Protective role of AKT-glycogen synthase kinase-3β signaling pathway in the N2a cell with SOD1G93Amutation

doi:10.16098/j.issn.0529-1356.2016.04.001

Abstract

Abstract:

Objective To explore the role and mechanism of AKT-GSK-3β signaling pathway in the N2a cells with SOD1^G93A mutation. Methods Mouse neuroblastoma cell line N2a was transfected with pEGFP-WT-SOD1 and pEGFP-G93A-SOD1 plasmids. The expressions changes of AKT, GSK-3β and cyclin D1 were detected in the cell model using RT-PCR and Western blotting technique. The expressions of GSK-3β and cyclin D1 were detected in the N2a cells with SOD1^G93A mutation regulated by knockdown of AKT, and the changes of cell proliferation and survival were determined by MTS assay. Results No significant changes were observed in AKT and GSK-3β total protein in the N2a cells transfected with pEGFP-G93A-SOD1, compared with those transfected with pEGFP-WT-SOD1. However, the phospho-AKT(Ser473), phospho-GSK-3β(Ser 9) and cyclinD1 protein in the N2a cells transfected with pEGFP-G93A-SOD1 were up-regulated significantly compared with N2a cells transfected with pEGFP-WT-SOD1 at 24 hours and 48 hours after transfection. The immunouorescence staining showed that the expressions of p-AKT(Ser473)、p-GSK-3β(Ser 9) and cyclin D1 were increased in the N2a cells transfected with pEGFP-G93A-SOD1 at 24 hours and 48 hours. AKT, p-AKT(Ser473), GSK-3β, p-GSK-3β (Ser9) and cyclin D1 protein were all downregulated significantly at 48 hours and 72 hours after knockdown of AKT compared with the negative control. MTS assay showed that N2a cell proliferation and viability were significantly decreased at 72 hours, 96 hours and 120 hours after transfection. Conclusion The mutation of SOD1 affects the phosphorylated modification of AKT and GSK-3β after translation and the expression of cyclin D1 in the N2a cells. AKT may affect the proliferation and survival of N2a cells with SOD1^G93A mutation by regulating the expression of GSK-3β and cyclin D1.

Key words: Amyotrophic lateral sclerosis, Neuroblastoma cell N2a, AKT, Glycogen synthase kinase-3β, Cyclin D1, Western blotting, Mouse

CHEN Yan-chun GUAN Ying-jun* LIU Yong-xin ZHOU Feng-hua LIU Huan-cai WANG Qiao-zhen MA Xiao-jun ZHAO Chun-yan. Protective role of AKT-glycogen synthase kinase-3β signaling pathway in the N2a cell with SOD1^G93Amutation[J]. AAS, 2016, 47(4): 433-441.

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Protective role of AKT-glycogen synthase kinase-3β signaling pathway in the N2a cell with SOD1^G93Amutation

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