All Title Author
Keywords Abstract

PLOS ONE  2011 

Effects of Heparin and Enoxaparin on APP Processing and Aβ Production in Primary Cortical Neurons from Tg2576 Mice

DOI: 10.1371/journal.pone.0023007

Full-Text   Cite this paper   Add to My Lib


Background Alzheimer's disease (AD) is caused by accumulation of Aβ, which is produced through sequential cleavage of β-amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE1) and γ-secretase. Enoxaparin, a low molecular weight form of the glycosaminoglycan (GAG) heparin, has been reported to lower Aβ plaque deposition and improve cognitive function in AD transgenic mice. Methodology/Principal Findings We examined whether heparin and enoxaparin influence APP processing and inhibit Aβ production in primary cortical cell cultures. Heparin and enoxaparin were incubated with primary cortical cells derived from Tg2576 mice, and the level of APP and proteolytic products of APP (sAPPα, C99, C83 and Aβ) was measured by western blotting. Treatment of the cells with heparin or enoxaparin had no significant effect on the level of total APP. However, both GAGs decreased the level of C99 and C83, and inhibited sAPPα and Aβ secretion. Heparin also decreased the level of β-secretase (BACE1) and α-secretase (ADAM10). In contrast, heparin had no effect on the level of ADAM17. Conclusions/Significance The data indicate that heparin and enoxaparin decrease APP processing via both α- and β-secretase pathways. The possibility that GAGs may be beneficial for the treatment of AD needs further study.


[1]  Allan LM, Ballard CG, Rowan EN, Kenny RA (2009) Incidence and prediction of falls in dementia: a prospective study in older people. PLoS One 4: e5521.
[2]  Goedert M, Spillantini MG (2006) A century of Alzheimer's disease. Science 314: 777–781.
[3]  Kidd M (1964) Alzheimer's Disease–an Electron Microscopical Study. Brain 87: 307–320.
[4]  Terry RD, Gonatas NK, Weiss M (1964) Ultrastructural Studies in Alzheimer's Presenile Dementia. Am J Pathol 44: 269–297.
[5]  Glenner GG, Wong CW (1984) Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120: 885–890.
[6]  Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, et al. (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A 82: 4245–4249.
[7]  Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, et al. (1987) The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325: 733–736.
[8]  Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, et al. (1999) Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286: 735–741.
[9]  Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, et al. (1999) Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402: 533–537.
[10]  Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, et al. (1999) Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402: 537–540.
[11]  Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, et al. (2001) BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4: 233–234.
[12]  Haass C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A, et al. (1992) Amyloid beta-peptide is produced by cultured cells during normal metabolism. Nature 359: 322–325.
[13]  Sisodia SS, Koo EH, Beyreuther K, Unterbeck A, Price DL (1990) Evidence that beta-amyloid protein in Alzheimer's disease is not derived by normal processing. Science 248: 492–495.
[14]  Sisodia SS (1992) Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A 89: 6075–6079.
[15]  Esch FS, Keim PS, Beattie EC, Blacher RW, Culwell AR, et al. (1990) Cleavage of amyloid beta peptide during constitutive processing of its precursor. Science 248: 1122–1124.
[16]  Lammich S, Kojro E, Postina R, Gilbert S, Pfeiffer R, et al. (1999) Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad Sci U S A 96: 3922–3927.
[17]  Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL, et al. (1998) Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem 273: 27765–27767.
[18]  Koike H, Tomioka S, Sorimachi H, Saido TC, Maruyama K, et al. (1999) Membrane-anchored metalloprotease MDC9 has an alpha-secretase activity responsible for processing the amyloid precursor protein. Biochem J 343 Pt 2: 371–375.
[19]  Endres K, Fahrenholz F (2011) The Role of the Anti-Amyloidogenic Secretase ADAM10 in Shedding the APP-Like Proteins. Curr Alzheimer Res.
[20]  Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, et al. (1998) Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 95: 6448–6453.
[21]  Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol 8: 101–112.
[22]  Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, et al. (1999) Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J Neurosci 19: 8876–8884.
[23]  Kim HJ, Chae SC, Lee DK, Chromy B, Lee SC, et al. (2003) Selective neuronal degeneration induced by soluble oligomeric amyloid beta protein. FASEB J 17: 118–120.
[24]  Small DH, Losic D, Martin LL, Turner BJ, Friedhuber A, et al. (2004) Alzheimer's disease therapeutics: new approaches to an ageing problem. IUBMB Life 56: 203–208.
[25]  Hirsh J (1991) Heparin. N Engl J Med 324: 1565–1574.
[26]  Weitz JI (1997) Low-molecular-weight heparins. N Engl J Med 337: 688–698.
[27]  Kadusevicius E, Kildonaviciute G, Varanaviciene B, Jankauskiene D (2010) Low-molecular-weight heparins: pharmacoeconomic decision modeling based on meta-analysis data. Int J Technol Assess Health Care 26: 272–279.
[28]  Leveugle B, Ding W, Laurence F, Dehouck MP, Scanameo A, et al. (1998) Heparin oligosaccharides that pass the blood-brain barrier inhibit beta-amyloid precursor protein secretion and heparin binding to beta-amyloid peptide. J Neurochem 70: 736–744.
[29]  Ma Q, Dudas B, Hejna M, Cornelli U, Lee JM, et al. (2002) The blood-brain barrier accessibility of a heparin-derived oligosaccharides C3. Thromb Res 105: 447–453.
[30]  Pollack SJ, Sadler , II , Hawtin SR, Tailor VJ, Shearman MS (1995) Sulfonated dyes attenuate the toxic effects of beta-amyloid in a structure-specific fashion. Neurosci Lett 197: 211–214.
[31]  Pollack SJ, Sadler , II , Hawtin SR, Tailor VJ, Shearman MS (1995) Sulfated glycosaminoglycans and dyes attenuate the neurotoxic effects of beta-amyloid in rat PC12 cells. Neurosci Lett 184: 113–116.
[32]  Bergamaschini L, Rossi E, Storini C, Pizzimenti S, Distaso M, et al. (2004) Peripheral treatment with enoxaparin, a low molecular weight heparin, reduces plaques and beta-amyloid accumulation in a mouse model of Alzheimer's disease. J Neurosci 24: 4181–4186.
[33]  Sandwall E, O'Callaghan P, Zhang X, Lindahl U, Lannfelt L, et al. (2010) Heparan sulfate mediates amyloid-beta internalization and cytotoxicity. Glycobiology 20: 533–541.
[34]  Rose M, Dudas B, Cornelli U, Hanin I (2004) Glycosaminoglycan C3 protects against AF64A-induced cholinotoxicity in a dose-dependent and time-dependent manner. Brain Res 1015: 96–102.
[35]  Rose M, Dudas B, Cornelli U, Hanin I (2003) Protective effect of the heparin-derived oligosaccharide C3, on AF64A-induced cholinergic lesion in rats. Neurobiol Aging 24: 481–490.
[36]  Timmer NM, van Dijk L, van der Zee CE, Kiliaan A, de Waal RM, et al. (2010) Enoxaparin treatment administered at both early and late stages of amyloid beta deposition improves cognition of APPswe/PS1dE9 mice with differential effects on brain A beta levels. Neurobiol Dis 40: 340–347.
[37]  Small DH, Nurcombe V, Reed G, Clarris H, Moir R, et al. (1994) A heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regulation of neurite outgrowth. J Neurosci 14: 2117–2127.
[38]  Mok SS, Sberna G, Heffernan D, Cappai R, Galatis D, et al. (1997) Expression and analysis of heparin-binding regions of the amyloid precursor protein of Alzheimer's disease. FEBS Lett 415: 303–307.
[39]  Clarris HJ, Cappai R, Heffernan D, Beyreuther K, Masters CL, et al. (1997) Identification of heparin-binding domains in the amyloid precursor protein of Alzheimer's disease by deletion mutagenesis and peptide mapping. J Neurochem 68: 1164–1172.
[40]  McLaurin J, Fraser PE (2000) Effect of amino-acid substitutions on Alzheimer's amyloid-beta peptide-glycosaminoglycan interactions. Eur J Biochem 267: 6353–6361.
[41]  Williamson TG, Mok SS, Henry A, Cappai R, Lander AD, et al. (1996) Secreted glypican binds to the amyloid precursor protein of Alzheimer's disease (APP) and inhibits APP-induced neurite outgrowth. J Biol Chem 271: 31215–31221.
[42]  Fraser PE, Nguyen JT, Chin DT, Kirschner DA (1992) Effects of sulfate ions on Alzheimer beta/A4 peptide assemblies: implications for amyloid fibril-proteoglycan interactions. J Neurochem 59: 1531–1540.
[43]  McLaurin J, Franklin T, Zhang X, Deng J, Fraser PE (1999) Interactions of Alzheimer amyloid-beta peptides with glycosaminoglycans effects on fibril nucleation and growth. Eur J Biochem 266: 1101–1110.
[44]  Scholefield Z, Yates EA, Wayne G, Amour A, McDowell W, et al. (2003) Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase. J Cell Biol 163: 97–107.
[45]  Beckman M, Holsinger RM, Small DH (2006) Heparin activates beta-secretase (BACE1) of Alzheimer's disease and increases autocatalysis of the enzyme. Biochemistry 45: 6703–6714.
[46]  Klaver DW, Wilce MC, Gasperini R, Freeman C, Juliano JP, et al. (2010) Glycosaminoglycan-induced activation of the beta-secretase (BACE1) of Alzheimer's disease. J Neurochem 112: 1552–1561.
[47]  Leveugle B, Ding W, Durkin JT, Mistretta S, Eisle J, et al. (1997) Heparin promotes beta-secretase cleavage of the Alzheimer's amyloid precursor protein. Neurochem Int 30: 543–548.
[48]  Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, et al. (1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274: 99–102.
[49]  Ando K, Oishi M, Takeda S, Iijima K, Isohara T, et al. (1999) Role of phosphorylation of Alzheimer's amyloid precursor protein during neuronal differentiation. J Neurosci 19: 4421–4427.
[50]  Dovey HF, John V, Anderson JP, Chen LZ, de Saint Andrieu P, et al. (2001) Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J Neurochem 76: 173–181.
[51]  Sahin U, Weskamp G, Kelly K, Zhou HM, Higashiyama S, et al. (2004) Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands. J Cell Biol 164: 769–779.
[52]  Kveiborg M, Instrell R, Rowlands C, Howell M, Parker PJ (2011) PKCalpha and PKCdelta regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways. PLoS One 6: e17168.
[53]  Mendelson K, Swendeman S, Saftig P, Blobel CP (2010) Stimulation of platelet-derived growth factor receptor beta (PDGFRbeta) activates ADAM17 and promotes metalloproteinase-dependent cross-talk between the PDGFRbeta and epidermal growth factor receptor (EGFR) signaling pathways. J Biol Chem 285: 25024–25032.
[54]  Racchi M, Mazzucchelli M, Pascale A, Sironi M, Govoni S (2003) Role of protein kinase Calpha in the regulated secretion of the amyloid precursor protein. Mol Psychiatry 8: 209–216.
[55]  Klaver D, Hung AC, Gasperini R, Foa L, Aguilar MI, et al. (2010) Effect of heparin on APP metabolism and Abeta production in cortical neurons. Neurodegener Dis 7: 187–189.
[56]  Xia W, Ray WJ, Ostaszewski BL, Rahmati T, Kimberly WT, et al. (2000) Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid beta-protein generation. Proc Natl Acad Sci U S A 97: 9299–9304.
[57]  Hoey SE, Williams RJ, Perkinton MS (2009) Synaptic NMDA receptor activation stimulates alpha-secretase amyloid precursor protein processing and inhibits amyloid-beta production. J Neurosci 29: 4442–4460.
[58]  Kawarabayashi T, Younkin LH, Saido TC, Shoji M, Ashe KH, et al. (2001) Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease. J Neurosci 21: 372–381.
[59]  Barten DM, Guss VL, Corsa JA, Loo A, Hansel SB, et al. (2005) Dynamics of {beta}-amyloid reductions in brain, cerebrospinal fluid, and plasma of {beta}-amyloid precursor protein transgenic mice treated with a {gamma}-secretase inhibitor. J Pharmacol Exp Ther 312: 635–643.
[60]  Abramowski D, Wiederhold KH, Furrer U, Jaton AL, Neuenschwander A, et al. (2008) Dynamics of Abeta turnover and deposition in different beta-amyloid precursor protein transgenic mouse models following gamma-secretase inhibition. J Pharmacol Exp Ther 327: 411–424.
[61]  Cai XD, Golde TE, Younkin SG (1993) Release of excess amyloid beta protein from a mutant amyloid beta protein precursor. Science 259: 514–516.
[62]  Johnston J, O'Neill C, Lannfelt L, Winblad B, Cowburn RF (1994) The significance of the Swedish APP670/671 mutation for the development of Alzheimer's disease amyloidosis. Neurochem Int 25: 73–80.
[63]  Zhu H, Yu J, Kindy MS (2001) Inhibition of amyloidosis using low-molecular-weight heparins. Mol Med 7: 517–522.
[64]  Kisilevsky R, Lemieux LJ, Fraser PE, Kong X, Hultin PG, et al. (1995) Arresting amyloidosis in vivo using small-molecule anionic sulphonates or sulphates: implications for Alzheimer's disease. Nat Med 1: 143–148.
[65]  Reinhard C, Hebert SS, De Strooper B (2005) The amyloid-beta precursor protein: integrating structure with biological function. EMBO J 24: 3996–4006.
[66]  Klafki HW, Wiltfang J, Staufenbiel M (1996) Electrophoretic separation of betaA4 peptides (1-40) and (1-42). Anal Biochem 237: 24–29.


comments powered by Disqus