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Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway

Nature Neuroscience volume 19, pages 55–64 (2016)Cite this article

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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.

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Figure 1: OptiCAB: an assay to detect APP–BACE-1 interactions in situ.
Figure 2: APP–BACE-1 interactions in the trans-Golgi network.
Figure 3: Subcellular sites of APP–BACE-1 interaction in dendrites of hippocampal neurons.
Figure 4: APP–BACE-1 interaction in axons and presynaptic boutons of hippocampal neurons.
Figure 5: Axonal transport of APP and BACE-1.
Figure 6: Kinetics of APP/BACE-1 BiFC particles.
Figure 7: Fate of internalized APP in neurons.
Figure 8: Attenuated APP–BACE-1 interactions with an Alzheimer's disease protective mutation.

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Acknowledgements

We thank G. Banker (Oregon Health and Science University) for the NPYss:mCherry and TfR:mCherry/GFP constructs, G. Patterson and J. Lipincott-Schwartz (NIH) for the GalT:mCherry/GFP constructs, Z.-H. Sheng (NIH) for the synaptophysin-mRFP construct, W. Almers (Oregon Health and Science University) for the broken CMV promoter and D. Boehning (The University of Texas Health Science Center) for the APP(Lon):GFP construct. Constructs from Addgene and investigators are acknowledged in the Online Methods. U.D. was partly supported by a pilot award from the UCSD Alzheimer's Center (P50 AG005131). This work was supported by grants from the NIH/NIA (R01AG048218 and NIH/NINDS (R01NS075233) to S.R.

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Authors and Affiliations

  1. Department of Pathology, University of California, San Diego, La Jolla, California, USA.,

    Utpal Das, Lina Wang, Archan Ganguly & Subhojit Roy

  2. Department of Neurosciences, University of California, San Diego, La Jolla, California, USA.,

    Utpal Das, Lina Wang, Archan Ganguly, Junmi M Saikia, Steven L Wagner, Edward H Koo & Subhojit Roy

  3. Department of Medicine and Physiology, National University of Singapore, Yong Loo Lin School of Medicine, Singapore

    Edward H Koo

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Contributions

U.D. and S.R. designed the assays and wrote the paper. U.D. designed and performed most of the experiments and data analysis. L.W. designed and performed the synaptic targeting and some of the axonal transport experiments. A.G. and L.W. helped with neuronal cultures and J.M.S. performed and analyzed some of the axonal transport experiments. S.L.W. and E.H.K. provided key reagents and technical advice.

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Integrated supplementary information

Supplementary Figure 1 Expression levels of various constructs used in this study.

(a) Neurons were transfected with the indicated plasmids and soluble mCherry (volume filler), fixed ~ 5-6 h posttransfection, and immunostained with a polyclonal anti-GFP antibody (that detects all VN/VC fragments, visualized using an anti-Rabbit-647 antibody). Note comparable expression with all expressed proteins, including the various APP mutants.

(b) HEK293T cells were co-transfected with APP:VN and BACE-1:VC (incubated with or without the γ-secretase inhibitor BMS-299897) and analyzed by Western blotting with two anti-APP antibodies. Note that omission of BMS-299897 led to a shift in the ~ 30kDa band (β-cleavage product of APP:VN, red arrowheads) to ~ 26kDa (blue arrowheads). This 4kDa shift is likely due to further cleavage of the APP:VN-β-CTF into a ~ 26kDa AICD-VN fragment and a ~ 4kDa Aβ fragment (which will be likely secreted). Also note that in these gels, a ~ 11kDa band is greatly attenuated upon omission of BMS- 299897. This fragment probably represents endogenous β-CTF’s – generated from the endogenous APP present in these cells – and upon BMS-299897 removal, these are further cleaved into Aβ that is secreted (the ~ 6kDa endogenous AICD probably turns over too rapidly in the absence of BMS-299897). These results further confirm that APP:VN is cleaved by BACE-1:VC in the OptiCAB assay. Also note holo-APP is better recognized by the CT15 antibody (brackets).

Supplementary Figure 2 Complementation of familial AD mutants and experiments with ‘crippled CMV promoter’.

(a) BACE-1 null fibroblasts were co-transfected with BACE-1:VC and WT-APP:VN (or VN-tagged APP Arctic and London mutations) Note that with both mutants there is an increase in levels of the ~ 30kDa fragment likely corresponding to bCTF:VN (as noted by other studies 12, 13); though there was no change in the levels of mutant-APP/BACE-1 complementation in neurons after ~ 5-6h (b).

(c) Left: APP:VN and BACE-1:VC were sub-cloned into “crippled” CMV promoters (ΔAPP:VN and ΔBACE:VC), known to express lower protein-levels (see “methods”; note that ~ 48h of expression was necessary to visualize fluorescence by light microscopy). Right: Untransfected or transfected neurons (constructs indicates on X-axis) were fixed and immunostained with an anti-APP antibody (A4) to detect total APP fluorescence in soma (Y-axis). Note that fluorescence-levels in ΔAPP:VN/ΔBACE:VC are only slightly higher than untransfected neurons.

(d) ΔAPP:VN/ΔBACE-1:VC transfected neurons were either incubated with Alexa-transferrin (or co-transfected with the stated organelle markers), and imaged as described in “methods”. Note colocalization of ΔAPP/ΔBACE-1 BifC with

endosomes.

Supplementary Figure 3 Identity of axonal APP and BACE-1 carriers.

(a,b) Neurons were co-transfected with APP:GFP (or BACE-1:GFP) and RFP-tagged endocytic markers (Rab5 and Rab11) as indicated; and imaged live by simultaneous dual-cam imaging. Note little co-transport of APP or BACE-1 with endocytic markers.

Supplementary Figure 4 Internalization of APP in neuronal soma.

(a, b) Neurons were co-transfected with BBS-APP and various organelle markers, incubated with BTX-594, and internalized APP was evaluated in cell bodies by colocalization analyses (see ‘methods’). Colocalization of internalized BTX-594 with organelle markers was as follows: TfR (41.6 %), Rab5 (49.9), GalT (44.1), NPYss (31.2 %), and LAMP-1 (79.1 %). 10-18 neurons from two separate cultures were analyzed; * p < 0.05.

Supplementary Figure 5 Trafficking parameters of protective APP(Ice) mutant.

(a) Construct design and experimental strategy to evaluate internalization of APP.

(b) Similar internalization of WT and APP(Ice) mutants in neurons, quantified on right (26-28 neurons from three independent experiments were analyzed).

(c) Comparing vesicle transport kinetics of APP WT and APP(Ice) in dendrites. Neurons were transfected with APP:GFP or APP(Ice):GFP, and were imaged live with fast image acquisition (1 frame/sec for 200 secs), 5-6 h post-transfection. Note that various transport parameters – frequency, velocity and run length – are largely similar between the two groups, though a slight decrease in anterograde particle frequency was seen with APP(Ice) mutant. ~ 450 particles from 28-36 neurons – from three separate experiments – were analyzed; * p < 0.05.

Supplementary Figure 6 OptiCAB assay in HEK293T cell lines.

(a) HEK293T cells were transfected with indicated constructs and visualized 5-6 h post-transfection. Note in the APP:VN/BACE-1:VC group, complemented APP/BACE-1 fluorescence was widespread and punctate (zoomed inset). However, diminished fluorescence was seen in in APP:VN/BACE-1:VC transfected cells treated with an inhibitor of clathrin-dependent endocytosis (Dynasore), or in cells transfected with APPYENPTY:VN/BACE-1:VC. Fluorescence complementation was also markedly attenuated when cells were transfected with APP(Ice):VN/BACE-1:VC.

(b)Western blot of HEK293T cells transfected with APP:VN/BACE-1:VC (in presence of a γ-secretase inhibitor) shows the expected ~30 kDa β-cleavage product of APP:VN (data from 2-3 independent cultures).

Supplementary Figure 7 Neuronal recycling endosomes are acidic.

Neurons were co-transfected with TfR:pHluorin and soluble mCherry for ~5-6 h and imaged live. TfR:pHluorin is the recycling endosome marker (TfR) tagged to a pHsensitive probe (pHluorin). Note that the TfR:pHluorin fluorescence increases upon incubation in an alkaline media – indicating that these vesicles are acidic (middle panel) – and quenches in an acidic media (bottom panel), with little change in the soluble mCherry fluorescence (right panels).

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Das, U., Wang, L., Ganguly, A. et al. Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway. Nat Neurosci 19, 55–64 (2016). https://doi.org/10.1038/nn.4188

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