Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOX increased significantly compared to the control. Finally, analysis using a new mitochondrial specific fluorescence dye, MitoPeDPP, indicated that treatment with ionomycin significantly increased the mitochondrial lipid hydroperoxide production in N1E-115 cells. The fluorescence emissions of Fluo-4 AM and MitoPeDPP were detected in the cell soma and neurite regions in ionomycin-treated N1E-115 cells. However, the emissions of neurites were much lower than those of the cell soma. TBARS values of ionomycin-treated cells significantly increased compared to the control. These results indicate that ionomycin induces calcium influx into the intracellular region and reactive oxygen species production in N1E-115 cells. Lipid hydroperoxide production was induced in ionomycin-treated N1E-115 cells. Calcium influx into the intracellular region is a possible activator of neurite degeneration.

Reactive oxygen species attack several living organs and induce cell death. Previously, we found axonal/dendrite degeneration before the induction of cell death in hydrogen peroxide treated neuro blastoma: N1E 115 cells and primary neurons. This phenomenon may be connected with membrane oxidation, microtubule desta bilization and disruption of intracellular calcium homeostasis. However, its detailed mechanisms are not fully understood. Here, we identified proteins after treatment with hydrogen peroxide using isolated neurites by liquid chromatography matrix assisted laser desorption/ionization time of flight/time of flight analysis. Twenty one proteins were increased after treatment with hydro gen peroxide. Specifically, 5 proteins which were secretogranin 1, heat shock protein family D member 1, Brain acid soluble protein 1, heat shock 70 kDa protein 5 and superoxide dismutase 1, were identified of all experiments and increased in isolated neurites of hydrogen peroxide treated cells compared to the controls. Further more, secretogranin 1 and heat shock protein family D member 1 protein expressions were significantly increased in normal aged and Alzheimer's transgenic mice brains. These results indicate that secretogranin 1 and heat shock protein family D member 1 might contribute to reactive oxygen species induced neurite degeneration. Both proteins have been related to neurodegenerative disorders, so their study may shed light on neurite dysfunction.

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