Characterization of Cerebrospinal Fluid BACE1 Species
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.
KeywordsBACE1 β-Secretase Cerebrospinal fluid Biomarker Alzheimer’s disease
β-site amyloid precursor protein cleaving enzyme 1
amyloid precursor protein
enzyme-linked immunosorbent assay
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.
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.
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
Conflict of Interest
The authors declare that they have no conflict of interest.
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