The Role Of Cdk5-mediated Drp1 Phosphorylation In Regulating Mitochondrial Dynamics In Aβ_1-42-induced Neuron Apoptosis

Objective:In the present study,we focused on the role of Cdk5-mediated Drp1phosphorylation in regulation of mitochondrial dynamics and Alzheimer’s disease.Method:In our study,primary cortical neurons were treated with Aβ_1-42to induce neuron damage to mimic Alzheimer’s disease（AD）.In order to alter the activity of Cdk5,Cdk5 RNAi and the inhibitor of Cdk5 Roscovitine were used respectively.The phosphorylation level of Drp1 in each experimental group was detected by Western Blot.In order to observe the changes of mitochondrial morphology,the Mito-Ds Red plasmid was transfected into neurons and the mitochondrial changes were observed under fluorescence microscope.In order to further detect the effect of the phosphorylation site of Drp1 on mitochondrial morphology,the cells were transfected with GFP-Drp1-S579A or GFP-Drp1-S579D and Mito-Ds Red simultaneously,and the changes of mitochondrial morphology were observed under fluorescence microscope.In the detection of neuronal apoptosis,the level of Cleaved Caspase-3 was detected by Western Blot and Heochest 33258 staining was used to observe the changes of nuclei.Results:The results of Western Blot showed that Aβ_1-42could increase the level of phosphorylation of Drp1 Ser579 in the neurons.The use of Cdk5 RNAi and Roscovitine could effectively inhibit the increase of Aβ_1-42-induced phosphorylation of Drp1 Ser579.The mitochondrial morphology results showed that Aβ_1-42resulted in increased mitochondrial division,while Cdk5 RNAi and Roscovitine inhibited mitochondrial cleavage caused by Aβ_1-42.Further detection of the effect of mutations in the phosphorylation site of Drp1 on mitochondria found that GFP-Drp1-S579A attenuated mitochondrial division induced by Aβ_1-42,whereas GFP-Drp1-S579D resulted in increased mitochondrial division.By detecting the apoptosis of neurons,Aβ_1-42could increase the level of Cleaved Caspase-3 which could reversed by Cdk5RNAi and Roscovitine.Heochest 33258 staining also showed that Cdk5 RNAi and Roscovitine could inhibite the apoptosis of neurons induced by Aβ_1-42.Conclusion:Cdk5-mediated phosphorylation of Drp1 Ser579 is involved in Aβ_1-42-induced mitochondrial division,which provides a new theoretical basis for the future prevention and treatment of AD and provides new drug targets.