UCHL1 Regulated TrkB Deubiquitination And Played An Important Role In Learning And Memory

BackgroudIt has been well known that Brain derived neurotrophic factor (BDNF) plays an important role in regulation of neuronal survival, growth and synaptic plasticity via activating its receptor TrkB and downstream signaling pathways. By binding with BDNF. TrkB could be activated by phosphorylation, and consequently activating several signaling pathways. It has been reported that BDNF could induce dramatic increase of another posttranslational modification-ubiquitination of TrkB. However how TrkB ubiquitination is regulated and its biological functions remain unknown. In the preliminary studies we have found that Ubiquitin C-terminal hydrolase LI (UCHLl) could interact with TrkB in vitro, indicating that UCHLl as a deubiquitinating enzyme for TrkB regulating its ubiquitination level and functions. This project will be the first study as we know demonstrate that UCHLl as a deubiquitinating enzyme for TrkB, and deepen our understanding in TrkB ubiquitination and UCHLl functions in nervous system.MethodsTo study whether UCHLl could regulate surface TrkB receptors ubiquitination level and influence the surface TrkB endocytosis and transport after endocytosis, we designed the immune fluorescence quantitative analysis, the membrane surface cracking biotin to detect the distribution of receptor and endocytosis and recycling experiment, analyzing that UCHLl regulates TrkB deubiquitination and playing important role in recycling and other biological effects. We further carried out the CFC or open-field test to investigate whether UCHLl played important role in the learning and memory or capacity of mice via regulating TrkB ubiquitination invivo.Results:1. UCHL1 associates with TrkB-FL independent of TrkB kinase activityEarly mass spectrometry analysis found that UCHL1 associated with intracellular TrkB in the 60 min, and we further used immune coprecipitation technology, respectively analyzed the interaction of exogenous and endogenous UCHL1 and TrkB, we found UCHL1 may interact with TrkB-FL, TrkB-KD and TrkB-Tl. JM1 in TrkB and 75-85 aa in UCHL1 play important roles in TrkB/UCHLl interaction.2. UCHL1 regulates TrkB deubiquitination via interaction with TrkBBy using antibody P4D1 and antibody FK1, we found that the main ubiquitination form of TrkB was multimonoubiquitination. By using CO-IP assay, we obsered that UCHL1 could deubiquitin TrkB. By constracting various site mutants of TrkB and CO-IP assay, we found that UCHL1 mainly regulate the deubiquitination of TrkB K488 site.3. TAT-UCHL175-85 facilitates TrkB degradation via promoting the ubiquitination of TrkBThrough a cracking biotin technology, we found that UCHL1 participated in TrkB-FL endocytosis and trafficking after endocytosis process further impact on TrkB and its downstream signaling pathways.4. TAT-UCHL175-85 could decrease learning memory ability of C57 mice via inhibiting the combination of UCHL1 and TrkBThrough the CFC test we interestingly found that TAT-UCHL175-85 could affect the acquisition of of C57 mice.Conclusion:We found UCHL1 could regulate the ubiquitination of TrkB via interacting with TrkB which further influencing the endocytosis and recycling or degradation after endocytosis, affecting BDNF-TrkB and its down stream signaling pathways, finally regulating the learning and memory ability of mice.