Schuichi Koizumi

P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury

Pain after nerve damage is an expression of pathological operation of the nervous system, one hallmark of which is tactile allodynia—pain hypersensitivity evoked by innocuous stimuli. Effective therapy for this pain is lacking, and the underlying mechanisms are poorly understood. Here we report that pharmacological blockade of spinal P2X4 receptors (P2X4Rs), a subtype of ionotropic ATP receptor, reversed tactile allodynia caused by peripheral nerve injury without affecting acute pain behaviours in naive animals. After nerve injury, P2X4R expression increased strikingly in the ipsilateral spinal cord, and P2X4Rs were induced in hyperactive microglia but not in neurons or astrocytes. Intraspinal administration of P2X4R antisense oligodeoxynucleotide decreased the induction of P2X4Rs and suppressed tactile allodynia after nerve injury. Conversely, intraspinal administration of microglia in which P2X4Rs had been induced and stimulated, produced tactile allodynia in naive rats. Taken together, our results demonstrate that activation of P2X4Rs in hyperactive microglia is necessary for tactile allodynia after nerve injury and is sufficient to produce tactile allodynia in normal animals. Thus, blocking P2X4Rs in microglia might be a new therapeutic strategy for pain induced by nerve injury.

UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis

Microglia, brain immune cells, engage in the clearance of dead cells or dangerous debris, which is crucial to the maintenance of brain functions. When a neighbouring cell is injured, microglia move rapidly towards it or extend a process to engulf the injured cell. Because cells release or leak ATP when they are stimulated or injured, extracellular nucleotides are thought to be involved in these events. In fact, ATP triggers a dynamic change in the motility of microglia in vitro and in vivo, a previously unrecognized mechanism underlying microglial chemotaxis; in contrast, microglial phagocytosis has received only limited attention. Here we show that microglia express the metabotropic P2Y6 receptor whose activation by endogenous agonist UDP triggers microglial phagocytosis. UDP facilitated the uptake of microspheres in a P2Y6-receptor-dependent manner, which was mimicked by the leakage of endogenous UDP when hippocampal neurons were damaged by kainic acid in vivo and in vitro. In addition, systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in messenger RNA encoding P2Y6 receptors that colocalized with activated microglia were observed. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and could function as a sensor for phagocytosis by sensing diffusible UDP signals, which is a previously unknown pathophysiological function of P2 receptors in microglia.

Activation of p38 Mitogen-Activated Protein Kinase in Spinal Hyperactive Microglia Contributes to Pain Hypersensitivity Following Peripheral Nerve Injury

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen‐activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho‐p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho‐p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho‐p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4‐(4‐fluorophenyl)‐2‐(4‐methylsulfonylphenyl)‐5‐(4‐pyridyl)‐1H‐imidazole (SB203580), suppresses development of the nerve injury‐induced tactile allodynia. Taken together, our results demonstrate that nerve injury‐induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.

Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures

Originally ascribed passive roles in the CNS, astrocytes are now known to have an active role in the regulation of synaptic transmission. Neuronal activity can evoke Ca2+ transients in astrocytes, and Ca2+ transients in astrocytes can evoke changes in neuronal activity. The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between astrocytes and neurons. We demonstrate here that ATP, a primary mediator of intercellular Ca2+ signaling among astrocytes, also mediates intercellular signaling between astrocytes and neurons in hippocampal cultures. Mechanical stimulation of astrocytes evoked Ca2+ waves mediated by the release of ATP and the activation of P2 receptors. Mechanically evoked Ca2+ waves led to decreased excitatory glutamatergic synaptic transmission in an ATP-dependent manner. Exogenous application of ATP does not affect postsynaptic glutamatergic responses but decreased presynaptic exocytotic events. Finally, we show that astrocytes exhibit spontaneous Ca2+ waves mediated by extracellular ATP and that inhibition of these Ca2+ responses enhanced excitatory glutamatergic transmission. We therefore conclude that ATP released from astrocytes exerts tonic and activity-dependent down-regulation of synaptic transmission via presynaptic mechanisms.

The TRPV4 Cation Channel Mediates Stretch-evoked Ca2+ Influx and ATP Release in Primary Urothelial Cell Cultures

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD) evoked Ca2+ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca2+ concentration and ATP release. Stretch stimulation evoked intracellular Ca2+ increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca2+ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca2+ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4α-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca2+. These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

Ca2+ waves in keratinocytes are transmitted to sensory neurons: the involvement of extracellular ATP and P2Y2 receptor activation.

ATP acts as an intercellular messenger in a variety of cells. In the present study, we have characterized the propagation of Ca2+ waves mediated by extracellular ATP in cultured NHEKs (normal human epidermal keratinocytes) that were co-cultured with mouse DRG (dorsal root ganglion) neurons. Pharmacological characterization showed that NHEKs express functional metabotropic P2Y2 receptors. When a cell was gently stimulated with a glass pipette, an increase in [Ca2+]i (intracellular Ca2+ concentration) was observed, followed by the induction of propagating Ca2+ waves in neighbouring cells in an extracellular ATP-dependent manner. Using an ATP-imaging technique, the release and diffusion of ATP in NHEKs were confirmed. DRG neurons are known to terminate in the basal layer of keratinocytes. In a co-culture of NHEKs and DRG neurons, mechanical-stimulation-evoked Ca2+ waves in NHEKs caused an increase in [Ca2+]i in the adjacent DRG neurons, which was also dependent on extracellular ATP and the activation of P2Y2 receptors. Taken together, extracellular ATP is a dominant messenger that forms intercellular Ca2+ waves in NHEKs. In addition, Ca2+ waves in NHEKs could cause an increase in [Ca2+]i in DRG neurons, suggesting a dynamic cross-talk between skin and sensory neurons mediated by extracellular ATP.

Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP‐evoked production of interleukin‐6 (IL‐6) and its intracellular signals were examined using a mouse microglial cell line, MG‐5. ATP, but not its metabolites, produced IL‐6 in a concentration‐dependent manner. Although ATP activated two mitogen‐activated protein kinases, i.e. p38 and extracellular signal‐regulated protein kinase, only p38 was involved in the IL‐6 induction. However, the activation of p38 was not sufficient for the IL‐6 induction because 2′‐ and 3′‐O‐(4‐benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL‐6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL‐6 production by the cells. The ATP‐evoked IL‐6 production was attenuated by Gö6976, an inhibitor of Ca2+‐dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX‐insensitive P2Y receptors activates p38 and Ca2+‐dependent PKC, thereby resulting in the mRNA expression and release of IL‐6 in MG‐5. This is a novel pathway for the induction of cytokines in microglia.

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia.

ATP (10–100 μm), but not glutamate (100 μm), stimulated the release of plasminogen from microglia in a concentration‐dependent manner during a 10 min stimulation. However, neither ATP (100 μm) nor glutamate (100 μm) stimulated the release of NO. A one hour pretreatment with BAPTA‐AM (200 μm), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 μm). The Ca2+ ionophore A23187 induced plasminogen release in a concentration‐dependent manner (0.3 μm to 10 μm). ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration‐dependent manner which was very similar to the ATP‐evoked plasminogen release, whereas glutamate (100 μm) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μm) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). The ATP‐evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2‐Methylthio ATP was active (7 out of 7 cells), but α,β‐methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 μm) was shown not to inhibit the ATP‐evoked increase in [Ca2+]i (20 out of 20 cells). 2′‐ and 3′‐O‐(4‐Benzoylbenzoyl)‐adenosine 5′‐triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long‐lasting increase in [Ca2+]i even at 1 μm, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′‐triphosphate‐2′, 3′‐dialdehyde (oxidized ATP, 100 μm), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 μm). These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.

The role of nucleotides in the neuron--glia communication responsible for the brain functions.

Accumulating findings indicate that nucleotides play an important role in cell‐to‐cell communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (7 types; P2X1–P2X7) contain intrinsic pores that open by binding with ATP. P2Y (8 types; P2Y1, 2, 4, 6, 11, 12, 13, and 14) are activated by nucleotides and couple to intracellular second‐messenger systems through heteromeric G‐proteins. Nucleotides are released or leaked from non‐excitable cells as well as neurons in physiological and pathophysiological conditions. One of the most exciting cells in non‐excitable cells is the glia cells, which are classified into astrocytes, oligodendrocytes, and microglia. Astrocytes express many types of P2 purinoceptors and release the ‘gliotransmitter’ ATP to communicate with neurons, microglia and the vascular walls of capillaries. Microglia also express many types of P2 purinoceptors and are known as resident macrophages in the CNS. ATP and other nucleotides work as ‘warning molecules’ especially through activating microglia in pathophysiological conditions. Microglia play a key role in neuropathic pain and show phagocytosis through nucleotide‐evoked activation of P2X4 and P2Y6 receptors, respectively. Such strong molecular, cellular and system‐level evidence for extracellular nucleotide signaling places nucleotides in the central stage of cell communications in glia/CNS.

Purinergic receptors in microglia: Functional modal shifts of microglia mediated by P2 and P1 receptors

Microglia are sensitive to environmental changes and are immediately transformed into several phenotypes. For such dynamic “modal shifts”, purinergic receptors have central roles. When microglia sense ATP/ADP leaked from injured cells by P2Y12 receptors, they are transformed into a moving phenotype, showing process extension and migration toward the injured sites. Microglia upregulate adenosine A2A receptors, by which they retract the processes showing an amoeboid‐shaped, activated phenotype. Microglia also upregulate P2Y6 receptors, and if they meet UDP leaked from dead cells, microglia start to engulf and eat the dead cells as a phagocytic phenotype. The P2Y12 receptor‐mediated responses are modulated by other P2 or P1 receptors. In contrast, the P2Y6 receptor‐mediated responses were not influenced by P2Y12 receptors and vice versa. Microglia appear to use purinergic signals either cooperatively or distinctively to cause their modal shifts.