We have studied the effects of peptide aldehyde protease inhibitors on the secretion of ␤-amyloid peptide 1-40 (A␤(1-40)) and A␤(1-42) by HEK 293 and COS-1 cells expressing ␤-amyloid precursor protein with the Swedish double mutation. A multiphasic SDS-polyacrylamide gel electrophoresis system was used for the discrimination of A␤(1-40) and A␤(1-42). Calpain inhibitor I, carbobenzoxyl-Leu-Leu-leucinal, and calpeptin were found to reduce the amount of A␤(1-40) released into the medium in a dose-dependent manner. The reduction of A␤(1-40) after treatment with 50 M calpain inhibitor I or 5 M carbobenzoxyl-Leu-Leu-leucinal was accompanied by a slight increase of A␤(1-42) released into the medium. These observations suggest that the cleavages at residues 40 and 42 are accomplished by different enzyme activities.Characteristic pathological findings in the brains of Alzheimer's disease (AD) 1 patients include amyloid deposits containing the ␤-amyloid peptide (A␤) as a major component. A␤ is a proteolytic fragment of the ␤-amyloid precursor protein (APP), a large transmembrane glycoprotein with a single membranespanning region that exists in different isoforms (1). Proteolytic cleavage of APP just outside the transmembrane region within the A␤ sequence (2) by an unknown enzyme called ␣-secretase releases the soluble ectodomain of APP (3). This cleavage precludes A␤ production. Soluble A␤ peptides are generated by an alternative proteolytic processing pathway during normal metabolism of cells expressing APP (4 -6). They can also be detected in cerebrospinal fluid (7). A double mutation within the APP gene (NL670/671) found in a Swedish early onset AD family (8) increases A␤ production (9 -12). The enzymes cleaving APP to produce the amino and carboxyl termini of A␤ are referred to as ␤-and ␥-secretases, respectively (13). None of these secretases has yet been identified.Analyses of the A␤ peptides in cerebrospinal fluid (14), in conditioned media from HEK 293 cells transfected with an APP cDNA containing the Swedish double mutation (15) and from IMR 32 cells (16) revealed a series of A␤ peptides of various lengths. A minor portion of those has a longer carboxyl terminus and ends at amino acid residue 42 or 43 of the A␤ sequence. Point mutations located in codon 717 of the APP gene (APP770 numbering) found in some familial forms of AD increase the proportion of the longer A␤ peptides generated by transfected cells (17,18). The longer peptides aggregate more rapidly (19) and were reported to be deposited preferentially in amyloid plaques (20 -22) and at an early stage (23). Taken together, these findings strongly suggest that the peptides ending at residue 42 or 43 are critical for amyloidogenesis in AD.Recent findings indicate that A␤ production can be reduced by inhibition of ␥-secretase cleavage of APP. Higaki et al. (24) observed inhibition of A␤ formation with carbobenzoxyl-ValPhe-alaninal in Chinese hamster ovary cells transfected with a wild type APP cDNA. We previously reported the reduction of A␤ secretion from...

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BackgroundThe quantification of amyloid-beta (Aβ) peptides in blood plasma as potential biomarkers of Alzheimer’s disease (AD) is hampered by very low Aβ concentrations and the presence of matrix components that may interfere with the measurements.MethodsWe developed a two-step immunoassay for the simultaneous measurement of the relative levels of Aβ38, Aβ40 and Aβ42 in human EDTA plasma. The assay was employed for the study of 23 patients with dementia of the Alzheimer’s type (AD-D) and 17 patients with dementia due to other reasons (OD). We examined relationships with the clinical diagnosis, cerebral Aβ load as quantified by amyloid-positron emission tomography, apolipoprotein E genotype, Aβ levels and Tau protein in cerebrospinal fluid.ResultsPreconcentration of plasma Aβ peptides by immunoprecipitation substantially facilitated their immunological measurements. The Aβ42/Aβ40 and Aβ42/Aβ38 ratios were statistically significantly lower in the AD-D patients than in the OD group. The areas under the receiver operating characteristic curves reached 0.87 for the Aβ42/Aβ40 ratio and 0.80 for the Aβ42/Aβ38 ratio.ConclusionsThe measurement of plasma Aβ peptides with an immunological assay can be improved by preconcentration via immunoprecipitation with an antibody against the Aβ amino-terminus and elution of the captured peptides by heating in a mild detergent-containing buffer. Our findings support the Aβ42/Aβ40 ratio in blood plasma as a promising AD biomarker candidate which correlates significantly with the validated core biomarkers of AD. Further studies will be needed for technical advancement of the assay and validation of the biomarker findings.Electronic supplementary materialThe online version of this article (10.1186/s13195-018-0448-x) contains supplementary material, which is available to authorized users.

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βA4 secretion occurs upon processing of amyloid protein precursor (APP) by β‐secretase (N‐terminus of βA4) and γ‐secretase (C‐terminus). To determine the sequence of these activities and the processing intermediate of βA4, we expressed several truncated APP molecules in human HEK‐293 cells. Immunofluorescence and biotinylation studies indicated that full‐length APP or APP lacking the cytosolic domain both were located intracellularly, associated with the cell surface and secreted. APPs truncated after amino acid 40, 42, or 43 of βA4 were not inserted into cell membranes, were found intracellularly but not on the cell surface, and were efficiently secreted into the culture medium. The secretion of APP truncated at amino acid 40 of βA4 occurred without proteolytic processing. Neither βA4 nor P3 (the product of the α‐secretase) was secreted from any of the APP molecules truncated at the γ‐secretase sites. In sharp contrast to this, when the C‐terminal 100 amino acids of APP were expressed (APP truncated at the N‐terminus of βA4), a robust βA4 secretion was observed. Thus, the C‐terminal fragment of APP produced by β‐secretase activity is likely to be the processing intermediate of βA4. © 1996 Wiley‐Liss, Inc.