Amyloid precursor protein truncated at any of the ?-secretase sites is not cleaved to ?-amyloid

Abstract

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.

Dynamics of the Microglial/Amyloid Interaction Indicate a Role in Plaque Maintenance

“…Immediately after the last imaging session, mice were transcardially perfused with ice-cold 4% paraformaldehyde in PBS, and the brains were removed, postfixed overnight in the same fixative, cryoprotected in increasing concentrations of glycerol (10 -20%) in phosphate buffer, frozen in 2-propanol (Merck, Darmstadt, Germany), and stored at Ϫ80°C until sectioning on a freezing-sliding microtome (40 m sections at a plane parallel to the window orientation). Sections were either observed after washing without additional manipulation or were immunostained using antibodies to visualize microglia (rat polyclonal Iba-1; Waco, Richmond, VA), A␤ [mouse monoclonal ␤1 or rabbit polyclonal NT12 (Paganetti et al, 1996;Sturchler-Pierrat et al, 1997;Pfeifer et al, 2002); kindly provided by P. Paganetti and M. Staufenbiel, Novartis Pharma, Basel, Switzerland], astrocytes (rabbit anti-GFAP; Dako, High Wycombe, UK), and/or lysosomes [mouse monoclonal lamp-1 (lysosome-associated membrane protein 1) (BD Biosciences, San Diego, CA) and rat monoclonal lamp-1 (Abcam, Cambridge, UK)]. Visualization was via the appropriate fluorescent-coupled secondary antibodies (Alexa 568/594/633; Invitrogen) or via biotinylated antibodies, an avidinbiotin reaction and Vector SG Blue.…”

A New Functional Screening System for Identification of Regulators for the Generation of Amyloid β-Protein

“…This chimeric cDNA was inserted into pCxN at KpnI and NotI sites, which have been introduced at the EcoRI site of pCxN (26) (designated as pCxN-C53NICD). The resulting C53NICD does not contain a signal peptide similar to C100 construct without a signal peptide, which has been well characterized (27)(28)(29)(30)(31). A plasmid named pHES1-pac, which carries a puromycin resistance gene (pac), driven by the HES-1 promoter (25,32), was constructed as follows.…”

Noninvasive Magnetic Resonance Imaging Detection of Cerebral Amyloid Angiopathy-Related Microvascular Alterations Using Superparamagnetic Iron Oxide Particles in APP Transgenic Mouse Models of Alzheimer's Disease: Application to Passive Aβ Immunotherapy

“…Male mice were passively immunized weekly by intraperitoneal injections of 0.5 mg of ␤1 mouse monoclonal IgG2a antibody that recognizes amino acids 3-6 of human A␤ (Paganetti et al, 1996). Age-matched control mice received weekly intraperitoneal injections of 0.5 mg of a control antibody (monoclonal mouse anti-wheat auxin IgG2a antibody; AMS Biotechnology).…”

“…NT12 antiserum raised against A␤ 40 (Paganetti et al, 1996) was diluted 1:2000 in PBS with 3% goat serum and incubated overnight at 4°C. After rinsing, sections were incubated for 1 h with biotinylated anti-rabbit IgG secondary antibody (BA1000, Vector Laboratories) diluted 1:200 in PBS.…”