Regulation Of TrkB Receptor Recycling By Neuronal Activity

The neurotrophin brain-derived neurotrophic factor (BDNF) plays an important role in the activity-dependent regulation of synaptic structure and function. BDNF, released in an activity dependent manner, binds and activates the tropomyosin related kinase TrkB receptor. TrkB activation by BDNF initiates three major cascades of signalling pathways:phospholipase Cy (PLCy), phosphatidylinositol3-kinase (PI3K), and extracellular signal-regulated kinases (Erk). BDNF has emerged as a key regulator of activity-dependent synaptic plasticity. In the hippocampus, substantial evidence suggests that BDNF facilitates both early-phase and late-phase LTP (E-LTP and L-LTP), which is mediated by the binding and activation of TrkB receptor. Interestingly, TrkB-FL and TrkB.T1traverse divergent recycling pathways after endocytosis. TrkB.T1receptors, which lack the kinase domain but contains short isoform-specific cytoplasmic domain in its place, predominantly recycle back to the cell surface by a "default" mechanism. However, endocytosed TrkB-FL receptors recycle to a lesser extent in a sequence-directed recycling pathway which relies on its tyrosine kinase activity. It will be interesting to compare the recycling levels of TrkB-FL and TrkB.T1in response to LTP stimuli.Therefore,in the present study biotinylation assay and fluorescence ratio microscopy assay were used to measure TrkB receptors postendocytic recycling. We want to know whether neuronal activity could enhance TrkB-FL recycling and its mechanism. The results are summarized as follows:1. Neuronal activity increases surface expression of TrkB-FL after BDNF treatmentCells were first biotinylated then given BDNF-induced internalization to test activity-dependent degradation, we found that less TrkB-FL was sorted into degradation pathway after neuronal activity. Glycine-induced cLTP enhances levels of surface biotinylated TrkB-FL receptors, but not TrkB.T1. And RT-PCR further confirmed that the transcriptional levels of TrkB-FL or TrkB.T1were changed during cLTP.2. More TrkB-FL was sorted into recycling pathway following cLTP stimuliCleavable biotinylation and live cell ratiometric fluorescence results prove that glycine treatment induced more internalized TrkB-FL receptors recycling back to the cell surface.3. Glycine-induced TrkB-FL recycling increase requires the kinase activity of TrkBFirst, we constructed TrkB deletion mutants:△CT (C terminal was deleted),△TK (tyrosine kinase and C terminal were both deleted) and KD [with a point mutation in the kinase domain established to abolish kinase activity (K571A)]. We found TrkB△CT but not△TK or KD could be regulate by neuronal activity, which suggests that the kinase activity of TrkB is necessary for cLTP induced TrkB-FL recycling.4. TrkB-FL and TrkB.T1are sorted into distinct recycling pathways via Rab11and Rab4respectivelyWe demonstrate that Rab11could co-immunoprecipitate with TrkB-FL. However, we discovered Rab11could not be immunoprecipitated by TrkB.T1or TrkB.KD. Co-immunoprecipitate assays show that Rab11S25N could bind to TrkB-FL insignificantly higher amounts compared with Rab11wt, but Rab11Q70L interacted only weakly with TrkB-FL. Moreover, we found that most recycled TrkB-FL was co-localized with Rab11-containing vesicles, but not Rab4-containing vesicles. We also found that Rab11regulate TrkB-FL recycling and Rab4regulate TrkB.T1recycling.5. Increased recycling upon cLTP stimuli enhances BDNF triggered TrkB downstream signalingGlycine pretreatment significantly enhanced the BDNF induced ERK1/2and AKT activation at the1hr time point. In contrast, in neurons transfected with Rab11siRNA, the phosphorylation levels of ERK1/2and AKT were attenuated compared with control group, which underscores the importance of Rab11-dependent TrkB recycling for efficient BDNF-induced signaling. Moreover, knockdown of Rab11abolished the cLTP stimuli enhanced BDNF downstream signaling.6. Effects of neuronal activity on TrkB recycling endosome distributionTo determine the spatial relation of TrkB recycling endosomes distribution in spines, we transfected hippocampal neurons with GFP and analyzed its localization with endogenous TrkB endosomes by immunocytochemical assay. TrkB recycling endosomes distribute into the spines by BDNF. We found that the recycled TrkB level in postsynaptic region in neuronal processes was increased after glycine treatment. However, co-transfect Rab11S25N-GFP construct, recycled TrkB exhibited much less overlap with PSD95either under basal condition or glycine-stimulate condition.7. Active form of Rab11mediates the interaction between TrkB-FL and PSD95We found overexpression of Rab11Q70L significantly increased the association between TrkB and PSD-95, conversely, overexpression of Rab11S25N or siRNA knock-down of Rab11decreased the association between TrkB and PSD-95. We found that neuronal activity affects the cytosol-membrane cycle of Rab11. We treated hippocampal slice cultures (P6+9DIV) with recombinant BDNF for1h either in glycine stimuli condition or not, and then assessed TrkB/PSD95association. We found BDNF treatment led to enhanced the interaction between TrkB and PSD95, however, under the cLTP condition, this interaction became more strongly.Conclusion:Our results provide neuronal activity enhances TrkB-FL but not TrkB.T1endocytic recycling. Moreover, we identified kinase domain is essential for neuron activity enhanced TrkB-FL recycling. In addition, we found that the recycling of TrkB-FL and TrkB.TI were respectively mediated by the Rab11and Rab4dependent pathway, which may explain their differential response to cLTP stimuli. Finally, we demonstrated the increased TrkB-FL recycling could facilitate TrkB-FL translocation to postsynaptic density during LTP..