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