BACE1 deletion in the adult mouse reverses preformed amyloid deposition and improves cognitive functions

Abstract

BACE1 initiates the generation of the β-amyloid peptide, which likely causes Alzheimer's disease (AD) when accumulated abnormally. BACE1 inhibitory drugs are currently being developed to treat AD patients. To mimic BACE1 inhibition in adults, we generated BACE1 conditional knockout (BACE1^fl/fl) mice and bred BACE1^fl/fl mice with ubiquitin-Cre^ER mice to induce deletion of BACE1 after passing early developmental stages. Strikingly, sequential and increased deletion of BACE1 in an adult AD mouse model (5xFAD) was capable of completely reversing amyloid deposition. This reversal in amyloid deposition also resulted in significant improvement in gliosis and neuritic dystrophy. Moreover, synaptic functions, as determined by long-term potentiation and contextual fear conditioning experiments, were significantly improved, correlating with the reversal of amyloid plaques. Our results demonstrate that sustained and increasing BACE1 inhibition in adults can reverse amyloid deposition in an AD mouse model, and this observation will help to provide guidance for the proper use of BACE1 inhibitors in human patients.

Graphical abstract

Figure 1.

Characterization of Cre recombinase expression in UBC-Cre/ER^T2 mice. (A) Gt(ROSA)26Sor^tm1Sor/J Cre-reporter mice were bred with UBC-Cre/ER^T2 mice to monitor expression of Cre recombinase in the brain, as detected by expression of β-galactosidase. Expression of LacZ, as detected by X-Gal, in P15 brains was sporadic and not readily detected. LacZ was detectable at P45 and was more prominent at P90 in compound mice. Bar, 200 µm. (B) Confocal staining was conducted to monitor the expression of β-galactosidase in neurons (labeled by NeuN antibody), microglia (Iba1 antibody), or astrocytes (Smi22 for GFAP) in P90 compound mice or control littermates. Bar, 100 µm.

Figure 2.

Sequential deletion of BACE1 in adult mice. (A) BACE1^fl/fl mice were crossed with UBC-Cre/ER^T2 mice to obtain BACE1^{fl/fl/UbcCreER} mice, and expression of BACE1 in BACE1^{fl/fl/UbcCreER} mice was examined at different ages (P7–P120). BACE1 substrates such as APP, Nrg1, and Jag1, as well as selected relevant downstream molecules such as Notch intracellular domain (NICD), MBP, PLP, and BLBP, were also examined in parallel. Molecular mass is indicated in kilodaltons. Antibody to β-actin was used to verify equal loading. (B) Bar graphs show the arbitrary levels of specified proteins in different age groups (n = 3 independent experiments; two or three animals in each age group were compared side by side; *, P < 0.05, **, P < 0.01; two-tailed Student’s t test). (C) Ratios of BACE1 levels in BACE1^{fl/fl/UbcCreER} mice to BACE1^fl/fl mice in different age groups are plotted (n = 6–9 animals in each group). BACE1 levels were reduced by ∼80% in P120 BACE1^{fl/fl/UbcCreER} mice. Values are expressed as mean ± SEM.

Figure 3.

Reversal of amyloid deposition in an adult AD mouse model produced by sequentially increasing deletion of BACE1. (A) Fixed brain sections from different age groups of the indicated genotypes of mice were stained with antibody 6E10 to label amyloid plaques. A sequential increase in amyloid plaque load in BACE1^fl/fl/5xFAD mice from P75, P120, P190, and P300 was visible. Enlarged views show differences in neurons and amyloid plaques, which are indicated by red arrows. No amyloid plaques were observed in P300 BACE1^{fl/fl/UbcCreER/5xFAD} mice, whereas a high load of amyloid plaques was observed in P120 BACE1^{fl/fl/UbcCreER/5xFAD} mice. Bar, 200 µm. (B and C) Numbers of 6E10-positive plaques in the cortex (B) and hippocampus (C) were quantified from six animals in each age group, and mean numbers per section are plotted for comparison (**, P < 0.01; ***, P < 0.001; two-tailed Student’s t test). Plaque load was significantly reduced in BACE1^{fl/fl/UbcCreER/5xFAD} mice older than P120 compared with BACE1^fl/fl/5xFAD littermates. Values are expressed as mean ± SEM.

Figure 4.

Sequentially increased deletion of BACE1 reduces APP processing and Aβ generation. (A) APP processing products were examined by Western blot analyses. C99 is a BACE1-cleaved APP C-terminal fragment, which was detected by both antibody 6E10 and A8717, which recognize the APP C terminus. Antibody A8717 also detects C83, which is a product resulting from α-secretase cleavage of APP. Antibody to β-actin was used to verify equal loading. Blot measurements in kilodaltons. (B) Relative levels of BACE1 in different age groups are plotted for comparison (equal to at least six animals in each genotype and age group). Significantly less C83 and C99 were observed in BACE1^{fl/fl/UbcCreER/5xFAD} mice compared with BACE1^fl/fl/5xFAD mice beginning at P120. (C and D) Insoluble Aβ₄₀ and Aβ₄₂ from mouse hippocampal regions were extracted and measured by standard ELISA methods (n = 6 pairs of animals; **, P < 0.01; ***, P < 0.001; two-tailed Student’s t test). Values are expressed as mean ± SEM.

Figure 5.

Reversal of gliosis and neuritic dystrophy is associated with removal of amyloid plaques. (A) Fixed brain sections from P75, P120, and P300 mice were stained with antibody 6E10 to label amyloid plaques and Iba1 to label microglia. (B) Similar brain sections were stained with an IBL antibody specific to Aβ₄₂ to label core amyloid plaques. Astrocytes were labeled by Smi22 antibody, which is specific to GFAP. (C) Although amyloid plaques were labeled by antibody 6E10, dystrophic neurites were labeled by antibody R458, which is specific to the C terminus of RTN3. Dystrophic neurites were formed in correlation with amyloid plaque density in older 5xFAD mice but were essentially absent when plaques were cleared in P300 BACE1^{fl/fl/UbcCreER/5xFAD} mice. All images were captured from hippocampal subiculum. Bars, 40 µm. White arrows indicate the dispersed dystrophic neurites, which were labeled by R458 antibody.

Figure 6.

BACE1 deletion in the adult impacts LTP. (A–D) LTP was recorded on horizontal hippocampal slices from four genotypes of 10–12-mo-old mice using the MED64 system, and Schaffer collaterals to CA1 synapses were analyzed for LTP assays. Comparison between BACE1^fl/fl mice and BACE1^fl/fl/5xFAD littermates is shown in A. (B) LTP was also recorded on horizontal hippocampal slices from 4–5-mo-old (labeled as P140+) BACE1^fl/fl/5xFAD mice and compared with that from 10–12-mo-old (P300+) BACE1^fl/fl/5xFAD mice (n = 8–10 slices). Comparison between BACE1^fl/fl/5xFAD mice and BACE1^{fl/fl/UbcCreER/5xFAD} mice is shown in C (Student’s t test). There was no significant difference in LTP between BACE1^{fl/fl/UbcCreER} mice and BACE1^{fl/fl/UbcCreER/5xFAD} mice (D). (E and F) Comparisons of all four genotypes of mice are shown in E, and all five groups are shown in F. *, P < 0.05; **, P < 0.01; Student’s t test. ns, no significance. Values are expressed as mean ± SEM.

Figure 7.

BACE1 deletion in the adult 5xFAD mouse model ameliorates learning and behavioral impairments. A fear conditioning assay was conducted for 3 d using standard procedures. There were no differences in the percentage of freeze time during the day 1 preconditioning test. On day 2, contextual fear learning and memory of the mice were analyzed. Changes in total freeze time on day 2 are reflected in their contextual learning ability. BACE1^fl/fl/5xFAD mice (n = 20) showed significantly less freezing time compared with BACE1^{fl/fl/UbcCreER/5xFAD} mice (n = 25; *, P < 0.05). Day 3 measured tone-mediated cue memory by comparing freezing during the presentation of tones in a different chamber, which is more related to amygdala function, and no significant differences were noted among the four genotypes of mice. Values are expressed as mean ± SEM.