Login Register Cart 0
Generic placeholder image

Current Alzheimer Research

Editor-in-Chief

ISSN (Print): 1567-2050
ISSN (Online): 1875-5828

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe Translate in Chinese
Research Article

A Novel Cell-based β-secretase Enzymatic Assay for Alzheimer’s Disease

Author(s): Bruno De Araujo Herculano, Zhe Wang and Weihong Song*

Volume 16, Issue 2, 2019

Page: [128 - 134] Pages: 7

DOI: 10.2174/1567205016666181212151540

Price: $65

Abstract

Background: Deposition of the amyloid β protein (Aβ) into neuritic plaques is the neuropathological hallmark of Alzheimer’s Disease (AD). Aβ is generated through the cleavage of the Amyloid Precursor Protein (APP) by β-secretase and γ-secretase. Currently, the evaluation of APP cleavage by β-secretase in experimental settings has largely depended on models that do not replicate the physiological conditions of this process.

Objective: To establish a novel live cell-based β-secretase enzymatic assay utilizing a novel chimeric protein that incorporates the natural sequence of APP and more closely replicates its cleavage by β-secretase under physiological conditions.

Methods: We have developed a chimeric protein construct, ASGβ, incorporating the β-site cleavage sequence of APP targeted by β-secretase and its intracellular trafficking signal into a Phosphatase-eGFP secreted reporter system. Upon cleavage by β-secretase, ASGβ releases a phosphatase-containing portion that can be measured in the culture medium, and an intracellular fraction that can be detected through Western Blot. Subsequently, we have generated a cell line stably expressing ASGβ that can be utilized to assay β-secretase in real time.

Results: ASGβ is specifically targeted by β-secretase, being cleaved exclusively at the site responsible for the generation of Aβ. Dosage response to β-secretase inhibitors shows that β-secretase activity can be positively correlated to phosphatase activity in culture media.

Conclusion: Our findings suggest this system could be a high-throughput tool to screen compounds that aim to modulate β-secretase activity and Aβ production under physiological conditions, as well as evaluating factors that regulate this cleavage.

Keywords: Alzheimer's disease, BACE1, β-secretase, secreted alkaline phosphatase, enzymatic assay, Amyloid Precursor Protein (APP).

« Previous Next »
[1]
Kang Y, Zhang Y, Feng Z, Liu M, Li Y, Yang H, et al. Nutritional deficiency in early life facilitates aging-associated cognitive decline. Curr Alzheimer Res 14: 841-9. (2017).
[2]
Liu F, Zhang Y, Liang Z, Sun Q, Liu H, Zhao J, et al. Cleavage of potassium channel Kv2.1 by BACE2 reduces neuronal apoptosis. Mol Psychiatry 23(7): 1542-54. (2018).
[3]
Zhang S, Cai F, Wu Y, Bozorgmehr T, Wang Z, Zhang S, et al. A presenilin-1 mutation causes Alzheimer disease without affecting Notch signaling. Mol Psychiatry (2018).
[http://dx.doi.org/10.1038/s41380-018-0101-x]
[4]
Li Y, Zhou W, Tong Y, He G, Song W. Control of APP processing and Abeta generation level by BACE1 enzymatic activity and transcription. FASEB J 20(2): 285-92. (2006).
[5]
Deng Y, Wang Z, Wang R, Zhang X, Zhang S, Wu Y, et al. Amyloid-beta protein (Abeta) Glu11 is the major beta-secretase site of beta-site amyloid-beta precursor protein-cleaving enzyme 1(BACE1), and shifting the cleavage site to Abeta Asp1 contributes to Alzheimer pathogenesis. Eur J Neurosci 37(12): 1962-9. (2013).
[6]
Zhang S, Wang Z, Cai F, Zhang M, Wu Y, Zhang J, et al. BACE1 Cleavage Site selection critical for amyloidogenesis and Alzheimer’s pathogenesis. J Neurosci 37(29): 6915-25. (2017).
[7]
Sun X, He G, Qing H, Zhou W, Dobie F, Cai F, et al. Hypoxia facilitates Alzheimer’s disease pathogenesis by up-regulating BACE1 gene expression. Proc Natl Acad Sci USA 103(49): 18727-32. (2006).
[8]
Sun X, Tong Y, Qing H, Chen CH, Song W. Increased BACE1 maturation contributes to the pathogenesis of Alzheimer’s disease in Down syndrome. FASEB J 20(9): 1361-8. (2006).
[9]
Ly PT, Wu Y, Zou H, Wang R, Zhou W, Kinoshita A, et al. Inhibition of GSK3β-mediated BACE1 expression reduces Alzheimer-associated phenotypes. J Clin Invest 123(1): 224-35. (2013).
[10]
Oh M, Kim SY, Oh YS, Choi DY, Sin HJ, Jung IM, et al. Cell-based assay for beta-secretase activity. Anal Biochem 323(1): 7-11. (2003).
[11]
Pietrak BL, Crouthamel MC, Tugusheva K, Lineberger JE, Xu M, DiMuzio JM, et al. Biochemical and cell-based assays for characterization of BACE-1 inhibitors. Anal Biochem 342(1): 144-51. (2005).
[12]
Tomasselli AG, Qahwash I, Emmons TL, Lu Y, Leone JW, Lull JM, et al. Employing a superior BACE1 cleavage sequence to probe cellular APP processing. J Neurochem 84(5): 1006-17. (2003).
[13]
Volbracht C, Penzkofer S, Mansson D, Christensen KV, Fog K, Schildknecht S, et al. Measurement of cellular beta-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems. Anal Biochem 387(2): 208-20. (2009).
[14]
Zhou W, Li X, Huang D, Li T, Song W. No significant effect of 7,8-dihydroxyflavone on APP processing and Alzheimer-associated phenotypes. Curr Alzheimer Res 12(1): 47-52. (2015).
[15]
Attallah C, Etcheverrigaray M, Kratje R, Oggero M. A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species. Protein Expr Purif 132: 27-33. (2017).
[16]
Kober L, Zehe C, Bode J. Optimized signal peptides for the development of high expressing CHO cell lines. Biotechnol Bioeng 110(4): 1164-73. (2013).
[17]
Bai Y, Markham K, Chen F, Weerasekera R, Watts J, Horne P, et al. The in vivo brain interactome of the amyloid precursor protein. Mol Cell Proteomics 7(1): 15-34. (2008).
[18]
Perreau VM, Orchard S, Adlard PA, Bellingham SA, Cappai R, Ciccotosto GD, et al. A domain level interaction network of amyloid precursor protein and Abeta of Alzheimer’s disease. Proteomics 10(12): 2377-95. (2010).
[19]
Russo C, Venezia V, Repetto E, Nizzari M, Violani E, Carlo P, et al. The amyloid precursor protein and its network of interacting proteins: physiological and pathological implications. Brain Res Brain Res Rev 48(2): 257-64. (2005).
[20]
Mullan M, Crawford F, Axelman K, Houlden H, Lilius L, Winblad B, et al. A pathogenic mutation for probable Alzheimer’s disease in the APP gene at the N-terminus of beta-amyloid. Nat Genet 1(5): 345-7. (1992).
[21]
Citron M, Oltersdorf T, Haass C, McConlogue L, Hung AY, Seubert P, et al. Mutation of the beta-amyloid precursor protein in familial Alzheimer’s disease increases beta-protein production. Nature 360(6405): 672-4. (1992).
[22]
Thinakaran G, Teplow DB, Siman R, Greenberg B, Sisodia SS. Metabolism of the “Swedish” amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the “beta-secretase” site occurs in the golgi apparatus. J Biol Chem 271(16): 9390-7. (1996).
[23]
Koo EH, Squazzo SL. Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J Biol Chem 269(26): 17386-9. (1994).
[24]
Ben Halima S, Mishra S, Raja KMP, Willem M, Baici A, Simons K, et al. Specific inhibition of beta-secretase processing of the alzheimer disease amyloid precursor protein. Cell Reports 14(9): 2127-41. (2016).
[25]
Gibson Wood W, Eckert GP, Igbavboa U, Muller WE. Amyloid beta-protein interactions with membranes and cholesterol: causes or casualties of Alzheimer’s disease. Biochim Biophys Acta 1610(2): 281-90. (2003).
[26]
Scott JD, Li SW, Brunskill AP, Chen X, Cox K, Cumming JN, et al. Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A beta-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer’s Disease. J Med Chem 59(23): 10435-50. (2016).
[27]
May PC, Willis BA, Lowe SL, Dean RA, Monk SA, Cocke PJ, et al. The potent BACE1 inhibitor LY2886721 elicits robust central Abeta pharmacodynamic responses in mice, dogs, and humans. J Neurosci 35(3): 1199-210. (2015).
[28]
Alzheimer’s A. 2016 Alzheimer’s disease facts and figures. Alzheimers Dement 12(4): 459-509. (2016).
[29]
Zhang Y, Song W. Islet amyloid polypeptide: another key molecule in Alzheimer’s pathogenesis? Prog Neurobiol 153: 100-20. (2017).
[30]
Qing H, He G, Ly PT, Fox CJ, Staufenbiel M, Cai F, et al. Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer’s disease mouse models. J Exp Med 205(12): 2781-9. (2008).
[31]
Zeng J, Chen L, Wang Z, Chen Q, Fan Z, Jiang H, et al. Marginal vitamin A deficiency facilitates Alzheimer’s pathogenesis. Acta Neuropathol 133(6): 967-82. (2017).
[32]
Vassar R. BACE1 inhibitor drugs in clinical trials for Alzheimer’s disease. Alzheimers Res Ther 6(9): 89. (2014).
[33]
Koelsch G. BACE1 Function and inhibition: implications of intervention in the amyloid pathway of Alzheimer’s disease pathology. Molecules 22(10): E1723. (2017).
[34]
Egan MF, Kost J, Tariot PN, Aisen PS, Cummings JL, Vellas B, et al. Randomized trial of verubecestat for mild-to-moderate Alzheimer’s disease. N Engl J Med 378(18): 1691-703. (2018).
[35]
Lahiri DK, Maloney B, Long JM, Greig NH. Lessons from a BACE1 inhibitor trial: off-site but not off base. Alzheimers Dement 10(5)(Suppl.): S411-9. (2014).
[36]
Blume T, Filser S, Jaworska A, Blain JF, Koenig G, Moschke K, et al. BACE1 inhibitor MK-8931 alters formation but not stability of dendritic spines. Front Aging Neurosci 10: 229. (2018).
[37]
Zhu K, Peters F, Filser S, Herms J. Consequences of pharmacological bace inhibition on synaptic structure and function. Biol Psychiatry 84(7): 478-87. (2018).
[38]
Citron M, Westaway D, Xia W, Carlson G, Diehl T, Levesque G, et al. Mutant presenilins of Alzheimer’s disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. Nat Med 3(1): 67-72. (1997).

Rights & Permissions Print Cite
Article Metrics
71
6
1
© 2024 Bentham Science Publishers | Privacy Policy