Entacapone promotes hippocampal neurogenesis in mice

Abstract

Entacapone, a catechol-O-methyltransferase inhibitor, can strengthen the therapeutic effects of levodopa on the treatment of Parkinson's disease. However, few studies are reported on whether entacapone can affect hippocampal neurogenesis in mice. To investigate the effects of entacapone, a modulator of dopamine, on proliferating cells and immature neurons in the mouse hippocampal dentate gyrus, 60 mice (7 weeks old) were randomly divided into a vehicle-treated group and the groups treated with 10, 50, or 200 mg/kg entacapone. The results showed that 50 and 200 mg/kg entacapone increased the exploration time for novel object recognition. Immunohistochemical staining results revealed that after entacapone treatment, the numbers of Ki67-positive proliferating cells, doublecortin-positive immature neurons, and phosphorylated cAMP response element-binding protein (pCREB)-positive cells were significantly increased. Western blot analysis results revealed that treatment with tyrosine kinase receptor B (TrkB) receptor antagonist significantly decreased the exploration time for novel object recognition and inhibited the expression of phosphorylated TrkB and brain-derived neurotrophic factor (BDNF). Entacapone treatment antagonized the effects of TrkB receptor antagonist. These results suggest that entacapone treatment promoted hippocampal neurogenesis and improved memory function through activating the BDNF-TrkB-pCREB pathway. This study was approved by the Institutional Animal Care and Use Committee of Seoul National University (approval No. SNU-130730-1) on February 24, 2014.

Keywords: brain-derived neurotrophic factor; entacapone; hippocampus; neurogenesis; neurotrophic factor; phosphorylated cAMP response element-binding protein; tyrosine kinase receptor B receptor.

Figure 1

Exploration time (familiar and new object) and the discrimination index (the test trial) in the novel object recognition test in the vehicle-treated (control) and 10, 50, and 200 mg/kg entacapone-treated (E10, E50, and E200) mice. Data are expressed as the mean ± SD from n = 10 mice per group. *P < 0.05, vs. the familiar object; ^#P < 0.05, control group; ^†P < 0.05, vs. E10 group (one-way analysis of variance followed by Tukey’s multi-range test). The bars indicate the standard deviation.

Figure 2

Immunohistochemistry for Ki67 in the mouse dentate gyrus of the vehicle-treated (control, A) and 10 (B), 50 (C), and 200 mg/kg (D) entacapone-treated (E10, E50, and E200) groups. More Ki67-positive cells (arrows) are detected in the E50 and E200 groups compared with those in the control or E10 group. Scale bar: 50 μm. (E) The number of Ki67-positive cells per section in all groups (n = 5 per group). ^#P < 0.05, vs. control group; ^†P < 0.05, vs. E10 group (one-way analysis of variance followed by Tukey’s multi-range test). The bars indicate the standard deviation. GCL: Granule cell layer; ML: molecular layer; PoL: polymorphic layer.

Figure 3

Immunohistochemistry for doublecortin in the mouse dentate gyrus of the vehicle-treated (control, A) and 10 (B), 50 (C), and 200 mg/kg (D) entacapone-treated (E10, E50, and E200) groups. In the E50 and E200 groups, doublecortin-immunoreactive cell bodies (arrows) and dendrites are increased compared with those in the control group. Scale bar: 50 μm. (E) Relative optical densities (RODs) in the dentate gyrus per section for each group are depicted as a percentile value (n = 5 per group). ^#P < 0.05, vs. control group; ^†P < 0.05, vs. E10 group (one-way analysis of variance followed by Tukey’s multi-range test). The bars indicate the standard deviation. GCL: Granule cell layer; ML: molecular layer; PoL: polymorphic layer.

Figure 4

Immunohistochemistry for phosphorylated cAMP response element-binding protein in the mouse dentate gyrus of the vehicle-treated (control, A) and 10 (B), 50 (C), and 200 mg/kg (D) entacapone-treated (E10, E50, and E200) groups. pCREB-positive nuclei (arrows) were abundantly observed in the E50 and E200 groups compared with those in the control or E10 group. Scale bar: 50 μm. (E) Number of the pCREB-positive cells per section in all groups (n = 5 per group). ^#P < 0.05, vs. control group; ^†P < 0.05, vs. E10 group; (one-way analysis of variance followed by Tukey’s multi-range test). The bars indicate the standard deviation. GCL: Granule cell layer; ML: molecular layer; PL: polymorphic layer.

Figure 5

Western blot analysis of the BDNF and pTrkB in the hippocampi of the vehicle-treated (control) and 10, 50, and 200 mg/kg entacapone-treated (E10, E50, and E200) mice. All experiments were triplicated and relative optical density (ROD) in entacapone-treated groups versus that in the control group is presented in percentages (n = 5 per group). ^#P < 0.05, vs. control group (one-way analysis of variance test followed by Tukey’s multi-range test). The bars indicate the standard deviation. BDNF: Brain-derived neurotrophic factor; pTrkB: phosphorylated tyrosine kinase receptor B.

Figure 6

Object exploring time in training and testing trials and the discrimination index in the testing trial in the vehicle-treated (control) group, 2.5 μL TrkB receptor antagonist ANA-12-treated (ANA-12 alone)group, 50 mg/kg entacapone-treated (E50 alone) group, and E50 + ANA-12 treatment (E50 + ANA-12) group. n = 5 per group. *P < 0.05, vs. the familiar object; ^#P < 0.05, vs. control group; ^$P < 0.05, vs. ANA-12 alone group; ^‡P < 0.05, vs. E50 alone group (one-way analysis of variance test followed by Tukey’s multi-range test). The bars indicate the standard deviation. ANA-12: N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]benzo[b]thiophene-2-carboxamide.

Figure 7

Western blot analysis of the mature form of BDNF and pTrkB in the vehicle-treated (control) group, 2.5 μL TrkB receptor antagonist ANA-12-treated (ANA-12 alone) group, 50 mg/kg entacapone-treated (E50 alone) group, and E50 + ANA-12 (E50 + ANA-12) group. All experiments are triplicated and ROD in all groups are demonstrated as a percentile value vs. control group (n = 5 per group). ^#P < 0.05, vs. control group; ^$P < 0.05, vs. ANA-12 group (one-way analysis of variance followed by Tukey’s multi-range test). The bars indicate the standard deviation. BDNF: Brain-derived neurotrophic factor; ROD: relative optical density; pTrkB: phosphorylated tyrosine kinase receptor B.