7,8-dihydroxyflavone, a small-molecule TrkB agonist, reverses memory deficits and BACE1 elevation in a mouse model of Alzheimer's disease

Abstract

Increasing evidence suggests that reductions in brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) may have a role in the pathogenesis of Alzheimer's disease (AD). However, the efficacy and safety profile of BDNF therapy (eg, gene delivery) remains to be established toward clinical trials. Here, we evaluated the effects of 7,8-dihydroxyflavone (7,8-DHF), a recently identified small-molecule TrkB agonist that can pass the blood-brain barrier, in the 5XFAD transgenic mouse model of AD. 5XFAD mice at 12-15 months of age and non-transgenic littermate controls received systemic administration of 7,8-DHF (5 mg/kg, i.p.) once daily for 10 consecutive days. We found that 7,8-DHF rescued memory deficits of 5XFAD mice in the spontaneous alternation Y-maze task. 5XFAD mice showed impairments in the hippocampal BDNF-TrkB pathway, as evidenced by significant reductions in BDNF, TrkB receptors, and phosphorylated TrkB. 7,8-DHF restored deficient TrkB signaling in 5XFAD mice without affecting endogenous BDNF levels. Meanwhile, 5XFAD mice exhibited elevations in the β-secretase enzyme (BACE1) that initiates amyloid-β (Aβ) generation, as observed in sporadic AD. Interestingly, 7,8-DHF blocked BACE1 elevations and lowered levels of the β-secretase-cleaved C-terminal fragment of amyloid precursor protein (C99), Aβ40, and Aβ42 in 5XFAD mouse brains. Furthermore, BACE1 expression was decreased by 7,8-DHF in wild-type mice, suggesting that BDNF-TrkB signaling is also important for downregulating baseline levels of BACE1. Together, our findings indicate that TrkB activation with systemic 7,8-DHF can ameliorate AD-associated memory deficits, which may be, at least in part, attributable to reductions in BACE1 expression and β-amyloidogenesis.

Figure 1

Reductions of mature BDNF in 5XFAD mice. (a) Immunoblot analysis of hippocampal lysates from 5XFAD and age-matched wild-type control mice at different ages. (b) Immunoreactive bands for mature BDNF (14 kDa) were quantified and expressed as the percentage of wild-type control levels at 3 months of age (n=5–6 mice per group). Hippocampal mature BDNF levels are significantly reduced as early as 3 months of age in 5XFAD mice (^*p<0.05 vs wild-type). All data are presented as mean±SEM.

Figure 2

Effects of systemic 7,8-DHF on memory deficits in 5XFAD mice. 5XFAD and wild-type control mice at 12–15 months of age received repeated administration of 7,8-DHF (5 mg/kg, i.p.) or vehicle (17% DMSO) once daily for 10 consecutive days. Two hours after the last injection, the mice were tested for memory using the spontaneous alternation Y-maze task. (a) Spatial working memory, as assessed by the spontaneous alternation performance, is impaired (around 50% chance levels) in 5XFAD mice compared with wild-type controls (^*p<0.05). Note that 7,8-DHF-treated 5XFAD mice are rescued completely back to wild-type levels of alternation performance (^#p<0.05 vs vehicle-treated 5XFAD). n=5–12 mice per group. (b) Total number of arm entries reflecting exploratory activities of mice in the Y-maze does not differ between the groups, suggesting that the effect of 7,8-DHF is memory-specific. n=5–12 mice per group. All data are presented as mean±SEM.

Figure 3

Effects of systemic 7,8-DHF on BDNF-TrkB signaling in 5XFAD mice. 5XFAD and wild-type control mice at 12–15 months of age received repeated administration of 7,8-DHF or vehicle (once daily for 10 consecutive days), and were killed 2 h after the last injection. (a) Immunoblot analysis of hippocampal lysates from wild-type and 5XFAD mice treated with 7,8-DHF or vehicle. (b–d) Immunoreactive bands for phosphorylated TrkB (b), total TrkB (c), and mature BDNF (d) were quantified and expressed as the percentage of vehicle-treated wild-type control levels (n=6–9 mice per group). Note that hippocampal phospho-TrkB levels are dramatically reduced in vehicle-treated 5XFAD mice (^*p<0.05 vs wild-type, vehicle), and this reduction is completely rescued by 7,8-DHF treatments (^#p<0.05 vs 5XFAD, vehicle). In fact, phospho-TrkB levels in 7,8-DHF-treated 5XFAD mice are significantly higher than those of wild-type vehicle controls (^*p<0.05). Moreover, repeated 7,8-DHF treatments also rescues the downregulation of total TrkB protein levels in 5XFAD mice (^#p<0.05); however, it shows a trend toward reducing TrkB receptors in wild-type controls (p=0.15). Meanwhile, 7,8-DHF treatments do not affect baseline levels of mature BDNF in the hippocampus of wild-type mice or BDNF reductions in 5XFAD mice. All data are presented as mean±SEM.

Figure 4

Immunofluorescence labeling of phosphorylated TrkB in the hippocampus of 5XFAD mice. 5XFAD mice at 12–15 months of age received repeated administration of 7,8-DHF or vehicle (once daily for 10 consecutive days), and were perfused for immunostaining 2 h after the last injection (n=3 mice per group). Shown are representative photomicrographs of hippocampal CA1 (a–c) and CA3 (d–f) regions. Note that phospho-TrkB staining is dramatically reduced in vehicle-treated 5XFAD mice (b, e) compared with vehicle-treated wild-type controls (a, d), whereas TrkB activation is restored back to wild-type levels in 7,8-DHF-treated 5CFAD mice (c, f). Scale bar=50 μm.

Figure 5

Effects of systemic 7,8-DHF on APP processing and Aβ levels in 5XFAD mice. 5XFAD and wild-type control mice at 12–15 months of age received repeated administration of 7,8-DHF or vehicle (once daily for 10 consecutive days), and were killed 2 h after the last injection. (a) Immunoblot analysis of hemibrain lysates from wild-type and 5XFAD mice treated with 7,8-DHF or vehicle. (b, c) Immunoreactive bands for BACE1 (b) and C99 (c) were quantified and expressed as the percentage of vehicle-treated wild-type and 5XFAD levels, respectively (n=6–9 mice per group). Note that BACE1 levels are significantly elevated in vehicle-treated 5XFAD mice (^*p<0.05 vs wild-type, vehicle), and this upregulation is suppressed by 7,8-DHF treatments (^#p<0.05 vs 5XFAD, vehicle). Moreover, repeated 7,8-DHF treatment also significantly reduces baseline levels of BACE1 in wild-type mice (^*p<0.05). Consistent with the BACE1 reduction, C99 levels are significantly lowered by 7,8-DHF treatments in 5XFAD mice (^#p<0.05). (d, e) Levels of total Aβ40 (d) and Aβ42 (e) were quantified by sandwich ELISAs of guanidine extracts of hemibrain samples and expressed as the percentage of vehicle-treated 5XFAD levels (n=6–9 mice per group). Repeated 7,8-DHF treatment almost equivalently reduced Aβ40 and Aβ42 levels in 5XFAD mouse brains (^#p<0.05 vs 5XFAD, vehicle). All data are presented as mean±SEM.

Figure 6

Effects of acute 7,8-DHF application on BACE1 expression and enzymatic activity. (a, b) Wild-type mice received single administration of 7,8-DHF (5 mg/kg, i.p.) or vehicle, and were killed 2 h later for the analysis. Immunoblot analysis shows that acute 7,8-DHF dramatically increases hippocampal phospho-TrkB levels (^*p<0.05 vs vehicle) without affecting total TrkB (a). Note that a single injection of 7,8-DHF is sufficient to significantly reduce baseline levels of BACE1 in wild-type mouse brains (^*p<0.05 vs vehicle) (b). n=4 mice per group. Data are presented as mean±SEM. (c) In vitro β-secretase activity assay was conducted by coincubating recombinant BACE1 with different concentrations of 7,8-DHF or vehicle. 7,8-DHF (1–10 μM) has no direct inhibitory effect on BACE1 activity.