Carboxypeptidase E Is Invovled In Neuronal Activity-dependent TrkB Intracellular Trafficking In Hippocampal Neurons

BackgroundNeuronal activity can modify neural synapses, this is commonly referred to as synaptic plasticity. Synaptic plasticity not only plays the important role in neural circuits in the developmental stage, but also involved in the control of the cognitive function of the brain and complex behavior in adulthood. Brain-derived neurotrophic factor (BDNF) is a important member of neurotrophin family. It can not only promote neuronal survival, differentiation and growth of dendrites and axons, but also play a key role in regulating synaptic plasticity, Moreover, the function of BDNF was regulated by the neural activity. There are two types of functional receptor for BDNF:p75neurotrophin receptor and TrkB tyrosine kinase receptor. TrkB tyrosine kinase receptor is a key molecule with high affinity to BDNF. Therefore, the surface distribution of TrkB receptor in hippocampal neuron is very important for BDNF-induced function. A large number of studies have shown that the activity stimulation can significantly inchance the surface levels of the TrkB receptor, such as high K+stimulation, electrical stimulation, and Glycine stimulation further confirmed in our previous study It tell us why the function of BDNF was regulated by the neural activity at a certain extent. Because of the high activity-induced surface inertion of TrkB receptor, it makes the neuron easier to accept the signal of BDNF. However, the regulatory mechanism is not very clear. Our previous findings indicated that the juxtamembrane area of the TrkB receptor (Juxtamembrane domain,JM) play a vital role in the process. At the same time, the previous study have screened the CPE molecules interacted with intracellular domain of TkB, interfering with the expression of CPE can inhibit the the neurons activity-dependent TrkB receptor surface inertion increase. Whether this interaction between CPE and TrkB dose regulate the activity-dependent intracellar transports.ObjectiveThe purpose of this study is to further confirmed CPE does regulating TrkB receptor activity-dependent surface inertion, which helping our in-depth understanding of the mechanisms of TrkB activity-induced surface inertion and and thus to provide a reference for clinical diagnosis on mental illness mediated by BDNF.Methods1. Total internal reflection fluorescence(TIRF) microscopy. The TIRF microscopy techniques are widely used in dynamic observation of surface molecules near cell. The hippocampal neurons co-transfected with two plasmids of the CPE-RFP and TrkB-GFP, were observed by the TIRF microscope and recorded their dynamic co-localization in the vicinity of the surface before and after the Glycine stimulation in living cells.2. Immunofluorescence experiments. Immunofluorescence is according to the specificity of antigen-antibody reaction by tracing or check the corresponding antigen with fluorescence antibody. Investigation of CPE whether regulates cLTP-enhanced TrkB translocate from dendrite shaft into spine. The effect of knocking down the expression of CPE on the relative distribution of TrkB in spines and the corresponding dendritic shaft of any impact, before and after Glycine stimulation. And The effect of knocking down the expression of CPE on the BDNF-TrkB mediated dendritic spine growth and morphological plasticity.3. Western Blot experiments. Western Blot is that the proteins were transferred to a membrane, and then using the corresponding antibody to detect. In this study,it was used to detect a range of co-immunoprecipitation, surface biotinylation and the activity-dependent BDNF-induced downstream signaling pathway.4. Surface biotinylation assay. In order to detect the effect CPE on the activity-dependent endogenous TrkB receptor surface insertion. Before collecting of cell lysates, We first treatment the cell with Glycine and then use biotin to labeling the surface protein molecules of hippocampal neurons transfected with Ctr siRNA or CPE siRNA.5. Co-immunoprecipitation experiments. Co-immunoprecipitation experiments is a classic method that based on the specific interaction between antibody and antigen and used to study protein-protein interaction.To immunoprecipitation the specific molecules, the corresponding antibody conjugates with agarose beads to specific recognition of different molecules was used. For exogenous Co-immunoprecipitation, the lysates of cells exogenous overexpression corresponding CPE or TrkB mutant plasmids was extracted, for endogenous Then combined with the molecular would precipitate, Western blot technology was used to detection the results of Co-immunoprecipitation.6. Surface ratiometric fluorescence assay. Immuocytochemistry experiment investigating the activity-induced TrkB surface insertion. In reference to the ratiometric fluorescence assay established in published articles by Zhao Ling teacher.We made a FLAG-TrkB-GFP construct that contained a FLAG epitope just after the signal peptide (extracellular) and a GFP tag fused to the C-terminal (intracellular) of TrkB receptor. When we transfected FLAG-TrkB-GFP into hippocampal neurons; surface TrkB was measured by staining with anti-FLAG M2antibody, under non-permeabilized conditions. The GFP fluorescence throughout the cell represented the total TrkB levels. If the CPEsiRNA or CPE DN plasmid were co-transfected with FLAG-TrkB-GFP, these influence on activity-dependent insertion of TrkB into the neuronal surface can be detected.ResultsPreviously, the findings of the Institute of Neurobiology, Shandong University, Zhao Ling teacher has confirmed that the combination of CPE and TrkB receptor molecules by co-immunoprecipitation and immunofluorescence experiments. Meanwhile, through interference the expression of CPE, it is confirmed that the the CPE molecule does affect the neuronal activity-dependrnt TrkB receptor expression on the surface. Here we have another experiment for this proof to enrichment,and further develop the mechanisms and functional experiments, as follows:1. CPE regulates endogenous TrkB receptor activity-dependent on surface insertion.Previously, we have proved CPE can affect exogenous overexpression of Flag-TrkB activity-dependent surface insertion in hippocampal neurons. Here, through the surface biotinylation methods, we studies of the effect of CPE on endogenous TrkB receptor activity-dependent surface insertion in hippocampal neurons. At the physiological level, CPE also has a regulatory role.on endogenous TrkB receptor activity-dependent surface insertion in hippocampal neurons which further confirmed the effect of CPE.2. CPE and TrkB have activity-dependent co-mobilization on the surface adjacent region.Hippocampal neurons cotansfectied with CPE-RFP and TrkB-GFP plasmids, was observed and recorded at live cells before and after Glycine stimulation. Activity stimulation inchanced CPE-RFP and TrkB-GFP vesicles activity-dependent co-mobilization on the surface adjacent region.3. CPE regulates cLTP-enhanced TrkB translocate from dendrite shaft into spine.To test whether CPE is involved in a local step of TrkB translocation from dendritic spines to the adjacent dendritic shafts. Line plots of fluorescence intensity were generated across the spine head and the adjacent dendritic shaft in cultured hippocampal neurons transfected with CtrsiRNA or CPE-siRNA construct. To assess local TrkB delivery to spines, we quantified TrkB accumulation at spine heads and adjacent dendritic shafts using confocal microscopy and line plots of fluorescence intensity We then compared the average spine/dendrite ratios of TrkB for the different conditions. In control group, Glycine stimulation led to a marked accumulation of TrkB at spines compared with the adjacent dendritic shaft. In contrast, overexpression of CPE siRNA significantly abolished the enhancement of TrkB accumulation in the spine head. Thus, this result indicated that CPE regulates cLTP-enhanced TrkB translocation into spine.4. The Box2domain of juxtamembrane region of TrkB is necessary and sufficient for cLTP-induced TrkB surface insertion.After immunoprecipitation of TrkB mutants with Flag antibodies, we found that without extracellular domain, CPEC25-RFP still have an association with ΔEXTrkB, and RFP alone did not interact with TrkB, as assessed by Western blot analysis using Myc antibodies. This result suggests the intracellular domain are the key binding domain. To explore the potential region in intracellular domain of TrkB responsible for the interaction, additional coimmunoprecipitation experiments were performed with a series of two deletion mutants of TrkB receptor (ΔEXTrkBΔJM, ΔEXTrkBΔTK and ΔEXTrkBΔCT). We found that ΔEXTrkBΔJM abolished the TrkB/CPE interaction.This result suggests that the JM domain is necessary for TrkB binding with CPE. Three deletion different box mutants of ΔEXTrkB were constructed, and their ability to bind to TrkB-FL also was analyzed in HEK293cells by coimmunoprecipitation studies. This results indicated that the JM box2domain is the key domain for the association of TrkB and CPE.To define whether the box2region in the TrkB receptor functioned for its activity-dependent plasma membrane insertion, we next fused a GFP tag to a series of FLAG-TrkBΔboxs construct, which allowed us to measure their surface recruitment by ratiometric fluorescence assay. Together, all the results indicated that the TrkB juxtamembrane box2domain are not only necessary but also sufficient for the rapid activity-dependent recruitment of TrkB to the neuronal surface.5. The462-466amino acid regions of CPE is the key binding region that mediate its interaction with TrkB.We constructed a series of deletion mutants from CPEC25and tested their ability to bind to TrkBΔEX in HEK293cells by coimmunoprecipitation studies. We found only CPEC25Δ15could not be immunoprecipitated by XTrkBΔE, which confirms the essential role of the C-terminal462to466amino acids of CPEC25in the TrkB/CPE association. Next, We examined whether CPE DN of (CPEC25without462to466amino acids) could compete with wild-type CPE to bind with TrkB by ratiometric fluorescence assay. CPE DN could be used as a dominant-negative construct to evaluate the role of CPE in regulating TrkB-FL activity-dependent surface insertion.6. CPE regulates activity-dependent BDNF-induced downstream signaling.We next studied the effect of CPE on TrkB function by investigating BDNF signaling in cultured hippocampal neurons transfected with scrambled or CPE siRNA. Addition of BDNF to hippocampal neurons for10min induced a activation of TrkB and its downstream targets ERK1/2, Akt of TrkB activation. In the control group (transfected with scrambled siRNA), glycine+BDNF significantly enhanced the BDNF-induced TrkB, ERK1/2and Akt activation compared with only treatment with BDNF group. In contrast, knocking down of CPE, the BDNF-induced phosphorylation levels of TrkB, ERK1/2and Akt didn’t change with glycine pretreatment compared with control group.7. CPE regulates the function of activity-dependent BDNF-induced spine growth and plasticity in hippocampal neurons.We examine another role of CPE on TrkB function by investigating the spine growth in cultured hippocampal neurons by transfected with scrambled or CPE siRNA. As shown in results, glycine pretreatment significantly enhanced the BDNF-induced spine density increase and spine head enlargement in scrambled RNA group. However, in overexpression of CPEsiRNA group, glycine pretreatment didn’t initiate BDNF-induced spine density or spine headwidth change compared with neurons treatment with BDNF alone. These results indicate that CPE is important for reglating BDNF-TrkB originated spine function in hippocampal neurons in cLTP.ConclusionCPE molecules may be involved in regulating multiple process before TrkB receptor activity-dependent surface insertion. The C-terminal penultimate10-15amino acids of intracellular domain of CPE is the critical regions that mediates its conjunction with TrkB intracellular domain; Simultaneously, the BOX2region of juxtamembrane region of the TrkB intracellular domain is critical region that mediate its binding with the CPE intracellular domain. CPE regulates TrkB receptor activity-dependent intracellular transports and thus affects the activity-dependent surface expression levels of TrkB, and has a distinct role for TrkB receptor-induced the downstream signaling pathways and TrkB receptor-mediated the growth functions of dendritic spines under the activity conditions.