BDNF Influences AMPAR Local Translocation By Regulating The Level Of Rab8-GTP In Postsynaptic Membrane

BackgroudAs an important member of neurotrophic factors, Brain-derived neurotrophic factor (BDNF) could not only promote neurons survival and differentiation, dendritic and axonal growth, but also play an important role in regulating synaptic plasticity. Synaptic plasticity is often considered as the molecular basis of learning and memory, which is crucial in regulating the transmission of informations between neurons. In presynaptic membrane, BDNF regulates the presynaptic plasticity through promoting the release of neurotransmitter; but in postsynaptic membrane, BDNF regulates the postsynaptic plasticity through regulating the distribution and numbers of ionotropic glutamate receptors especially the Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR). Although it has been reported that BDNF could regulate AMPAR levels in total membrane, but in the local of postsynaptic membrane, especially in the dendritic spine, whether BDNF could regulate AMPAR protein levels is still unclear.Members of the Rab family of small GTPases are essential regulators of intracellular membrane sorting at postsynaptic terminals. Rab8 GTPase, as a member of the Rab GTPase family of small G-proteins, has been reported to contribute to the membrane trafficking from the TGN to the plasma membrane. In neurons, Rab8 is essential for polarized neuronal transport. It has been reported that Rab8 was required for AMPAR delivery into the spine surface, especially for AMPAR insertion into the postsynaptic membrane in hippocampus. However, the role of Rab8 in BDNF-regulated AMPAR translocation at postsynaptic terminals is still unclear. Which Rabs are needed to regulate BDNF-dependent AMPAR translocation, and its molecular mechanism are valueable.Methods1. Plasmid Constructions.2. Hippocampal Neuronal Cultures and Transfection.3. Local translocation assay of internalized and surface AMPAR in spines.4. Subcellular fractionation.5. GST-pull down and Western Blot.Results1. BDNF upregulated the level of GluAl in spine surface.By Local translocation assay of internalized and surface AMPAR in spines, we found that acute BDNF stimulation not only significantly increase the total protein level of GluAl, but also upregulate the surface level of GluAl in spines compared to in dendrites.2. BDNF had no influence on the regulation of GluA2 in spine surface.By Local translocation assay of internalized and surface AMPAR in spines, we found that acute BDNF stimulation could significantly increase the total protein level of GluA2, but could not upregulate the surface level of GluA2 in spines compared to in dendrites.3. BDNF upregulated the level of GluAl through influencing the switch of Rab8.By Local translocation assay of internalized and surface AMPAR in spines, we found that with knocking down of Rab8 GTPase, BDNF could not upregulate the surface level of GluAl, but could increase the total protein level of GluAl in spines compared with in dendrites; we also found that with knocking down of Rabll GTPase, BDNF could upregulate the surface level of GluAl, but could not increase the total protein level of GluAl in spines compared with in dendrites.4. BDNF upregulated the GTP-form of Rab8.By subcellular fractionation assay and GST pull down assay, we found that BDNF stimulation for 30 min could upregulate the protein level of Rab8-GTP.5. BDNF upregulated the activity of Rab8 required Rabin3 proteins.By GST JFC1 pull down assay, we found that knocking down of Rabin3, BDNF could not upregulate Rab8-GTP, while knocking down of TBC1D17 or AS 160 have no influence in BDNF-dependent increase of Rab8-GTP.6. BDNF-induced increase of Rab8-GTP protein was regulated by TrkB-PLCY-PKC signal pathway.By GST JFC1 pull down assay, we found that pretreated with inhibitor of TrK(K252a) could block BDNF-dependent increase of Rab8-GTP, then we found that pretreated with inhibitor of PLCy signal pathway(U73122) could also block BDNF-dependent increase of Rab8-GTP. In addition, we fould that pretreated with inhibitor of PKC signal pathway(CHE) could also block BDNF-dependent increase of Rab8-GTP. These results suggested that BDNF increase the Rab8-GTP through TrkB-PLCy-PKC signal pathway.7. BDNF upregulated the Rab8-GTP via PLCy-Rabin3 signal pathway.By GST JFC1 pull down assay, with knocking down of Rabin3, we found that pretreated with agonist of PKC signal pathway(TPA) could block BDNF-dependent increase of Rab8-GTP.ConclusionOur study provided that BDNF could upregulate the level of GluAl in spine surface through upregulating Rab8-GTP. We also found that BDNF increase Rab8-GTP through PLCy-Rabin3 signal pathway.