with microglial NLRP3 activation in PD .…”

“…The cytotoxicity was also a result of P2X7-mediated mitophagy and cathepsin B activation in microglial cells. 74 Moreover, the loss of activity of 2 mitophagy regulators, Parkin or PINK1, is directly linked with microglial NLRP3 activation in PD. 75,76 In bone marrow-derived macrophage (BMDM), activation of the AIM2 and NLRP3 inflammasomes has been found to trigger caspase-1-dependent mitochondrial damage through attenuation of mitophagy.…”

P2X7R-mediated Autophagic Impairment Contributes to Central Sensitization in a chronic Migraine Model with Recurrent Nitroglycerin Stimulation in Mice

“…Numerous studies have indicated the involvement role of P2X7R in autophagy regulation [32,33]. Shortterm P2X7R activation has been shown to stimulate autophagic ux, while persistent P2X7R stimulation leads to autophagic dysfunction [36].…”

“…Autophagy is regulated by immune signals [31]. Numerous studies have indicated the critical role of P2 × 7R in autophagy regulation [32][33][34][35][36][37]. The P2 × 7R-mediated autophagy modulation has been con rmed to participate in the pathophysiological process of amyotrophic lateral sclerosis [36], Duchenne muscular dystrophy [37] and status epilepticus [35].…”