Effects Of ERK Pathway On GRASP65and Golgi Morphology In Oxidative Stress

Background and Objective:Oxidative Stress is a pathophysiological reaction originated from a significant increase in free radicals, such as reactive nitrogen and reactive oxygen species, which can finally result in cells or tissue injury. Oxidative stress is considered to be responsible for the development of a variety of diseases and the body aging. In recent years, increasing attentions has been paid to its role in neurological diseases such as neurodegenerative diseases, stroke, etc. Golgi apparatus is an important organelle functioning in cell metabolism. More and more evidences indicate that the Golgi apparatus is involved in oxidative stress, assisting in the stress signal transduction. In the meanwhile, the Golgi apparatus also shows a variety of morphological alterations in many physiological and pathophysiological processes and neurodegenerative diseases, mechanisms in which oxidative stress is strongly involved. Furthermore, Golgi fragmentation has been found to be classical feature in neurodegenerative diseases, and is even considered to be an early causative step in neural apoptosis. Since that, neuronal GA morphology may be a reliable index of activity of degeneration and thus should be studied further. Golgi reassembly and stacking proteins (GRASPs) are important components of the Golgi vesicle membranes, which are involved in many cell behaviors such as Golgi stacking, cell migration, cell division, apoptosis, and cargo secretion. GRASPs consist of two proteins, termed GRASP65and GRASP55. GRASP65, which is mainly located at the cis-Golgi, has been well studied and is found to be of great significance in Golgi morphological alterations in a variety of physiological and pathological physiological processes, such as the Golgi and centrosome orientation in cell migration, Golgi deoligomerization and disassembly in mitosis, as well as Golgi fragmentation in cell apoptosis.Extracellular signal-regulated kinase (ERK) is an important member in mitogen-activated protein kinase (MAPKs) system, which is involved in a variety of cellular functions such as cell proliferation, cell survival, cell apoptosis and cell migration. Considerable evidence has revealed that the MAPK/ERK pathway is activated in oxidative stress and contributes to the neuronal damage induced by oxidative stress. At the same time, studies have found that the phosphorylatioon of GRASP65by ERK is required for Golgi disassembly at interphase. Little attention, however, has been paid to the role of ERK pathway on Golgi apparatus and Golgi associated proteins in oxidative stress, in which Golgi apparatus is strongly involved. In this study, we will use the mice neuroma N2a cells in H2O2-induced oxidative stress, exploring the effects of MAPK/ERK pathway on GRASP65and Golgi morphology in oxidative stress.Methods:1. Establish a H2O2-induced oxidative stress model in vitro. The neuronal injury was measured by the MTT assay. Select the appropriate concentration of H2O2according to the inhibition rate, and observe the cell morphological changes under Light Microscopes.2. MTT assay to detect the effect of U0126on cell viability in H2O2-induced oxidative stress. Select the appropriate time for H2O2.3. In order to evaluate the role of ERK in oxidative stress, U0126was used to inhibit the ERK activation. Experimental grouping was designed as follows:control group; H2O2injury group; U0126inhibited group. The latter two groups were further divided into three different subsets for various exposed H2O2injury time (lh,3h,6h).4. Western blot to detect the expression of ERK, pERK and GRASP65, pGRASP65.5. Immunofluorescence laser scanning confocal to observe the morphological alterations of Golgi apparatus, as well as the expression of GRASP65and pGRASP65.6. The rate of cell apoptosis was measured by flow cytometric analysis.Results:1. MTT test showed the H2O2-induced N2a cell injury is concentration-and time-dependent, the higher of the H2O2concentration and the longer of the injury time, the stronger of the damage effect to cells; N2a cells exhibited obvious morphologic changes in H2O2-induced oxidative stress.2. Cell viability in H2O2injury group and U0126inhibited group both decreased with a time-dependent manner. U0126obviously promoted the cell survival in lh,3h compared with that in H2O2injury group (P<0.01); While cell viability in6h,12h,24h between U0126inhibited group and H2O2injury group has no significant difference (P>0.05). These results showed that the inhibition of ERK activation has a protective effect on oxidative stress mediated cell damage not more than6h.3. Western Blot results showed that expression of ERK has no significant difference between that in control group and in experimental groups. Meanwhile, expression of pERK significantly increased (P<0.01) at each time point in H2O2injury group compared with the control group, indicating that ERK activation occured in oxidative stress, and the pERK expression reaches peak at3h in H2O2injury group; U0126intervention reduced the pERK expression at each time point compared with that in H2O2injury group (P<0.01), showing that U0126intervention succesfully supressed the ERK activation in oxidative stress.4. Expression of GRASP65has no significant difference between that in control group and in experimental groups (P>0.05).5. Expression of pGRASP65significantly increased at each time point in H2O2injury group, compared with that in control group (P<0.01), indicating that GRASP65was phosphorylated in oxidative stress, and expression of pGRASP65shared a same trend with that of pERK; After the intervention of U0126, expression of pGRASP65significantly decreased (P<0.01) compared with that in H2O2injury group, indicating that the supression of ERK activation inhibited GRASP65phosphorylation.6. Immunofluorescence confocal:In control group, GRASP65labeled Golgi apparatus is compact, crescent-shaped, locating at the perinuclear region, pGRASP65has no significant expression. In H2O2injury group, expressioon of GRASP65showed no significant difference with that in control group; while pGRASP65expression was significantly increased, and the expression region coincide with that of GRASP65; Golgi apparatus exhibited scattered structure and irregular morphological changes at6h; In U0126inhibited group, there is no significant changes of GRASP65expression than that in H2O2injury group, and pGRASP65expression was significantly weakened at each time points compared with H2O2injury group, which share the same expression region to GRASP65; Golgi apparatus showed similar morphological alterations with that in H2O2injury group at6h, and the neurons with Golgi morphological alterations showed no significant differeces compared to that in H2O2injury group(P>0.05).7. Flow cytometric analysis results showed no significant early stage cell apoptosis both in H2O2injury group and in U0126inhibited group (apoptotic rate<0.1%), and each group showed no significant difference with the control group (P=NS). However, cell mortality (late stage apoptotic/necrotic cells) increased both in H2O2injury group and in U0126inhibited group, with significant differences compared to that in control group (P<0.01), and U0126obviously reduced cell mortality at each time point compared with that in H2O2injury group (P<0.05). These results showed that the inhibition of ERK activation rescued N2a from cell death in oxidative stress.Conclusion:1. ERK pathway was activated in oxidative stress and was involved in oxidative stress-mediated cell death; inhibition of ERK activation could play a protective role in this process;2. GRASP65was phosphorylated in oxidative stress by ERK;3. Golgi apparatus experienced irregular morphological alterations in H2O2-induced oxidative stress, but which may not be associated with ERK mediated GRASP65phosphorylation or cell death.