amyloid-beta-peptides and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Cerebrovascular accumulation of amyloid-β protein (Aβ) aggregates in Alzheimer's disease (AD) is proposed to contribute to disease progression and brain inflammation as a result of Aβ-induced increases in endothelial monolayer permeability and stimulation of the endothelium for cellular adhesion and transmigration. These deficiencies facilitate the entry of serum proteins and monocyte-derived microglia into the brain. In the current study, a role for nuclear factor-κB (NF-κB) in the activation of cerebral microvascular endothelial cells by Aβ is explored.Quantitative immunocytochemistry is employed to demonstrate that Aβ(1-40) preparations containing isolated soluble aggregates elicit the most pronounced activation and nuclear translocation of NF-κB. This rapid and transient response is observed down to physiological Aβ concentrations and parallels phenotypic changes in endothelial monolayers that are selectively elicited by soluble Aβ(1-40) aggregates. While monomeric and fibrillar preparations of Aβ(1-40) also activated NF-κB, this response was less pronounced, limited to a small cell population, and not coupled with phenotypic changes. Soluble Aβ(1-40) aggregate stimulation of endothelial monolayers for adhesion and subsequent transmigration of monocytes as well as increases in permeability were abrogated by inhibition of NF-κB activation. Together, these results provide additional evidence indicating a role for soluble Aβ aggregates in the activation of the cerebral microvascular endothelium and implicate the involvement of NF-κB signaling pathways in Aβ stimulation of endothelial dysfunction associated with AD.

Topics: Amyloid beta-Peptides; Analysis of Variance; Anti-Inflammatory Agents; Brain; Cell Adhesion; Cell Line, Transformed; Cell Movement; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium; Enzyme Inhibitors; Humans; Hydrocortisone; Leupeptins; NF-kappa B; Peptide Fragments; Permeability; Signal Transduction; Time Factors

Recent studies of gamma-secretase have pointed out that it may be comprised of a multisubunit complex with presenilin 1 and presenilin 2 as central components. Elucidation of the biochemical mechanism of this enzymatic activity will provide important information for developing gamma-secretase inhibitors in Alzheimer's disease therapy. Here we describe the biochemical characterization of gamma-secretase activities using a sensitive, membrane-based assay system. Membranes were isolated from 293 cells expressing C99, the substrate of gamma-secretase. Upon incubation at 37 degrees C, C99 is cleaved by the endogenous gamma-secretase, and Abeta peptides are liberated. Abeta40 and Abeta42 gamma-secretase activities are very similar in terms of their kinetic profiles and pH dependence, supporting the notion that a single enzyme is involved in both Abeta40 and Abeta42 production. Pepstatin A inhibited Abeta40 and Abeta42 gamma-secretase activities with similar potency. Peptide difluoroketone and peptide aldehyde inhibitors inhibited Abeta40 production in a dose-dependent fashion, enhanced Abeta42 production at low concentrations, and inhibited Abeta42 production at high concentrations. Although the selective increase of Abeta42 by low concentrations of peptide difluoroketone and peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occurs in a membrane-based assay system suggests that these compounds increase Abeta42 by a direct effect on gamma-secretase. The ability of these compounds to increase Abeta42 production may reflect allosteric modulation of the gamma-secretase complex by a mechanism related to that responsible for the increase of Abeta42 production by mutations in presenilins.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Brefeldin A; Cell Line; Cell Membrane; Cell-Free System; Endopeptidases; Enzyme Activation; Humans; Hydrolysis; Leupeptins; Membrane Proteins; Oligopeptides; Peptide Fragments; Protease Inhibitors; Protein Transport; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity

We have studied the effects of peptide aldehyde protease inhibitors on the secretion of beta-amyloid peptide 1-40 (Abeta(1-40)) and Abeta(1-42) by HEK 293 and COS-1 cells expressing beta-amyloid precursor protein with the Swedish double mutation. A multiphasic SDS-polyacrylamide gel electrophoresis system was used for the discrimination of Abeta(1-40) and Abeta(1-42). Calpain inhibitor I, carbobenzoxyl-Leu-Leu-leucinal, and calpeptin were found to reduce the amount of Abeta(1-40) released into the medium in a dose-dependent manner. The reduction of Abeta(1-40) after treatment with 50 microM calpain inhibitor I or 5 microM carbobenzoxyl-Leu-Leu-leucinal was accompanied by a slight increase of Abeta(1-42) released into the medium. These observations suggest that the cleavages at residues 40 and 42 are accomplished by different enzyme activities.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; COS Cells; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Glycoproteins; Humans; Leupeptins; Peptide Fragments; Tumor Cells, Cultured