Mechanism Underlying Activity Dependent TrkB Neuronal Surface Insertion

「Background and objective」Neurotrophins are best known for their ability to promote neuronal survival and differentiation,Brain-derived neurotrophic factor(BDNF) is an important member in neurotrophins family which has a novel function in synapse development and plasticity,particularly in the central nervous system.It suggests that BDNF facilitates both early-phase and late-phase LTP is necessary for LTP.Remarkably,neuronal activity could influence the effectiveness of BDNF,for example,BDNF cannot enhance the survival of retinal ganglion neurons unless depolarized by high K~+ or glutamate agonists,or increased intracellular cAMP.Previous studies have shown that active neurons/synapses might respond better to BDNF than less active one,a plausible mechanism would be local synthesis and/or secretion of BDNF at the active synapse or active neurons/synapses might respond better to BDNF than less active ones, and this could be achieved by activity dependent control of BDNF signaling. Neuronal activity dependent signaling of BDNF is virtually all attributed to TrkB receptor tyrosine kinase.Neuronal activity could increase the number of TrkB receptors on cell surface or enhance the signaling transduction for BDNF.Depolarization or Electric stimulation has been shown to increase the levels of the TrkB receptor on the cell surface of neurons. Activity-dependent enhancement of the quantity of surface TrkB receptor may define an important mechanism by which the specificity of BDNF activity-dependant modulation is achieved.However the detailed cellular and molecular mechanism underlying this effect is unclear.For further provide insights into the mechanistic link between activity-dependent and neurotrophic modulation of CNS neurons and synapses,acquire of the intracellular trafficking mechanisms of TrkB is the key research point.For finding out the molecules which could not only interact with TrkB but also assistant with TrkB's intracellular trafficking,we performed Yeast Two-hybrid assay and found TrkB could interact with the carboxypeptidase E,a proneuropeptide/prohormone processing enzyme which is also a sorting receptor associated with BDNF vesicles and other neuropeptide or prohormone targeting into the regulated secretory pathway (RSP).It is proved the C-terminal cytoplasmic tail of CPE functioned as the key region of the membrane binding and sorting.There is also work suggested the 4 or 15-residue sequence in the C-terminal region of CPE is necessary to sorting of CPE to the RSP.Considering for there are some similar characters between TrkB knock out mice and CPE knock out mice, we hypothesize CPE participates in the regulated progress of TrkB activity dependent membrane surface insertion.Here we employed a dual tag TrkB fluorescence assay to investigate the molecular and cellular mechanisms which regulate the trafficking progress of TrkB activity dependent membrane surface insertion to provided new insights regarding neuronal activity dependent surface delivery of TrkB receptor,which will advance our understanding the role of TrkB in synaptic plasticity modulation. 「Methods and results」1.Ratiometric fluorescence assay to measure surface TrkB receptor levelTo employed the FLAG-TrkB-GFP construct and the "surface FLAG-TrkB-GFP fluorescence ratiometric assay" to monitor the TrkB quick recruitment to the plasma membrane.With this assay to provided temporal, structure and spatial information regarding neuronal activity dependent surface delivery of TrkB receptor,Through this assay we found chemical LTP(glycine) induced the most robust increase(1.82±0.08 fold) in surface TrkB levels and 200μM glycine was then chosen as the stimulation condition in our subsequent studies.2.Plasma surface TrkB fraction measurementTo measure the percentage of surface TrkB receptor in total TrkB at basal state,we stained FLAG tag of FLAG-TrkB-GFP under permeabilized and non-permeabilized conditions.From plotting the anti-FLAG fluorescence signal in the whole cell region inpermeabilized neurons against the GFP fluorescence signal we got the relationship between the anti-FLAG and the GFP signals by immunofluorescence(IF) is 1.01.With this constant we calculate the fraction of TrkB on hippocampal neurons surface is about 34%.By this assay we found activity dependent cell surface TrkB increase was much more obvious in neuronal processes than in cell body(1.35 vs.1.98 fold) which could be used to understand the mechanisms of BDNF's function in synapse plasticity.3.Juxtamembrane domain of TrkB is necessary and sufficient for cLTP induced TrkB plasma membrane insertionTo define the potential region in TrkB receptor that is required for its activity-dependent plasma membrane insertion,we generated a series of constructs in which various TrkB domains(juxtamembrane,kinase,and C terminal) were deleted from TrkBFL or different TrkB domains (juxtamembrane,kinase,and C terminal) were grafted to the C-terminal of T1 receptor(a truncated TrkBFL isoform).All the mutant constructs (TrkBΔJM,TrkBΔTK,TrkBΔCT,T1JM,T1TK and T1CT) contain the same FLAG and GFP epitope tag.With the ratiometric fluorescence assay we found only the TrkBΔJM abolished glycine-induced TrkB receptor surface insertion in the deletion constructs and only T1JM shown activity-dependent surface recruitment upon cLTP stimuli in the grafting constructs.It is suggested that TrkB cytoplasmic juxtamembrane region is not only necessary for efficient TrkB surface recruitment but also sufficient to promote rapid insertion of TrkB to plasma membrane upon cLTP stimulation.4.Cdk5 but not TrkB kinase activity is involved in cLTP induced TrkB plasma membrane insertionProtein kinases play important roles in neuronal activity and mediating BDNF signaling and biological activity.By the ratiometric fluorescence assay together with selective inhibitor of Cyclin-dependent kinase 5(Cdk5) Roscovitin and Cdk5 siRNA,the data shown the cLTP-induced TrkB surface recruitment was significantly abrogated.Furthermore TrkBS478A (ser478 mutant to alanine) and T1JMS478A neuronal surface level could not increase in response to cLTP stimuli.And cLTPstimuli could lead to an acute Cdk5 phosphorylation at Tyr15 and Ser159 site.It suggested Cdk5 is crucial in activity dependent TrkB plasma membrane insertion.From the results of both pre-treatment of Trk inhibitor K252a and kinase dead(KD) TrkB construct retained the ability to insert into surface in response to glycine stimuli,it suggested tyrosine kinase activity is not required for this behavior.5.cLTP induced TrkB surface recruitment occurs more efficiently in postsynaptic regionWith the ratiometric fluorescence assay combined with transfection with the constructs of PSD-95-mCherry and FLAG-TrkBFL-GFP or FLAG-TrkBΔJM-GFP it present that the neuronal activity could facilitate more TrkBFL receptors trafficking to the postsynaptic region which shown it dramatically insertion into the PSD-95(postsynaptic marker) positive region than in PSD-95 negative region(2.13 vs.1.75 fold),but the TrkBΔJM receptor abolished this process which suggested the JM domain is essential for TrkB's activity-dependent intracellular trafficking.6.CPE cytoplasmic tail interact with TrkB receptor's intracellular domainFor investigating the molecule regulating TrkB activity-dependent insertion into membrane surface,Yeast Two-hybrid assay and Co-immunoprecipitation(CO-IP) were performed and found TrkB could interact with carboxypeptidase E.This result was confirmed by the images of confocal fluorescent microscope.Established constructs of MYC-CPE25AA-RFP(intracellular domain) and FLAG-TrkB IC,which only has the intracellular domain of CPE or TrkB and further performed CO-IP,it is suggested that c-terminal 25aa of CPE could associate with TrkB intracellular domain.7.CPE is involved into the regulating of cLTP induced TrkB membrane surface insertionDesign and generate siRNA of CPE together with the ratiometric fluorescence assay to test the activity dependent cell surface TrkB increase was significantly restrained.This result confirmed us cLTP induced TrkB surface insertion is regulated by the biologic function of CPE.「Conclusion」1.Established the "Surface TrkB receptor fluorescence ratiometric assay" to investigate the mechanism underlying TrkB plasma membrane recruitment upon chemical LTP(cLTP) stimuli.2.Calculated and defined the percentage of surface TrkB receptor in total TrkB at basal state is 34%.3.Revealed the juxtamembrane(JM) domain of TrkB is necessary and sufficient for its activity-dependent plasma membrane insertion. 4.The kinase activity of Cyclin-dependent kinase 5(Cdk5) is crucial to cLTP induced TrkB surface recruitment;and the Tyr478 of TrkB JM domain which could be phosphorylated by Cdk5 is the most key site.5.cLTP induced TrkB surface recruitment occurs more efficiently on neuronal processes especially at the postsynaptic membrane,which may present a mechanism for rapid enhancement of postsynaptic sensitivity to incoming BDNF signaling.6.The C-terminal 25aa of Carboxypeptidase E was confirmed associate with the intracellular domain of TrkB and this interaction is crucial in regulating LTP induced TrkB surface recruitment.