Bdnf Signaling Pathways Regulating Formed Synapses And Study The Mechanism Of Neurotransmitter Release

Background and Aims:Synapse, consisted of presynaptic membrane, synaptic cleft and postsynapticmembrane, is a specialized structure between neurons. Correct synapse formation isthe basis of the structure and function of the nervous system and could be regulatedby many factors including neurotropic factors, integrins and astrocytes etc. BDNF(brain derived neurotropic factor), a member of neurotropic factor family, isinvolved in regulating and maintaining the development and function of nervoussystem, especially for synapse formation.GIT1(G protein coupled receptor kinase interacting protein-1) is an intracellularmultidomain scaffold protein with regulating receptor internalization and trafficking,cell adhesion and migration, synapse formation and maintain while CaMKII, aserine/threonine kinase widespread in nervous system also could control synapsematuration and function.Our study aims to investigate whether BDNF affects premature synapse formationby accelerating the interaction between GIT1and CaMKIIβ in primary corticalneurons through gene point-mutation, gene transfection and immunoprecipitationetc.Methods:1. Isolation, culture and identification of rat primary cortical neurons. 2. Investigate the influences of GIT1and CaMKIIβ on synapse formation by usingGIT1-siRNA and KN-93respectively.3. Construct HA-GIT1(wild type), HA-GIT1(S419A) and Flag-CaMKIIβ plasmids.The above plasmids were transfected into HEK293T and cortical neuronsrespectively.4. Immunoblotting, immunofluorescence and immunoprecipitation are used todetect proteins expressions and interactions.Results:1. The cells (Day in vitro7) were confirmed to be cortical neurons by β-tubulin IIIand NeuN staining.2. Comparing with the control group, BDNF significantly enhanced synpaseformation and the level of p-CaMKIIthr286(P<0.05) while GTI1-siRNA andKN-93significantly suppressed the BDNF promoted synaptogenesis (P<0.05).GIT1-siRNA could reduced the the level of p-CaMKIIthr286(P<0.05).3. Comparing with GIT1-WT, the interaction and co-localization between GIT1(S419A) and CaMKIIβ were decreased in HEK293T cells and cortical neurons(P<0.05).4. Comparing with GIT1-WT, GIT1(S419A) significantly reduced the synapseformation (P<0.05).Conclusions:1. GIT1and CaMKIIβ were signaling downstream of BDNF-induced synapseformation.2. GIT1serine419influenced: the BDNF-mediated interaction between GIT1andCaMKIIβ in cortical neurons, CaMKIIβ phosphorylation and synapse formation. Background and Aims:BDNF participates no only in synapse formation but also in synaptic function.Neurotransmitter release as one of the synaptic function, is the basis of neuronalcommunications. The release of glutamate, a common neurotransmitter, is regulatedby BDNF, but the exact mechanism is still unclear. Recent researches suggest thatthere are interrelations between BDNF and Src in the regulation of nervous systemfunction. Src, as a member of the non-receptor tyrosine kinase family, is alsoinvolved in glutamate release but with controversies. PLCγ1is a member of PLCserine/threonine kinase family and can activate neurotransmitter release after itstyrosine783phosphorylated. There is mutual regulation between Src and PLCγ1, forexample Src controls PLCγ1activation through GIT1. However, the relationshipbetween Src and PLCγ1in BDNF signaling is poorly unknown. In this study, weused primary cortical neurons in vitro to investigate the signaling pathway of Trkb,Src and PLCγ1in BDNF-induced glutamate release.Methods:1Isolation and culture of rat primary cortical neurons.2Specific inhibitors of TrkB, Src, PLCγ1, Akt, and MEK1/2(i.e., K252a, PP2,U73122, LY294002, and PD98059, respectively) were used to treat cortical neurons and to detect the glutamate release from cortical neurons stimulatedwith BDNF.3Immunoblotting and immunoprecipitation are used to detect proteinsinteractions.Reuslts:1. BDNF significantly increased glutamate release, and simultaneously enhancedphosphorylation levels of TrkB, Src, PLCγ1, Akt, and Erk1/2(P <0.05).2. For BDNF-stimulated cortical neurons, K252a inhibited glutamate release andinhibited the phosphorylation levels of TrkB, Src, PLCγ1, Erk1/2, and Akt (P <0.05).3. PP2reduced the glutamate release from BDNF-stimulated cortical neurons (P <0.05) and inhibited the phosphorylation levels of TrkB and PLCγ1(P <0.05).However, PP2had no effect on the phosphorylation levels of Erk1/2or Akt (P>0.05).4. U73122inhibited the glutamate release from BDNF-stimulated cortical neurons,but had no influence on the phosphorylation levels of TrkB, Src, Erk1/2, or Akt(P>0.05).5. LY294002and PD98059did not affect the BDNF-stimulated glutamate releaseand did not inhibit the phosphorylation levels of TrkB, Src, or PLCγ1(P>0.05).Conclusions:1. BDNF stimulated the glutamate release from cortical neurons via the TrkB/Src/PLCγ1signaling pathway. 2. For BDNF-stimulated cortical neurons, Src was activated by TrkB and it couldpromote the full activation of TrkB. Following the full activation, TrkBpromoted the release of glutamate by increasing the level of PLCγ1phosphorylation.