The novel squamosamide derivative FLZ enhances BDNF/TrkB/CREB signaling and inhibits neuronal apoptosis in APP/PS1 mice

Abstract

Aim: The aim of this study was to study the effects of compound FLZ, a novel cyclic derivative of squamosamide from Annona glabra, on brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)/cAMP response element-binding protein (CREB) signaling and neuronal apoptosis in the hippocampus of the amyloid precursor protein (APP)/presenilin-1 (PS1) double transgenic mice.

Methods: APP/PS1 mice at the age of 5 months and age-matched wild-type mice (WT) were intragastrically administered FLZ (150 mg/kg) or vehicle [0.05% carboxymethyl cellulose sodium (CMC-Na)] daily for 20 weeks. The levels of BDNF in the hippocampus of WT and APP/PS1 mice were then measured by immunohistochemistry and Western blot analysis. Neuronal apoptosis in mouse hippocampus was detected by Nissl staining. Expression of NGF, NT3, pTrkB (Tyr515)/TrkB, pAkt (Ser473)/Akt, pERK/ERK, pCREB (Ser133)/CREB, Bcl-2/Bax, and active caspase-3 fragment/caspase-3 in the hippocampus of WT and APP/PS1 mice was detected by Western blot analysis.

Results: Compared with vehicle-treated APP/PS1 mice, FLZ (150 mg/kg) significantly increased BDNF and NT3 expression in the hippocampus of APP/PS1 mice. In addition, FLZ promoted BDNF high-affinity receptor TrkB phosphorylation and activated its downstream ERK, thus increasing phosphorylation of CREB at Ser133 in the hippocampus of APP/PS1 mice. Moreover, FLZ showed neuroprotective effects on neuronal apoptosis by increasing the Bcl-2/Bax ratio and decreasing the active caspase-3 fragment/caspase-3 ratio in the hippocampus of APP/PS1 mice.

Conclusion: FLZ exerted neuroprotection at least partly through enhancing the BDNF/TrkB/CREB pathway and inhibiting neuronal apoptosis in APP/PS1 mice, which suggests that FLZ can be explored as a potential therapeutic agent in long-term Alzheimer's disease therapy.

Figure 1

FLZ treatment increased the expression of BDNF in the hippocampus of APP/PS1 mice. Five-month-old mice were treated with vehicle (0.05% CMC-Na) or FLZ (150 mg/kg) daily for 20 weeks. (A) Representative immunohistochemistry images of BDNF in the hippocampus of wild type (WT) and APP/PS1 (Tgs) mice (4 mice per group). BDNF immunoreactivity was quantification by Image Pro Plus 6.0 software. IOD, integrated optical density. (B) Representative Western blot bands showing BDNF expression in the hippocampus of WT and Tgs mice treated with vehicle or FLZ (150 mg/kg). n=5–6 mice per group. Three representative Western blots for each group are shown. The integrated density value was determined using densitometry. IDV, integrated density value. WT, wild-type mice; Tgs, APP/PS1 mice treated with 0.05 CMC-Na; FLZ-treated Tgs, APP/PS1 mice treated with FLZ (150 mg/kg). Results are expressed as means±SEM. ^bP<0.05 vs WT. ^eP<0.05 vs Tgs.

Figure 2

FLZ treatment increased pTrkB (Tyr515) levels and the pTrkB (Tyr515)/TrkB ratio in the hippocampus of APP/PS1 mice. Five-month-old mice were treated with vehicle (0.05% CMC-Na) or FLZ (150 mg/kg) for 20 weeks. Levels of TrkB and pTrkB (Tyr515) were detected by Western blot and analyzed by Gel Pro Plus 4.0. n=5–6 mice per group. Three representative Western blots for each group are shown. IDV, integrated density value. Data are expressed as means±SEM. ^bP<0.05 Tgs vs WT. ^eP<0.05 FLZ-treated Tgs vs Tgs.

Figure 3

FLZ treatment activated ERK but not Akt in the hippocampus of APP/PS1 mice. Five-month-old mice were treated with vehicle (0.05% CMC-Na) or FLZ (150 mg/kg) for 20 weeks. The levels of pERK, total ERK, pAkt (Ser473) and Akt (1/2) were detected by Western blot and analyzed by Gel Pro Plus 4.0. Figure A, Representative Western blot bands of pERK and ERK in the hippocampus of WT and Tgs mice. Figure B, Representative Western blot bands of pAkt and Akt in the hippocampus of different groups treated with vehicle or FLZ (150 mg/kg). IDV, integrated density value. n=5−6 mice per group. Three representative Western blot for each group are shown. Means±SEM. ^bP<0.05 vs WT. ^eP<0.05 vs Tgs.

Figure 4

FLZ increased pCREB (Ser133) expression in the hippocampus of APP/PS1 mice. Five-month-old mice were treated with vehicle (0.05% CMC-Na) or FLZ (150 mg/kg) for 20 weeks. pCREB (Ser133) levels were detected by Western blot and analyzed by Gel Pro Plus 4.0. n=5−6 mice per group. Three representative Western blots for each group are shown. Data are expressed as means±SEM. ^cP<0.01 Tgs vs WT. ^eP<0.05 FLZ-treated Tgs vs Tgs.

Figure 5

FLZ treatment increased NT3 expression but not NGF expression in the hippocampus of APP/PS1 mice. Five-month-old mice were treated with vehicle (0.05% CMC-Na) or FLZ (150 mg/kg) for 20 weeks. (A) Representative Western blot bands of NGF and NT3 in the hippocampus of WT and Tgs mice. (B) Densitometric quantification of NGF and NT3 expression in the hippocampus of WT and Tgs mice. IDV, integrated density value. n=5−6 mice per group. Three representative western blots for each group are shown. Results are expressed as means±SEM. ^cP<0.01 Tgs vs WT. ^fP<0.01 FLZ-treated Tgs vs Tgs.

Figure 6

FLZ treatment inhibited neuronal apoptosis in the hippocampus of APP/PS1 mice by Nissl staining. Five-month-old mice were treated with vehicle (0.05% CMC-Na) or FLZ (150 mg/kg) for 20 weeks. (A) Representative Nissl staining images showing Nissl bodies in the hippocampal CA1 regions of WT and Tgs mice. (B) Quantification of Nissl bodies in the hippocampus of WT and Tgs mice (4 mice per group) by Image Pro Plus 6.0 software. IOD, integrated optical density. ^bP<0.05, Tgs vs WT. ^eP<0.05, FLZ-treated Tgs vs Tgs.

Figure 7

FLZ treatment increased the Bcl-2/Bax ratio and decreased the active caspase-3 fragment/caspase-3 ratio in the hippocampus of APP/PS1 mice. Five-month-old mice were treated with vehicle (0.05% CMC-Na) or FLZ (150 mg/kg) for 20 weeks. (A) Representative Western blot bands of Bcl-2 and Bax in the hippocampus of WT and Tgs mice. (B) Representative Western blot bands of active caspase-3 fragment and caspase-3 in the hippocampus of different groups. IDV, integrated density value. n=5−6 mice per group. Three representative Western blots for each group are shown. Data are expressed as means±SEM. ^bP<0.05, ^cP<0.01 Tgs vs WT. ^eP<0.05, FLZ-treated Tgs vs Tgs.