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

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.