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

This study aimed to investigate the effects of HO-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 HO 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. HO 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 HO treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after HO 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 HO treatment, while its role in HO-induced Golgi morphological changes may be complex.