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

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

Journal Name: Current Alzheimer Research

Volume 16 , Issue 2 , 2019

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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).

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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Alzheimer’s A. 2016 Alzheimer’s disease facts and figures. Alzheimers Dement 12(4): 459-509. (2016).
Zhang Y, Song W. Islet amyloid polypeptide: another key molecule in Alzheimer’s pathogenesis? Prog Neurobiol 153: 100-20. (2017).
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).
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).
Vassar R. BACE1 inhibitor drugs in clinical trials for Alzheimer’s disease. Alzheimers Res Ther 6(9): 89. (2014).
Koelsch G. BACE1 Function and inhibition: implications of intervention in the amyloid pathway of Alzheimer’s disease pathology. Molecules 22(10): E1723. (2017).
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).
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).
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).
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).
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).

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Article Details

Year: 2019
Published on: 04 February, 2019
Page: [128 - 134]
Pages: 7
DOI: 10.2174/1567205016666181212151540
Price: $65

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