Protective effect of autophagy inhibition on ischemia-reperfusioninduced injury of N2a cells

Abstract

Autophagy is a conserved and programmed catabolic process that degrades damaged proteins and organelles. But the underlying mechanism and functions of autophagy in the ischemiareperfusion (IR)-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 (a key molecule of autophay complex, Beclin-1/class III PI3K) and LC-3II (an autophagy marker) were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases p70S6K and mTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Administration of 3-methyladenine (3MA), an inhibitor of class III PI3K, abolished autophagy during reperfusion, while employment of rapamycin, an inhibitor of mTORC1 (normally inducing autophagy), surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the decline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K complex-dependent and mTORC1-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.

Keywords

ischemia/reperfusion / autophagy / LC-3 / Beclin-1 / mTORC1 / 3-methyladenine / rapamycin

References

[1] WenYD, ShengR, ZhangLS, et al.. Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways. Autophagy, 2008, 4(6): 762-769

[2] DanielJK, ScottDE. Autophagy as a regulated pathway of cellular degradation. Science, 2000, 290(5497): 1717-1721

[3] EskelinenEL, SaftigP. Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta, 2009, 1793(4): 664-673

[4] RamiA, LanghagenA, SteigerS. Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death. Neurobiol Dis, 2008, 29(1): 132-141

[5] AdhamiF, LiaoG, MorozovYM, et al.. Cerebral ischemia-hypoxia induces intravascular coagulation and autophagy. Am J Pathol, 2006, 169(2): 566-583

[6] PuyalJ, ClarkePG. Targeting autophagy to prevent neonatal stroke damage. Autophagy, 2009, 5(7): 1060-1061

[7] ZhengYQ, LiuJX, LiXZ, et al.. RNA interference-mediated downregulation of Beclin1 attenuates cerebral ischemic injury in rats. Acta Pharmacol Sin, 2009, 30(7): 919-927

[8] CarloniS, BuonocoreG, BalduiniW. Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury. Neurobiol Dis, 2008, 32(3): 329-339

[9] ParkHK, ChuK, JungKH, et al.. Autophagy is involved in the ischemic preconditioning. Neurosci Lett, 2009, 451(1): 16-19

[10] GuoYC, WangJF, WangZQ, et al.. Melatonin protects N2a against ischemia/reperfusion injury through autophagy enhancement. J Huazhong Univ Sci Technol [Med Sci], 2010, 30(1): 1-7

[11] KabeyaY, MizushimaN, UenoT. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J, 2000, 19(21): 5720-5728

[12] MizushimaN. Methods for monitoring autophagy. Int J Biochem Cell Biol, 2004, 36(12): 2491-2502

[13] MatsuiY, TakagiH, QuX, et al.. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res, 2007, 100(6): 914-922

[14] KiharaA, KabeyaY, OhsumiY, et al.. Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep, 2001, 2(4): 330-335

[15] ZhongY, WangQJ, LiX, et al.. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat Cell Biol, 2009, 11(4): 468-476

[16] LiangC, FengP, KuB, et al.. Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nat Cell Biol, 2006, 8(4): 688-699

[17] TakahashiY, CoppolaD, MatsushitaN, et al.. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nat Cell Biol, 2007, 9(10): 1142-1151

[18] LevineB, SinhaS, KroemerG. Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy, 2008, 4(5): 600-606

[19] PattingreS, TassaA, QuX, et al.. Bcl-2 antiapoptotic proteins inhibit Beclin-1-dependent autophagy. Cell, 2005, 122(6): 927-939

[20] JungCH, JunCB, RoSH, et al.. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell, 2009, 20(7): 1992-2003

[21] GanleyIG, LamduH, WangJ, et al.. ULK1-ATG13-FIP200 complex mediates mTOR signaling and is essential for autophagy. J Biol Chem, 2009, 284(18): 12 297-12 305

[22] BanasiakKJ, XiaY, HaddadGG. Mechanisms underlying hypoxia-induced neuronal apoptosis. Prog Neurobil, 2000, 62(3): 215-249

[23] HosokawaN, HaraT, KaizukaT, et al.. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell, 2009, 20(7): 1981-1991

[24] de TorresC, MunellF, FerrerI, et al.. Identification of necrotic cell death by the TUNEL assay in the hypoxic-ischemic neonatal rat brain. Neurosci Lett, 1997, 230(1): 1-4

[25] ColbourneFT, SutherlandGR, AuerRN, et al.. Electron microscopic evidence against apoptosis as the mechanism of neuronal death in global ischemia. J Neurosci, 1999, 19(11): 4200-4210

[26] SorianoMA, FerrerI, Rodríguez-FarréE, et al.. Apoptosis and c-Jun in the thalamus of the rat following cortical infarction. Neuroreport, 1996, 7(2): 425-428

[27] HaraA, MoriH, NiwaM. Novel apoptotic evidence for delayed neuronal death in the hippocampal CA1 pyramidal cells after transient ischemia. Stroke, 2000, 31(1): 236-238

[28] WeiL, YingDJ, CuiL, et al.. Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats. Brain Res, 2004, 1022(1–2): 54-61

[29] GustafssonAB, GottliebRA. Recycle or die: the role of autophagy in cardioprotection. J Mol Cell Cardiol, 2008, 44(4): 654-661

[30] GozuacikD, KimchiA. Autophagy and cell death. Curr Top Dev Biol, 2007, 78: 217-245

[31] WeiY, PattingreS, SinhaS, et al.. JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Mol Cell, 2008, 30(6): 678-688

[32] JungCH, RoSH, CaoJ, et al.. mTOR regulation of autophagy. FEBS Lett, 2010, 584(7): 1287-1295

[33] MeijerAJ, CodognoP. Regulation and role of autophagy in mammalian cells. Int J Biochem Cell Biol, 2004, 36(12): 2445-2462

[34] ScottRC, JuhaszG, NeufeldTP. Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr Biol, 2007, 17(19): 1-11

[35] LeeSB, KimS, LeeJ, et al.. ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase. EMBO Rep, 2007, 8: 360-365

[36] ZengXH, KinsellaTJ. Mammalian target of rapamycin and S6 kinase 1 positively regulate 6-thioguanine-induced autophagy. Cancer Res, 2008, 68(7): 2384-2390