Background: Alzheimer�s disease (AD) is characterized by cerebral deposition of ?-amyloid peptide (A?). A? is\nproduced by sequential cleavage of the Amyloid Precursor Protein (APP) by ?- and ?-secretases. Many studies have\ndemonstrated that the internalization of APP from the cell surface can regulate A? production, although the exact\norganelle in which A? is produced remains contentious. A number of recent studies suggest that intracellular\ntrafficking also plays a role in regulating A? production, but these pathways are relatively under-studied. The\ngoal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular\nAPP processing.\nResults: We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein\n(paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan\nFluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the\nGolgi to downstream compartments identified with compartment markers tagged with red fluorescent protein\n(mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because\n?-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of\nfluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that\nAPP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the\nhighly selective ?-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is\nbeing cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal\nAPP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3\n(AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we\nare able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces A? production by more\nthan a third.\nConclusion: These data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a\nlikely site of A? production.
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