Effects of suppressed autophagy on mitochondrial dynamics and cell cycle of N2a cells

Abstract

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 G²/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.

Keywords

cdc2 / cdk4 / Fis1 / LC3 / Mfn2

References

[1] LevineB, KroemerG. Autophagy in the pathogenesis of disease. Cell, 2008, 132(1): 27-42

[2] BenardG, BellanceN, JamesD, et al.. Mitochondrial bioenergetics and structural network organization. J Cell Sci, 2007, 120(Pt5): 838-848

[3] Scherz-ShouvalR, ElazarZ. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci, 2011, 36(1): 30-38

[4] TwigG, ElorzaA, MolinaA J, et al.. Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J, 2008, 27(2): 433-446

[5] SantosRX, CorreiaSC, WangX, et al.. A synergistic dysfunction of mitochondrial fissio/fusion dynamics and mitophagy in Alzheimer’s disease. J Alzheimers Dis, 2010, 20(Suppl2): S401-S412

[6] WongE, CuervoAM. Autophagy gone awry in neurodegenerative diseases. Nat Neurosci, 2010, 13(7): 805-811

[7] PickfordF, MasliahE, BritschgiM, et al.. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. J Clin Invest, 2008, 118(6): 2190-2199

[8] HiraiK, AlievG, NunomuraA, et al.. Mitochondrial abnormalities in Alzheimer’s disease. J Neurosci, 2001, 21(9): 3017-3023

[9] WangX, SuB, LeeHG, et al.. Impaired balance of mitochondrial fission and fusion in Alzheimer’s disease. J Neurosci, 2009, 29(28): 9090-9103

[10] BossyB, PetrilliA, KlinglmayrE, et al.. S-Nitrosylation of DRP1 does not affect enzymatic activity and is not specific to Alzheimer’s disease. J Alzheimers Dis, 2010, 20(Suppl2): S513-S526

[11] VincentI, JichaG, RosadoM, et al.. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer’s disease brain. J Neurosci, 1997, 17(10): 3588-3598

[12] VincentI, BuB, HudsonK, et al.. Constitutive Cdc25B tyrosine phosphatase activity in adult brain neurons with M phase-type alterations in Alzheimer’s disease. Neuroscience, 2001, 105(3): 639-650

[13] DranovskyA, VincentI, GregoriL, et al.. Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer’s disease pathology. Neurobiol Aging, 2001, 22(4): 517-528

[14] HarrisH, RubinszteinDC. Control of autophagy as a therapy for neurodegenerative disease. Nat Rev Neurol, 2012, 8(2): 108-117

[15] WilliamsA, SarkarS, CuddonP, et al.. Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway. Nat Chem Biol, 2008, 4(5): 295-305

[16] ChanDC. Mitochondrial fusion and fission in mammals. Annu Rev Cell Dev Biol, 2006, 22: 79-99

[17] ZhuX, PerryG, SmithMA, et al.. Abnormal mitochondrial dynamics in the pathogenesis of Alzheimer’s disease. J Alzheimers Dis, 2013, 33(Suppl1): S253-S262

[18] BuB, LiJ, DaviesP, et al.. Deregulation of cdk5, hyperphosphorylation, and cytoskeletal pathology in the Niemann-Pick type C murine model. J Neurosci, 2002, 22(15): 6515-6525