GSK3β Could Promotes Mitochondrial Fission Through Phosphorylating Drp1in Neurons

Mitochondrial dynamics is that mitochondria are not static organelle, they are stay in a fission and fusion balance process which is main regulated by dynamin-related protein1(Drpl) and mitochondrial fusion protein MFN1/2, OPA1. Recent studies suggests that the mitochondrial dynamics being disturbed is a key role in neurodegenerative disease especially Alzheimer’s disease (AD) and Huntington disease (HD). The disturbance of mitochondrial dynamics in these disease results in the excessive fission of mitochondria which could influence the ATP production, promote neuronal apoptosis and reduced the synaptic formation. Glycogen synthesis kinase3β(GKS3β) is a key regulating kinase in neuronal system and widely involved in the neuronal development and differentiation. The formal studies have proved that the activity of GSK3P is elevated in Alzheimer’s disease, however, whether GSK3β could regulate the mitochondrial dynamics is still unclear. It is been proved that GSK3β could interact with Drp1in Yeast which suggests that this interaction may exists in neuronal system and play important roles in the maintenance of neuronal function.In order to find out whether GSK3β could regulate mitochondrial dynamics in neuronal system, we built Immunofluorescence and Western Blotting methods to investigate the molecular mechanism. Then we block the interaction of GSK3β and Drpl to observe if this will correct the learning and memory deficient of APP/PS1mutant mouse which is a model of Alzheimer’s disease. The results are summarized as follows:1. GSK3β promotes mitochondrial fission in neuronsIn contrast to control, overexpression of GSK3β results in shortage of mitochondria and promotes mitochondrial fission in DIV7neurons; while using GSK3β inhibitor AR-A014418would lead to excessive fusion of mitochondria and reduces mitochondrial fission in neurons.2. Drp1mediated GSK3β induced Mitochondrial FissionIn condition of degradation of Drpl by using interfere RNA, GSK3β could not facilitate mitochondrial fragmentation while overexpression OPA1has no effects proved that GSK3P induce mitochondrial fission through Drpl. Then we use immunofluorescence and immunoprecipitation to verify the interaction of GSK3β and Drp1.3. Phosphorylation of Drpl at Ser40and Ser44involved in GSK3β induced Mitochondrial fragmentationFirstly we predicted the potential sites on Drp1that could be phosphorylated by GSK3β by using bioinformatics tools, GPS2.1; then we using gene cloning technology to mutate these sites and mimic the phosphorylation state or non-phosphorylation state. In morphology observation experiment we found Ser40and Ser44were key amino acids in GSK3β induced mitochondrial fission.4. GSK3β phosphorylate Drpl at Ser40and Ser44through different mechanismsTo find the molecular mechanisms of the phosphorylation process, we made the specific phosphorylation antibody to Ser40and Ser44. We found that Ser44could be directly phosphorylated by GSK3β while Ser40phosphorylation needs a priming site which means its phosphorylation dependent on Ser44being phosphorylated first.5. Phosphorylation of Drpl regulate its GTPase activity and localization to mitochondria but not the dimerization of DrplUsing GTPase activity detection kit, we found the phosphorylation of Drpl will enhance its GTPase activity; then we extracted the mitochondria component and the western blotting experiment showed phosphorylated Drpl localized to mitochondria fraction more while non-phosphorylated Drpl stay in cytoplasm fraction more. Otherwise, the phosphorylation of Drp1has no effect on its dimerization.6. Phosphorylation of Drpl reduces ATP production and spine density The morphology study showed phosphorylation of Drp1results in excessive mitochondrial fission. Furthermore, using biochemical detection method we found the phosphorylation of Drpl at Ser40and Ser44will reduce the ATP production and spine formation.7. Tat-Drp1DD block the interaction between GSK3p and Drp1In the basis of former work, we designed a peptides Tat-Drp1DD to block the GSK3β and Drp1interaction. Using Tat-Drp1DD treat GSK3βoverexpression neurons could reverse GSK3β induced mitochondrial fission.8. Tat-Drp1DD injectionreduces the phosphorylation level of Drpl at Ser40and Ser44We microinjected the polypeptides into hippocampi of APP/PS1mice. The results showed that the phosphorylation of Drpl at Ser40and Ser44were significantly reduced in Tat-Drp1DD injected mice. Meanwhile, the activity of caspase3has also reduced and the level of synaptophysin elevated. Notably, the amyloid burden were also relieved in Tat-Drp1DD injected mice.9. Hippocampus injection of Tat-Drp1DD improved learning and memory ability of APP/PS1miceWe choose12months old APP/PS1mice that had dementia symptom outcomes. Experimental group inject in hippocampus with Tat-Drp1DD then analysis with novel object recognition and Morris Water Maze test. Contrast to control mice, Tat-Drp1DD injected mice show significant improving in learning and memory ability.Conclusion:Our results provide that GSK3P could promote mitochondrial fragmentation by phosphorylated Drpl at Ser40and Ser44. Moreover, we found out the phosphorylation of Drpl would enhance its GTPase activity, it also promotes its localization to mitochondrial fraction is the reason why its phosphorylation could results in excessive mitochondrial fission. Then we designed a polypeptide Tat-Drp1DD to block the GSK3p and Drpl interaction and this peptide would correct the learning and memory deficient of AD mouse model.