Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid β protein
1 Department of Pathology and Laboratory Medicine, Boston University School of Medicine, 715 Albany Street, Rm. L808, Boston MA 02118, USA
2 Gemeinnützige Salzburger Landeskliniken Betriebsgesellschaft mbH, Universitätsklinik für Innere Medizin III, Paracelsus Medizinische Privatuniversität, Müllner Hauptstrasse 48, A-5020 Salzburg, Austria
BMC Cell Biology 2005, 6:30 doi:10.1186/1471-2121-6-30Published: 11 August 2005
The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β- and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.
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 (sAPPα), accumulation of the C-terminal α-secretase product C83, and a reduction in the release of Aβ. 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 α-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 α-secretase inhibitor TAPI-1.
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 Aβ production.