Fluoxetine attenuates the impairment of spatial learning ability and prevents neuron loss in middle-aged APPswe/PSEN1dE9 double transgenic Alzheimer's disease mice

Abstract

Selective serotonin reuptake inhibitors (SSRIs) have been reported to increase cognitive performance in some clinical studies of Alzheimer's disease (AD). However, there is a lack of evidence supporting the efficacy of SSRIs as cognition enhancers in AD, and the role of SSRIs as a treatment for AD remains largely unclear. Here, we characterized the impact of fluoxetine (FLX), a well-known SSRI, on neurons in the dentate gyrus (DG) and in CA1 and CA3 of the hippocampus of middle-aged (16 to 17 months old) APPswe/PSEN1dE9 (APP/PS1) transgenic AD model mice. We found that intraperitoneal (i.p.) injection of FLX (10 mg/kg/day) for 5 weeks effectively alleviated the impairment of spatial learning ability in middle-aged APP/PS1 mice as evaluated using the Morris water maze. More importantly, the number of neurons in the hippocampal DG was significantly increased by FLX. Additionally, FLX reduced the deposition of beta amyloid, inhibited GSK-3β activity and increased the level of β-catenin in middle-aged APP/PS1 mice. Collectively, the results of this study indicate that FLX delayed the progression of neuronal loss in the hippocampal DG in middle-aged AD mice, and this effect may underlie the FLX-induced improvement in learning ability. FLX may therefore serve as a promising therapeutic drug for AD.

Keywords: APP/PS1 mice; Alzheimer’s disease; Gerotarget; cognition; fluoxetine; neuron.

Figure 1. Assessment of the effect of FLX on learning and memory impairment in middle-aged APP/PS1 transgenic mice tested in the Morris water maze and the effect of FLX on body weight of middle-aged APP/PS1 mice

FLX was administered (10 mg/kg/day, i.p.) for 4 weeks prior to training in the Morris water maze. Treatment was continued while the mice were submitted to the test. A. Mean escape latencies of the three groups (WT, APP/PS1 and APP/PS1+FLX) in the hidden platform tests, which were conducted for 5 consecutive days. B. The frequencies at which the mice in the three groups crossed the platform location (on the 6th day) during the probe test. C. The swimming tracks the mice in the three groups made in the water tank on the last day of the test. The circle in the left lower quadrant represents the location of the hidden platform, while the curves indicate the different swimming strategies of the three groups of mice. D. Body weight of mice in the three groups. The body weight was monitored on a daily basis. Data are presented as the means ± S.E.M. n = 10-13/group. ##, P < 0.01, vs. WT group. *, P < 0.05, vs. APP/PS1 group.

Figure 2. Deposition of beta amyloid in the hippocampus of mice in the WT, APP/PS1 and APP/PS1+FLX groups

Immunohistochemical staining for beta amyloid in the hippocampus of mice from the three groups. Enlargements show a higher magnification of the indicated areas of interest. Black scale bars = 500 μm; white scale bars = 200 μm; green scale bars = 80 μm.

Figure 3. Comparison of accurately determined numbers of neurons in the DG and in CA1 and CA3 of the hippocampus among the WT, APP/PS1 and APP/PS1+FLX groups

A. representative photographs of toluidine blue-stained tissues were used to show the general distribution of the numbers of neurons in the DG and in CA1 and CA3 of the hippocampus in the WT, APP/PS1 and APP/PS1+FLX groups. B. The hippocampal DG in the WT and APP/PS1+FLX mice contained significantly more total neurons than did that in the APP/PS1 mice. C. The hippocampal CA1 in the WT mice contained significantly more total neurons than did that in the APP/PS1 and the APP/PS1+FLX mice. D. The total numbers of neurons in CA3 of the hippocampus did not significantly differ among the three groups. Data are presented as the mean ± SD. n = 5-6/group. #, P < 0.05, vs. WT group. *, P < 0.05, vs. APP/PS1 group. ^, P < 0.05, vs. WT group.

Figure 4. Involvement of the canonical Wnt signaling pathway in the effect of FLX

FLX increases the level of phosphorylated of GSK-3β and the level of β-catenin in middle-aged APP/PS1 mice. A. Western blots showing the expression levels of p-GSK-3β and total GSK-3β in FLX-treated middle-aged APP/PS1 mice. GAPDH was used as an internal control. B.-D. Quantification of the levels of GSK-3β and p-GSK-3β, along with the p-GSK-3β/GSK-3β ratio, in middle-aged APP/PS1 mice. B. The level of p-GSK-3β was significantly increased in the FLX-treated mice. C. There were no significant differences in the levels of total GSK-3β among the three groups. D. The p-GSK-3β/GSK-3β ratio was significantly increased in FLX-treated mice. E. Western blots showing the expression level of β-catenin in middle-aged APP/PS1 mice after FLX treatments. GAPDH was used as an internal control. F. Quantification of the level of β-catenin in middle-aged APP/PS1 mice. The level of total β-catenin was markedly increased after FLX treatment of middle-aged APP/PS1 mice. Data are presented as the mean ± SD. n = 5-6/group. ##, P < 0.01, vs. WT group. #, P < 0.05, vs. WT group. ** P < 0.01, vs. APP/PS1 group. *, P < 0.05, vs. APP/PS1 group.

Figure 5. The experimental design

A. The mice began to receive FLX treatment at 64-68 weeks (16-17 months) of age, and the treatment was continued until they reached 69-73 weeks (17.25-18.25 months) of age, with the FLX administration lasting for 5 weeks. The Morris water maze was conducted when mice were at 68-72 weeks (17-18 months) of age, after being administered fluoxetine for 4 weeks. B. Six mice each were randomly chosen from the WT group and the APP/PS1 group, and five mice were randomly chosen from the APP/PS1+FLX group for the subsequent stereological investigation and immunohistochemistry. In each group, those mice not used for the stereological investigation and immunohistochemistry were deeply anesthetized and then killed. Then, the hippocampus was obtained from each brain and homogenized for western blotting analysis.

Figure 6. The contours of the DG, CA1, and CA3 of the hippocampus and the method of counting neurons

A. Coronal histological section showing the contours of the DG (red), CA1 (green), and CA3 (blue) in the hippocampus. B.-C. The method of counting neurons. The focal depth in micrometers (measured using a microcator attached to the microscope stage) is indicated on the focal depth bar, where 0 m represents the top surface of the section. An unbiased counting frame is included in B. and C. The red line of the frame and its extension represent the exclusion lines, and the green line of the frame represents the inclusion line. Neurons were counted when their nuclei first came into focus if they were completely inside of the counting frame or partly inside the counting frame but only touching the inclusion (green) line. B. When the microscopic field of vision was clearly focused for the first time, its location on the Z axis was set to 0 μm. Then, as the microscope was adjusted, the neurons within the guard zones were not counted. C. Thereafter, as the Z axis moved below the guard zones, focusing downward to 8.0 μm brought 1 nucleolus into focus, as indicated by the arrow.