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Enhanced cleavage of APP by co-expressed Bace1 alters the distribution of APP and its fragments in neuronal and non-neuronal cells

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Abstract

Background

Alzheimer’s disease amyloid-beta peptides (Aβ) are generated via sequential cleavage of the amyloid precursor protein (APP) by β-secretase (Bace1) and γ-secretase. Though the precise subcellular location(s) of Bace1-mediated APP cleavage remains unresolved, current models suggest APP internalization into Bace1-containing endosomes is a critical step. However, direct evidence for this model is lacking, and previous reports that probed the APP/Bace1 interaction (using co-expressed APP and Bace1 differentially labeled with fluorescent protein tags) did not determine if APP fluorescence originated from full-length APP (fl-APP) molecules that had internalized from the cell surface pool.

Methods

We adapted the bungarotoxin-ligand (BTX) system to label surface APP and track internalized fluorescent APP/BTX puncta in rodent primary neurons co-expressing fluorescently-tagged Bace1. Subsequently, we employed imaging and biochemical-based approaches to measure N- and C-terminal APP epitope levels in primary neurons, N2a neuroblastoma, and HeLa cell lines.

Results

We hypothesized that surface-labeled APP/BTX puncta would, upon internalization, colocalize with fluorescently-tagged Bace1. Unexpectedly, we observed a dramatic loss of internalized APP in co-transfected neurons and ~ 80–90% loss of surface-resident fl-APP, which we also observed in HeLa and N2a cells. Loss of surface fl-APP could be reversed by a Bace1 inhibitor, suggesting that enhanced Bace1-mediated APP cleavage was responsible for the altered processing and mis-sorting. Importantly, in a C-terminally-tagged APP construct, the majority of C-terminal fluorescence was preserved in HeLa cells despite the loss of N-terminal APP signal. This phenomenon was not only recapitulated in cultured neurons, but also showed a progressive disappearance of the APP N-terminal tag, reflecting continual cleavage of fl-APP by Bace1 away from the cell body.

Conclusions

Our results strongly suggested that in APP/Bace1 co-expression approaches, there was significant early and aberrant Bace1-mediated APP cleavage that perturbed fl-APP trafficking from the secretory pathway onwards, resulting in a substantial loss of surface fl-APP, which in turn led to a marked reduction in APP internalization. In C-terminally-tagged APP constructs, a large fraction of the APP fluorescence signal therefore likely arose from fluorescently-tagged β-C-terminal-fragment (β-CTF) or downstream proteolytic derivatives instead of fl-APP. Thus, care is needed in interpreting results where APP is detected only with a C-terminal tag in the presence of Bace1 co-expression, and previous findings may need to be reinterpreted if it is unclear whether fl-APP is present in normal physiological levels.

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Data availability

All data generated or analyzed during this study are included in the published article [and its supplementary information files].

Abbreviations

β-CTF:

β-C-terminal fragment

A647:

Alexa-fluor-647

Aβ:

Amyloid-beta

AD:

Alzheimer’s disease

AICD:

APP intracellular domain

APP:

Amyloid precursor protein

APP-CTF:

APP-C-terminal-fragment

Βace1:

β-Secretase

BBS:

α-Bungarotoxin binding site

BFP:

Blue fluorescent protein

BSI:

Bace1-inhibitor

BTX:

α-Bungarotoxin

CME:

Clathrin-/dynamin-mediated endocytosis

DIV:

Days-in-vitro

FBS:

Fetal bovine serum

FL-APP:

Full-length APP

FRET:

Forster resonance energy transfers

FWHM:

Full-width half-maximum

GFP:

Green fluorescent protein

HRP:

Horse-radish peroxidase

mCh:

MCherry

MEM-NEAA:

Minimum Essential Medium non-essential amino acids

Nb-A:

Neurobasal-A

NH4Cl:

Ammonium chloride

PBS:

Phosphate-buffered saline

PDL:

Poly-d-lysine

PFA:

Paraformaldehyde

RFP:

Red fluorescent protein

ROIs:

Regions-of-interest

sAPPβ:

Soluble-APP-β fragment

SEM:

Standard error of measurement

SEP:

Super-ecliptic pHluorin

TBST:

Tris-buffered saline with Tween

TGN:

Trans-Golgi-network

References

  1. Selkoe DJ, Hardy J (2016) The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med 8(6):595–608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Nhan HS, Chiang K, Koo EH (2015) The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes. Acta Neuropathol 129(1):1–19

    Article  CAS  PubMed  Google Scholar 

  3. Haass C, Kaether C, Thinakaran G, Sisodia S (2012) Trafficking and proteolytic processing of APP. Cold Spring Harb Perspect Med. 2(5):a006270

  4. Rajendran L, Annaert W (2012) Membrane trafficking pathways in Alzheimer’s disease. Traffic 13(6):759–770

    Article  CAS  PubMed  Google Scholar 

  5. Toh WH, Gleeson PA (2016) Dysregulation of intracellular trafficking and endosomal sorting in Alzheimer’s disease: controversies and unanswered questions. Biochem J 473(14):1977–1993

    Article  CAS  PubMed  Google Scholar 

  6. Toh WH, Tan JZ, Zulkefli KL, Houghton FJ, Gleeson PA (2017) Amyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b- and AP4-dependent pathway. Traffic 18(3):159–175

    Article  CAS  PubMed  Google Scholar 

  7. Tam JH, Seah C, Pasternak SH (2014) The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid. Mol Brain 7:54

    Article  PubMed  PubMed Central  Google Scholar 

  8. Kuhn PH, Wang H, Dislich B, Colombo A, Zeitschel U, Ellwart JW et al (2010) ADAM10 is the physiologically relevant, constitutive alpha-secretase of the amyloid precursor protein in primary neurons. EMBO J 29(17):3020–3032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P et al (1999) Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286(5440):735–741

    Article  CAS  PubMed  Google Scholar 

  10. Cole SL, Vassar R (2007) The Basic Biology of BACE1: A Key Therapeutic Target for Alzheimer’s Disease. Curr Genomics 8(8):509–530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sathya M, Premkumar P, Karthick C, Moorthi P, Jayachandran KS, Anusuyadevi M (2012) BACE1 in Alzheimer’s disease. Clin Chim Acta 414:171–178

    Article  CAS  PubMed  Google Scholar 

  12. Yan R, Vassar R (2014) Targeting the β-secretase BACE1 for Alzheimer’s disease therapy. Lancet Neurol 13(3):319–329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tan J, Evin G (2012) β-site APP-cleaving enzyme 1 trafficking and Alzheimer’s disease pathogenesis. J Neurochem 120(6):869–880

    CAS  PubMed  Google Scholar 

  14. Hemming ML, Elias JE, Gygi SP, Selkoe DJ (2009) Identification of beta-secretase (BACE1) substrates using quantitative proteomics. PLoS One. 4(12):e8477

  15. Ben Halima S, Mishra S, Raja KMP, Willem M, Baici A, Simons K et al (2016) Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein. Cell Rep 14(9):2127–2141

    Article  CAS  PubMed  Google Scholar 

  16. Zhang X, Song W (2013) The role of APP and BACE1 trafficking in APP processing and amyloid-β generation. Alzheimers Res Ther 5(5):46

    Article  PubMed  PubMed Central  Google Scholar 

  17. Perez RG, Soriano S, Hayes JD, Ostaszewski B, Xia W, Selkoe DJ et al (1999) Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42. J Biol Chem 274(27):18851–18856

    Article  CAS  PubMed  Google Scholar 

  18. Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA et al (2007) The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci 27(29):7817–7826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Carey RM, Balcz BA, Lopez-Coviella I, Slack BE (2005) Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid beta protein. BMC Cell Biol 6:30

    Article  PubMed  PubMed Central  Google Scholar 

  20. Zhu L, Su M, Lucast L, Liu L, Netzer WJ, Gandy SE et al (2012) Dynamin 1 regulates amyloid generation through modulation of BACE-1. PLoS One. 7(9):e45033

  21. Cirrito JR, Kang JE, Lee J, Stewart FR, Verges DK, Silverio LM et al (2008) Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron 58(1):42–51

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Das U, Scott DA, Ganguly A, Koo EH, Tang Y, Roy S (2013) Activity-induced convergence of APP and BACE-1 in acidic microdomains via an endocytosis-dependent pathway. Neuron 79(3):447–460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. von Arnim CA, von Einem B, Weber P, Wagner M, Schwanzar D, Spoelgen R et al (2008) Impact of cholesterol level upon APP and BACE proximity and APP cleavage. Biochem Biophys Res Commun 370(2):207–212

    Article  Google Scholar 

  24. Marquer C, Devauges V, Cossec JC, Liot G, Lécart S, Saudou F et al (2011) Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis. FASEB J 25(4):1295–1305

    Article  CAS  PubMed  Google Scholar 

  25. Das U, Wang L, Ganguly A, Saikia JM, Wagner SL, Koo EH et al (2016) Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway. Nat Neurosci 19(1):55–64

    Article  CAS  PubMed  Google Scholar 

  26. Kinoshita A, Fukumoto H, Shah T, Whelan CM, Irizarry MC, Hyman BT (2003) Demonstration by FRET of BACE interaction with the amyloid precursor protein at the cell surface and in early endosomes. J Cell Sci 116(Pt 16):3339–3346

    Article  CAS  PubMed  Google Scholar 

  27. von Einem B, Schwanzar D, Rehn F, Beyer AS, Weber P, Wagner M et al (2010) The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1. Exp Neurol 225(1):85–93

    Article  Google Scholar 

  28. von Einem B, Weber P, Wagner M, Malnar M, Kosicek M, Hecimovic S et al (2012) Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells. Int J Mol Sci 13(12):15801–15812

    Article  Google Scholar 

  29. Sekine-Aizawa Y, Huganir RL (2004) Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors. Proc Natl Acad Sci U S A 101(49):17114–17119

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mammen AL, Huganir RL, O’Brien RJ (1997) Redistribution and stabilization of cell surface glutamate receptors during synapse formation. J Neurosci 17(19):7351–7358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hu YB, Dammer EB, Ren RJ, Wang G (2015) The endosomal-lysosomal system: from acidification and cargo sorting to neurodegeneration. Transl Neurodegener 4:18

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kopec CD, Li B, Wei W, Boehm J, Malinow R (2006) Glutamate receptor exocytosis and spine enlargement during chemically induced long-term potentiation. J Neurosci 26(7):2000–2009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Muresan V, Varvel NH, Lamb BT, Muresan Z (2009) The cleavage products of amyloid-beta precursor protein are sorted to distinct carrier vesicles that are independently transported within neurites. J Neurosci 29(11):3565–3578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sisodia SS (1992) Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A 89(13):6075–6079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Parvathy S, Hussain I, Karran EH, Turner AJ, Hooper NM (1999) Cleavage of Alzheimer’s amyloid precursor protein by alpha-secretase occurs at the surface of neuronal cells. Biochemistry 38(30):9728–9734

    Article  CAS  PubMed  Google Scholar 

  36. Small SA, Petsko GA (2015) Retromer in Alzheimer disease, Parkinson disease and other neurological disorders. Nat Rev Neurosci 16(3):126–132

    Article  CAS  PubMed  Google Scholar 

  37. Lee EB, Zhang B, Liu K, Greenbaum EA, Doms RW, Trojanowski JQ et al (2005) BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo. J Cell Biol 168(2):291–302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Caudano F, Montalto G, Passalacqua M, Pronzato MA, Fedele E, Ricciarelli R (2020) cGMP favors the interaction between APP and BACE1 by inhibiting Rab5 GTPase activity. Sci Rep 10(1):1358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Corish P, Tyler-Smith C (1999) Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng 12(12):1035–1040

    Article  CAS  PubMed  Google Scholar 

  40. Bustamante HA, Rivera-Dictter A, Cavieres VA, Muñoz VC, González A, Lin Y et al (2013) Turnover of C99 is controlled by a crosstalk between ERAD and ubiquitin-independent lysosomal degradation in human neuroglioma cells. PLoS One. 8(12):e83096

  41. Burgos PV, Mardones GA, Rojas AL, daSilva LL, Prabhu Y, Hurley JH et al (2010) Sorting of the Alzheimer’s disease amyloid precursor protein mediated by the AP-4 complex. Dev Cell 18(3):425–436

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kaether C, Schmitt S, Willem M, Haass C (2006) Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface. Traffic 7(4):408–415

    Article  CAS  PubMed  Google Scholar 

  43. Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T et al (2003) APP processing and synaptic function. Neuron 37(6):925–937

    Article  CAS  PubMed  Google Scholar 

  44. Greicius MD, Srivastava G, Reiss AL, Menon V (2004) Default-mode network activity distinguishes Alzheimer’s disease from healthy aging: evidence from functional MRI. Proc Natl Acad Sci U S A 101(13):4637–4642

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Dr. Utpal Das and Dr. Subhojit Roy for providing the APP-GFP and Bace1-mCherry plasmids. We gratefully acknowledge members of the Koo lab for technical and administrative assistance, as well as for many helpful discussions and advice. We would also like to acknowledge the National University of Singapore Centre for Bioimaging Sciences (NUS-CBIS) for use of their confocal spinning disk microscopy system.

Funding

This work was funded in part by grants to EHK from the Singapore National Medical Research Council (NMRC, NMRC/StaR/009/2012) and the National Institutes of Health (NIH, NS84324). GT is funded by grants AG054223 and AG056061 from the National Institutes of Health. JA is funded by an Open-Fund Young Investigator’s Research Grant from NMRC (OF-YIRG, MOH-000237–00) and the Genome Institute of Singapore. TRH is supported by an Integrated Science and Engineering Programme scholarship from the National University of Singapore.

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Author notes

  1. Jonathan Aow and Tzu-Rung Huang contributed equally.

Authors and Affiliations

  1. Agency for Science, Technology and Research (A*STAR), Genome Institute of Singapore, Singapore, Singapore

    Jonathan Aow

  2. Department of Medicine, National University of Singapore, Singapore, Singapore

    Jonathan Aow & Edward H. Koo

  3. Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore

    Tzu-Rung Huang & Edward H. Koo

  4. USF Health Byrd Alzheimer’s Center and Research Institute and Department of Molecular Medicine, University of South Florida, Tampa, FL, USA

    Gopal Thinakaran

  5. Department of Neurosciences, University of California San Diego, San Diego, CA, USA

    Edward H. Koo

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  1. Jonathan Aow
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Contributions

JA, TRH and EK devised the project and planned experiments. JA and TRH carried out experiments and analyzed data. JA and TRH wrote the first draft of the manuscript. EK supervised the project and edited the manuscript with input from GT. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jonathan Aow or Edward H. Koo.

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All animal experiments were performed with the National University of Singapore Institutional Animal Care and Use Committee (IACUC) approval.

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Aow, J., Huang, TR., Thinakaran, G. et al. Enhanced cleavage of APP by co-expressed Bace1 alters the distribution of APP and its fragments in neuronal and non-neuronal cells. Mol Neurobiol 59, 3073–3090 (2022). https://doi.org/10.1007/s12035-022-02733-6

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