Inhibition of UDP/P2Y sub(6) purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo

• Published in: Glia 2014-09, Vol.62 (9), p.1463-1475
• Main Authors: Neher, Jonas J, Neniskyte, Urte, Hornik, Tamara, Brown, Guy C
• Format: Article
• Language: English
• ISSN: 0894-1491; 1098-1136
• DOI: 10.1002/glia.22693

Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y sub(6) receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y sub(6) signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. 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 P2Y sub(6) receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y sub(6) receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 mu M peroxynitrite, or 50 mu M 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y sub(6) 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. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y sub(6) receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y sub(6) signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons. GLIA 2014; 62:1463-1475 Main Points: * Inhibition of UDP/P2Y sub(6) receptor prevents death of neurons induced by A[beta], LPS, LTA, or peroxynitrite in vitro. * UDP induces microglial phagocytosis and loss of viable neurons. * LPS-induced neuronal loss in vivo is prevented by blocking P2Y sub(6) receptor.