The Mechanism Of BDNF In Regulating VGLUT3 And VMAT2 In Primary Sensory Neurons

The vesicular glutamate transporters(VGLUTs)pack the neurotransmitter into synaptic vesicles so that it can be released into the synapse.VGLUTs are dependent on the proton gradient that exists in the secretory system.By now,three main types of VGLUTs are known,vesicular glutamate transporter VGLUT 1-3.Compared to VGLUT1 and 2,VGLUT3 has a more restricted distribution,and less is known about its function.It also has been shown that VGLUT3,contributes to glutamate signaling by serotonergic neurons in a classic transporting manner,via the vesicular monoamine transporter 2(VMAT2)and then facilitate 5-hydroxytryptamine(5-HT)transmission.The transportation of 5-HT into synaptic vesicles and presynaptic membrane also relies on the expression pattern and function of VMAT2.The co-existence of VGLUT3 and VMAT2 illustrated the synergy mechanism inside the transportation of transmitters.Dorsal root ganglion(DRG)neurons and spiral ganglion neurons(SGN)are the essential two kinds of primary afferent neurons.Particularly,DRG neurons take their parts in transmitting somatic and visceral sensation from the peripheral target organs to the central nervous system(CNS),while SGN neurons transmits the hearing signals from hair cells into the CNS,relying on the transmitting,loading and release mechanisms of neurotransmitter.In the meanwhile,the expression pattern and function maintain of neurotransporters are essential parts in the process.So far,the mechanism of actions for VGLUT3 and VMAT2 in regulating and expression pattern in DRG and SGN neurons are not fully understood.Brain-derived neurotrophic factor(BDNF)is one of the members of the neurotrophin(NT)family and is one of the most profound known regulators of survival in the developing peripheral nervous system(PNS).Through binding to its receptor,tyrosine kinase receptor B(TrkB),BDNF activates the mitogen-activated protein kinase(MAPK),phosphatidylinositol 3-kinase(PI3K),and phospholipase C-gamma(PLC?)signaling pathways.However,whether BDNF plays important roles in regulating VGLUT3 and VMAT2 in DRG neuron and SGN,need to be clarified and its roles in regulating the neurite outgrowth and expression pattern of neurotransporters also should be involved in the following researches.Based on the background,we decided to use primary cultured DRG neurons and SGN neurons as the representatives of primary afferent neurons,to further investigate the mechanism in BDNF signaling regulating transporters VGLUT3 and VMAT2 via its downstream pathway extracellular signal-regulated protein kinase 1/2(ERK1/2),PI3K/Akt and PLCy pathways and further participate in the process of BDNF stimulating neurite outgrowth via the interfering design of VGLUT3 and VMAT2 small interfering RNA(siRNA).Furthermore,in order to confirm the mechanism of transcriptional factors Etv4 and Etv5 in BDNF stimulating neurite outgrowth,we also have designed the Etv4 and Etv5 siRNAs to inferring their expression levels or over-expressed their expression level via plasmid transfection experiments,to further demonstrate their roles in BDNF regulating neurite growth.Part ? BDNF's roles in regulating transporters VGLUT3 and VMAT2 in DRG neuronsIn this part we have involved the researches in BDNF and its downstream pathways in regulating VGLUT3 and VMAT2,as well as its relationship with neurite outgrowth.The main results demonstrated that the incubation with external BDNF could significantly improve the growth status of neuron and elevate the expression of GAP-43 protein,in a dose-dependent manner.In addition,BDNF exposure could stimulate the up-regulation of VGLUT3 through PLCy pathway in DRG neurons.Even though BDNF exposure could not have the ability to elevate another neurotransporter VMAT2's expression,the inhibition of PI3K/Akt and PLC? pathways could decreased the effects of BDNF toward VMAT2.The results illustrated the possible participation of BDNF downstream pathways in regulating VMAT2 even though the effects are not significant as VGLUT3.In addition,the siRNA interfering of VGLUT3 and VMAT2 could not significantly disturb external BDNF's roles in stimulating the neural growth status and upregulating the expression of GAP-43,which demonstrated the potential variable mechanism of BDNF's regulation in glutamate pathways.In other aspect,the upregulation of VGLUT3 might be involved in the regulation of BDNF's roles in neuropathological pain transmission.In all,this part revealed the possible mechanism of the biological regulation of neurotransporters of BDNF in primary sensory neurons,which could also provides new insights into the mechanism of BDNF regulating in neuropathological pain.Part ? The transcriptional factor Etv4 and Etv5's roles in BDNF regulating the expression of VGLUT3 and the neuron outgrowthIn this part,we have mainly focused on the roles of transcriptional factors Etv4 and Etv5 and their expression pattern towards regulations of BDNF signaling pathways.We further analyzed the the regulation of BDNF and its downstream pathways towards growth related protein GAP-43 and neuron structural protein medium neurofilament(NF-M)and light neurofilament(NF-L),in conditions of Etv4 and Etv5 siRNA interfering and overexpression via expression vector transfection experiments,to further clarify the mechanism of DRG neuron outgrowth and regeneration in specified conditions.In addition,we have analyzed the expression patterns of VGLUT3 and VMAT2 in Etv4 and Etv5 siRNA interfering experiments in BDNF incubation,to further reveal the expression pattern of Etv4 and Etv5 in DRG neurite outgrowth and VGLUT3 and VMAT2 regulation.The results showed that external BDNF could elevate the expression of Etv4 and Etv5,including their mRNA levels,possibly via the activation of downstream ERK1/2 pathway.In the presence of external BDNF,Etv4 and Etv5 siRNAs interfering could downregulate the expression of GAP-43,NF-M and NF-L,which is in accordance with the relative short of neurite length in DRG neurons;While the over expression of Etv4 and Etv5 could upregulate the expression of GAP-43,NF-M and NF-L,which is accordance with relative elongation of neurite length in DRG neurons.In addition,Etv4 and Etv4 siRNA could partly block the VGLUT3 upregulation in presence of BDNF,instead of VMAT2.In all,the results demonstrated that BDNF could upregulate the expression of Etv4 and Etv5,thus elevate the expression of GAP-43 and NF-M and NF-L,which participate in the DRG neuron outgrowth and neuron maturation.Meanwhile,BDNF could evoke the upregulation of VGLUT3 via elevate the expression of Etv4 and Etv5,which could be one the possible mechanism of BDNF strengthens the transportation efficiency of glutamate signals via upregulation of VGLUT3,which is also accordance with the BDNF potential roles in transporting mechanical pain signals and hyperalgesia.As the key mediators of BDNF and its downstream signals in regulating neurite outgrowth and VGLUT3 upregulation,Etv4 and Etv5 as well as their potential roles in neurite outgrowth and VGLUT3 expression,could provide additional proof for the mechanism of BDNF signaling pathways in neurite outgrowth,and the mechanism in VGLUT3-mediated glutamate signaling transportation.Part ? The mechanism of BDNF's signaling pathway in regulating VGLUT3 and VMAT2 in spiral ganglion neuronsIn this part,we have mainly focused on the external BDNF's roles in VGLUT3 and VMAT2 regulations and possible mechanisms involved,in spiral ganglion neurons(SGN),and trying to verify the relationship between the regulations and SGN survival and neurite outgrowth.In all,the results showed that external BDNF could significantly elevate the expression level of VGLUT3 in SGN via activation of downstream PI3K/Akt,while it could also be beneficial to SGN neuron survival and neuron outgrowth.However,external BDNF could not regulate the expression pattern of VMAT2 in SGN in the specialized experiment condition.Moreover,the unremarkable relationship between BDNF's facilitation in SGN neuron survival and neuron outgrowth and the expression pattern of VGLUT3 and VMAT2 revealed the possible various mechanisms of BDNF regulations towards SGN neuron survival and outgrowth.The regulation of external BDNF towards VGLUT3 in SGN could be one of the mechanisms among the BDNF signaling pathways in regulating glutamate signaling transportation.In the meantime,the roles of BDNF in regulating SGN neuron outgrowth and survival could be crucial in maintenance of hearing function in cochlea.