Partial reduction of BACE1 improves synaptic plasticity, recent and remote memories in Alzheimer's disease transgenic mice

Abstract

beta-Site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates amyloid-beta (Abeta) generation that is central to the pathophysiology of Alzheimer's disease (AD). Therefore, lowering Abeta levels by BACE1 manipulations represents a key therapeutic strategy, but it remains unclear whether partial inhibition of BACE1, as expected for AD treatments, can improve memory deficits. In this study, we used heterozygous BACE1 gene knockout (BACE1+/-) mice to evaluate the effects of partial BACE1 suppression on different types of synaptic and cognitive dysfunctions in Alzheimer's transgenic mice (5XFAD model). We found that approximately 50% BACE1 reductions rescued deficits of 5XFAD mice not only in hippocampus-dependent memories as tested by contextual fear conditioning and spontaneous alternation Y-maze paradigms but also in cortex-dependent remote memory stabilization during 30 days after contextual conditioning. Furthermore, 5XFAD-associated impairments in long-term potentiation (a synaptic model of learning and memory) and declines in synaptic plasticity/learning-related brain-derived neurotrophic factor-tyrosine kinase B signaling pathways were prevented in BACE1+/-.5XFAD mice. Finally, these improvements were related with reduced levels of beta-secretase-cleaved C-terminal fragment (C99), Abeta peptides and plaque burden in relevant brain regions of BACE1+/-.5XFAD mice. Therefore, our findings provide compelling evidence for beneficial effects of partially BACE1-inhibiting approaches on multiple forms of functional defects associated with AD.

Fig. 1

Effects of heterozygous BACE1 deletion on APP processing and Aβ levels in brains of 5XFAD mice at 6 months of age. (a) Immunoblot analysis of protein extracts from hemibrain homogenates of wild-type mice and 5XFAD mice with BACE1^+/+ or BACE1^+/− genotype. (b–e) Intensities of immunoreactive bands on blots for BACE1 (b), full-length APP (c), sAPPβ (d) and C99 (e) were quantified by phosphorimaging and expressed as percentage of 5XFAD levels (n = 5–8 mice per group). Note that ~50% elimination of BACE1 expression results in ~51% and ~55% reductions in sAPPβ and C99, respectively (* p < 0.05 versus 5XFAD), while APP overexpression levels are not changed in BACE1^+/−·5XFAD mice compared to 5XFAD mice. (f–g) Levels of total Aβ40 (f) and Aβ42 (g) were quantified by sandwich ELISAs of guanidine extracts of hemibrain samples and expressed in nanograms per milligram of total protein (n = 5–7 mice per group). Note that excessive levels of Aβ40 and Aβ42 are significantly reduced in BACE1^+/−·5XFAD mice as compared with 5XFAD mice (by ~66% and ~57%, respectively; * p < 0.05). All data are presented as mean ± SEM.

Fig. 2

Effects of heterozygous BACE1 deletion on deficits in hippocampus-dependent memory in 5XFAD mice at 6 months of age. (a) Mice were trained with two CS-US pairings for contextual fear conditioning. 5XFAD mice show significantly lower levels of contextual freezing than wild-type mice when tested 1 day after training (* p < 0.05). Note that BACE1^+/−·5XFAD are rescued completely back to wild-type control levels of contextual memory (^# p < 0.05 versus 5XFAD). n = 20–38 mice per group. (b) Spatial working memory of mice was tested by spontaneous alternation performance in the Y-maze. Note that 5XFAD mice perform poorly (only slightly above 50% chance levels) compared to wild-type controls (* p < 0.05), while BACE1^+/−·5XFAD are improved completely back to wild-type levels of alternation performance (^# p < 0.05 versus 5XFAD). n = 11–21 mice per group. (c) Total number of arm entries reflecting exploratory activities of mice in the Y-maze does not differ between the four groups. n = 11–21 mice per group. All data are presented as mean ± SEM.

Fig. 3

Effects of heterozygous BACE1 deletion on deficits in remote memory stabilization in 5XFAD mice at 6 months of age. (a) To ensure initial hippocampus-dependent memory formation in 5XFAD mice, they were trained by more intensive protocols with three CS-US pairings for contextual fear conditioning. 5XFAD mice show levels of contextual freezing equivalent to those of wild-type mice when tested 1 day after the stronger training. n = 15–22 mice per group. (b) 5XFAD mice exhibit significantly lower levels of freezing than wild-type controls when tested 30 days after training with three CS-US pairings (* p < 0.05). Note that BACE1^+/−·5XFAD are rescued completely back to wild-type levels of 30-day remote contextual memory (^# p < 0.05 versus 5XFAD). n = 12–17 mice per group. All data are presented as mean ± SEM.

Fig. 4

Effects of heterozygous BACE1 deletion on amyloid deposition in the hippocampus and anterior cingulate cortex of 5XFAD mice at 6 months of age. (a–f) Brain sections from wild-type control (a, d), 5XFAD (b, e) and BACE1^+/−·5XFAD (c, f) mice were immunostained with the 6E10 anti-APP/Aβ antibody. Shown are representative photomicrographs of the hippocampus (a–c) and anterior cingulate cortex (d–f) (areas enclosed by the dashed line). Scale bar = 1 mm. (g–h) Percentage area covered by amyloid plaque in the hippocampus (g) and anterior cingulate cortex (h) was measured for quantification. Note that BACE1^+/−·5XFAD mice show significantly lower levels of Aβ plaque burden in both brain regions compared to those of 5XFAD mice (* p < 0.05). n = 7 mice per group. All data are presented as mean ± SEM.

Fig. 5

Effects of heterozygous BACE1 deletion on hippocampal synaptic dysfunctions in 5XFAD mice at 6 months of age. (a) The amplitudes of presynaptic fiber volleys are plotted against the slopes of corresponding fEPSPs in response to different stimulation strengths (20–100 µA) to compare input/output (I/O) curves at Schaffer collateral-CA1 synapses. (b) The summary bar graph shows that basal synaptic transmission as measured by the average slope of I/O curves is significantly impaired in both 5XFAD and BACE1^+/−·5XFAD mice (* p < 0.05 versus wild-type controls). n = 8–14 mice per group. (c) LTP was induced by 3-TBS protocols at Schaffer collateral-CA1 synapses. Each point indicates the fEPSP slope normalized to the average baseline response before 3-TBS is applied at time 0. (d) The summary bar graph shows that LTP as measured by the average normalized fEPSP slopes during 35–40 min after 3-TBS is significantly impaired in 5XFAD mice (* p < 0.05 versus wild-type controls), while BACE1^+/−·5XFAD mice are rescued almost completely back to wild-type control levels of LTP (^# p < 0.05 versus 5XFAD). n = 8–12 mice per group. Traces are the average of fEPSPs recorded during baseline and 35–40 min after 3-TBS. Calibration: 0.5 mV, 10 ms. All data are presented as mean ± SEM.

Fig. 6

Effects of heterozygous BACE1 deletion on impaired hippocampal BDNF-TrkB signaling in 5XFAD mice at 6 months of age. (a) Immunoblot analysis of protein extracts from hippocampal homogenates of wild-type mice and 5XFAD mice with BACE1^+/+ or BACE1^+/− genotype. (b–c) Intensities of immunoreactive bands on blots for BDNF (b) and phosphorylated TrkB (p-TrkB) (c) were quantified by phosphorimaging and expressed as percentage of wild-type levels (n = 5–7 mice per group). Note that both BDNF and p-TrkB levels are significantly reduced in the hippocampus of 5XFAD mice (* p < 0.05 versus wild-type), while these BDNF-TrkB signaling defects are increased back to wild-type control levels in BACE1^+/−·5XFAD mice (^# p < 0.05 versus 5XFAD). All data are presented as mean ± SEM.