Roles Of The New P2Y-like G Protein-coupled Receptor 17 In Ischemic Brain Injury

BackgroundsIschemic brain injury is a complex series of events. In the acute phase, neuronal injury is dominant responses; while in the later (subacute/chronic) phases, one of the important ischemic responses is activation of microglia/macrophages (microgliosis) that release bioactive molecules and remove cellular debris. The responses in both the acute and later phases are regulated by various bioactive molecules, including cysteinyl leukotrienes (CysLTs, namely LTC4, LTD4 and LTE4), and extracellular nucleotides (ATP, ADP), uracil and sugar nucleotides (such as UTP, UDP, UDP-glucose and UDP-galactose). These two important signaling molecules are involved in many physiological processes, and their dysfunctions are associated with various diseases including cerebral ischemia.Recently, the previously orphan receptor GPR17 has been reported to functionally link the nucleotides and cysLTs in ischemic/inflammatory responses. The new P2Y-like G protein-coupled receptor GPR17 is phylogenetically related to the already known P2Y receptors and the CysLT1 and CysLT2 receptors. This receptor seems to respond to both uracil nucleotides (UDP, UDP-glucose and UDP-galactose) and cysLTs (LTD4, LTC4 and LTE4). Thus, GPR17 had been considered as a dual receptor, but it has not been termed as a CysLT receptor recently.GPR17 is highly expressed in organs that undergo ischemic injury such as brain, heart and kidney. In the normal brain, GPR17 is normally localized in in neurons, oligodendrocytes precurors, and ependymal cells. GPR17 is not expressed in normal or ischemic astrocytes. Currently, the main focus is on the roles of GPR17 in regulating oligodendrocyte precursor cell maturation or as a cell-intrinsic timer of myelination. In cerebral ischemic injury, GPR17 is up-regulated after cerebral ischemia; its blockade (?) antisense oligonucleotides prevents the progression of acute permanent brain ischemic injury. Similar GPR17 expression and modulation have been reported in traumatic spinal cord injury. GPR17 is also expressed in several other embryonically distinct cell types including oligodendrocytes as well as microglia/macrophages after permanent cerebral ischemia in mice, indicating a potential role of GPR17 during chronic ischemic injury. However, whether GPR17 mediates acute neuronal injury and post-ischemic microgliosis is still unknown which needs detailed investigation.AimsTo clarify the roles of GPR17 in ischemic neuronal injury and microglial activation, in the present study we performed the following experiments:(1) We examined the spatio-temporal properties of GPR17 expression and cellular localization in the brain after focal cerebral ischemia in rats. Furthermore, we investigated the role of GPR17 in ischemic neuronal injury and microgliosis by RNA interference strategy after ischemic brain injury in rats.(2) We also investigated the role of GPR17 in oxygen-glucose deprivation (OGD) induced neuronal injury and microglial activation in the mixed cultures of rat cortical cells.MethodsIschemic brain injury was induced by middle cerebral arterial occlusion (MCAO). The spatio-temporal properties of GPR17 expression were determined by immunohistochemistry, Western blot and RT-PCR analysis; and the cellular distribution of GPR17 was assessed by double immunofluorescence. Because no GPR17 selective antagonist is currently available, RNA interference strategy was used in vivo to evaluate the regulatory role of GPR17. Acute and subacute/chronic injury after focal cerebral ischemia was assessed by TTC staining, cresyl violet staining and immunohistochemistry (Iba-1 and ED1 staining).In the mixed cultures of primary rat cortical cells, ischemic injury was induced by OGD. The gene expression of GPR17 was silenced by a targeted siRNA. Then, the effect of RNA interference on OGD-induced neuronal injury and microglia activation was assessed in the mixed cultures of rat cortical cells.ResultsPart I Increased expression of GPR17 in rat brain mediates acute neuronal injury and microglia activation after focal cerebral ischemia.Western blot analysis showed that in the ischemic core, GPR17 protein expression increased initially at 12 and 24 h, then recovered at 3 days, and increased again at 7 and 14 days after reperfusion. In the boundary zone, the expression was only increased at 7 and 14 d after reperfusion. In accordance with Western blot results, in the ischemic core, GPR17 mRNA expression was increased with two peaks at 24 h and 7-14 d after reperfusion; however, in the boundary zone the increase was significant but relatively low at 24 h and 3 and 14 d after reperfusion. Immunohistochemical analysis showed that the number of GPR17-immunoreactive cells in the ischemic core was significantly increased at 12 and 24 h, then recovered at 3 d, and increased again 5,7,14 and 28 d after reperfusion. Interestingly, GPR17 was translocated from the cell membrane to the cytoplasm and nucleus after cerebral ischemia in the acute phase. In contrast, in the boundary zone, the number of GPR17-immunoreactive cells began to increase 24 h after reperfusion, and progressively increased thereafter. The expression in the contralateral cortex did not significantly change over 28d after reperfusion. Double immunofluorescence results showed that GPR17 was primarily localized in neurons and oligodendrocytes precurors in intact cortex. GPR17 immunoreactivity was also found in ependymal cells around the ventricles. After MCAO, in the ischemic core, GPR17 was primarily localized in neurons and few in microglia and oligodendrocyte precurors at 24 h, but it was restricted to microglia, and some in oligodendrocyte precurors at 14 d after reperfusion. Whereas, in the boundary zone, it was primarily localized in neurons, some in microglia and oligodendrocyte precurors 14 d after reperfusion.To determine the role of GPR17 in ischemic brain injury,'RNA interference strategy was used in vivo. In a preliminary study, the rat GPR17 siRNA sequence effectively suppressed GPR17 mRNA expression by about 78% in HEK293 cells transfected with rGPR17, thus it was used in the in vivo study. Western blot and immunoreactivity results showed that GPR17 siRNA successfully down-regulated GPR17 protein expression in the ischemic cortex at both 24 h and 14 d after reperfusion. These results confirmed a substantial and stable GPR17 knockdown.The ischemic infarction and moderate edema in the ischemic hemisphere at 24 h, and atrophy at 14 d after reperfusion were significantly attenuated by GPR17 siRNA. In addition, compared to vehicle or negative control sequence, GPR17 siRNA significantly decreased the neurological deficit score and infarct volume 24 h after reperfusion. It also attenuated post-ischemic edema at 24 h and brain atrophy at 14 ds after reperfusion.The reduction in the number of neurons was ameliorated by GPR17 siRNA in the ischemic core at 24 h, and in the boundary zone at 24 h and 14 d after reperfusion, while neurons completely disappeared in the ischemic core 14 d after reperfusion. In the boundary, GPR17 siRNA slightly reduced the number of Iba-1 positive the microglia 24 h after reperfusion, though microglia in the ischemic core did not significantly changed. However,14 d after reperfusion, GPR17 siRNA significantly reduced both Iba-1 and ED1 posistive microglia in the ischemic core and boundary zone.Therefore, these findings suggest that the increased expression of GPR17 may mediate acute neuronal damage, subacute/chronic microglia activation.Part II GPR17 mediates OGD induced acute neuronal injury and microglia activation in the mixed cultures of rat cortical cellsIn the mixed cultures of rat cortical cells, immunofluorescence and Western blot results confirmed that GPR17 siRNA successfully inhibited GPR17 protein expression. OGD/R (OGD 1 h, recovery 24 h) increased LDH release, increased cell necrosis, and decreased cell viability. After treatment with GPR17 siRNA, OGD-induced LDH release was inhibited, the number of necrotic cells was decreased and the cell viability was increased. Double immunofluoresence showed that the main necrotic cells were neurons, and few was astrocytes, microglia or oligodendrocytes. GPR17 siRNA significantly decreased neuronal death. Immunofluorescence staining showed that GPR17 siRNA inhibited morphological changes of microglial activation. After inhibition of GPR17 expression, CysLTi receptor expression was significantly up-regulated.These findings suggest GPR17 may mediate neuronal injury and microglial activation in the mixed culture of rat cortical cells. There may be a possible interaction between GPR17 and CysLT1 receptor in regulation of neuronal injury and microglial activation.Conclusions:1. The expression of GPR17 is up-regulated in the brain after focal cerebral ischemia. GPR17 siRNA attenuates the neuronal injury and inhibits the microgliosis. These findings show that GPR17 mediates acute neuronal injury and subacute/chronic microgliosis after focal cerebral ischemia.2. In the mixed cultures of rat cortical cells, the results of GPR17 siRNA show that GPR17 mediates OGD induced neuronal injury and microglial activation. Interference of GPR17 expression up-regulates CysLT1 receptor expression, suggesting a possible interaction between two receptors in regulation of the neuronal injury and microglial activation.