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. 2016 Jan;19(1):55-64.
doi: 10.1038/nn.4188. Epub 2015 Dec 7.

Visualizing APP and BACE-1 Approximation in Neurons Yields Insight Into the Amyloidogenic Pathway

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Free PMC article

Visualizing APP and BACE-1 Approximation in Neurons Yields Insight Into the Amyloidogenic Pathway

Utpal Das et al. Nat Neurosci. .
Free PMC article

Abstract

Cleavage of amyloid precursor protein (APP) by BACE-1 (β-site APP cleaving enzyme-1) is the rate-limiting step in amyloid-β (Aβ) production and a neuropathologic hallmark of Alzheimer's disease; thus, physical approximation of this substrate-enzyme pair is a crucial event with broad biological and therapeutic implications. Despite much research, neuronal locales of APP and BACE-1 convergence and APP cleavage remain unclear. Here we report an optical assay, based on fluorescence complementation, for visualizing in cellulo APP-BACE-1 interactions as a simple on/off signal. Combining this with other assays tracking the fate of internalized APP in hippocampal neurons, we found that APP and BACE-1 interacted in both biosynthetic and endocytic compartments, particularly along recycling microdomains such as dendritic spines and presynaptic boutons. In axons, APP and BACE-1 were cotransported, and they also interacted during transit. Finally, our assay revealed that the Alzheimer's disease-protective 'Icelandic' mutation greatly attenuates APP-BACE-1 interactions, suggesting a mechanistic basis for protection. Collectively, the data challenge canonical models and provide concrete insights into long-standing controversies in the field.

Figures

Figure 1
Figure 1. OptiCAB: An assay to detect APP/BACE-1 interactions in-situ
(a) The principle: APP and BACE-1 were tagged to complementary (VN- or VC-) fragments of VFP. Note that interaction of APP/BACE-1 leads to reconstitution of VFP fluorescence. Cloning strategies on right, numbers denote amino-acid residues and light-grey box represents the transmembrane domain. (b) Hippocampal neurons were cotransfected with APP:VN and BACE-1:VC, and time course of complementation was evaluated. Post-transfection times (in hours) shown on upper right, intensities quantified below (N=7 neurons). Note the gradual increase in somatic and neuritic fluorescence over time. (c) Neurons transfected with APP:VN/BACE-1:VC and soluble mCherry (volume filler); note punctate fluorescence in soma and dendrites (top panels, zoomed in inset). Attenuated complementation was seen with an APP C-terminus mutant (APPSTOP40, middle panels, also see ‘results’). Individually transfected VN/VC fragments were non-fluorescent (bottom panels). (d) Cleavage of APP:VN by BACE-1:VC. BACE-1 knock out fibroblasts were transfected with APP:VN alone or APP:VN + BACE-1:VC (with a γ-secretase inhibitor, “GSI”) and APP-cleavage products were analyzed by Western blotting. Note that in presence of BACE-1:VC, APP:VN was cleaved to generate a fragment of ∼30 kDa (red arrowhead) – the expected β-cleavage product of APP:VN. Corresponding BACE-1 blot confirms knockdown. (e) Neuro2A cells were co-transfected with APP (APP:GFP, APP:VN or APP:VC) and BACE-1:VC in presence of a γ-secretase inhibitor and APP-cleavage products were analyzed by Western blotting. Note that the major bands are consistent with CTF:GFP (∼40 kDa), CTF:VN (∼30 kDa), and CTF:VC (∼22 kDa) respectively, indicating that β-cleavage is not influenced by APP/BACE-1 complementation.
Figure 2
Figure 2. APP/BACE-1 interactions at the trans-Golgi network
(a) Schematic: Hippocampal neurons were co-transfected with APP:VN/BACE-1:VC (to mark APP/BACE-1 interaction sites) and NPYss-mCherry (to label Golgi-derived organelles, see ‘results’); and a subset of neurons were incubated at 20 °C, known to ‘trap’ vesicles at the TGN. (b) Note colocalization of complemented APP/BACE-1 puncta with NPYss – enhanced upon temperature block. Complemented APP/BACE-1 also extensively colocalized with the TGN marker GalT. Fluorescence intensities are quantified in (C); * p = 0.0002
Figure 3
Figure 3. Subcellular sites of APP/BACE-1 interaction in dendrites of hippocampal neurons
(a) Fluorescence puncta representing APP/BACE-1 interaction sites were seen throughout the dendritic shafts and spines (insets show complementation in spine head, neck and base). Note that many puncta are within spine heads or bases. (b) Quantification showing that ∼ 40% of APP/BACE-1 complementation occurs in close proximity to spines (283 spines from 12 neurons were analyzed). (c, d) To determine colocalization of APP/BACE-1 interaction sites with dendritic organelles, neurons were co-transfected with APP:VN/BACE-1:VC and the indicated organelle markers, and colocalization was determined quantitatively (see ‘methods’). Note that complemented APP/BACE-1 particles most conspicuously colocalized with the recycling endosome markers TfR and Rab11 (∼ 60%); and there was lesser (∼ 30%) colocalization with early endosomal (Rab5), lysosomal (LAMP-1), and Golgi-vesicle markers (NPYss). 20-24 dendrites from 12-18 neurons (two separate cultures) were analyzed; * p < 0.0001 (e) APP/BACE-1 complementation is also attenuated upon inhibiting clathrin-dependent endocytosis (by Dynasore) or by mutating an endocytosis motif in APP (APP-YENPTY, see ‘results’). 18-25 dendrites from 10-14 neurons (two separate cultures) were analyzed; * p = . 0.0148
Figure 4
Figure 4. APP/BACE-1 interaction in axons and presynaptic boutons of hippocampal neurons
(a, b) To determine colocalization of APP/BACE-1 interaction sites with axonal organelles, neurons were co-transfected with APP:VN/BACE-1:VC and the indicated organelle markers, and colocalization was determined quantitatively (see ‘methods’). Low power view of APP/BACE-1 BifC (arrowheads mark axon and section magnified in inset). (c) Complemented APP/BACE-1 particles most conspicuously colocalized with NPYss – a marker for Golgi-derived vesicles – while colocalization with endosomal markers was limited. 20-25 axons from two separate cultures were analyzed; * p = 0.0001 (d, e) To determine if presynapses are sites of APP/BACE-1 interaction, neurons were co-transfected with APP:VN/BACE-1:VC and synaptophysin:mRFP (to mark boutons). ∼ 96 % contained APP/BACE-1 BifC. 257 boutons from 15 neurons from two separate cultures were analyzed. (f-h) Quantitative analyses of APP/BACE-1 BifC presynaptic targeting, compared to a known presynaptic protein α-synuclein. Neurons were co-transfected with the respective BifC/YFP and soluble mCherry constructs, fluorescence at boutons was compared to flanking axons, normalizing for variations in expression (see “results” for details). Reprsentative images shown in (G); all quantified data shown in (H). Note presynaptic targeting of APP/BACE-1 BifC at boutons is significantly higher than soluble YFP, though expectedly not as high as α-synuclein (∼ 370-395 boutons from 20 neurons and two separate cultures were analyzed); p = 0.0001
Figure 5
Figure 5. Axonal transport of APP and BACE-1
(a) Neurons were transfected with either APP:GFP (left panels) or BACE-1:mCherry (right panels), and axonal transport was analyzed at a high temporal resolution (see “methods”). Schematic on top; representative kymographs with quantification shown below. Note similar kinetics of APP and BACE-1 transport.. Estimated velocities of APP vesicles: anterograde – 1.74 ± 0.03 μ/s, retrograde – 1.51 ± 0.06 μ/s and of BACE-1 vesicles: anterograde – 2.1 ± 0.06 μ/s, retrograde – 1.76 ± 0.07 μ/s. (∼ 360 APP and 300 BACE-1 vesicles from 20-22 neurons and two separate cultures were analyzed). (b) Neurons were co-transfected with APP:GFP (or BACE-1:GFP) and various RFP-tagged organelle markers as noted, and imaged live by simultaneous dual-cam imaging. Schematic on top; representative kymographs with quantification shown below. Note co-transport with NPYss, a marker for Golgi-derived vesicles. For APP/NPYss pair ∼ 320 APP and 390 NPYss particles (16-18 neurons from two separate cultures). For BACE-1/NPYss pair ∼ 410 BACE-1 and 500 NPYss (18-20 neurons from two separate cultures) were analyzed. (c) Neurons were co-transfected with APP:GFP and BACE-1:mCherry and imaged live by simultaneous dual-cam imaging. Note significant co-transport
Figure 6
Figure 6. Kinetics of APP/BACE-1 BifC particles
(a) Neurons were co-transfected with APP:VN and BACE-1:VC, and kinetics of complemented YFP particles were imaged live. (b) Representative kymographs (left) and quantification (right) of APP/BACE-1 BifC movements in axons. ∼200 (DIV7) and 160 (DIV14) vesicles from 12-14 axons from two separate cultures were analyzed, p= 0.7488 (between the mobile groups) and p = 0.6634 (between the stationary groups). (c) Representative kymographs (left) and quantification (right) of APP/BACE-1 BifC movements in dendrites. Note restricted movement of APP/BACE-1 BifC in regions around dendritic spines (also see text). ∼200 (DIV7) and 170 (DIV14) vesicles from 12-14 dendrites (two separate cultures) were analyzed. Image at bottom shows APP/BACE-1 BifC in a dendrite. p= 0.0002 (between the mobile groups) and p = 0.0002 (between the stationary groups). (d) APP/BACE-1 BifC particles in presynaptic boutons were largely stationary (85.19 ± 2.53 %) compared to the mobile particles (14.79 ± 2.53 %) as shown in the representative kymograph (right). ∼150 YFP vesicles from 10-12 neurons (two separate cultures) were analyzed, * p =0.0001)
Figure 7
Figure 7. Fate of internalized APP in neurons
(a) Strategy of APP internalization assay. Neurons were transfected with an APP construct containing a bungarotoxin (BTX)-binding site (BBS) at the luminal N-terminus (BBS-APP), and cells were incubated with Alexa-594 labeled BTX (BTX-594). Upon recycling, BTX-594 binds to BBS-APP and internalized Alexa-594 dye indicates APP endocytosis. (b) Schematic shows experimental design; a representative neuron with internalized APP shown below. Note APP internalization throughout somatodendritic compartments, magnified in inset. (c) Specificity of the BTX-594 uptake assay. BTX-594 internalization was seen in BBS-APP:GFP transfected neurons but not in neurons transfected with APP:GFP (lacking BBS domain). Representative of 5-7 neurons in each group. (d) Internalized dendritic BBS-APP particles were largely stationary (54.8 %) along with bi-directionally moving particles comprising anterograde (24.7 %) and retrograde (20.2 %). N = 128 particles from 12 neurons were analyzed; * p = 0.0001. (e, f) Colocalization of internalized BTX-594 with organelle markers. Neurons were co-transfected with BBS-APP and the stated organelle marker; and colocalization was analyzed in dendrites. Internalized BBS-APP predominantly colocalized with recycling endosomes and lysosomes (48.9% colocalization with TfR, 29% with Rab5, 26.7% with NPYss, and 71.9% with LAMP-1; 18-22 dendrites from 15-18 neurons – two separate cultures – were analyzed; * p = 0.0001).
Figure 8
Figure 8. Attenuated APP/BACE-1 interactions with an AD protective mutation
(a) Schematic showing location of AD protective mutation [APP(Ice)] and experimental strategy. (b, c) Neurons were transfected with APP:VN [WT or APP(Ice)] and BACE-1:VC, and fluorescence was analyzed. Complementation of APP(Ice) with BACE-1 was significantly lower in soma (normalized fluorescence intensity of APP(Ice):VN + BACE-1:VC was 0.22 ± 0.06 compared to APP:VN + BACE-1:VC – 1.0 ± 0.17; p = 0.0001) and dendrites (normalized fluorescence intensity of APP(Ice):VN + BACE-1:VC was 0.37 ± 0.07 compared to APP:VN + BACE-1:VC – 1.0 ± 0.18; p = 0.0042). 22-24 dendrites from 14-18 neurons – three separate cultures – were analyzed. (d) BACE-1 knock out fibroblasts were co-transfected with APP:VN [WT or APP(Ice)] and BACE-1:VC, and APP fragments were analyzed by Western blotting (in presence of γ-secretase inhibitor). Note that the ∼30 kDa band corresponding to the expected β-cleavage product of APP:VN (red arrowhead) is greatly attenuated in the APP(Ice) lane (performed four independent experiments); p = 0.0272.

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