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The role of Golgi reassembly and stacking protein 65 phosphorylation in H2O2-induced cell death and Golgi morphological changes

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Abstract

This study aimed to investigate the effects of H2O2-induced oxidative stress on cell viability and survival, as well as changes in the distribution of Golgi apparatus and in the level of Golgi reassembly and stacking protein 65 (GRASP65). Cell viability of cultured N2a cells treated with H2O2 was measured by the MTT assay. Apoptosis was measured by flow cytometry analyses. Cells labeled by indirect immunofluorescence were observed under confocal microscope to detect any Golgi morphological alterations; electron microscopy of Golgi apparatus was also done. Expression of GRASP65 and phospho-GRASP65 was examined by immunoblotting. H2O2 treatment reduced the cell viability and raised the cell mortality of N2a cells in a time-dependent manner. Notable changes were only observed in the distribution and morphology of Golgi apparatus at 6 h after H2O2 treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after H2O2 treatment, peaked at 3 h, and finally dropped at 6 h. Taken together, GRASP65 phosphorylation may have a critical role in inducing cell death at the early stage after H2O2 treatment, while its role in H2O2-induced Golgi morphological changes may be complex.

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Acknowledgments

We would like to thank Prof. Nobuhiro Nakamura (Kyoto Sangyo University) for his kind offering of anti-phospho-S277 GRASP65 antibody. This article was supported by the National Natural Science Foundation (Grant 81171239), China.

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Authors and Affiliations

  1. Department of Neurology, Second Hospital of Hebei Medical University, Shijiazhuang, 050000, People’s Republic of China

    Guang Ji & Moyuan Quan

  2. Department of Neurology, Second Xiangya Hospital, Central South University, Changsha, 410011, People’s Republic of China

    Guang Ji, Weiwei Zhang, Yang Chen, Hui Qu & Zhiping Hu

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  1. Guang Ji
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  2. Weiwei Zhang
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  3. Moyuan Quan
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  4. Yang Chen
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Correspondence to Zhiping Hu.

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Ji, G., Zhang, W., Quan, M. et al. The role of Golgi reassembly and stacking protein 65 phosphorylation in H2O2-induced cell death and Golgi morphological changes. Med Mol Morphol 49, 217–223 (2016). https://doi.org/10.1007/s00795-016-0138-3

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  • DOI: https://doi.org/10.1007/s00795-016-0138-3

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