All procedures were performed in accordance with the Guide for and Use of Medical Laboratory Animals (Ministry of Health China, 1998) (16) and were approved by the Shanghai University of Traditional Chinese Medicine (Shanghai, China) Laboratory Animal Care and Use Committee (no. PZSHUTCM19011807).

In total, 18 male APP/PS1 transgenic mice (age: 24 weeks; weight: 26±3 g) and 6 male C57BL/6 mice (age: 24 weeks; weight: 27±2 g) were used in the present study. The APP/PS1 mouse strain is a double-transgenic hemizygote that expresses a chimeric mouse/human amyloid precursor protein and mutant human presenilin-1. These transgenic mice were used as they develop behavioral and pathology features of AD similar to patients and have been used in previous AD studies worldwide (17–19). Separately, C57BL/6 mice were used as age-matched controls. The APP/PS1 transgenic mice were purchased from the Model Animal Research Center of Nanjing University, while C57BL/6 mice were obtained from the Experimental Animal Center of Shanghai Academy. The NAD⁺ group were intraperitoneally injected with NAD⁺ (30 mg/kg, Sigma-Aldrich; Merck KGaA) at 20 weeks of age and once every other day for 4 weeks. The FK866 (NAMPT inhibitor) group were intraperitoneally injected with FK866 (1 mg/kg, Sigma-Aldrich; Merck KGaA) at 20 weeks of age and once every other day for 4 weeks. The mice were housed in a controlled specific-pathogen-free environment (22±3°C; 60% relative humidity; 12-h light/dark cycle) with ad libitum access to water and food.

Spatial learning and memory of the mice were assessed using a Morris water maze test (MWM) according to a previous study (20) with minor modifications. The water was made opaque with titanium dioxide and water temperature was kept at 22±2°C. A platform was placed 1 cm under the surface of the water. In the hidden platform experiment, mice received 4 training trials per day for 5 consecutive days. On the first trial of the first day, mice were placed on the platform for 10 sec, after which they were placed in the water. The pool area was conceptually divided into four quadrants of equal size. Taking the quadrant of the platform as the first quadrant, the second and fourth quadrants were taken as the starting point for 2 trials and the mice were placed facing the pool wall. If a mouse failed to find the platform in 70 sec, it was gently guided to the platform location and allowed to stay on it for 30 sec. The time to find the platform was recorded as the escape latency. The experiments were recorded with a camera connected a video recorder and a computerized tracking system, with the automatic timer set to 70 sec.

In the probe trials, at 24 h after the last training trial, the platform was removed and the mice were placed at the second and fourth quadrants, and the time of the target quadrant (first quadrant) and the number of times crossing the location that previously contained the platform within 70 sec were recorded.

Mice were anesthetized with 5% chloral hydrate at a dose of 400 mg/kg intraperitoneally and saline solution was used for perfusion through the heart, which was followed by 4% paraformaldehyde. The brains of the mice were then removed, fixed in 4% paraformaldehyde for 24 h at room temperature and immersed in 30% sucrose until they sank. The brain tissue was serially sectioned at a 30-µm thickness using a HM1950 freezing microtome. The sections were permeabilized in xylene for 10 min, dehydrated using anhydrous ethanol for 5 min, and then stained with 1% thioflavin S (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. The stained samples were differentiated in 70% alcohol solution for 5 min, mounted with glycerol gel, and observed using fluorescence microscopy (magnification, 200×; Olympus BX51; Olympus Corporation).

A NAD⁺/NADH quantification kit was used to determine NAD⁺/NADH levels (cat. no. k337-100; BioVision, Inc.), according to the manufacturer's instructions. Hippocampus tissue (20 mg) was washed with precooled phosphate-buffered saline (PBS), homogenized in 400 µl of the NAD⁺/NADH extraction buffer and then centrifuged for 5 h at 18,000 × g at 4°C. The resulting supernatant was labeled as total NAD⁺ sample (NAD⁺t). Subsequently, 200 µl of the NAD⁺t sample was heated at 60°C for 30 min (to consume all NAD⁺ from the sample, leaving only NADH to be analyzed). Following cooling on ice, the sample was centrifuged at 12,000 × g for 30 sec at 4°C and the resulting supernatant was labeled as the NADH sample. NAD⁺/NADH extraction buffer was added to bring the volume to 50 µl. Subsequently, 100 µl of enzyme reaction mix and 10 µl of NADH developer were added to each well, the mixture was incubated at room temperature for 2 h and the absorbance was measured using a microplate reader (wavelength: 450 nm).

Cortical or hippocampus tissue (10 mg) was washed with precooled PBS, homogenized in 100 µl of lysis buffer (cat. no. P0013B: Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g at 4°C for 5 min. The resulting supernatant was transferred to a new Eppendorf tube and then the reaction solution (ATP Assay Buffer, ATP probe, ATP Converter and Develop Mix; Sigma-Aldrich; Merck KGaA) was added to bring the volume to 50 µl. The reaction solution was shaken in a shaker for 30 min at room temperature. To correct for the background of the samples, a well per sample was used as the sample background well. The absorbance was measured at a wavelength of 570 nm using a microplate reader.

Following a behavioral test, three mice were randomly selected in each group. Following anesthesia, the brains were perfused with 4% paraformaldehyde for 4 h and then transferred to a 30% sucrose gradient at room temperature. After the brains had sunk to the bottom, continuous coronal sectioning was performed (30 µm thickness) using a HM1950 freezing microtome. The sections were permeabilized in xylene for 10 min and dehydrated using anhydrous ethanol for 5 min. The sections were washed with 0.01 M of PBS and blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Then the sections were incubated with polyclonal anti-NAMPT antibody (1:200; cat. no. ab45890; Abcam) overnight at 4°C. Following rinsing with PBS, sections were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:500; cat. no. SA00001-2; ProteinTech Group, Inc.) for 1 h at 37°C, washed with PBS, and streptavidin-biotin complex (Beyotime Institute of Biotechnology) was added. Then the sections were developed with 3,3′-diaminobenzidine for 10 min at room temperature and dehydrated with gradient alcohol, cleared with xylene, sealed with neutral gum and observed with using a light microscope (magnification, ×200; Olympus BX51; Olympus Corporation).

The isolated hippocampus tissue was collected, homogenized in lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology), lysed on ice for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. Total protein concentration was determined using a Micro BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Then, 30 µg of protein sample was heated at 95°C for 5 min and separated by 10% SDS-polyacrylamide gel electrophoresis, after which it was transferred onto a PVDF membrane. The membrane was blocked with 3% BSA at 37°C for 1 h. Following blocking, the membranes were incubated with primary antibodies (anti-NAMPT 1:500, cat. no. ab45890, Abcam; anti-SIRT1, 1:1,000, cat. no. ab110304, Abcam; and GAPDH, 1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.) overnight at 4°C. The membrane was washed with TBST three times for 10 min and then treated with HRP-labeled goat anti-rabbit secondary antibody (1:2,000; cat. no. SA00001-2; ProteinTech Group, Inc.) and HRP-labeled goat anti-mouse secondary antibody (1:2,000; cat. no. SA00001-1; ProteinTech Group, Inc.) for 1 h at 37°C. Blots were washed three times for 10 min in TBST. After incubation with enhanced chemiluminescence chromogenic solution (cat. no. P0018s; Beyotime Institute of Biotechnology) for 1 min at room temperature, quantification of the proteins of interest was determined relative to GAPDH using ImageJ software (version 1.4; National Institutes of Health).

Statistical analysis was performed using GraphPad software 6.0 (GraphPad Software, Inc.). Measurement data are presented as the mean ± standard error of the mean. Two-way ANOVA followed by Bonferroni post hoc test was used to analyze group differences in the escape latency. A one-way ANOVA followed by LSD multiple-range test was used in the probe trial. For the non-normally distributed data or for data with heterogeneous variance, a Kruskal-Wallis test was used. P<0.05 was considered to indicate a statistically significant difference.

Memory impairment in APP/PS1 transgenic mice was determined using the MWM test in terms of the escape latency. The results demonstrated that, with the increase in the number of training days, the escape latency gradually shortened. On the fifth day, the escape latency of the APP/PS1 group (AD) mice was significantly longer compared with the blank control group (P<0.01), suggesting that the learning and memory abilities of AD mice were significantly impaired. Furthermore, the escape latency of NAD⁺ mice was significantly shorter than that of APP/PS1 mice after intraperitoneal injection of NAD⁺ (P<0.01); while, after FK866 intervention, there was no significant change in escape latency in the FK866 mice compared with the APP/PS1 mice (Fig. 1A).

In the space exploration experiment on the sixth day, after removing the platform it was found that APP/PS1 mice significantly reduced the target quadrant residence time (P<0.01) compared with control group mice, indicating that the spatial memory ability of AD mice was impaired. Following NAD⁺ treatment, the NAD⁺ mice spent more time in the target quadrant compared with the APP/PS1 mice (P<0.01); however, there was no significant change in the target quadrant residence time of the mice in the APP/PS1 group compared with the FK866 group (Fig. 1B). In comparison with the control group, the number of platform crossings was significantly reduced in the APP/PS1 group (P<0.05) and the NAD⁺ mice exhibited an increased number of platform crossings compared with the APP/PS1 mice, but these findings were not statistically significant (P>0.05). Overall, there was no significant change between the APP/PS1 group and FK866 group (Fig. 1C).

Staining with Thioflavin S for senile plaques in the cortex and hippocampus was conducted. As expected, the AD mice presented numerous plaques at 6 months of age as compared with the control mice in cortex and hippocampus; however, following NAD⁺ treatment, scattered green fluorescent plaques were observed in the cortex and hippocampus of every group and, after FK866 injection, multiple green fluorescent plaques were also seen in the cortex and hippocampus in AD mice (Fig. 2).

ATP content in the cortex and hippocampus was measured in each group of mice. The results demonstrated that, compared with the control group, the ATP content in the cortex in the APP/PS1 group was significantly decreased (P<0.05), while, in comparison with the APP/PS1 model group, the ATP content in the cortex in the NAD⁺ group was significantly increased (P<0.05). Following FK866 intervention, the ATP content of the cortex decreased compared with APP/PS1 mice, but without statistical significance (P>0.05). Additionally, in comparison with the control group, the ATP content in the hippocampus in the APP/PS1 group was significantly decreased (P<0.05), while, compared with the APP/PS1 model group, the ATP content in the hippocampus in the NAD⁺ group was significantly increased (P<0.05). Following FK866 intervention, there was no significant change in the NAD⁺ group compared with APP/PS1 model group (Fig. 3).

NAD⁺ and NADH are essential coenzymes for cell energy metabolism. Under normal physiological conditions, the NAD⁺/NADH ratio reflects the redox state of cells. When the energy metabolism of the cells is deregulated, the levels of NAD⁺ and NADH will change, which is reflected as an imbalance in the NAD⁺/NADH ratio (21). The present study detected NAD⁺ and NADH levels in the cortex and hippocampus. The NAD⁺ content in the cortex in the APP/PS1 group was significantly decreased compared with the control group (P<0.05) and the NAD⁺/NADH ratio was similarly decreased (P<0.01), although the NADH level was not significantly changed. Compared with the APP/PS1 model group, the NAD⁺ content of the cortex was significantly increased in the NAD⁺ group (P<0.05) and the NAD⁺/NADH ratio was significantly increased (P<0.01), while the NADH level was not significantly changed. Separately, in comparison with APP/PS1 mice, NAD⁺ content in the cortex decreased significantly in the FK866 group (P<0.05) and the NAD⁺/NADH ratio decreased (P<0.01), although the NADH level did not change significantly between the two groups.

Compared with the control group, the NAD⁺ content in the hippocampus in the APP/PS1 group was significantly decreased (P<0.05), while the NADH level was not significantly changed and the NAD⁺/NADH ratio was decreased (P<0.01). Furthermore, compared with the APP/PS1 model group, the NAD⁺ content in the hippocampus in the NAD⁺ group was significantly increased (P<0.05) and the NAD⁺/NADH ratio was significantly increased (P<0.01), while the NADH level was not significantly different. Regarding the NAD⁺ content, NADH level and NAD⁺/NADH ratio, there was no significant change in the hippocampus in the FK866 group compared with the other mice (Fig. 4).

In order to confirm the expression of NAMPT proteins in the cortex and hippocampus in APP/PS1 transgenic mice, immunohistochemical staining was performed. It was observed that the brown positive expression of NAMPT protein in the cortex and hippocampus in APP/PS1 group was decreased compared with the control group (P<0.05). Following intraperitoneal injection of NAD⁺, the positive expression of NAMPT protein increased compared with the APP/PS1 group (P<0.05), while there was no significant change in NAMPT levels in the cortex and hippocampus in FK866 mice compared with APP/PS1 mice (P>0.05; Fig. 5).

Protein extracts were also prepared and analyzed by western blotting to determine protein levels. The results demonstrated that the protein expression of NAMPT and SIRT1 in the cortex in the APP/PS1 group was significantly decreased when compared with the control group (P<0.01). Intraperitoneally injecting NAD⁺ increased the expression of NAMPT in the cortex in the NAD⁺ mice (P<0.05). There was no significant change in NAMPT protein levels in the FK866 mice compared with in APP/PS1 mice (P>0.05). Consistent with the changes in the cortex, the results demonstrated that the NAMPT and SIRT1 level in the hippocampus in the APP/PS1 group was decreased compared with the control group (P<0.01). NAMPT expression levels in the hippocampus in the NAD⁺ group were also significantly increased after intraperitoneal injection of NAD⁺ (P<0.05); however, injecting NAD⁺ did not increase SIRT1 expression levels. Finally, there was no significant change in NAMPT and SIRT1 protein expression levels in the hippocampus in FK866 mice, compared with in APP/PS1 mice (P>0.05; Fig. 6).