A fluorescence assay for detecting amyloid-β using the cytomegalovirus enhancer/promoter
Keywords:amyloid-beta, Alzheimer, cytomegalovirus, fluorescent reporter, high-throughput
Robust assays for detecting the effects of elevated concentrations of amyloid-β (Aβ) may facilitate Alzheimer’s disease research. An appropriate assay would be high-throughput and enable identification of drugs and genetic mutations that block the effects of Aβ, potentially leading to treatments for Alzheimer’s disease. We discovered that the commonly used cytomegalovirus (CMV) enhancer/promoter is sensitive to the effects of Aβ. By combining the CMV enhancer/promoter with a fluorescent protein, we created a reporter system that produces changes in intracellular fluorescence in response to Aβ. Using hippocampal neurons, we quantified the ability of a CMV-fluorescent protein recombinant reporter to detect both exogenously applied and overexpressed Aβ. This is the first report of a high-throughput enhancer/promoter-based Aβ detection method. The reporter is able to detect the effects of elevated concentrations of Aβ in a high-throughput fashion, providing a new tool for Alzheimer’s disease research and important knowledge about the commonly used CMV enhancer/promoter.
Sery O, Povova J, Misek I, Pesak L, Janout V (2013) Molecular mechanisms of neuropathological changes in Alzheimer's disease: a review. Folia Neuropathol 51: 1-9.
Querfurth HW, LaFerla FM (2010) Alzheimer's disease. N Engl J Med 362: 329-344.
Sadigh-Eteghad S, Sabermarouf B, Majdi A, Talebi M, Farhoudi M, et al. (2015) Amyloid-beta: a crucial factor in Alzheimer's disease. Med Princ Pract 24: 1-10.
Lacor PN, Buniel MC, Furlow PW, Clemente AS, Velasco PT, et al. (2007) Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer's disease. J Neurosci 27: 796-807.
Mucke L, Selkoe DJ (2012) Neurotoxicity of amyloid beta-protein: synaptic and network dysfunction. Cold Spring Harb Perspect Med 2: a006338.
Wilcox KC, Lacor PN, Pitt J, Klein WL (2011) Abeta oligomer-induced synapse degeneration in Alzheimer's disease. Cell Mol Neurobiol 31: 939-948.
Haes AJ, Chang L, Klein WL, Van Duyne RP (2005) Detection of a biomarker for Alzheimer's disease from synthetic and clinical samples using a nanoscale optical biosensor. J Am Chem Soc 127: 2264-2271.
Herskovits AZ, Locascio JJ, Peskind ER, Li G, Hyman BT (2013) A Luminex assay detects amyloid beta oligomers in Alzheimer's disease cerebrospinal fluid. PLoS One 8: e67898.
Holtta M, Hansson O, Andreasson U, Hertze J, Minthon L, et al. (2013) Evaluating amyloid-beta oligomers in cerebrospinal fluid as a biomarker for Alzheimer's disease. PLoS One 8: e66381.
Pitschke M, Prior R, Haupt M, Riesner D (1998) Detection of single amyloid beta-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy. Nat Med 4: 832-834.
Zhang YW, Thompson R, Zhang H, Xu H (2011) APP processing in Alzheimer's disease. Mol Brain 4: 3.
Honarnejad K, Daschner A, Giese A, Zall A, Schmidt B, et al. (2013) Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease. PLoS One 8: e80645.
Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, et al. (1999) Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284: 1811-1816.
Haas K, Sin WC, Javaherian A, Li Z, Cline HT (2001) Single-cell electroporation for gene transfer in vivo. Neuron 29: 583-591.
Otmakhov N, Lisman J (2012) Measuring CaMKII concentration in dendritic spines. J Neurosci Methods 203: 106-114.
Aow J, Dore K, Malinow R (2015) Conformational signaling required for synaptic plasticity by the NMDA receptor complex. Proc Natl Acad Sci U S A 112: 14711-14716.
Dore K, Aow J, Malinow R (2015) Agonist binding to the NMDA receptor drives movement of its cytoplasmic domain without ion flow. Proc Natl Acad Sci U S A 112: 14705-14710.
Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193-199.
Brooks AR, Harkins RN, Wang P, Qian HS, Liu P, et al. (2004) Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle. J Gene Med 6: 395-404.
Prosch S, Stein J, Staak K, Liebenthal C, Volk HD, et al. (1996) Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro. Biol Chem Hoppe Seyler 377: 195-201.
Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, et al. (2010) Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5: e10611.
Duan B, Cheng L, Gao Y, Yin FX, Su GH, et al. (2012) Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter. Theriogenology 78: 793-802.
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