title: Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid beta protein.
abstract: BACKGROUND
  The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by alpha-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of beta- and gamma-secretases generates the amyloid beta protein (Abeta). In this study, we investigated the effects of modulators of endocytosis on APP processing.
  
  
  RESULTS
  Human embryonic kidney cells were transfected with a dominant negative mutant of dynamin I, an important mediator of clathrin-dependent endocytosis, and APP proteolysis was analyzed. Overexpression of the mutant dynamin (dyn I K44A) resulted in increased shedding of the APP ectodomain (sAPPalpha), accumulation of the C-terminal alpha-secretase product C83, and a reduction in the release of Abeta. Levels of mature APP on the cell surface were increased in cells expressing dyn I K44A, and internalization of surface-immunolabeled APP, assessed by fluorescence microscopy, was inhibited. Dynamin is a substrate for protein kinase C (PKC), and it was hypothesized that activators of PKC, which are known to stimulate alpha-secretase-mediated cleavage of APP, might exert their effects by inhibiting dynamin-dependent endocytosis. However, the internalization of surface-biotinylated APP was unaffected by treatment of cells with phorbol 12-myristate 13-acetate in the presence of the alpha-secretase inhibitor TAPI-1.
  
  
  CONCLUSIONS
  The results indicate that APP is internalized by a dynamin-dependent process, and suggest that alterations in the activity of proteins that mediate endocytosis might lead to significant changes in Abeta production.
authors.affiliation: Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA 02118, USA. rmcarey@bu.edu
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Carey
authors.forename: Brigitte A
authors.initials: BA
authors.lastname: Balcz
authors.forename: Ignacio
authors.initials: I
authors.lastname: Lopez-Coviella
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Slack
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000096:biosynthesis*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D034284:Dynamin I / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D011493:Protein Kinase C; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D034284:Dynamin I