Elsevier

Clinical Biochemistry

Volume 41, Issue 12, August 2008, Pages 986-996
Clinical Biochemistry

Decrease in age-adjusted cerebrospinal fluid β-secretase activity in Alzheimer's subjects

https://doi.org/10.1016/j.clinbiochem.2008.04.022Get rights and content

Abstract

Objectives

To develop a novel cerebrospinal fluid (CSF) β-secretase-1 activity assay and evaluate β-secretase-1 (BACE-1) activity as a potential biomarker in human Alzheimer's disease.

Methods

The assay consisted of an enzymatic reaction of CSF samples with an optimized β-secretase peptide substrate and the cleavage products were detected using a neo-epitope specific antibody.

Results

The CSF BACE-1 activity assay described exhibits time, temperature, dose, and pH dependence, with sensitivity down to < 1 pM of recombinant BACE-1 enzyme, and is completely blocked by BACE-1 inhibitors. The endogenous BACE-1 enzyme in CSF appears to exist as a c-terminally truncated protein, based on both western blotting and capture-based activity assays. In a small cohort of human subjects, an age-dependent increase in CSF BACE activity was observed (~ 1.0 pM/year, p < 0.05). In Alzheimer's disease subjects, a significant decline in age-adjusted CSF BACE activity was observed compared to controls (56% in the log-transformed scale, p = 0.02).

Conclusion

We have developed a robust assay to measure CSF BACE-1 activity which could serve as a potential biomarker in human Alzheimer's disease subjects.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia and characterized histopathologically by amyloid plaques and neurofibrillary tangles in the brain. Amyloid-beta (Aβ) is the major component of neuritic plaques and is generated from beta-amyloid precursor protein (APP) following sequential cleavage by beta- and gamma-secretase enzymes. β-secretase or β-site APP cleaving enzyme 1 (BACE-1), is the initial rate-limiting enzyme in cutting APP into a large secreted APP fragment (sAPPβ) and a small membrane-bound fragment (c-99) which is further cleaved by γ-secretase resulting in Aβ generation [1], [2]. Extensive evidence suggests that the accumulation of Aβ peptide, especially Aβ42, in the brain may have a causal relationship with AD. Therefore, there is an intense effort to develop compounds that can inhibit β or γ -secretase enzymes to lower Aβ as a potential treatment for AD [3].

BACE-1 is a 501-amino acid-long preproprotein and a type I transmembrane aspartyl protease and is considered to be the primary β-site cleaving enzyme in the brain. The extra cellular domain contains two active aspartyl residues at amino acid positions 93 and 289. Genetic knockdown of BACE-1 in mice has been shown to completely abolish brain Aβ production [4], [5], [6]. Further, these mice are viable and show no obvious behavioral or pathological phenotypes [6], [7] although there is controversy over mild phenotypic effects in mice generated from different groups.

In human subjects with sporadic AD, an increase in BACE protein and BACE enzymatic activity was observed in frontal cortex from postmortem specimens, as compared to control subjects and appears correlated with amyloid load [8]. Brain BACE activity also appears to increase in humans with sporadic AD [9], [10]. Preliminary studies indicate an increase in CSF BACE enzymatic activity in AD subjects compared to control subjects [11], [12]. A 70 kDa protein corresponding to full-length BACE-1 has been suggested as the source of CSF BACE activity in human cerebrospinal fluid (CSF) [13]. In contrast, a soluble truncated form of BACE-1 has been proposed as the active enzymatic species in human CSF [12], and demonstrated modest increase in BACE activity in AD subjects compared to controls. The exact nature of BACE-1 in human CSF samples still remains to be clarified.

Previously, BACE-1 activity in CSF was measured using a solution-based assay with an internally quenched fluorescent peptide substrate containing APP Swedish β-site cleavage sequence [14]. This assay did not use a BACE standard curve to determine BACE concentration in control and human subjects. Recently, an elegant method was developed whereby the switch from inactive proenzyme caspase 3 to an active caspase 3 was mediated via an engineered BACE cleavage site mimicking APP and the activity of caspase 3 was detected using a chromogenic substrate [12]. The potential limitations of this method are the expression of an engineered procaspase 3 and the optimization of two enzymatic reactions with different kinetics.

In the present work we describe a new, sensitive and specific assay for measuring BACE-1 activity in both human and rhesus monkey CSF. We use a novel optimized BACE substrate, which after cleavage the product can be detected using a neo-epitope antibody and a sensitive luminescence based enzyme-linked immunosorbent assay (ELISA). Our studies indicate that BACE in human and rhesus CSF is c-terminally truncated. In addition BACE activity, after adjusting for age appears to be reduced in AD subjects compared to control subjects, in apparent contradiction to earlier reports [11], [12], [13].

Section snippets

Materials and antibodies

Expression, purification and characterization of soluble c-terminally truncated recombinant BACE-1 (aa1–460) in baculovirus were described previously [15]. Full-length BACE-1 cDNA was transiently transfected and expressed in HEK293 cells (human embryonic kidney cells). The cell membranes were harvested, dissolved in BACE suspension buffer (50 mM NaOAc, 0.01% bovine serum albumin (BSA), 15 mM EDTA, 0.2% CHAPS, 1 mM Deferoxamine Mesylate, (pH 4.5) and characterized by western blotting and BACE-1

CSF BACE activity assay

In order to determine the best assay format for assaying BACE enzymatic activity in biological extracts or fluids such as CSF, we initially tested a solution-based assay and compared it with either a BACE capture assay format or a substrate capture assay format (see Supplemental Information) to determine the signal and background properties of these different assays. In the solution-based BACE enzymatic assay, which was superior to the capture-based assay formats (Fig. 1), a biotin labeled 15

Discussion

We have developed a novel and sensitive solution-based BACE-1 activity assay in CSF by using an optimized BACE-1 substrate (NFEV) and a novel detection method utilizing an antibody to the neo-epitope generated following BACE cleavage of the substrate. This assay has a lower limit of reliable quantitation of < 1 pM which was confirmed by both recombinant BACE standard curves and serial dilution of rhesus CSF samples. This assay has a signal to noise window of ~ 20 fold after all non-BACE aspartyl

References (27)

  • S. Sinha et al.

    Cellular mechanisms of beta-amyloid production and secretion

    Proc Natl Acad Sci U S A

    (1999)
  • H. Cai et al.

    BACE-1 is the major beta-secretase for generation of Abeta peptides by neurons

    Nat Neurosci

    (2001)
  • Y. Luo et al.

    Mice deficient in BACE-1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation

    Nat Neurosci

    (2001)
  • Cited by (43)

    • The β-Secretase BACE1 in Alzheimer's Disease

      2021, Biological Psychiatry
      Citation Excerpt :

      A recent study showed an association between plasma BACE1 concentrations and amyloid-positron emission tomography quantitative measures in a cohort of cognitively healthy individuals at risk for AD (86). By contrast, some studies showed no acceptable diagnostic performance of BACE1 biomarkers (90–92) or no association between them and AD established biomarkers. Moreover, lower levels of BACE1 were found in advanced dementia stages of AD, potentially owing to advanced neuronal and synaptic loss (93–95).

    • The standardization of cerebrospinal fluid markers and neuropathological diagnoses brings to light the frequent complexity of concomitant pathology in Alzheimer's disease: The next challenge for biochemical markers?

      2019, Clinical Biochemistry
      Citation Excerpt :

      Novel candidate markers are proposed by different groups, for example, to differentiate AD from FTLD or to diagnose AD from plasma samples. However these groups struggle to get further validation of their findings because of the lack of fully characterized cohorts of patients [90–94]. Strikingly, diagnoses in the field of degenerative diseases and in the field of brain tumors took opposite directions.

    • Search for biomarkers of Alzheimer‘s disease: Recent insights, current challenges and future prospects

      2019, Clinical Biochemistry
      Citation Excerpt :

      In addition, the authors found a correlation between MCP-1 levels and the pace of cognitive decline in the patients. Several studies [59–62] have also focused on β-site amyloid precursor protein cleaving enzyme 1 (BACE1). This enzyme is responsible for the production of Aβ by specifically decomposing the amyloid precursor protein.

    • Cerebrospinal fluid BACE1 activity and markers of amyloid precursor protein metabolism and axonal degeneration in Alzheimer's disease

      2014, Alzheimer's and Dementia
      Citation Excerpt :

      We did not find any CSF BACE1 activity differences between the control group and any of the patient groups. This aspect of our research is in line with one study [6] but in contrast to other previous studies with partly contradictory findings, showing increased BACE1 activity in MCI but not AD [7,8], increased activity in MCI and AD [9], or even decreased activity in AD [5]. Part of the discrepancy may be explained by the different properties of the applied laboratory assays, the characteristics of the study samples, and the definitions of patient groups, but the wide range of BACE1 activity measurements and the large overlap between the groups may also have a significant effect.

    View all citing articles on Scopus
    View full text