BACE1 and BACE2 enzymatic activities in Alzheimer's disease

Abstract

beta-Secretase is the rate limiting enzymatic activity in the production of the amyloid-beta peptide (Abeta) and is thought to be involved in Alzheimer's disease (AD) pathogenesis. Although BACE1 (beta-site APP Cleaving Enzyme 1, EC 3.4.23.46) has received significant attention, the related BACE2 (EC 3.4.23.45) has not. Though BACE2 is also expressed in the brain, its potential role in AD has not been resolved. In this study, we compared the activities of both BACE1 and BACE2, which were isolated from the same samples of frontal cortex from both AD-affected individuals and age-matched controls. BACE1 activity showed a significant positive correlation with the amount of extractable Abeta, and BACE1 protein and activity were significantly increased in AD cases. Unexpectedly, there were substantial total amounts of BACE2 protein and enzymatic activity in the human brain. BACE2 activity did not change significantly in the AD brain, and was not related to Abeta concentration. These data indicate that BACE1 likely accounts for most of the Abeta produced in the human brain, and that BACE2 activity is not a likely contributor. However, as both forms of BACE compete for the same substrate pool, even small changes in BACE2 activity could have consequences for human disease.

Figure 1

The majority of BACE1 protein (as determined by ELISA) does not track with the majority of enzymatic cleavage activity against the APPΔNL substrate when brain homogenate is separated on a discontinuous sucrose gradient. The bulk of BACE1 protein is located in the third (32% sucrose) fraction, whereas the bulk of activity is found in last (43% sucrose) fraction.

Figure 2

Specificity of the BACE immunocapture method for measuring enzymatic activity. Isolated activity can be blocked by the addition of a commercially available specific inhibitor (β-secretase inhibitor IV; EMD biosciences) selective against both forms of BACE (BACE1 IC₅₀, 15 nM; BACE2 IC₅₀, 230 nM). For example, this compound will not effectively inhibit the highly abundant kidney aspartyl protease renin (IC50 >50 μM), and still blocks all of the activity isolated by either BACE1 or BACE2 antibody pull down. Data are expressed as base fluorescence units over two hours, corrected to background fluorescence.

Figure 3

Immunocaptured BACE1 and BACE2 prefer the APPΔNL sequence over the corresponding hexameric sequence (P3′ to P3) from wild type human APP, as shown by time dependent cleavage of both substrates at 37°C. Data are expressed as base fluorescence units corrected to background (control wells, identical except with antibody omitted) at each time point. Capture at either the N- or C-terminus gave similar results, although N-terminal capture usually resulted in greater total activity. Antibodies: MAB931 (N-terminal) or MAB5308 (C-terminal) for BACE1; Ab1 (N-terminal) or Ab5670 (C-terminal) for BACE2.

Figure 4

(A) BACE1 (MAB931) and (B) BACE2 (Ab1) are both readily detected in human brain (recombinant protein in ng/lane). Comparison between multiple immunoblots with either recombinant BACE1 or BACE2 protein indicated that the amount of each enzyme in human brain, at least in the frontal cortex, is approximately equal. The predominant form of BACE2 in brain (consistent with that of rodent kidney), based on its apparent molecular weight and pattern of epitope reactivity, is likely splice form C (exon 7 omitted). (C) Differences between control cases and AD were difficult to detect by immunoblot alone, although BACE1 was slightly higher (p<0.1). Additional evaluation by ELISA confirmed that BACE1 was in fact elevated in the frontal cortex in AD brain (* = p<0.05) whereas BACE2 was unchanged.

Figure 5

BACE1 activity is significantly elevated in AD cases as compared to controls (* = p<0.05) when specific activity is determined either by standardization to total protein (A) or to BACE1 protein as measured by ELISA (B). BACE2 did not change significantly.

Figure 6

The total amount of Aβ increases in AD (* = p<0.01), as measured in both the RIPA and FA extracted fractions from human brain (note the exponential scale). The amount of Aβ in each fraction was correlated (p<0.05) with BACE1, but not BACE2, activity.