Effect of disruption of mitochondrial and endoplasmic reticulum calcium homeostasis on neurites in hydrogen peroxide- and ionomycin-treated cells.

Abstract

Neurite degeneration is seen in the early stages of many neurodegenerative diseases, and is strongly related to oxidative damage. Possible mechanisms underlying this morphological change include dysruption of calcium homeostasis, increased membrane oxidation, and destabilization of cytoskeletal proteins. However, the detailed mechanisms leading to neuronal damage has not been elucidated. Calcium plays an important role in neuronal changes caused by oxidative stress. Mitochondria and endoplasmic reticulum (ER) play roles in intracellular calcium storages. One mechanism of neurite degeneration associated with oxidative stress may be related to calcium-mediated interactions between mitochondria and ER. In this study, we evaluated intracellular calcium homeostasis, mitochondria, and ER localization when neurite degeneration was induced in neuroblastoma cells that had extended neurites. Treatment with hydrogen peroxide (H₂O₂) and the calcium ionophore ionomycin induced mitochondria-dependent superoxide production and membrane oxidation. When examining the involvment of calcium efflux from the ER and mitochondria, treatment with a ryanodine receptor inhibitor ruthenium red significantly reduced intracellular calcium concentrations in ionomycin-treated cells. Electron microscopy in neurite degeneration areas revealed numerous fragmented mitochondria in ionomycin-treated cells, and mitochondrial swelling was observed in the same area of H₂O₂-treated cells. Next, we investigated proteins related to fusion and fission by western blotting. However, mitochondrial dysfunction occurs in both cases and is therefore thought to be associated with neurite degeneration. Our results suggest that H₂O₂ and ionomycin cause neurite degeneration via different mechanisms. Interactions between mitochondria and the ER through unknown crosstalk pathways and calcium transfer may play an important role in maintaining neurite function.