) Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidantinduced apoptosis of cultured neuroblastoma cells, Autophagy, 7:12, 1528-1545 To link to this article: https://doi.org/10.4161/auto.7.12.18051
Do not distribute.Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells Keywords: Alzheimer disease, amyloid β-protein, amyloid precursor protein, apoptosis, autophagy, lysosomes, oxidative stressAbbreviations: 3MA, 3-methyladenine; AD, Alzheimer disease; ATG5, autophagy-related protein 5; Aβ, amyloid β-protein; APP, amyloid precursor protein; NH4Cl, ammonium chloride; APPwt, wild-type APP695; APPswe, Swedish KM670/671NL double mutation; α-APP, α-secretase processed APP; DAPT, LY-374973, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; DCF, carboxy-H 2 DCFDA; DAPI, 4' 6-diamidino-2-phenylindole; E64d, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, EST; HEK, human embryonic kidney; LAMP-2, lysosomal associated membrane protein-2; LC3, microtubule-associated protein 1 light chain 3; NFT, neurofibrillary tangles; RA, retinoic acid; ROS, reactive oxygen speciesIncreasing 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 additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.