P2X4-Receptor-Mediated Synthesis and Release of Brain-Derived Neurotrophic Factor in Microglia Is Dependent on Calcium and p38-Mitogen-Activated Protein Kinase Activation

Abstract

Microglia in the dorsal horn of the spinal cord are increasingly recognized as being crucial in the pathogenesis of pain hypersensitivity after injury to a peripheral nerve. It is known that P2X4 purinoceptors (P2X4Rs) cause the release of brain-derived neurotrophic factor (BDNF) from microglia, which is necessary for maintaining pain hypersensitivity after nerve injury. However, there is a critical gap in understanding how activation of microglial P2X4Rs leads to the release of BDNF. Here, we show that stimulating P2X4Rs with ATP evokes a biphasic release of BDNF from microglia: an early phase occurs within 5 min, whereas a late phase peaks 60 min after ATP stimulation. Concomitant with the late phase of release is an increased level of BDNF within the microglia. Both phases of BDNF release and the accumulation within the microglia are dependent on extracellular Ca 2ϩ . The late phase of BDNF release and accumulation, but not the early phase of release, are suppressed by inhibiting transcription and translation, indicating that activation of P2X4R causes an initial release of a pre-existing pool of BDNF followed by an increase in de novo synthesis of BDNF. The release of BDNF is abolished by inhibiting SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated exocytosis. Furthermore, we find that the P2X4R-evoked release and synthesis of BDNF are dependent on activation of p38-mitogen-activated protein kinase (MAPK). Together, our findings provide a unifying mechanism for pain hypersensitivity after peripheral nerve injury through P2X4R-evoked increase in Ca 2ϩ and activation of p38-MAPK leading to the synthesis and exocytotic release of BDNF from microglia.

Microglia in the spinal cord and neuropathic pain

“…It was shown that activation of microglial P2X4Rs stimulates the synthesis and release of brain‐derived neurotrophic factor (BDNF)52, 57, and that BDNF then causes an alteration of transmembrane anion gradient in a subpopulation of dorsal horn lamina I neurons presumably through the downregulation of the neuronal chloride transporter, KCC2, which in turn renders γ‐aminobutyric acid and glycine effects depolarizing, rather than hyperpolarizing, in these neurons (Figure 3). Furthermore, Keller et al 58.…”

P2Y purinergic receptors: New targets for analgesic and antimigraine drugs

“…Activation of the P2X4 receptor leads eventually to the activation of p38 and release of BDNF [65,66]. Up-regulation of the P2X4 receptor in spinal cord microglia was later demonstrated to require the CCL21 chemokine, which is expressed only in damaged neurons [67], and turned out to be necessary for the development of nerve injury-induced tactile allodynia and, to a lesser extent, peripheral inflammation [68].…”

P2X receptor antagonists for pain management: examination of binding and physicochemical properties

“…This increased expression appears to be specific since P2X4 receptor protein expression is not increased in neurons or astrocytes following trauma or injury [27,41]. Several intracellular signaling pathways have been implicated in the upregulation of P2X4 receptor expression including the PI3K/AKT pathway [42], and lyn kinase via an associated fibronectin/integrin mechanism [43][44][45][46][47][48][49].…”

“…This action appears to be mediated by increased intracellular Cl − concentrations in lamina I neurons that is dependent on brain-derived neurotrophic factor (BDNF) and neurotropin (TrkB) receptor signaling pathways [45,47]. P2X4 receptor stimulation results in down-stream signaling involving PIP2, PIP3, and p38 MAPK and subsequent release of BDNF [48,49]. Microglial P2X4 receptor stimulation also leads to phosphorylation of the NMDA receptor NR1 on subunit spinal neurons [45] that is likely mediated by P2X7-dependent release of interleukin-1β (IL-1β).…”

“…Agonist stimulation of P2X7 receptors opens a nonselective cation channel and also leads to the rapid maturation and release of IL-1β [53][54][55]. Prolonged (>60 s) receptor activation leads to the formation of large cytolytic pores in the cell membrane that are produced, at least in part, by receptor-mediated down-stream signaling events linked to the recruitment of hemichannels to the cell surface [5,[48][49][50][51][52][53][54][55][56][57]. P2X7(−/−) mice exhibit disrupted cytokine signaling cascades and attenuated ATP-induced processing of pro-IL-1β [58].…”