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Current Alzheimer Research, 2013, 10, 000-000
BACE1 Levels Correlate with Phospho-Tau Levels in Human Cerebrospinal Fluid Soraia Barão1,2, Lujia Zhou1,2, Katarzyna Adamczuk3, Tine Vanhoutvin1,2, Fred van Leuven5, David Demedts5, Anne-Catherine Vijverman6, Xavier Bossuyt4, Rik Vandenberghe3,6 and Bart De Strooper1,2,* From the 1VIB Center for the Biology of Disease, Leuven, Belgium; 2Center for Human Genetics and LIND, KULeuven and University Hospitals, Leuven, Belgium; 3Laboratory for Cognitive Neurology, KULeuven, Department of Neurology, Leuven, Belgium; 4Experimental Laboratory Immunology, Department of Microbiology and Immunology, KULeuven, Leuven, Belgium; 5Laboratory of Experimental Genetics and Transgenesis, KULeuven, Leuven, Belgium; 6 Neurology Department, University Hospitals Leuven, Belgium Abstract: Previous studies have investigated the activity and protein levels of BACE1, the -secretase, in the brain and cerebrospinal fluid (CSF) of Alzheimer’s disease (AD) patients, however, results remain contradictory. We present here a highly specific and sensitive BACE1 ELISA, which allows measuring accurately BACE1 levels in human samples. We find that BACE1 levels in CSF of AD patients and other neurological disorder (OND) patients are slightly increased when compared to those of a non-neurological disorder control group (NND). BACE1 levels in CSF were well correlated with total-tau and hyperphosphorylated tau levels in the CSF, suggesting that the recorded alterations in BACE1 levels correlate with cell death and neurodegeneration.
Keywords: Alzheimer’s disease; BACE1; biomarker; human CSF; Neurodegeneration; Sandwich ELISA. INTRODUCTION In Alzheimer’s disease (AD) patients, pathological changes in amyloid- peptide (A), total tau (t-tau) and hyperphosphorylated tau (p-tau) in cerebrospinal fluid (CSF) can be recorded many years before neurodegeneration and clinical signs of dementia are observed [1-6]. New additional biomarkers, apart from A and tau, would be of great value as they might help to diagnose the disease in an early stage, or could help to stratify patients, or be used as surrogate markers of treatment efficacy. Such biomarkers can come from unbiased analysis of CSF, blood or other tissues, but also from testing candidate proteins based on knowledge of molecular processes relevant to the disease. One of the major pathways in the disease is the amyloidogenic processing of the Amyloid Precursor Protein (APP) towards the amyloid peptide by two consecutive cleavages carried out by beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and -secretase [7-10]. BACE1, the rate limiting enzyme in the generation of A from APP, is a ~70kDa glycosylated type I transmembrane endoprotease [7, 10-12]. It belongs to the aspartyl protease family and shares sequence and structure similarities with its homolog BACE2, pepsin, renin, cathepsin D and cathepsin E [11-13]. However, there are seven loops and helices on the surface of the BACE1 ectodomain that structurally distinguish BACE1 from other aspartyl proteases and we have raised highly selective antibodies against *Address correspondence to this author at the VIB Center for the Biology of Disease and Center for Human Genetics, KULeuven, Herestraat 49, 3000 Leuven, Belgium; Tel: (32) 16-373-102; Fax: (32) 16-330-827; Email: [email protected]
those domains [14-18]. BACE1 was first described in 1999 and since then extensive efforts have clarified its biology and function and led to the development of inhibitors that are potentially of therapeutic use. Elevated BACE1 levels and activity have been shown in the brain of patients with sporadic AD [19-23]. Since the CSF is in direct contact with the central nervous system, it is reasonable to speculate that such increase might also be monitored in the CSF, especially since BACE1 is shed from membranes by unknown mechanisms [18, 24-26]. Previous studies have shown that CSF specific markers of AD, such as A1-42, t-tau and p-tau, are well correlated with the brain pathology and constitute reliable predictors of AD [3, 27]. Therefore, changes of BACE1 levels in the CSF have also been considered as a possible biomarker of the disease (reviewed by Hampel, H. ). Previous investigations measuring the activity or protein levels of -secretase in CSF have resulted in controversial conclusions [25, 26, 29-35]. Some studies observed a small increase in -secretase activity measured with an enzymatic assay in CSF of AD patients versus non-demented subjects as well as a higher -secretase activity in mild cognitive impaired (MCI) patients compared to AD patients [25, 29, 31, 35]. Others reported no significant differences between controls, MCI and AD patients , and finally even decreased activity of -secretase in CSF of AD patients was reported in one study , and also in a second study investigating multiple sclerosis patients . A last study reported again increased -secretase activity in AD subjects, and also in patients suffering from sporadic Creutzfeldt-Jakob disease . All these studies measured BACE1 enzymatic activity, not protein levels directly. One problem to be considered © 2013 Bentham Science Publishers
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with these assays is the lack of specificity of such reporter assays, as controls to demonstrate that the cleavage of the reporter proteins was unique to BACE1 were not provided. Recently, Gonzales et al. developed a new BACE1 sandwich ELISA and measured BACE1 levels in post-mortem human brain, CSF and plasma . However so far, only Zhong et al. and Ewers et al. measured CSF BACE1 levels directly in different patient groups using a combination of Western blot and ELISA and found, interestingly, significantly increased levels of soluble BACE1 in MCI subjects versus AD and non-demented controls [31, 35]. In these studies commercial available polyclonal antibodies were used and therefore, large scale application of such ELISA poses the problem of limited antibody availability. Given the controversies with regard to BACE1 as a biomarker for AD, and also the raising interest in BACE1 as a therapeutic target in AD, we decided to make use of our highly specific monoclonal antibodies to generate a new, sensitive and specific immunoassay to detect BACE1 levels in human CSF and other tissue samples . We use this assay here for an initial study of CSF BACE1 levels in Alzheimer’s disease (AD) patients, nonneurological disorder controls (NND) and controls with a neurological disorder other than AD, other dementia (OD) patients and other neurological disorder patients (OND). We find a mild increase in BACE1 levels in AD and OND samples. The change is not significantly different between the other groups and AD patients, and the difference between AD and controls is very small, suggesting that BACE1 CSF level is a poor diagnostic biomarker for AD. Interestingly, BACE1 levels were well correlated with the levels of tau and p-tau in the CSF indicating that the BACE1 levels recorded in the CSF might reflect cell death and neurodegeneration in the brain [32, 34]. EXPERIMENTAL PROCEDURES Antibodies and Reagents The anti-BACE1 monoclonal antibodies (mAbs) 5G7, 1A11 and 10B8 (Fig. 1A) were generated as described before . All these mAbs are highly specific for BACE1, without cross reactivity for BACE2 and other structurally related aspartyl proteases . These mAbs do not compete for each others binding to BACE1. The Peroxidase Labeling Kit (Roche) was used to prepare peroxidase labeled mAbs (5G7-HRPO, 1A11-HRPO and 10B8-HRPO). The human BACE1 ectodomain protein (amino acids 45-460) was purified from transfected HEK293 cell cultures by the Protein Service Facility (PSF), VIB, Belgium , was used to generate the standard curve in the BACE1 sandwich ELISA immunoassays. BACE1 Sandwich ELISA NUNC ninety-six-well plates (Life Technologies) were coated with 50 l/well of capture antibody (1A11, 10B8 or 5G7) dissolved in coating buffer (10mM Tris-HCl, 10mM NaCl, 10mM NaN3, pH 8.5) with a final concentration of 2 g/ml. After overnight incubation at 4˚C, the plates were washed with PBS+0.05% Tween 20 and blocked with 100 l/well of casein buffer (1 g casein in 1L PBS, pH7.4) for 4h at room temperature. The coating was always done the day before the actual experiment and we have no data on stability
Barão et al.
of the plates upon prolonged storage. Samples or standards were diluted in casein buffer and mixed with the detection antibody (1A11-HRPO, 5G7-HRPO or 10B8-HRPO, 10mg/ml) diluted 1:2000 in casein buffer. The mixtures were added to the ELISA plates and incubated overnight at 4˚C. Plates were washed and developed with 0.2 mg/ml of 3,5,3’,5’-tetramethyl-benzidine (TMB, Sigma) dissolved in 100 mM sodium acetate (NaAc, pH 4.9) supplemented with 0.03% H2O2. The reactions were allowed to proceed for maximum 15 minutes on a plate shaker at room temperature. The reactions were stopped by adding 2N H2SO4, 50l/well and the plates were read on a Perkin Elmer Envision 2103 multilabel reader at 450 nm. Mouse Brain Homogenates Each hemisphere from the brain of P7 wild-type or BACE1 knockout mouse , was homogenized in 300 l ice cold TBS buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl) by 12 strokes at 750 rpm using a glass-teflon homogenizer. Homogenates were centrifuged at 14 000 x g for 15 min to separate the supernatants and the cell pellets. The obtained cell pellets were lysed in RIPA buffer (50 mM TrisHCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) on ice for 20 min, and cleared by centrifugation at 14 000 x g for 10 min. 12l (1:5 dilution) of ~2g/l brain homogenates were used. All procedures were performed at 4˚C and all buffers were supplemented with complete protease inhibitor (Roche). Human CSF Samples CSF samples from 134 individuals, obtained for diagnostic purposes, were used in this study: 7 community-recruited cognitively intact healthy controls received the lumbar puncture for research purposes only. These cases were screened for the absence of significant neurological or psychiatric history, they had no subjective memory complaints, normal neuropsychological test scores and no structural MRI abnormalities. The remaining 127 cases were based on a retrospective case series from the memory clinic UZ Leuven who received the lumbar puncture for diagnostic purposes. In each case the clinical diagnostic protocol included neurological evaluation, neuropsychological assessment, CSF biomarkers A1-42, t-tau and p-tau, and structural MRI, as well as longitudinal follow-up. In 110 of these cases (n = 110 it also included fluorodeoxyglucose positron emission tomography. Blind to the BACE1 result, these cases were classified a priori based on clinical diagnosis: clinically probable AD (n = 53), amnestic mild cognitive impairment (MCI, n = 6), dementia other than AD (OD, total n = 22: clinically probable Lewy body dementia (n=7), clinically probable frontotemporal dementia or clinically probable primary progressive aphasie semantic or progressive nonfluent variant (n = 10), clinically probable vascular dementia (n = 4), alcoholic dementia (n = 1)), other neurological disorder (OND, total n = 22: epilepsy (n = 2), metabolic encephalopathy (n = 3), Parkinson’s disease (n = 1), autoimmune encephalitis (n = 3), trigeminus neuralgia (n = 1), posttraumatic (n = 1), post-irradiation leukoencephalopathy (n = 2), post-beta cell transplantation (n = 1), iatrogenic parkinsonism (n = 1), panhypopituitary insufficiency (n = 1), drug-induced cognitive
A New BACE1 Sandwich ELISA
deficits (n = 1), Duchenne (n = 1), transient amnesia (n = 1), cerebellar ataxia (n = 1), alcohol abuse (n = 1) and obstructive sleep apnea syndrome (n = 2)), and no evidence for neurological diseases (total n = 24). This last group will be pooled with the 7 cognitively intact healthy controls that underwent the lumbar puncture strictly for research purposes in the non-neurological disorder control group (NND, total n = 31). All subjects gave informed consent for the lumbar puncture. The healthy controls who received the lumbar puncture for scientific purposes provided written informed consent and the study was approved by the Ethics Committee UZ Leuven. The subjects who received the lumbar puncture for diagnostic purposes provided oral consent for the diagnostic procedure and for the storage and scientific use of the rest material in agreement with the EC approved UZ Leuven policy. CSF was sampled by lumbar puncture at the L3/L4 intervertebral space at a standardized time (8-12 AM) to avoid potential diurnal variation. CSF was collected in polypropylene tubes and stored in polypropylene cryotubes at -20˚C within 4 hours after sampling until further analysis. Aliquots of the CSF samples were used for routine analysis, measurement of Alzheimer’s disease biomarkers (A1-42, ttau and p-tau levels) and measurement of BACE1 levels in our assay. During the initial preliminary run of the new sandwich ELISA, BACE1 levels were measured in a large group of human CSF samples and two pools of human CSF, including samples with low and high concentration of BACE1 respectively, were mixed and aliquoted in order to have control samples that could be included in every run (QC sample low concentration and high concentration). We avoided to use samples contaminated with blood. Measurements of BACE1 were stable over three freeze-thaw cycles. Statistical Analysis Statistical analyses were performed using GraphPad Prism software (Prism; GraphPad Software San Diego, California). Results are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (One-way ANOVA) was used to detect a significant difference of CSF A1-42, t-tau, p-tau, A1-42/t-tau and BACE1 levels in the different groups (NND, MCI, OD, OND and AD). Bonferroni’s multiple comparison test was used to compare all groups and Dunnett’s multiple comparison test was used to compare all diagnose groups with the control group (NND). A Pearson correlation coefficient was calculated using STATISTICA software (STATISTICA, version 10, StatSoft, www.statsoft.com) to evaluate the possible correlation of CSF BACE1 levels with CSF A1-42, t-tau and p-tau levels as well as with the ratio of A1-42 over t-tau and age. P