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Molecular Neurobiology

, Volume 56, Issue 12, pp 8603–8616 | Cite as

Characterization of Cerebrospinal Fluid BACE1 Species

  • Inmaculada Lopez-FontEmail author
  • Claudia P. Boix
  • Henrik Zetterberg
  • Kaj Blennow
  • Javier Sáez-ValeroEmail author
Article

Abstract

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the main brain β-secretase responsible for the amyloidogenic processing of the amyloid precursor protein (APP). Previous studies have suggested that cerebrospinal fluid (CSF) β-secretase activity may be a candidate diagnostic biomarker for Alzheimer’s disease (AD), but biochemical characterization of BACE1 protein in CSF is needed. CSF samples from 19 AD patients and 19 age-matched non-AD controls (n = 19) were classified according to their Aβ42, total tau, and P-tau CSF biomarker levels. We found that β-secretase activity was higher in the CSF of AD subjects than in that of the controls. We found that the majority of the β-secretase activity in the CSF, measured using a peptide substrate homologous to the BACE1 cleavage site, was not inhibited by specific BACE1 inhibitors. We defined enzymatic activity attributable specifically to BACE1 as the activity that was blocked by the specific inhibitors, which is still higher in AD subjects. BACE1 protein levels were characterized by lectin binding, immunoprecipitation, blue native-PAGE, and western blotting using antibodies against specific protein domains. BACE1 was found to be present in human CSF as a mature form of ~ 70 kDa that probably comprised truncated and full-length species, and also as an immature form of ~ 50 kDa that retains the prodomain. CSF-BACE1 was found to assemble into hetero-complexes containing distinct species. Immunoblotting with an antibody against the C-terminus of BACE1 revealed significantly higher levels of the 70-kDa full-length BACE1, while the 50 kDa immature form remained unaltered. When the 70-kDa species was probed with an antibody against the N-terminus of BACE1 (which does not discriminate between truncated and full-length forms), no increase in immunoreactivity was observed, suggesting that truncated forms of BACE1 do not increase in AD. In conclusion, the complexity of BACE1 species in CSF has to be taken into consideration when determining BACE1 activity and protein levels in CSF as biomarkers of AD.

Keywords

BACE1 β-Secretase Cerebrospinal fluid Biomarker Alzheimer’s disease 

Abbreviations

CSF

cerebrospinal fluid

BACE1

β-site amyloid precursor protein cleaving enzyme 1

APP

amyloid precursor protein

AD

Alzheimer’s disease

NADC

non-AD controls

β-amyloid protein

P-tau

phospho-tau

T-tau

total tau

ELISA

enzyme-linked immunosorbent assay

Notes

Acknowledgments

We acknowledge Drs. B. De Strooper (VIB Center for Brain and Disease Research & Katholieke Universiteit Leuven, Leuven, Belgium) for the generous gift of human BACE1 cDNA, and Drs. A. Lleó and D. Alcolea (Hospital de Sant Pau & CIBERNED, Barcelona, Spain) for technical advice about determination of CSF β-secretase activity.

Authors’ Contributions

ILF, HZ, KB, and JSV were involved with the conception, design, and interpretation of data. ILF and CPB performed the experiments. ILF and JSV were involved with data analysis. HZ and KB collected the clinical material. ILF, HZ, KB, and JSV provided general overall supervision of the study, and acquired funding. All authors contributed to the drafting and critical revision of the manuscript and have given final approval of the version to be published.

Fundings

This study was funded in part by the Direcció General d’Universitat, Investigació i Ciència, GVA (AICO/2018/090), by the EU BIOMARKAPD-Joint Programme on Neurodegenerative Diseases (JPND) and the Instituto de Salud Carlos III (ISCIII grant PI11/03026), by the ISCIII (PI15/00665), co-financed by the Fondo Europeo de Desarrollo Regional under the aegis of JPND), and through CIBERNED, ISCIII (FEDER, “Investing in your future”). HZ is a Wallenberg Academy Fellow supported by grants from the European Research Council, the Swedish Research Council and the UK Dementia Research Institute at UCL. KB holds the Torsten Söderberg Professorship in Medicine at the Royal Swedish Academy of Sciences, and is supported by the Swedish Research Council (#2017-00915), the Swedish Alzheimer Foundation (#AF-742881), Hjärnfonden, Sweden (#FO2017-0243), and a grant (#ALFGBG-715986) from the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement.

Compliance with Ethical Standards

Ethics Approval and Consent to Participate

This study was approved by the ethic committee at the Miguel Hernandez University and it was carried out in accordance with the Declaration of Helsinki. The samples after de-identification were used according to the Swedish Biobank law (Biobanks in Medical Care Act) and the Swedish ethical legislation (The Act concerning the Ethical Review of Research Involving Humans).

Consent for Publication

Not applicable.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2019_1677_Fig6_ESM.png (188 kb)
Supplemental Figure 1

Detection of β-secretase activity in human CSF in presence of the β-secretase inhibitor C3. The activity of β-secretase was measured using a fluorogenic peptide substrate as in Fig. 1. (A) β-Secretase activity was detectable in the same human CSF that in Fig. 1 (19 age-matched non-AD controls (NADC) and 19 ADs) in the absence (No inh) and presence of the β-secretase inhibitor C3 at 2 μM. The results are presented as means ± SEM. (B) Box and scatter plots of the β-secretase activity levels attributable to BACE1 estimated in the 19 NADC (closed symbol) and 19 AD (open symbol) cases after subtracting the activity in the presence of the β-secretase inhibitor C3 at 2 μM. p values are indicated. (PNG 188 kb)

12035_2019_1677_MOESM1_ESM.tif (233 kb)
High resolution image (TIF 233 kb)
12035_2019_1677_Fig7_ESM.png (97 kb)
Supplemental Figure 2

Specificity of the anti-BACE1 antibodies for the BACE1 species present in HEK cells over-expressing human BACE1. Western blotting of the cell extracts from wild-type (−) and HEK cells over-expressing human BACE1 (+), were resolved with the indicated anti-BACE1 antibodies. Note that the ~ 70-kDa band was detected with the ectodomain, N- terminus, and C-terminus (B0806) antibodies, but not with the prodomain antibody (the prodomain is proteolytic cleaved in mature forms). The ~ 50-kDa band was detected with all the antibodies. (PNG 96 kb)

12035_2019_1677_MOESM2_ESM.tif (918 kb)
High resolution image (TIF 917 kb)
12035_2019_1677_Fig8_ESM.png (156 kb)
Supplemental Figure 3

Albumin interferes in the electrophoretic migration of the 70-kDa BACE1 species of CSF-BACE1. Representative western blot of human CSF sample before (non-depleted) and after albumin immunodepletion (depleted) using the antibodies anti-BACE1 N-terminus (green) and anti-albumin (red). When albumin is not present in the CSF (depleted), a double BACE1-immunoreactive band can be observed at ~ 70 kDa (arrow head). The 70-kDa doublet-bands are also shown in an over-exposed image. (PNG 156 kb)

12035_2019_1677_MOESM3_ESM.tif (2 mb)
High resolution image (TIF 2059 kb)
12035_2019_1677_Fig9_ESM.png (114 kb)
Supplemental Figure 4

Mature and immature full-length form of BACE1, retaining the C-terminal domain are present in the conditioned medium of CHO cells. CHO cell-conditioned medium (Total, T) were immunoprecipitated using the N-terminus antibody and the precipitated proteins (IP) were immunoblotted with the C-terminus antibody B0806. No bands were observed in the absence of capture antibody (IPc). (PNG 114 kb)

12035_2019_1677_MOESM4_ESM.tif (1.5 mb)
High resolution image (TIF 1553 kb)
12035_2019_1677_Fig10_ESM.png (267 kb)
Supplemental Figure 5

Levels of BACE1 in AD CSF. Representative western blots of human CSF samples from NADC (closed symbol; n = 19) and AD (open symbol; n = 19) subjects using the propeptide (A) and the ectodomine (B) antibodies (same samples analyzed in Fig. 4). The densitometric quantification of the 50-kDa specie (propeptide) and 70-kDa specie (ectodomain) are represented as box and scatter plots. Calculations were performed in duplicate. n.s.: non-significant p value. (PNG 267 kb)

12035_2019_1677_MOESM5_ESM.tif (4.9 mb)
High resolution image (TIF 4983 kb)
12035_2019_1677_MOESM6_ESM.docx (17 kb)
Supplemental Table 1 (DOCX 16 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Instituto de Neurociencias de AlicanteUniversidad Miguel Hernández-CSICSant Joan d’AlacantSpain
  2. 2.Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)Sant Joan d’AlacantSpain
  3. 3.Clinical Neurochemistry LaboratorySahlgrenska University HospitalMölndalSweden
  4. 4.Department of Neuroscience and PhysiologyThe Sahlgrenska Academy at the University of GothenburgMölndalSweden
  5. 5.Department of Neurodegenerative DiseaseUCL Institute of NeurologyLondonUK
  6. 6.UK Dementia Research Institute at UCLLondonUK

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