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.
Highlights
P2X7 is ubiquitously expressed in myeloid cells and regulates the pathophysiology of inflammatory diseases
P2X7 induces Ca/calmodulin-dependent protein kinase kinase (CaMKK)- and reactive oxygen species (ROS)-dependent 5’ AMP-activated protein kinase (AMPK) activation in microglia and macrophages In order to link the energy-sensing kinase AMPK and P2X7 signaling, we first determined the effects of two P2X7 agonists, Adenosine 5′-triphosphate (ATP) and 2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (BzATP), on AMPK activation
Similar to BV-2 cells, BzATP activated AMPK in mouse primary microglial cells (Fig. 1b) and bone marrow-derived macrophages (BMDM) (Fig. 1c), and this effect of BzATP was abolished by P2X7 knockout (P2X7-/-) (Fig. 1b and c) and/or A438079 (Fig. 1b)
Summary
P2X7 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. Accumulated findings reveal that in response to brain injury or immunological stimuli, microglial cells become activated and migrate to the site of injury and secrete numerous chemokines, reactive oxygen species (ROS), and pro-inflammatory cytokines. Microglial activation-associated inflammatory response is believed to be the major cause to induce progressive neuronal death [2, 3]. Aberrant mitophagy has been linked to cell death and various neurodegenerative diseases [7]
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