The Role Of Activation Of TRPV4 Receptor In The Regeneration Of Adult Hippocampal Dentate Nerve And Its Molecular Mechanism

BackgroundNeurogenesis plays an important role in adult hippocampal function.Adult hippocampal neurogenesis is a complex process that begins with the proliferation of stem cells,followed by commitment to a neuronal phenotype,with newborn neurons finally integrating into the hippocampal circuitry after three to four weeks of morphological and physiological maturation.Calcium(Ca2+)is an important second messenger that regulates many cellular processes of neurogenesis,including cell proliferation,development,migration,invasion and survival.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.As it is permeable to calcium,activation of TRPV4 induces the inward current and results in an increase of intracellular free calcium concentration([Ca2+]i)in hippocampal neurons.TRPV4 activation,accompanied by elevations in[Ca2+]i,participates in the regulation of cell proliferation in cultured porcine endothelial cells,esophageal epithelial cells and human brain capillary endothelial cells.Ca2+ has been proven to be an important regulator of neurite growth.Axon outgrowth and dendritic development occur within the optimal[Ca2+]i level,and this process may be inhibited when the[Ca2+]i level is below or above this optimal[Ca2+]i level.A gain-of-function of TRPV4 mutation increased its Ca2+ channel activity,which underlies the pathogenesis of TRPV4-linked axonal neuropathies.By now,there is lack of evidence for the TRPV4-induced modulation of adult hippocampal neurogenesis.Mitogen-activated protein kinases(MAPKs)are a family of serine-threonine kinases that play an important role in regulating multiple biological processes,including proliferation,survival,differentiation and transformation.Previous studies in lung epithelial cells,trigeminal ganglion and dorsal root ganglion sensory neurons,capillary endothelial cells,adipocytes and hippocampi have shown that TRPV4 activation may enhance extracellular regulated protein kinases(ERK)and p38 mitogen-activated protein kinase(p38 MAPK)signaling.Activation of Adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)has been shown to regulate the hippocampal neuronal structure.Protein kinase B(Akt)plays an important role in modulating neurite outgrowth,which is mediated through downstream signals,such as mammalian target of rapamycin(mTOR)and glycogen synthase kinase 3?(GSK3?).Activation of TRPV4 has been reported to modulate the AMPK and Akt signaling pathways in vitro.Based on the above reports and our previous studies,the present study proposes that activation of TRPV4 may modulate the proliferation of stem cells and the neurite growth of newborn neurons through MAPK and/or AMPK-Akt signaling pathways.The results are expected to provide the theoretical basis for a new potential target that could modulate some process of adult hippocampal neurogenesisObjective1.To determine the effect of TRPV4 activation on the proliferation of stem cells in the adult hippocampal dentate gyrus and its underlying mechanisms.2.To determine the effect of TRPV4 activation on the neurite growth of newborn neurons in the adult hippocampal dentate gyrus and its underlying mechanisms.Part ?.Effect of TRPV4 activation on the proliferation of stem cells in the adult hippocampal dentate gyrus and its underlying mechanismsMaterials and Methods1.Drug treatment:different doses of TRPV4 agonist GSK1016790A was intracerebroventricularly(icv.)injected to activate TRPV4 in vivo.2.Histological examination:Mice received three intraperitoneal(ip.)injections of 5-bromo-2-deoxyuridine(BrdU,50 mg/kg)at 8h intervals for mitotic labeling.The mice were anesthetized with chloral hydrate(400 mg/kg,ip.)and transcardially perfused with 4%paraformaldehyde.The brains were removed and post-fixed overnight in 4%paraformaldehyde at 4 ?.Coronal sections were cut from the level of the hippocampus for BrdU and Ki67 staining to examine the effect of GSK1016790A on the proliferation of stem cells.3.Western blot:Western blot analysis was performed 5 d after GSK1016790A injection to examine the changes of protein expression caused by GSK1016790A injection.Results1.At doses ranging from 0.1 ?M/mouse to 5 ?M/mouse,icv.injection of GSK1016790A(GSK1016790A-injected mice)for 5 d dose dependently increased the number of 1-day-old BrdU immunopositive(1-day-old BrdU+)cells in the hippocampal dentate gyrus region.GSK1016790A application also increased the number of Ki67 immunopositive(Ki67+)cells and the hippocampal protein level of PNC A.2.GSK1016790A induced increases in the hippocampal protein levels of cyclin dependent kinase 6(CDK6),CDK2,cyclin E1 and cyclin A2 but did not affect CDK4 and cyclin D1 expression.The phosphorylated retinoblastoma protein(p-Rb)protein level in hippocampi was enhanced in GSK1016790A-injected mice compared with control mice.3.GSK1016790A treatment increased the phosphorylated ERK(p-ERK)and p38 MAPK(p-p38 MAPK)protein levels.4.GSK1016790A-enhanced proliferation was markedly blocked by a MAPK/ERK kinase(U0126)or p38 MAPK antagonist(SB203580).The increased protein levels of CDK2 and CDK6,as well as those of cyclin E1 and cyclin A2,in GSK1016790A-injected mice were markedly reduced by co-injection of U0126 or SB203580.ConclusionTRPV4 activation results in the proliferation of stem cells in the adult hippocampal dentate gyrus,which is mediated through ERK1/2 and p38 MAPK signaling to increase the expression of CDKs(CDK6 and CDK2)and cyclins(cyclin E1 and A2),phosphorylate Rb consequently and accelerate the cell cycle ultimately.Part ?.Effect of TRPV4 activation on the neurite growth of newborn neurons in the adult hippocampal dentate gyrus and its underlying mechanismsMaterials and Methods1.Drug treatment:different doses of TRPV4 agonist GSK1016790A was icv.injected to activate TRPV4 in vivo.2.Histological examination:The mice were anesthetized with chloral hydrate(400 mg/kg,ip.)and transcardially perfused with 4%paraformaldehyde.The brains were removed and post-fixed overnight in 4%paraformaldehyde at 4?.Coronal sections were cut from the level of the hippocampus for doublecortin(DCX)staining to examine the effect of GSK1016790A on the neurite growth of newborn neurons.3.Western blot:Western blot analysis was performed at different time points after GSK1016790A injection to examine the changes of protein expression caused by GSK1016790A injection.Results1.Icv.injection of the TRPV4 agonist GSK1016790A for 5 d(GSK1016790A-injected mice)reduced the number of DCX immunopositive(DCX+)cells and DCX+ fibers in the hippocampal dentate gyrus.2.The phosphorylated Adenosine 5'-monophosphate(AMP)-activated protein kinase(p-AMPK)protein level increased from 30 min to 2 h,and then decreased from 1 to 5 d after GSK1016790A injection.The phosphorylated p-Akt protein level decreased from 30 min to 5 d after GSK1016790A injection;this decrease was markedly attenuated by the AMPK antagonist compound C(CC),but not by the AMPK agonist AICAR.3.The phosphorylated mTOR and p70 ribosomal S6 kinase(p70S6K)protein levels were decreased by GSK1016790A,which was sensitive to 740 Y-P and CC.The phosphorylation of GSK3? at Y216 was increased by GSK1016790A,and this change was accompanied by increased phosphorylation of microtubule-associated protein 2(MAP2)and collapsin response mediator protein-2(CRMP-2),which was markedly blocked by 740 Y-P and CC.4.GSK1016790A-induced decrease of DCX+ cells and DCX+ fibers was markedly attenuated by 740 Y-P and CC,but was unaffected by AICAR.ConclusionTRPV4 activation impairs the neurite growth of newborn neurons in the adult hippocampal dentate gyrus through increasing AMPK and inhibiting Akt to inhibit mTOR-p70S6K pathway,activate GSK3? and thereby result in the inhibition of MAP2 and CRMP-2 function.