title: Intracellular production of beta A4 amyloid of Alzheimer's disease: modulation by phosphoramidon and lack of coupling to the secretion of the amyloid precursor protein.
abstract: The amyloid precursor protein (APP) undergoes abnormal metabolism in Alzheimer's disease, resulting in the accumulation of beta A4 amyloid in the brain. Normal APP metabolism includes the release of a truncated form (sAPP) which has been cleaved at the alpha-secretase site within the beta A4 amyloidogenic domain. However, intact forms of beta A4 protein may also be generated by the beta- and gamma-secretases. Soluble forms of beta A4 have been detected in various cell lines and in cerebrospinal fluid. Previous studies of protein kinase C activation have suggested a reciprocal relationship between sAPP secretion and beta A4 production and release. We find that phorbol ester activation of protein kinase C in untransfected SH-SY5Y neuroblastoma cells increases the release of sAPP without affecting beta A4 secretion. We provide further evidence for intracellular beta A4 production. Treatment of SY5Y cells with the protease inhibitor phosphoramidon results in a 2-fold increase in beta A4 secretion and an increase in the amount of beta A4 recovered from cell lysates, yet it does not affect sAPP secretion. The protease inhibitors thiorphan and N-[(RS)-2-carboxy-3-phenylpropanoyl]-L-leucine had no effect on beta A4 or sAPP secretion. The lysosomotropic agents chloroquine and NH4Cl decreased beta A4 secretion, providing additional evidence for the involvement of intracellular acidic compartments in the production of beta A4. Our results therefore demonstrate a double dissociation between the secretion of sAPP and beta A4 in the SH-SY5Y cell line. The effect of phosphoramidon supports previous studies which show that metalloproteases are involved in the biogenesis of beta A4.
authors.affiliation: Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: S J
authors.initials: SJ
authors.lastname: Fuller
authors.forename: E
authors.initials: E
authors.lastname: Storey
authors.forename: Q X
authors.initials: QX
authors.lastname: Li
authors.forename: A I
authors.initials: AI
authors.lastname: Smith
authors.forename: K
authors.initials: K
authors.lastname: Beyreuther
authors.forename: C L
authors.initials: CL
authors.lastname: Masters
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000096:biosynthesis*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases; D002738:Chloroquine / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D007163:Immunosorbent Techniques; D007700:Kinetics; D009447:Neuroblastoma / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D006020:Glycopeptides; D011480:Protease Inhibitors; D000643:Ammonium Chloride; D015240:Phorbol 12,13-Dibutyrate; D002738:Chloroquine; D011493:Protein Kinase C; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C008890:phosphoramidon