DAla2GIP-Glu-PAL treatment enhanced new object recognition ability in APP/PS1 mice
Before new object recognition test, the motor capability of mice was evaluated in the open field task. As shown in Fig. 1A, there was no significant difference in total moving distance among the four groups (APP/PS1: F(1,47) = 3.532, P = 0.067; DAla2GIP-Glu-PAL: F(1,47) = 0.204, P = 0.655; APP/PS1 × DAla2GIP–Glu-PAL interaction: F(1,47) = 1.813, P = 0.189). Thus, no evidence showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment affect the motor capability of the mice. Based on the instinct of mice to explore new things, new object recognition test was performed on the second day after the open field test. Two-way ANOVA showed that APP/PS1 gene mutation and GIP analogs treatment had significant main effects on new object recognition memory (APP/PS1: F(1,47) = 23.507, P < 0.001; DAla2GIP-Glu-PAL: F(1,47) = 4.626, P = 0.037; APP/PS1 × DAla2GIP-Glu-PAL interaction: F(1, 47) = 13.758, P < 0.001). Tukey’s post hoc tests (Fig. 1B) showed that the RI in the APP/PS1 + PBS mice (49.12 ± 0.05%) significantly lower than that in the WT + PBS group (61.99 ± 0.04%, P < 0.001), indicating that the APP/PS1 mice spent less time in exploring the novel object. However, the RI in the APP/PS1 + DAla2GIP-Glu-PAL group (58.45 ± 0.03%) had a significant increase compared with the APP/PS1 + PBS mice (P < 0.001).
DAla2GIP-Glu-PAL alleviated working memory deficits in APP/PS1 mice
The spontaneous alternation of mice in Y maze was tested to examine the working memory of animals. Two-way ANOVA showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment had significant main effects on the spontaneous alternation of the mice(APP/PS1: F(1, 47) = 7.223,P = 0.01; DAla2GIP-Glu-PAL: F(1, 47) = 11.910, P < 0.01; APP/PS1 × DAla2GIP-Glu-PAL interaction: (F(1, 47) = 4.626, P = 0.037). Tukey’s post hoc tests showed that the percentage of correct alternation in APP/PS1 + PBS (50.93 ± 1.97%) was significantly lower than that in WT + PBS group (61.51 ± 2.20%, P < 0.01). After treatment with DAla2GIP-Glu-PAL, the correct alternation percentage in APP/PS1 + DAla2GIP-Glu-PAL group (63.30 ± 2.64%) had a significant increase (P < 0.01) compared with APP/PS1 + PBS mice (Fig. 2A). Meantime, the total arm entries of mice did not show any significant difference among these groups (P > 0.05, Fig. 2B), suggesting that the differences in spontaneous alternation among groups were due to impairment of spatial working memory rather than a disability of locomotor activity.
DAla2GIP-Glu-PAL improved long term spatial learning and memory of APP/PS1 mice
The MWM was used to assess the long term spatial learning and memory of mice. The learning ability of mice to acquire spatial information was first assessed by a consecutive 5 days of hidden platform test. As shown in the table 1 and Fig. 3A, the escape latency in all groups gradually decreased with the increase of training days. Although there was no significant difference among groups on day 1 and day 2, the mean escape latency had a significant increase in the APP/PS1 + PBS group on days 3–5, compared with the WT + PBS group. Interestingly, the increased escape latency was significantly decreased in the APP/PS1 + DAla2GIP-Glu-PAL group on days 4–5. This result indicates that chronic DAla2GIP-Glu-PAL treatment could improve the spatial learning ability of APP/PS1 mice.
Table.1 Mean escape latencies of mice during 5 training days ( s, Mean ± S.E.M.)
Group
(n =12 in each group)
|
Day 1
|
Day 2
|
Day 3
|
Day 4
|
Day 5
|
WT+PBS
|
54.22±2.44
|
52.27±4.35
|
30.77±4.06
|
33.08±3.63
|
21.69±4.28
|
APP/PS1+PBS
|
56.71±1.94
|
55.52±1.90
|
54.60±2.39**
|
46.08±3.73**
|
44.62±3.89**
|
WT+DAla2GIP-Glu-PAL
|
52.26±2.18
|
43.33±3.06
|
36.16±4.15
|
28.45±4.69
|
29.59±4.51
|
APP/PS1+DAla2GIP-Glu-PAL
|
55.94±1.88
|
49.22±3.68
|
42.50±5.16
|
35.07±3.85##
|
29.47±4.93##
|
|
|
|
|
|
|
|
**P < 0.01 vs. WT+PBS group; ##P <0.01 vs. APP/PS1+PBS group.
To exclude the possibility that the difference in escape latency might be caused by drug-induced visual and locomotor deficits, a visual platform test was performed after the probe trials. Statistical analysis showed that there was no significant difference in the swimming speed in probe trials and the swimming time arriving the visible platform between groups (Fig. 3D and E, P > 0.05), suggesting that the changes in escape latency and swimming time percentage exactly resulted from the impairments of spatial learning and memory.
DAla2GIP-Glu-PAL reversed in vivo hippocampal L-LTP suppression in APP/PS1 mice
To clarify the possible mechanism underlying the neuroprotective effects of DAla2GIP-Glu-PAL on learning and memory, we investigated the effects of DAla2GIP-Glu-PAL on the long term synaptic plasticity by recording in vivo fEPSPs in the hippocampal CA1 region. After 30 min of stable basal fEPSP recording, three series of HFS were given to induce L-LTP. The potentiation of fEPSPs was compared at 60 min, 120 min, and 180 min after HFS (Fig. 4). Two-way ANOVA demonstrated that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment had significant main effects on the fEPSP slope at 60 min (APP/PS1: F(1,26) = 3.321, P = 0.028; DAla2GIP-Glu-PAL: F(1,26) = 4.380, P = 0.005; APP/PS1 × DAla2GIP-Glu-PAL interaction: F(1,26) = 4.917, P = 0.002), 120 min (APP/PS1: F(1,26) = 4.799, P = 0.003; DAla2GIP-Glu-PAL: F(1,26) = 4.215, P = 0.007; APP/PS1DAla2GIP-Glu-PAL interaction: F(1,26) = 6.096, P < 0.001) and 180 min (APP/PS1: F(1,26) = 6.802, P < 0.001; DAla2GIP-Glu-PAL: F(1,26) = 4.425, P = 0.005; APP/PS1 × DAla2GIP-Glu-PAL interaction: F(1,26) = 8.546, P < 0.001) post-HFS. Tukey’s post hoc test shown that the slopes of the fEPSPs immediately after HFS abruptly increased from 100% to 219.5 ± 1.16%, 202.34 ± 4.39%, 220.93 ± 2.74%, and 220.32 ± 4.19% in the WT + PBS, APP/PS1 + PBS, WT + DAla2GIP-Glu-PAL, and APP/PS1 + Dala2GIP-Glu-PAL groups, respectively, indicating that LTP was successfully induced in the four groups (Fig. 4A). However, the L-LTP values in the APP/PS1-PBS group obviously decreased from 40 min post-HFS. As shown in the Fig. 4A and 4C, compared with the WT + PBS mice, the L-LTP value was significantly suppressed at 60 min (131.92 ± 8.57%, P < 0.05), 120 min (121.76 ± 6.36%, P < 0.05) and 180 min (103.57 ± 1.67%, P < 0.05) in APP/PS1 + PBS mice. In contrast, there was a relative large maintenance of L-LTP in the APP/PS1 + DAla2GIP-Glu-PAL group at 60 min (177.28 ± 5.97%, P < 0.05), 120 min (165.58 ± 6.06%, P < 0.05) and 180 min (162.41 ± 7.08%, P < 0.05) compared with the APP/PS1 + PBS group.
Further, paired pulse facilitation (PPF) was examined to detect the possible involvement of presynaptic mechanism in the effects of DAla2GIP-Glu-PAL on the LTP (Fig. 4D). Application of paired pulses to the Schaffer collaterals always induced PPF (see inset of Fig. 4D). Two way ANOVA showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment had no significant main effects on PPF (APP/PS1: F(1,26) = 0.369, P = 0.550; DAla2GIP-Glu-PAL: F(1,26) = 0.003, P = 0.956; APP/PS1 × DAla2GIP-Glu-PAL interaction: F(1,26) = 0.298, P = 0.590). Thus, no evidence showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment affected presynaptic neurotransmitter release in the hippocampal CA1 region.
DAla2GIP-Glu-PAL treatment reduced Aβ plaques in the hippocampus of APP/PS1 mice
High-density Aβ plaques is typical pathological hallmarks in the AD brains. In the immunohistochemistry, we aimed to evaluate the histopathological changes in the hippocampus of mice. Aβ plaques were detected using specific antibodies 6E10 (Fig. 5). Two way ANOVA showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment had significant main effects on the numbers of 6E10 positive plaques (APP/PS1: F(1,23) = 222.732, P < 0.001; DAla2GIP-Glu-PAL: F(1,23) = 52.745, P < 0.001;APP/PS1 × DAla2GIP-Glu-PAL interaction: F(1, 23) = 52.745,P < 0.001). As shown in the Fig. 5A, 6E10 positive plaques could be easily seen in the hippocampus of APP/PS1 transgenic mice under low magnification. Tukey’s post hoc test showed that the number of Aβ plaques in APP/PS1 + PBS group (15.13 ± 0.89) was significantly more than that in WT + PBS group (0.33 ± 0.00, P < 0.001), while DAla2GIP-Glu-PAL treatment effectively reduced the number in the hippocampus of APP/PS1 mice (6.98 ± 0.46, P < 0.001). These results above demonstrated that DAla2GIP-Glu-PAL treatment could attenuate cerebral pathological changes in the APP/PS1 transgenic mice.
DAla2GIP-Glu-PAL attenuated inflammatory response in the hippocampus of APP/PS1 transgenic mice
In view of the fact that Aβ can promote astrocyte proliferation and trigger inflammatory reactions in the brain, we further observed the effects of DAla2GIP-Glu-PAL injection on the inflammatory responses in the APP/PS1 transgenic mice by using immunohistochemistry and immunofluorescence double labeling techniques (Fig. 6). Two way ANOVA showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment had significant main effects on intensity of GFAP, astrocyte specific antibody (APP/PS1: F(1,23) = 43.197,P < 0.001; DAla2GIP-Glu-PAL: F(1,23) = 13.259, P = 0.002;APP/PS1 × DAla2GIP-Glu-PAL interaction: F(1༌23) = 8.459༌P = 0.009). As shown in the Fig. 6A and C, Tukey’s post hoc test showed that the GFAP immunoreactivity had a significant increase in the APP/PS1 + PBS group (163.38 ± 12.21%) compared with the WT + PBS group (100.24 ± 4.71%) (P < 0.001), while the increased GFAP value has a significant reduction in DAla2GIP-Glu-PAL treated APP/PS1 group (120.14 ± 3.23%) (P < 0.001).
Because Aβ and phosphorylated Tau can stimulate astrocytes to synthesize and secrete inflammatory factors such as IL-1β, we further observed the double staining of GFAP-positive astrocytes and inflammatory factor IL-1β by immunofluorescence technique. As shown in the Fig. 6B, there were small number of GFAP (red) and IL-1β (green) immunopositive cells in the hippocampus of the WT + PBS and WT + DAla2GIP-Glu-PAL groups, while a lot of GFAP and IL-1β positive cells were found in the APP/PS1 + PBS group. The increased GFAP and IL-1β positive cells were significantly reduced after treatment with DAla2GIP-Glu-PAL. We further tested the content of IL-1β in the hippocampus by ELISA. Two way ANOVA showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment had significant main effects and interaction on IL-1β content (APP/PS1: F(1,23) = 145.079,P < 0.001; DAla2GIP-Glu-PAL: F(1,23) = 49.654, P < 0.001;APP/PS1 × DAla2GIP-Glu-PAL interaction༚F(1༌23) = 34.968༌P < 0.001). Tukey’s post hoc test showed that IL-1β OD values was significantly increased in the APP/PS1 + PBS group (331.61%±16.37%) compared with the WT + PBS group (126.77%±8.19%, P < 0.001), while treatment with DAla2GIP-Glu-PAL significantly reduced the values of IL-1β in the APP/PS1 + DAla2GIP-Glu-PAL group (183.78%±11.63%, P < 0.001, Fig. 6D).
In addition, considering that IL-1β and TNF-α can activate NF-κB to enter the nucleus and up-regulate the expression of inflammatory factors, and trigger the inflammatory cells proliferation [28, 29], we also examined the expression level of NF-κBp65 (Ser468) in the hippocampus by western blot (Fig. 6E and F). Two way ANOVA showed that APP/PS1 gene mutation and DAla2GIP-Glu-PAL treatment had significant main effects and interaction on NF-κBp65 (Ser468) expression level (APP/PS1: F(1,23) = 19.269,P < 0.001; DAla2GIP-Glu-PAL: F(1,23) = 14.293, P < 0.001;APP/PS1 × DAla2GIP-Glu-PAL interaction༚F(1༌23) = 4.964༌P = 0.038). Tukey’s post hoc test showed that the value of NF-κBp65 (Ser468) in the hippocampus of APP/PS1 + PBS mice (141.95%±8.31%) was significantly higher than that of other groups (P < 0.01), while DAla2GIP-Glu-PAL significantly reduced the expression level of NF-κBp65 in APP/PS1 + DAla2GIP-Glu-PAL group (103.86%±5.94%, P < 0.01). These results indicate that chronic i.p. injection of DAla2GIP-Glu-PAL attenuated inflammatory response in the hippocampus of APP/PS1 mice.
The DAla2GIP-Glu-PAL treatment up-regulated the expression levels of cAMP, S99p-PKA and S133p-CREB in the hippocampus of APP/PS1 transgenic mice
To investigate the possible mechanism of DAla2GIP-Glu-PAL in improving cognitive behavior and pathological features of APP/PS1 transgenic mice, we detected the expression levels of cAMP, S99p-PKA and S133p-CREB in the hippocampus. As shown in the Table 2, the two way ANOVA showed that the APP/PS1 gene mutation and DAla2GIP -Glu-PAL drug treatment have obvious main effects and interaction effects on each signal molecule. The typical Western-blotting bands and Tukey’s post hoc test (Fig. 7) showed that the relative gray values of cAMP, S99p-PKA, S133p-CREB were significantly decreased (P < 0.01) in APP/PS1 + PBS group compared with that in WT + PBS group. However, chronic i.p. injection of DAla2GIP-Glu-PAL effectively reversed the down-reglulation (P < 0.01). These results indicate that neuroprotective effects of DAla2GIP-Glu-PAL may be involved in the up-regulation of cAMP/ PKA/ CREB signaling pathway in the hippocampus of APP/PS1-PBS mice.
Table.2 Two-way ANOVA for the levels of cAMP, S99p-PKA and S133p-CREB in the hippocampus. (n=6 in each group)
Protein
|
Main effect
|
Interaction effect
|
APP/PS1
|
DAla2GIP-Glu-PAL
|
cAMP
|
38.243(P<0.001)
|
28.028(P<0.001)
|
11.052(P=0.003)
|
S99p-PKA
|
21.237(P<0.001)
|
11.584(P=0.003)
|
4.558(P=0.045)
|
S133p-CREB
|
32.412(P<0.001)
|
20.127(P<0.001)
|
8.669(P=0.008)
|