title: Evidence that secretase cleavage of cell surface Alzheimer amyloid precursor occurs after normal endocytic internalization.
abstract: Three different treatments (methylamine, colchicine, and 18 degrees C temperature block), known to disrupt normal endocytic internalization, significantly reduced the secretory cleavage of cell surface-derived Alzheimer amyloid precursor (APP) in non-transfected C6 cell cultures. Conversely, treatments with methylamine or colchicine had no significant effect on the secretion of total APP. Treatment of these cells with the lysosomotropic amine chloroquine resulted in a significant increase in the levels of both cell surface full-length APP and cell surface-derived secreted nexin II (NXII). Immunofluorescence analysis of C6 glioma cells transfected with APP751 indicated that under normal conditions, cell surface APP was internalized, and within 30 minutes was localized in discrete intracellular vesicles. These vesicles contained the endocytic tracer Texas red-conjugated ovalbumin and probably represented late endosomes or lysosomes. However, treatment of the transfected C6 cultures with methylamine or colchicine prevented localization of cell surface APP in intracellular vesicles, suggesting that these treatments altered the normal intracellular trafficking of cell surface-derived APP. Both the biochemical and immunofluorescence data are compatible with the suggestion that inhibition of normal endocytic internalization reduces the secretory cleavage of cell surface APP. Furthermore, our results suggest that following internalization, cell surface APP is cleaved by secretase(s) and secreted or routed to the lysosomes where it is degraded.
authors.affiliation: Department of Psychiatry, Mount Sinai School of Medicine, New York.
authors.forename: L M
authors.initials: LM
authors.lastname: Refolo
authors.forename: K
authors.initials: K
authors.lastname: Sambamurti
authors.forename: S
authors.initials: S
authors.lastname: Efthimiopoulos
authors.forename: M A
authors.initials: MA
authors.lastname: Pappolla
authors.forename: N K
authors.initials: NK
authors.lastname: Robakis
mesh_terms: D000544:Alzheimer Disease / Q000201:enzymology*; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D003080:Cold Temperature; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D010450:Endopeptidases / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005910:Glioma / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008744:Methylamines / Q000494:pharmacology; D011233:Precipitin Tests; D051381:Rats
chemical_list: D016564:Amyloid beta-Protein Precursor; D008744:Methylamines; D002738:Chloroquine; C027451:methylamine; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human