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Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

Neher JJ, Neniskyte U, Hornik T, Brown GC - Glia (2014)

Bottom Line: We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors).In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days.Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

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Exogenous UDP induces rapid, microglia-dependent neuronal loss through P2Y6 receptor activation. A: Microglia in mixed glial cultures treated acutely with 100 µM UDP show enhanced phagocytosis of fluorescent microbeads (micrographs show phagocytosis of 5 µm large microbeads; arrows). B: In mixed neuronal/glial cultures, the P2Y6 agonist UDP (100 μM) induces neuronal loss within 6 h, and this neuronal loss is blocked by co-application of the general phagocytosis inhibitor Cytochalasin D (100 nM). This UDP-induced neuronal loss is greater at 24 h, but is neither enhanced by the application of a second dose of UDP after an additional 24 h nor does it progress significantly further at 72 h. The synthetic P2Y6 agonist, MRS2693 induces neuronal loss equivalent to UDP. Importantly, UDP-induced neuronal loss is prevented by microglial depletion (with l-leucine methyl ester) or application of the selective P2Y6 inhibitor MRS2578 (MRS, 1 μM). Note that neither UDP nor MRS2693 affected microglial proliferation in any of the conditions (not shown). Data are presented as means ± s.e.m. for at least three independent experiments; */**/***P < 0.05/0.01/0.001 versus control, ##/###P < 0.01/0.001 versus UDP.
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fig04: Exogenous UDP induces rapid, microglia-dependent neuronal loss through P2Y6 receptor activation. A: Microglia in mixed glial cultures treated acutely with 100 µM UDP show enhanced phagocytosis of fluorescent microbeads (micrographs show phagocytosis of 5 µm large microbeads; arrows). B: In mixed neuronal/glial cultures, the P2Y6 agonist UDP (100 μM) induces neuronal loss within 6 h, and this neuronal loss is blocked by co-application of the general phagocytosis inhibitor Cytochalasin D (100 nM). This UDP-induced neuronal loss is greater at 24 h, but is neither enhanced by the application of a second dose of UDP after an additional 24 h nor does it progress significantly further at 72 h. The synthetic P2Y6 agonist, MRS2693 induces neuronal loss equivalent to UDP. Importantly, UDP-induced neuronal loss is prevented by microglial depletion (with l-leucine methyl ester) or application of the selective P2Y6 inhibitor MRS2578 (MRS, 1 μM). Note that neither UDP nor MRS2693 affected microglial proliferation in any of the conditions (not shown). Data are presented as means ± s.e.m. for at least three independent experiments; */**/***P < 0.05/0.01/0.001 versus control, ##/###P < 0.01/0.001 versus UDP.

Mentions: The endogenous agonist for P2Y6 receptors is thought to be UDP (Chang et al., 1995). Exogenous UDP (100 μM) has previously been reported to promote microglial phagocytosis via P2Y6 activation (Koizumi et al., 2007) and we confirmed here that acute treatment of mixed glial cultures with 100 μM UDP strongly enhanced microglial phagocytic activity as measured by their uptake of microbeads (1 or 5 μm diameter and negatively charged, Fig. 4A).


Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

Neher JJ, Neniskyte U, Hornik T, Brown GC - Glia (2014)

Exogenous UDP induces rapid, microglia-dependent neuronal loss through P2Y6 receptor activation. A: Microglia in mixed glial cultures treated acutely with 100 µM UDP show enhanced phagocytosis of fluorescent microbeads (micrographs show phagocytosis of 5 µm large microbeads; arrows). B: In mixed neuronal/glial cultures, the P2Y6 agonist UDP (100 μM) induces neuronal loss within 6 h, and this neuronal loss is blocked by co-application of the general phagocytosis inhibitor Cytochalasin D (100 nM). This UDP-induced neuronal loss is greater at 24 h, but is neither enhanced by the application of a second dose of UDP after an additional 24 h nor does it progress significantly further at 72 h. The synthetic P2Y6 agonist, MRS2693 induces neuronal loss equivalent to UDP. Importantly, UDP-induced neuronal loss is prevented by microglial depletion (with l-leucine methyl ester) or application of the selective P2Y6 inhibitor MRS2578 (MRS, 1 μM). Note that neither UDP nor MRS2693 affected microglial proliferation in any of the conditions (not shown). Data are presented as means ± s.e.m. for at least three independent experiments; */**/***P < 0.05/0.01/0.001 versus control, ##/###P < 0.01/0.001 versus UDP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4336556&req=5

fig04: Exogenous UDP induces rapid, microglia-dependent neuronal loss through P2Y6 receptor activation. A: Microglia in mixed glial cultures treated acutely with 100 µM UDP show enhanced phagocytosis of fluorescent microbeads (micrographs show phagocytosis of 5 µm large microbeads; arrows). B: In mixed neuronal/glial cultures, the P2Y6 agonist UDP (100 μM) induces neuronal loss within 6 h, and this neuronal loss is blocked by co-application of the general phagocytosis inhibitor Cytochalasin D (100 nM). This UDP-induced neuronal loss is greater at 24 h, but is neither enhanced by the application of a second dose of UDP after an additional 24 h nor does it progress significantly further at 72 h. The synthetic P2Y6 agonist, MRS2693 induces neuronal loss equivalent to UDP. Importantly, UDP-induced neuronal loss is prevented by microglial depletion (with l-leucine methyl ester) or application of the selective P2Y6 inhibitor MRS2578 (MRS, 1 μM). Note that neither UDP nor MRS2693 affected microglial proliferation in any of the conditions (not shown). Data are presented as means ± s.e.m. for at least three independent experiments; */**/***P < 0.05/0.01/0.001 versus control, ##/###P < 0.01/0.001 versus UDP.
Mentions: The endogenous agonist for P2Y6 receptors is thought to be UDP (Chang et al., 1995). Exogenous UDP (100 μM) has previously been reported to promote microglial phagocytosis via P2Y6 activation (Koizumi et al., 2007) and we confirmed here that acute treatment of mixed glial cultures with 100 μM UDP strongly enhanced microglial phagocytic activity as measured by their uptake of microbeads (1 or 5 μm diameter and negatively charged, Fig. 4A).

Bottom Line: We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors).In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days.Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

Show MeSH
Related in: MedlinePlus