Activation Of TRPV4 Receptors On Hippocampal Neurons And Its Molecular Mechanism

Background Transient receptor potential vanilloid 4(TRPV4) is a member of transient receptor potential superfamily. TRPV4 is widely expressed in the central nervous system, including hippocampus, cortex, thalamus and cerebellum, etc. TRPV4 is a multipolar-activated receptor that can be activated by cell edema, mild heat, arachidonic acid and its metabolism, etc. As it is permeable to calcium, activation of TRPV4 induces the inward current and results in an increase of intracellular free calcium concentration. It has been reported that application of TRPV4 agonist dosedependently induces the death of retinal ganglion cells and that TRPV4 mediates infrasound-induced neuronal injury. However, the mechanisms underlying TRPV4-induced neuronal death remain unclear. During the ischemia, the energy failure results in cytotoxic edema and membrane lipid metabolism disorder and these pathological changes can activate TRPV4. Our previous study reports that during the cerebral ischemia TRPV4 protein expression increases with ongoing reperfusion and TRPV4 blockage markedly reduces the brain infarction. In vitro studies also prove that TRPV4 blockers increase the viability of astrocytes exposed to oxidative stress and that closing TRPV4 is responsible for the neuroprotection against oxygen-glucose deprivation-induced injury. These reports suggest that TRPV4 is an important target of the cerebral ischemic injury. Mitogen-activated protein kinases(MAPK) play an important role in controlling cell proliferation, differentiation, survival and death. Phosphatidylinositol-3-kinase/ protein kinase B(PI3K/Akt) signaling has been proven to inhibit cell death by inactivating apoptogenic factors and to promote the survival. During the cerebral ischemia, MAPK signaling is over-activated, whereas Akt signaling is inhibited, which contributes to the cerebral ischemic injury. Activation of extracellular signalrelated kinase(ERK) can be promoted by TRPV4 signaling in capillary endothelialcells and adipocytes, whereas in cystic cholangiocytes, TRPV4 activation is associated with ERK1/2 inhibition and Akt phosphorylation. In our previous study, application of TRPV4 agonist enhances N-methyl-D-aspartate(NMDA)-mediated current, indicating that activation of TRPV4 enhances NMDA receptor function. Based on the above reports and our previous study, the present study proposes that activation of TRPV4 induces neuronal injury through modulating NMDA receptor and the downstream MAPK and/or PI3K/Akt signaling pathways, and thus is involved in cerebral ischemic injury. The results are expected to provide the theoretical basis for a new target of clinical treatment of cerebral ischemia injury.Objective 1. To determine the effect of TRPV4 activation on hippocampal neuronal survival and its underlying mechanisms. 2. To illuminate the mechanisms underlying TRPV4-mediated cerebral ischemia injury.Part I. Effect of TRPV4 activation on hippocampal neuronal survival and its underlying mechanismsMaterials and Methods 1. Drug treatment: different doses of TRPV4 agonist GSK1016790 A was intracerebroventricularly(icv.) injected to activate TRPV4 in vivo. 2. Histological examination: The brains were removed, placed in fixative overnight and then processed for paraffin embedding. Coronal sections were cut from the level of the hippocampus for toluidine blue staining to examine the effect of GSK1016790 A on hippocampal neuronal survival. 3. Western blot: Western blot analysis was performed on day 3 after GSK1016790A-injection to examine the changes of protein expression caused by GSK1016790A-injected.Results 1. At doses ranging from 0.1 μM/mouse to 5 μM/mouse, icv. injection of GSK1016790 A dose-dependently induced neuronal death in hippocampal CA1 and CA2/3 area. 2. Application of GSK1016790 A increased the phosphorylation of NR2 B subunit. 3. GSK1016790A-treatment increased the phosphorylation of ERK(p-ERK) protein and decreased the phosphorylation of Akt(p-Akt) protein, which was markedly blocked by an NR2 B subunit antagonist. 4. GSK1016790A-induced neuronal injury was markedly attenuated by an NR2 B subunit antagonist Ro 25-6981 or a PI3 K agonist 740 Y-P, but unaffected by an MAPK/ERK kinase(MEK) antagonist U0126.Conclusion Activation of TRPV4 induces neuronal injury through the enhancement of the phosphoryaltion of NR2 B subunit of NMDA receptor and the down-regluated Akt signaling pathway.Part II. Mechanisms underlying TRPV4-mediated cerebral ischemia injuryMaterials and methods 1. Preparation of the focal cerebral ischemia model: Transient focal cerebral ischemia was induced by middle cerebral artery occlusion(MCAO). After the mice were anesthetized, a poly-L-lysine-coated nylon monofilament thread was inserted through the external carotid artery and advanced into the internal carotidartery to occlude the origin of the middle cerebral artery for 60 min and then the thread was withdrawn for reperfusion. 2. Infarction volume measurement: At 24 h and 48 h post-MCAO, the brains were removed, sectioned into 5 equidistant coronal slices and then incubated with a 2% 2,3,5-triphenyle-tetrazoliumchloride(TTC) solution for 20 minutes. Brain infarction was visualized using image analysis software. 3. Hoechst staining: At 48 h post-MCAO, mice were anesthetized and perfused with ice-cold phosphate-buffered saline(PBS) followed by 4% paraformaldehyde. The hippocampal slices were stained with Hoechst-33342. Hoechst-positive(Hoechst+) cells were counted using a fluoresce microscope. 4. Western blot: Western blot analysis was performed at different time point postMCAO to examine the changes of protein expression.Results 1. The protein level of p-ERK in the ipsilateral hippocampus was up-regulated at 1 h to 24 h post-MCAO, but the increase of p-ERK protein level gradually decreased with ongoing reperfusion. 2. The protein level of p-Akt in the ipsilateral hippocampus was up-regulated at 1 h to 4 h post-MCAO and then down-regulated at 8 h to 24 h post-MCAO. 3. Icv. injection of TRPV4 antagonist HC-067047(10 μM/mouse) markedly reduced the brain infarction at 24 h and 48 h post-MCAO. Moreover, HC-067047 treatment rescued the increased p-ERK protein level and the decreased p-Akt protein level. 4. Icv. injection of TRPV4 antagonist HC-067047 markedly inhibited apoptosis in hippocampal CA1 area at 48 h post-MCAO, accompanied with the increase of Bcl-2/Bax protein ratio and the decrease of cleaved caspase-3 protein level. 5. Icv. injection of TRPV4 agonist GSK1016790 A led to the decrease of Bcl-2/Bax protein ratio and the increase of cleaved caspse-3 protein level, which could be attenuated by a PI3 K agonist 740 Y-P.Conclusion Combined with our previous study, the present study proposes that during the cerebral ischemia, the over-activation of TRPV4 may enhance NR2 B subunit of NMDA receptor to down-regulate Akt signaling, negatively shift Bcl-2/Bax protein ratio and activate caspase-3 at last, which is likely responsible for the apoptosis and the neuronal injury in cerebral ischemia.