Abstract
BackgroundAlzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing.ResultsWe have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces Aβ production by more than a third.ConclusionThese data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of Aβ production.
Highlights
Alzheimer’s disease (AD) is the leading cause of dementia in adults [1]
Amyloid Precursor Protein (APP)-paGFP can be followed as it traffics from the Golgi apparatus to LAMP1-labeled compartments In order to study the intracellular trafficking of APP from the Golgi apparatus in live cells, we generated expression constructs (Figure 1) containing full length APP (FL-APP) fused to an N-terminal HA epitope tag and photoactivatable Green Fluorescent Protein at its C-terminal cytoplasmic tail
We quantitated the fraction of fluorescent activated paGFP colocalized with LAMP-mRFP after 15 minutes of photoactivation, we found that 34.14 ± 5.10% (Mean ± SEM) of FL-APP and 34.70 ± 4.05% of βAPP was colocalized with LAMP1 (Figure 2c)
Summary
AD is the leading cause of dementia in adults [1]. A neuropathological hallmark of AD is the accumulation of β-amyloid (Aβ) in plaques in the brain [2]. Cleavage by β-secretase removes the N-terminal ectodomain, leaving a 99 residue C-terminal fragment (CTF) containing Aβ [3,4,5]. The CTF is processed by γ-secretase [6] to produce Aβ species ranging from 39–43 residues in length [7]. The 42 amino acid form of Aβ (Aβ42) has a higher propensity to aggregate, is more toxic in cell culture experiments and is the dominant component of amyloid plaques [8,9,10,11]. Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, the exact organelle in which Aβ is produced remains contentious. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing
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