High Activities of BACE1 in Brains with Mild Cognitive Impairment

Brain Samples for Research

Human tissue used in this study was collected with informed consent of participants or next of kin and with ethical approval from the institutional review boards of Sun Health Research Institute (Sun City, AZ). Details of the pathologic evaluation, including brain autopsy, and Braak staging, as well as the clinical evaluation, have been previously reported.⁹ All procedures that involved experiments on humans were performed in accordance with the ethical standards of the Committee on Human Experimentation of Sun Health Research Institute. Temporal cortex samples from AD, MCI, and ND patients with clinically diagnosed, neuropathologically confirmed conditions were frozen at autopsy and stored in vacuum-sealed plastic bags at −80°C until assayed. AD was diagnosed by National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related Disorders Association¹⁴ criteria and confirmed by autopsy using National Institute on Aging–Reagan Institute Working Group on Diagnostic Criteria for the Neurological Assessment of Alzheimer’s Disease.¹⁵ MCI was diagnosed according to the Petersen criteria¹⁶ (ie, MCI patients performed 1.5 SDs below the age-adjusted reference mean in memory scales, as assessed using the Consortium to Establish a Registry for Alzheimer’s Disease cognitive battery,¹⁷ including verbal learning, recognition, and recall tests). Global cognitive function and activities of daily living were unimpaired in the MCI subjects. A board-certified neuropathologist using Consortium to Establish a Registry for Alzheimer’s Disease criteria¹⁸ performed the neuropathologic evaluation. Postmortem intervals averaged <3 hours. We randomly selected 54 cases, consisting of 18 AD cases, 18 MCI cases, and 18 ND control cases. All of the patients had died from lung or kidney failure, and they did not have any other central nervous system (CNS) diseases, sudden death, immunologic disorders, or severe infectious diseases (based on clinical records and pathologic examination). These 3 groups were well matched with regard to sex, age, postmortem interval, and sample preparations (Table 1).

BACE1 Enzymatic Activity Assay

Activity assays of BACE1 were performed as described previously.¹⁹ In brief, samples of human temporal cortex were lyzed with a lysis buffer (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L Na₃VO₄, 10% glycerol, and 0.5% Triton X-100). BACE1 enzymatic activity assays were performed by using synthetic peptide substrates containing Swedish mutant BACE1 cleavage site (Calbiochem, San Diego, CA). BACE1 substrate was dissolved in dimethyl sulfoxide and mixed with a 100 mmol/L Tris-HCl and 100 mmol/L NaCl, pH 4.5 (pH adjusted with acetic acid), reaction buffer. An equal amount of protein was mixed with 100 μL of substrate, and fluorescence intensity was measured with a microplate reader (BioTek, Winooski, VT) at an excitation wavelength of 430 nm and an emission wavelength of 520 nm. For the inhibitor test experiment, human brain lysate or BACE1 stable cell lysate was preincubated with 10 nmol/L BACE1-specific inhibitor (BACE1 inhibitor IV; Millipore, Billerica, MA) or 1 μmol/L cathepsin D inhibitor pepstatin-A (Millipore) on ice for 30 minutes and then mixed with substrate for fluorescence intensity measurement.

Western Blot

The samples of human temporal cortex were individually homogenized in 1× radioimmunoprecipitation assay buffer, including a protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Protein concentration was measured by protein assay (Bio-Rad Laboratories, Hercules, CA), and 50 to 100 μg of total protein was subjected to SDS-PAGE (6% to 12% acrylamide). Separated proteins were then transferred onto polyvinylidene fluoride membranes. The blots were probed with the following antibodies: anti-BACE1 polyclonal antibody (Calbiochem), anti-BACE1 monoclonal antibody (R&D Systems, Minneapolis, MN), and anti–β-actin monoclonal antibody (1:5000; Sigma-Aldrich, St. Louis, MO). Western blot measurements were repeated three times independently.

ELISA

BACE1 protein levels were measured by enzyme-linked immunosorbent assay (ELISA) as described previously,^5,19 with some modifications. The capture antibody was anti-BACE1 monoclonal antibody (R&D Systems), and the detection antibody was biotinylated anti-BACE1 polyclonal antibody B280. TMB substrate (Thermo Scientific Pierce, Rockford, IL) was used to visualize the reaction product, which was read at OD 450 with a microplate reader (Sigma-Aldrich). BACE1 protein (Calbiochem) was used as a standard. Data are presented as means ± SD of three experiments.

Tumor necrosis factor (TNF)-α levels in the lyzed extracts were measured by a commercially available ELISA kit for human TNF-α (R&D Systems). The manufacturer instructions were used throughout. Dilution curve homogenate samples of the brain were parallel to the standard dilution curve. The absorbance was measured at 450 nm with an automatic wavelength correction of 540 nm, and each experiment was repeated three times.

Neurotypical Cell Line Culture

Human SH-SY5Y cells, which can be terminally differentiated into cells that bear numerous specific characteristics of neurons as described previously,²⁰ were cultured using 50% minimal essential medium plus 50% F12 medium, 10% heat-inactivated fetal calf serum, and 10 μmol/L retinoic acid. The culture medium was replaced every 3 days, and cells were differentiated for 6 days. Differentiated cells were seeded on 6-well plates that were precoated with 100 μg/mL of poly-l-lysine hydrobromide (Sigma-Aldrich) at a density of 1 × 10⁵ cells/cm². Differentiated neurotypical SH-SY5Y cells were grown with the medium based on Neurobasal A supplemented with 2% of B27, 2 mmol/L of l-glutamine, and 1% of N2 supplement (Invitrogen, Carlsbad, CA) at 37°C with 5% CO₂. Half volume of the medium was changed every 3 days. Differentiated SH-SY5Y cells were treated at 6 days with different concentration of TNF-α and IL-1β (R&D Systems). After 24 hours, cells were collected and lyzed for Western blot. Experiments were repeated three times.

Cell Transfection and Luciferase Assay

HEK293 cells were transfected with pB1P-A vector containing a BACE1 promoter (−1941 to +292) upstream from a luciferase reporter gene using lipofectamine (Invitrogen). After transfection, cells were treated with different concentrations of TNF-α, IL-8, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D Systems) or NF-κB inhibitor 6-amino-4(4-phenoxyphenylethylamino) quinazoline (Calbiochem). Cells were collected at different time points after treatment, and luciferase assay (Promega, Madison, WI) was performed and luminescence intensity measured as previously described.¹⁹

Statistical Analysis

Data are presented as means ± SD. Differences in means were determined by analysis of variance. P < 0.05 was considered statistically significant.

Elevated BACE1 Protein Levels in Brains with MCI

We and others have previously demonstrated that significantly increased protein levels of BACE1 are observed in the AD brain.^5,6,21,22 By using the same BACE1 antibodies as we previously described,^5,6 Western blots were used to measure BACE1 protein expression levels. As previously mentioned,⁶ the yield of total protein did not correlate with age at the time of death or with the postmortem interval for ND, MCI, or AD patients (data not shown). It was not surprising that similar and significant elevation of BACE1 protein levels were observed in AD temporal cortex samples compared with that of ND samples (Figure 1, A and B). Interestingly, the Western blot analysis in the samples also revealed a higher level of BACE1 protein expression in the temporal cortex of MCI patients than that in the same brain regions of ND controls. The statistical significance (P < 0.05) was reached after all cases were examined (n = 18) in each group (Figure 1, A and B). However, the difference between AD and MCI was not significant (Figure 1), which is consistent with our previous findings.^5,6 Similar significant differences were found in Western blots detected with additional antibodies as published (data not shown).

Increased BACE1 Enzymatic Activity Levels in MCI Brains

An increased BACE1 protein level is observed in MCI brains, suggesting similar BACE1 enzymatic activity in MCI brains as we observed in AD brains.^5,6 As for AD brains, fluorescent-labeled peptide bearing the APP containing the Swedish mutation (APPsw) sequence was used as a BACE1 enzymatic substrate to test whether the enzyme from the AD brain crude extracts could cleave the substrate. As expected, there was an approximately 37% increased fluorescent intensity in AD temporal cortex extracts compared with that in ND controls. Importantly, we found that the fluorescent intensity in MCI temporal cortex extracts was significantly increased by 27% (P < 0.05) compared with that of ND controls (Figure 1C). In a parallel test model for another positive control as previously reported,^5,6 we transiently transfected BACE1 cDNA into HEK293 cells stably expressing APPsw. We observed a high-fluorescent intensity in BACE1-transfected cells, suggesting that BACE1-overexpressing cells have higher BACE1 activity. In summary, a mixture of APPsw peptide and MCI and AD crude extracts generated higher fluorescent fragment products (127% and 137%, P < 0.05) than that of the mixture of APPsw peptide and ND extracts (Figure 1C). Our results suggest that elevated BACE1 enzymatic activity was observed not only in AD brains but also in MCI brains.

Association of BACE1 Activity with Amyloid Plaque Burden in the Temporal Cortex Not Only in AD but Also in MCI Patients

We previously reported that there is a correlation between BACE1 enzymatic activity and plaque numbers.⁶ Because plaques that contain Aβ are one of the established pathologic hallmarks in the AD brain, BACE1 is one of the major enzymes in Aβ production, and a high level of BACE1 was found in not only AD but also MCI brains (Figure 1), we examined a possibility of whether BACE1 activity was correlated with plaque burden in MCI brains. By using linear regression analysis, we observed a significant correlation between levels of BACE1 enzymatic activity and plaque numbers in MCI brains (Figure 2A). The data suggest that there is an active and dynamic AD-related pathologic process during the MCI status, an early stage of AD.

Selective Changes in Cortical BACE1 Activity as Cognitive Deficits Merge

We and others have reported the abnormal increase of BACE1 enzymatic activity in sporadic AD.^5,6,21 Moreover, we also discovered that CSF BACE1 activity was an indicator of the impaired cognitive function in MCI patients.⁷ We found increased BACE1 activity in MCI brains (Figure 1). We therefore performed within-subject correlational analyses to determine whether changes in the MCI brain BACE1 activity (Figure 1C) correspond to cognitive decline as assessed by the MMSE (Figure 2B). Among 18 MCI patients, 11 had last MMSE before death. Closer inspection of enzymatic activity levels across study participants sorted by cognitive status (Figure 2B) confirmed that MCI brain cortical BACE1 activity increases during the earliest stages of dementia. This increase was then followed by a precipitous decrease as cognitive decline progressed, which likely accounts for the slightly positive correlation between MCI brain BACE1 activity and MMSE scores (Figure 2B).

Increased Proinflammatory Cytokine TNF-α Protein Levels in MCI Brains

Because TNF-α is one of the major inflammatory cytokines in AD brains^19,23 and is required for amyloid protein–induced neuron death,¹⁹ it is essential to understand whether protein levels of TNF-α are changed in the MCI brain. TNF-α ELISA from identical brain regions of the same patients revealed a 26% and 36% increase in TNF-α in MCI and AD temporal tissues, respectively, when compared with that found in ND controls (Figure 3A). In addition, the TNF-α increase may not be entirely due to neuronal loss. The elevation of TNF-α levels may be due to the activation of glial cells that normally express TNF-α protein in affected regions of MCI and AD brains. However, no significant difference was observed in TNF-α protein levels between AD and MCI brains (Figure 3A).

TNF-α Up-Regulates BACE1 Protein Expression

Because both BACE1 and TNF-α protein levels were significantly increased in the brains of AD and MCI patients, the next question was whether there was any relationship between these two molecules. With exogenous TNF-α treatment on primary human neurons, BACE1 protein levels were significantly increased in a dose-dependent manner, whereas IL-1β appeared to have little effect on BACE1 protein expression (Figure 3B).

TNF-α Up-Regulates BACE1 Transcription through the NF-κB Pathway

To clarify how TNF-α up-regulates BACE1 protein expression, HEK293 cells were transfected with a BACE1 promoter luciferase reporter vector and then treated with different concentrations of TNFα, IL-8, Il-12, and GM-CSF, respectively. We found significantly increased BACE1 promoter activity in a concentration-dependent manner with exogenous TNF-α treatment (Figure 3C). However, there are little effects of IL-8, IL-12, and GM-CSF treatments on BACE1 promoter activity (Figure 4). Because NF-κB is one of the major mediators of TNF-α–activated signaling²⁴ and multiple NF-κB binding sites are located in the vicinity of the BACE1 promoter,²⁵ an NF-κB activation inhibitor was used to block NF-κB signaling in TNF-α–treated HEK293 cells transfected with the BACE1 promoter. Treating the cells with an NF-κB inhibitor significantly reduced TNF-α–induced BACE1 promoter activity (Figure 3C), suggesting that the up-regulation of BACE1 transcription may be through TNF-α–mediated activation of NF-κB.