The Protective Effects Of Dynaminl Phosphorylation-mediated TrkB Endocytosis In Alzheimer’s Disease

BackgroudBrain-derived neurotrophic factor (BDNF) plays critical roles in neuron development and function.The function of BDNF is initiated by binding to and activating tropomyosin related kinase B (TrkB) receptors. After BDNF binding to TrkB, it induces TrkB dimerizationand intrinsic tyrosine kinase activity, which further triggers activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol3-kinase (PI3K), and phospholipase Cy (PLCy) signaling pathways. Formation of BDNF-TrkB complex triggers TrkB receptors internalization, and this internalization has considering biological responses on signaling pathways and neuron development. Though some studies revealed that BDNF-dependent TrkB endocytosis is a clathrin-and dynamin-dependent manner, the specific mechanism governing BDNF-dependent TrkB endocytosis is still unclear.GSK3β is a critical kinase on neuron development and neurondegeneration. Recent studies demonstrated that GSK3P activity was extremely high, and it might the critical fator for AD. GSK3(3has been reported to regulate activity-dependent bulk endocytosis (ADBE) through phosphorylating dynamini on S-774. Whether GSK3β could regulate BDNF-dependent TrkB endocytosis, and how this regulation influences AD are valueable.Methods1. Plasmid Constructions2. Hippocampal Neuronal Cultures and Transfection 3. Surface TrkB Internalization Measurementby Fluorescence assay4. Cleavable Surface Biotinylation Internalization Assay5. TUNEL analysis6. Novel object recognition test and Morris water-maze testResults1. GSK3P regulates BDNF-dependent TrkB endocytosisWe found that overexpression of GSK3β3S9A could decrease BDNF-dependent TrkB endocytosis compared with the control group, while overexpression of GSK3(3R96A could facilitate it by cleavable biotinylation and live cell ratiometric fluorescence assays.2. GSK3P regulates BDNF-dependent TrkB endocytosis through phosphorylating Ser-774onDyn1By knocking down of Dyn1, we found that it could abolish the effects of GSK3β mutants on TrkB endocytosis, which suggested that GSK3P regulated TrkB endocytosis through Dyn1. Previous study demonstrated that GSK3β could phosphorylate Ser-774on Dyn1. We found that GSK3β activity could not further regulate TrkB endocytosis on the basis of overexpression of Dyn1S774D, which suggested that GSK3β regulates BDNF-dependent TrkB endocytosis through phosphorylating Ser-774on Dyn1.3. TAT-Dyn1SPS blockes GSK3β-Dyn1interactionTo inverstigate the effects of GSK3β-regulated TrkB edocytosis, we introduced a TAT-Dyn1SPS to specificly block GSK3β-Dyn1interaction. We found that TAT-Dyn1SPS could promote BDNF-dependent TrkB endocytosis and then enhanced BDNF-induced Akt activation.4. Aβ impairs BDNF-dependent TrkB endocytosisBy live cell ratiometric fluorescence assay, we found that BDNF-dependent TrkB endocytosis was impaired under Aβ exposure. AR-A014418and TAT-Dyn1SPS could partially rescue the impaired TrkB endocytosis under Aβ exposure, which suggested that Aβ impairs BDNF-dependent TrkB endocytosis through GSK3β.5. TAT-Dyn1SPS could rescue the Aβ-inhibited Akt activation of BDNF downstream signalingNeurons were added BDNF for15min, after treating with A(3and TAT-Dyn1SpS. Then the BDNF downstream signalings were detected. We found that TAT-Dyn1SpS could rescue the Aβ-inhibited Akt activation of BDNF downstream signaling.6. TAT-Dyn1SpS could facilitate the protective effects of BDNF on Aβ-induced cell deathRecent study demonstrated that BDNF had neuroprotective effects on Aβ-induced cell death. By cleaved caspase-3levels detection and a TUNNEL analysis, we found that TAT-Dyn1SpS could facilitate the protective effects of BDNF on Aβ-induced neurodegeneration.7. TAT-Dyn1SpS could facilitate the effects of BDNF on learning and memory in APP/PS1-transgenic miceWe injected TAT-Dyn1SpS into the hippocampal of APP/PS1transgenic mice. By Novel object recognition test and Morris water-maze test, we found that TAT-Dyn1SpS could promote the effects of BDNF on learning and memory in APP/PS1-trangenic mice.ConclusionOur study provided that GSK3β could regulate BDNF-dependent TrkB endocytosis through phosphorylating Ser-774on Dyn. In addition, we found that Aβ could impair BDNF-dependent TrkB endocytosis. Blocking GSK3β-Dyn1interaction by TAT-Dyn1SpS, we found that it could rescue the impaired TrkB endocytosis under Aβ exposure, and further resuce the inhibited Akt activation. Finally, we found that TAT-Dyn1SpS could enhance the neuronprotective effects of BDNF on Aβ toxicity.