Activated microglia are a feature of Alzheimer and other neurodegenerative diseases, but exactly what rouses these immune cells of the brain out of quiescence is unclear. Two recent publications point a finger at the P2X7 receptor, one of a family of purinergic receptors that have been linked to immune responses. In the March 25 Journal of Neuroscience, researchers led by David Williams at the University of Melbourne, Australia, report that upregulation of the P2X7 receptor (P2X7R) is enough to drive activation of microglia, and they pin this specifically to a pore-forming action of the receptor. In the March Journal of Immunology online, researchers led by Francesco Di Virgilio at the University of Ferrara, Italy, strengthen a link between P2X7R and AD. They report that amyloid-β-induced activation of microglia and their release of the proinflammatory cytokine interleukin 1β (IL-1β) require the purinergic receptor. Together, the two papers suggest that the P2X7R is a central player in microglial activation in AD and other neurodegenerative diseases.

P2X7 is one of seven purinergic receptors activated by ATP. The receptor has both ion channel and pore-forming activity, though how these two are related is not clear, Williams told ARF. It was also unknown whether upregulation of P2X7R drives or is itself driven by microglial activation. To address this, first author Matsura Monif and colleagues overexpressed the receptor in glia in rat primary hippocampal cultures (neurons and glia). “From that we showed that it was the P2X7 receptor that was driving activation of microglia, and their proliferation,” said Monif. Morphologically (activated microglia are large and have multiple lamellipodia) and immunohistochemically (they express CD68 and isolectin GS-IB4), cells overexpressing P2X7R appeared activated, and the P2X7R antagonist oxidized ATP (oxATP) prevented this.

To tease out whether the channel or the pore property of the receptor activated the cells, the researchers used a mutant receptor, G345Y, which they had previously found to retain ion channel activity but not form pores. “There are no pharmacological agents that can specifically inhibit the channel or specifically inhibit the pore,” explained Monif. When overexpressed in isolated hippocampal microglia, this receptor activated only about one-third as many cells as did the wild-type receptor, and the activated cells proliferated much less.

This work may represent a shift in how researchers view the P2X7R pore, which can open cells up to very large molecules. “Since its discovery, P2X7 was renowned for its ability to kill cells and in many papers has been described as cytolytic receptor, or a pro-apoptotic receptor. So it was surprising to find that it’s the pore component that is trophic for microglia,” said Monif. Now, this work suggests that the pore has a more subtle, regulatory function. Monif noted that one reason why P2X7 got its deadly reputation is that many experiments are done in exogenous settings, where the receptor is stimulated to its maximum and homeostasis and intracellular metabolites are ultimately lost. “Irrespective of how the pore was formed, if any cell is stimulated to such an extent, it would probably die,” she said.

The P2X7 receptor may not be apoptotic to microglia, but it may be bad news for other cells. “In microglia, P2X7 per se is trophic, but it is possible that those microglia, by releasing cytokines, chemokines, and reactive oxygen species, could…lead to neurodegeneration,” said Monif. In fact, there is some evidence that P2X7R may be linked to neurodegenerative disease. It is upregulated in multiple sclerosis (see Benveniste, 1997), AD, Parkinson disease, and more recently has been linked to epilepsy (see Avignone et al., 2008). In fact, James McLarnon and colleagues at the University of British Columbia reported that upregulated P2X7R is associated with amyloid plaques in a small number of AD cases and that amyloid-β causes increased expression of P2X7R activity in fetal human microglia, as measured by increased calcium influx (see McLarnon et al., 2006). Now, the work from Di Virgilio and colleagues supports the link between Aβ and the purinergic receptor.

First author Jana Sanz and colleagues found that commercially available Aβ (Aβ25-35 or Aβ1-42) induced Ca2+ uptake and release of ATP and lactate dehydrogenase from N13 microglial cell lines, but not from N13 cells devoid of the P2X7 receptor. Primary glial cultures from wild-type and P2X7R-/- mice yielded similar results.

Given the ability of P2X7R to form pores, the researchers looked to see if Aβ induced microglia to take up YO-PRO, a macromolecular fluorescent marker that can detect apoptotic cells. When incubated with Aβ25-35, wild-type but not P2X7R-negative microglia took up YO-PRO; this could be blocked by the receptor antagonist oxATP or by apyrase, an enzyme that metabolizes the purinergic receptor agonist ATP. The scientists did not report that this is necessarily detrimental to the microglia; in fact, they found that Aβ triggers release of IL-1β from microglial cells lines in a P2X7R-dependent manner, and that injection of Aβ into the hippocampus of wild-type, but not P2X7R knockout mice, caused a massive accumulation of IL-1β in the brain. “Though they haven’t shown whether it is the channel or the pore that is responsible for these effects, the article is basically in line with what we found,” said Monif.

Williams and colleagues plan to reproduce their findings in organotypic hippocampal slices. Interestingly, P2X7 antagonists improve recovery after spinal cord injury in rats (see Wang et al., 2004), but whether they might have therapeutic potential for AD remains to be seen. One problem right now is that no agents exist that can selectively inhibit the P2X7R pore. “It would be very interesting to develop a pharmacological agent that is capable of doing that, because then it might be possible to reduce the proliferation and activation of microglia and hence reduce the degree of neuroinflammation and degeneration,” suggested Monif.—Tom Fagan

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  1. Monif et al. have published an excellent paper. It provides independent confirmation of our earlier finding that the P2X7 receptor, far from being a mere "death receptor," is on the contrary a growth-promoting receptor (see Baricordi et al., 1999). We strengthened this finding in subsequent papers (Adinolfi et al., 2002; Adinolfi et al., 2005; Raffaghello et al., 2006). We also showed, in collaboration with Claudia Verderio and Maria Pia Abbracchio, that the P2X7 receptor had a crucial role in sustaining growth of microglia and discussed implications for neurodegenerative diseases (Bianco et al., 2006). In two recent papers we have further analyzed the molecular basis of the trophic effect of P2X7 (Adinolfi et al., 2009), and discussed the implications in cancer (Di Virgilio et al., 2009).

    See also:

    Di Virgilio F, Ferrari D, and Adinolfi E. (2009) P2X7: a growth-promoting receptor. Purinergic Signal in press.

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  2. Excellent work by Williams and DeVirgilio. But what is the stimulant to the P2X7 receptor that stirs the immune cells in glia to activate from quiescence in AD?

  3. Monif et al. have published an excellent paper. It provides independent confirmation of our earlier finding that the P2X7 receptor, far from being a mere "death receptor," is on the contrary a growth-promoting receptor (see Baricordi et al., 1999). We strengthened this finding in subsequent papers (Adinolfi et al., 2002; Adinolfi et al., 2005; Raffaghello et al., 2006). We also showed, in collaboration with Claudia Verderio and Maria Pia Abbracchio, that the P2X7 receptor had a crucial role in sustaining growth of microglia and discussed implications for neurodegenerative diseases (Bianco et al., 2006). In two recent papers we have further analyzed the molecular basis of the trophic effect of P2X7 (Adinolfi et al., 2009), and discussed the implications in cancer (Di Virgilio et al., 2009).

    View all comments by Francesco Di Virgilio

References

Paper Citations

  1. . Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis. J Mol Med (Berl). 1997 Mar;75(3):165-73. PubMed.
  2. . Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic signaling. J Neurosci. 2008 Sep 10;28(37):9133-44. PubMed.
  3. . Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus. J Neuropathol Exp Neurol. 2006 Nov;65(11):1090-7. PubMed.
  4. . P2X7 receptor inhibition improves recovery after spinal cord injury. Nat Med. 2004 Aug;10(8):821-7. PubMed.

Further Reading

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Primary Papers

  1. . The P2X7 receptor drives microglial activation and proliferation: a trophic role for P2X7R pore. J Neurosci. 2009 Mar 25;29(12):3781-91. PubMed.
  2. . Activation of microglia by amyloid {beta} requires P2X7 receptor expression. J Immunol. 2009 Apr 1;182(7):4378-85. PubMed.