Effects of suppressed autophagy on mitochondrial dynamics and cell cycle of N2a cells.
Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Meng-Cui
MC
Gui
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Bo
B
Chen
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Shan-Shan
SS
Yu
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. bubitao@tjh.tjmu.edu.cn.
Bi-Tao
BT
Bu
D000225:Adenine / Q000008:administration & dosage / Q000031:analogs & derivatives; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D016203:CDC2 Protein Kinase; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019926:Cyclin B / Q000096:biosynthesis; D018844:Cyclin-Dependent Kinases; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D063154:Mitochondrial Dynamics / Q000187:drug effects / Q000235:genetics; D024101:Mitochondrial Proteins / Q000096:biosynthesis; D009447:Neuroblastoma; D015398:Signal Transduction / Q000187:drug effects
D019926:Cyclin B; C476525:FIS1 protein, human; C477012:MAP1LC3A protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; C025946:3-methyladenine; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases; D000225:Adenine