Myo5a Mediates TrkB Receptors Recycling In Neurons

Brain-derived neurotrophic factor (BDNF) plays an important role in the neuronal survival, neurite generation and synaptic structure via tropomyosin related kinase B (TrkB) receptors activation. It has been proved that synaptic plasticity is regulated by BDNF-TrkB pathway under both basal and LTP situation in central nervous system. TrkB receptors perform endocytosis and recycling processes after BDNF stimuli. We have proved that TrkB full length (TrkB-FL) receptors but not the isoform T1(TrkB.T1) receptors recycled via a Rab11-dependent pathway, which is important for the BDNF-TrkB signaling pathway and synaptic plasticity. However, which motor mediates the actin-based local transfer of endocytosed TrkB receptors recycled back to plasma membrane is still unclear.To find out which motor protein participates in TrkB receptors recycling process, we set up several experiments using co-immunoprecipitation (CO-IP) assay, ratiometric fluorescence assay, biotinylation assay etc. The recults are summarized as follows:1. Myo5a associates with TrkB receptorsBy using CO-IP assay and immunofluorescence assay, we observed that TrkB-FL receptors but not TrkB.T1receptors associated with Myo5a and Myo5a might mediate TrkB-FL receptors trafficking.2. TrkB receptors kinase domain is necessary and sufficient for the interaction between Myo5a and TrkB receptorsBy using different TrkB mutants and CO-IP assay, we found out that the kinase activity was important for the interaction between Myo5a and TrkB-FL receptors.3. Myo5a is not involved in BDNF-dependent TrkB-FL endocytosis pathwayBy using mall interfering RNA, ratiometric fluorescence assay and biotinylation assay etc., we found that Myo5a did not affect TrkB receptors endocytosis. 4. Myo5a mediates BDNF-dependent postendocytic recycling process of TrkB-FL receptors but not TrkB.Tl receptorsBy using live cell ratiometric fluorescence-based recycling assay, small interfering RNA and biotinylation assay etc., we found that Myo5a participated in TrkB-FL receptors recycling process after its endocytosis.5. Rab11regulates the interaction between Myo5a and TrkBBy using small interfering RNA and CO-IP assay, we found that Rabll regulated the interaction between TrkB receptors and Myo5a only in the presence of Exon ABCEG.6. Identification of the key motif in Myo5a which mediated the assocation with Rab11By constructing mutant of Myo5a and using GST pull down assay etc. we proved that Myo5a ExonE55-66is necessary and sufficient for the interaction between Rab11and Myo5a.7. TAT-5aExon55-66reduces TrkB-FL receptors recycling levelsBy using TAT-5aExon55-66, the recycling levels of TrkB-FL and the surface expressing levels of TrkB-FL after BDNF stimuli are significantly decreased.8. Myo5a-mediated TrkB-FL postendocytic recycling contributes to the BDNF triggered TrkB-FL translocation to spine and the sustained downstream signaling.By inhibiting the Myo5a-mediated TrkB-FL postendocytic recycling, we found that TrkB-FL translocation to spine, BDNF-TrkB triggered downstream signaling were both inhibited, which suggests that the Myo5a-mediated TrkB-FL receptors recycling process contributes to the neuronal function.Conclusion:we found an actin-based motor protein, myosin Va (Myo5a), which was abundantly expressed in neuron, mediated TrkB-FL receptors recycling. Moreover, we also found that Rab11, which had been proved participating in TrkB-FL receptors recycling before, regulated this Myo5a mediated TrkB-FL receptors recycling process. Finally, when blocked the interaction between Rab11and Myo5a by using TAT-5aExon55-66, we discovered a clear reduction of BDNF-dependent TrkB-FL receptors recycling levels, and thus the inhibition of the BDNF-induced cell signaling and neuronal synaptogenesis. Through these findings we first reported the motor protein, Myo5a, mediates Rab11-dependent TrkB-FL receptors recycling process, which contributes to the BDNF-induced cell signaling.