Regulation Of TRPM7 By NGF/TrkA In Rat Hippocampus

Part 1 Effect of nerve growth factor on TRPM7 expression in ischemic rat hipppocampusAlthough the activity of transient receptor potential melastatin 7 (TRPM7) channels correlates with anoxic neuronal death in recent reports; the change or regulation of TRPM7 expression in response to ischemic insult has not been documented. Using in vivo (middle cerebral artery occlusion MCAO) and in vitro (oxygen-glucose deprivation OGD) models of cerebral ischemia, we explored the effect of nerve growth factor (NGF) on the expression of TRPM7 in rat hippocampus by means of RT-PCR and Western blot analyses. We also observed the staining pattern of TRPM7 and TrkA in hippocampal neurons by means of confocal laser scanning microscopy.The results obtained from in vivo research:(1) Compared with Sham and Normal groups TRPM7 mRNA expression in ipsilateral hippocampi of MCAO rats significantly increased (P<0.05) and became stable during 15~20 h after reperfusion. (2) Compared with Ischemia group, intracerebroventricular injection of NGF significantly reduced mRNA and protein expression of TRPM7 at 20 h after reperfusion (P<0.05), which could be abolished by co-application of TrkA inhibitor K252a.The results obtained from in vitro research:(1) The co-localization of TRPM7 and TrkA were found in cell bodies of the same hippocampal neurons.(2) OGD induced significant increase of LDH release and neuronal death, compared with control group (P<0.01). Pre-incubation of NGF markedly reduced extracellular LDH concentration and neuronal death, compared with OGD-treated cultures without NGF treatment (P<0.05); co-application with K252a fully abolished this protective effect of NGF (P>0.05).(3) Compared with control group, TRPM7 expression in hippocampal cultures was significantly increased at 20 h after OGD treatment (P<0.05). In contrast, pre-incubation of NGF markedly reduced TRPM7 expression, compared with OGD-treated cultures without NGF treatment (P<0.05); this inhibitory effect could be abolished by K252a.(4) When specific inhibitors were further introduced to selectively block phosphatidylinsitol-3 kinase (PI-3K), phospholipase C (PLC) or ras pathways, only PI-3K inhibitor Wortmannin abolished NGF effect on TRPM7 expression of OGD-treated cultures.All above results indicate that cerebral ischemia may induce highly expressed TRPM7 in hippocampus and TrkA, when activated by NGF, may prevent high expression of TRPM7 through PI-3K signal pathway. In light of the abundance of NGF in hippocampus that express both TrkA and TRPM7, these findings offers some clues for further investigation of potential roles of TRPM7 and NGF in hippocampal neurons during cerebral ischemia. Part 2 Regulation of a TRPM7-like current by NGF in rat CA1 hippocampal neuronsTRPM7 channels are widely expressed in the nervous system; however their function and regulation are largely unknown. This study aimed to explore whether the current mediated by TRPM7-like channels in rat CA1 hippocampal neurons could be modulated by NGF. Using whole-cell patch clamp recording techniques, we identified an outward-rectifying TRPM7-like current in CA1 hippocampal neurons freshly isolated from postnatal (10-day-old) rats. Tthe outward component of this current was reversibly reduced by NGF in dose- and time- dependent manners; and this effect was blocked by K252a. In addition, when U73122 was further introduced to inhibit the TrkA downstream effector phopholipase C in the presence of NGF, the reduction of the current was also abolished. These results suggest that NGF, by activing TrkA may regulate the function of TRPM7-like channels in hippocampal neurons via PLC pathway.Part 3 Effect of electroacupuncture on TRPM7 expression via TrkA in ischemic rat hipppocampusClinically, electroacupuncture (EA) has been shown to produce beneficial effect on stroke patients. However, the detailed mechanism underlying the beneficial effects of EA on stroke remains poorly understood. Our previous studies have shown that EA may up-regulate TrkA in brain tissues during cerebral ischemia and mediate neuroprotective effects. In this experiment, we found that EA treatment reduced high expression of TRPM7 in ischemic hippocampal tissues in the rat mode of middle cerebral artery occlusion. Using protein kinase inhibitors of specific TrkA signaling pathways, we found that this reduction appears to be mediated through PI-3K pathway. In view that TRPM7 has recently been found to play a key role in the death of anoxic neurons, our results may provide experimental evidence for the clinical application of EA treatment for stroke patients.