Oxidative stress is considered important for the pathogenesis of Alzheimer disease (AD), which is characterized by the formation of senile plaques rich in amyloid beta-protein (Aβ). Aβ cytotoxicity has been found dependent on lysosomes, which are abundant in AD neurons and are shown to partially co-localize with Aβ. To determine whether oxidative stress has any influence on the relationship between lysosomes and Aβ1-42 (the most toxic form of Aβ), we studied the effect of hyperoxia (40% versus 8% ambient oxygen) on the intracellular localization of Aβ1-42 (assessed by immunocytochemistry) in retinoic acid differentiated SH-SY5Y neuroblastoma cells maintained in serum-free OptiMEM medium. In control cells, Aβ1-42 was mainly localized to small non-lysosomal cytoplasmic granules. Only occasionally Aβ1-42 was found in large (over 1 μm) lysosomal-associated membrane protein 2 positive vacuoles, devoid of the early endosomal marker rab5. These large Aβ1-42-containing lysosomes were not detectable in the presence of serum (known to suppress autophagy), while their number increased dramatically (up to 24-fold) after exposure of cells to hyperoxia during 5 days. Activation of autophagy by hyperoxia was confirmed by transmission electron microscopy. Furthermore, an inhibitor of autophagic sequestration 3-methyladenine prevented the accumulation of Aβ1-42-positive lysosomes due to hyperoxia. In parallel experiments, intralysosomal accumulation of Aβ1-40 following oxidative stress has been found as well. The results suggest that Aβ can be autophagocytosed and its accumulation within neuronal lysosomes is enhanced by oxidative stress. © 2005 Elsevier Ireland Ltd. All rights reserved.

There is increasing evidence for the toxicity of intracellular amyloid beta-protein (A beta) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD. enhances macroautophagy and leads to intralysosomal accumulation of A beta in Cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of A that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with A beta-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal A beta content (Vector<APPwt<APPswe). Furthermore, the degree of apoptosis was positively correlated with lysosomal membrane permeabilization, whereas inhibitors Of macroautophagy and lysosomal function decreased oxidant-induced apoptosis and diminished the differences in apoptotic response between different cell lines. These results suggest that oxidative stress can induce neuronal death through macroautophagy of A beta and consequent lysosomal membrane permeabilization, which may help explain the mechanisms behind neuronal loss in AD.

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days, and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide aditional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.

Amyloid-beta peptide (A beta), the main component of Alzheimer's disease (AD) senile plaques, has been found to accumulate within the lysosomal compartment of AD neurons. We have previously shown that in differentiated SH-SY5Y neuroblastoma cells cultured under normal conditions, the majority of A beta is localized extralysosomally, while oxidative stress significantly increases intralysosomal A beta content through activation of macroautophagy. It is, however, not clear which cellular compartments contain extralysosomal A beta in intact SH-SY5Y cells, and how oxidative stress influences the distribution of extralysosomal A beta. Using confocal laser scanning microscopy and immunoelectron microscopy, we showed that in differentiated neuroblastoma cells cultured under normal conditions A beta (A beta(40), A beta(42), and A beta oligomers) is colocalized with both membrane-bound organelles (endoplasmic reticulum, Golgi complexes, multivesicular bodies/late endosomes, lysosomes, exocytotic vesicles and mitochondria) and non-membrane-bound cytosolic structures. Neuroblastoma cells stably transfected with A beta PP Swedish KM670/671NL double mutation showed enlarged amount of A beta colocalized with membrane compartments. Suppression of exocytosis by 5 nM tetanus toxin resulted in a significant increase of the amount of cytosolic A beta as well as A beta colocalized with exocytotic vesicles, endoplasmic reticulum, Golgi complexes, and lysosomes. Hyperoxia increased A beta localization in the endoplasmic reticulum, Golgi apparatus, mitochondria, and lysosomes, but not in the secretory vesicles. These results indicate that in SH-SY5Y neuroblastoma cells intracellular A beta is not preferentially localized to any particular organelle and, to a large extent, is secreted from the cells. Challenging cells to hyperoxia, exocytosis inhibition, or A beta overproduction increased intracellular A beta levels but did not dramatically changed its localization pattern.