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ATP-mediated cytotoxicity in microglial cells
Abstract:
Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop.
Insights
Microglial cells expressing the P2Z/P2X7 receptor are sensitive to ATP-mediated cell death. This study identifies the P2Z/P2X7 receptor
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglial cells, the immune cells of the central nervous system, express purinergic receptors for extracellular ATP.
- Both P2Y and P2X receptor subtypes are known to be present, with a particular focus on the pore-forming P2Z/P2X7 receptor.
Purpose of the Study:
- To identify and characterize the P2Z/P2X7 receptor in microglial cells (N9 and N13 cell lines).
- To investigate the role of the P2Z/P2X7 receptor in ATP-mediated cytotoxicity in microglial cells.
- To explore the mechanism of ATP-mediated cell death and ATP release in activated microglial cells.
Main Methods:
- Utilized a specific polyclonal antibody to identify the P2Z/P2X7 receptor in N9 and N13 microglial cell lines.
- Assessed ATP-mediated cytotoxicity in microglial cells with and without P2Z/P2X7 receptor expression.
- Transfected HEK293 cells with P2X7 receptor cDNA to confirm its role in cytotoxicity.
- Performed morphological and biochemical analyses to determine the mode of cell death.
Main Results:
- Confirmed the presence of the P2Z/P2X7 receptor in N9 and N13 microglial cells.
- Microglial cells expressing P2Z/P2X7 were highly sensitive to ATP-induced cell death, while resistant clones lacked the receptor.
- HEK293 cells transfected with P2X7 cDNA became susceptible to ATP cytotoxicity.
- ATP-dependent cell death in microglial cells was identified as apoptosis.
- Activated microglial cells released ATP via a non-lytic mechanism.
Conclusions:
- The P2Z/P2X7 receptor plays a critical role in mediating ATP-induced apoptosis in microglial cells.
- Microglial cells can release ATP through a non-lytic pathway upon activation by bacterial endotoxin.
- These findings suggest a purinergic autocrine/paracrine signaling loop in microglial activation and cell death.