Abstract
This chapter describes methods for establishing oxidative stress conditions that do not induce cell death in a neuronal cell culture model. We termed these conditions “mild oxidative stress,” as opposed to “severe oxidative stress,” which results in significant cell loss. Mild oxidative stress resembles more closely what happens in the aging brain than severe oxidative stress. The protocols we have delineated include the preparation and maintenance of mouse primary cortical cultures, the induction of oxidative stress by treatment with hydrogen peroxide, the assessment of cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the measurement of free radical production by the 2′,7′-dichlorofluorescein (DCF) assay, and western blot analysis of the amyloid precursor protein (APP) and β-site APP cleaving enzyme, BACE1, two key proteins associated with Alzheimer’s disease pathology and oxidative stress.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hussain I, Powell D, Howlett DR et al (1999) Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci 14:419–427
Lin X, Koelsch G, Wu S et al (2000) Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A 97:1456–1460
Sinha S, Anderson JP, Barbour R et al (1999) Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402:537–540
Vassar R, Bennett BD, Babu-Khan S et al (1999) Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286:735–741
Yan R, Bienkowski MJ, Shuck ME et al (1999) Membrane-anchored aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature 402:533–537
Tamagno E, Bardini P, Obbili A et al (2002) Oxidative stress increases expression and activity of BACE in NT2 neurons. Neurobiol Dis 10:279–288
Tamagno E, Guglielmotto M, Aragno M et al (2008) Oxidative stress activates a positive feedback between the gamma- and beta-secretase cleavages of the beta-amyloid precursor protein. J Neurochem 104:683–695
Tamagno E, Parola M, Bardini P et al (2005) Beta-site APP cleaving enzyme up-regulation induced by 4-hydroxynonenal is mediated by stress-activated protein kinases pathways. J Neurochem 92:628–636
Tamagno E, Guglielmotto M, Giliberto L et al (2009) JNK and ERK1/2 pathways have a dual opposite effect on the expression of BACE1. Neurobiol Aging 30:1563–1573
Guglielmotto M, Monteleone D, Giliberto L et al (2011) Amyloid-beta(4)(2) activates the expression of BACE1 through the JNK pathway. J Alzheimers Dis 27:871–883
Tong Y, Zhou W, Fung V et al (2005) Oxidative stress potentiates BACE1 gene expression and Abeta generation. J Neural Transm 112:455–469
Mouton-Liger F, Paquet C, Dumurgier J et al (2012) Oxidative stress increases BACE1 protein levels through activation of the PKR-eIF2alpha pathway. Biochim Biophys Acta 1822:885–896
Quiroz-Baez R, Rojas E, Arias C (2009) Oxidative stress promotes JNK-dependent amyloidogenic processing of normally expressed human APP by differential modification of alpha-, beta- and gamma-secretase expression. Neurochem Int 55:662–670
Kao SC, Krichevsky AM, Kosik KS, Tsai LH (2004) BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem 279:1942–1949
Kwak YD, Wang R, Li JJ et al (2011) Differential regulation of BACE1 expression by oxidative and nitrosative signals. Mol Neurodegener 6:17
Chen CH, Zhou W, Liu S et al (2011) Increased NF-kappaB signalling up-regulates BACE1 expression and its therapeutic potential in Alzheimer’s disease. Int J Neuropsychopharmacol 13:77–90
Jo DG, Arumugam TV, Woo HN et al (2010) Evidence that gamma-secretase mediates oxidative stress-induced beta-secretase expression in Alzheimer’s disease. Neurobiol Aging 31:917–925
Modarresi F, Faghihi MA, Patel NS et al (2011) Knockdown of BACE1-AS nonprotein-coding transcript modulates beta-amyloid-related hippocampal neurogenesis. Int J Alzheimers Dis 2011:929042
Perry G, Cash AD, Smith AI (2002) Alzheimer disease and oxidative stress. J Biomed Biotechnol 2:120–123
Swomley AM, Förster S, Keeney JT et al (2014) Abeta, oxidative stress in Alzheimer disease: evidence based on proteomics studies. Biochim Biophys Acta 1842:1248–1257
Wirz KT, Keitel S, Swaab DF et al (2014) Early molecular changes in Alzheimer disease: can we catch the disease in its presymptomatic phase? J Alzheimers Dis 38:719–740
Hardas SS, Sultana R, Clark AM et al (2013) Oxidative modification of lipoic acid by HNE in Alzheimer disease brain. Redox Biol 1:80–85
Tan J, Li QX, Ciccotosto G et al (2013) Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer’s disease amyloid precursor protein. PLoS One 8:e61246
Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. In: El-Gewely MR (eds) Biotech Annu Rev 11:127–152. Elsevier, New York. http://dx.doi.org/10.1016/S1387-2656(05)11004-7
Bass DA, Parce JW, Dechatelet LR et al (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 130:1910–1917
Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27:612–616
Brewer GJ, Torricelli JR, Evege EK, Price PJ (1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35:567–576
Lesuisse C, Martin LJ (2002) Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death. J Neurobiol 51:9–23
Acknowledgments
We thank Dr Joe Ciccotosto and Dr Laura Bica for sharing their expertise in the preparation of primary neuronal cultures, and Prof Sam Gandy for generously providing the 369 antibody. We also acknowledge support from the Judith Jane Mason and Harold Stannett Williams Memorial Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Tan, J., Li, QX., Evin, G. (2016). Effects of Mild and Severe Oxidative Stress on BACE1 Expression and APP Amyloidogenic Processing. In: Castrillo, J., Oliver, S. (eds) Systems Biology of Alzheimer's Disease. Methods in Molecular Biology, vol 1303. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2627-5_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2627-5_4
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2626-8
Online ISBN: 978-1-4939-2627-5
eBook Packages: Springer Protocols