The Effects of Oxidative Stress on the Subcellular Localization of Amyloid Precursor Protein in HTB-11 Neuroblastomas

Oxidative stress has been known to impede neuronal function. These impediments are associated with a variety of neurodegenerative disorders such as Alzheimer’s disease. Increased oxidants have been known to decrease the localization of amyloid precursor protein (APP) to the plasma membrane; however, it is unclear whether this is due to trafficking dysfunction or to downregulation of APP expression. I therefore aimed to explore this further using HTB-11 neuroblastomas as the model for this study. HTB-11 cells were grown in 100 nM retinoic acid (RA) to induce differentiation into neuronal phenotypes. Oxidative stress was induced with tert-butyl hydroperoxide at concentrations ranging from 1 μM to 31 μM. Cells were then immunostained using anti-APP; internal vesicles were concomitantly visualized with FITC-conjugated lectin. Confocal microscopy was used to image samples to obtain a 3D image of the cells and used to quantify the expression and the subcellular localization of APP; the Manders colocalization analysis was used to quantify the proportion of APP that colocalized to the membranes to determine if there were changes in the association of APP to membranes due to oxidative stress. Increased concentrations of tert-butyl hydroperoxide induced an increase in the expression of APP but did not cause a change in the colocalization of APP to the vesicle membranes. Lectin staining showed that vesicles tended to accumulate in the soma but their overall distribution along the axon was not affected. These results suggest that oxidative stress primarily increased endocytosis, resulting in concomitant accumulation of vesicular APP that did not localize to the plasma membrane without associated changes in the length of the projections of the cells. These results suggest that the intracellular APP accumulation is likely due to dysregulation of endosomal trafficking rather than stress-induced changes in the cytoskeleton. An understanding of the effects of oxidative stress on the trafficking of APP will help to increase our understanding of one of the fundamental aspects of Alzheimer’s disease pathology