The interplay of aryl hydrocarbon receptor/WNT/CTNNB1/Notch signaling pathways regulate amyloid beta precursor mRNA/protein expression and effected the learning and memory of mice

Abstract

The amyloid beta precursor protein (APP) plays a pathophysiological role in the development of Alzheimer's disease as well as a physiological role in neuronal growth and synaptogenesis. The aryl hydrocarbon receptor (AhR)/WNT/Catenin Beta 1 (CTNNB1)/Notch signaling pathways stamp in many functions, including development and growth of neurons. However, the regulatory role of AhR-/WNT-/CTNNB1-/Notch-induced APP expression and its influence on hippocampal-dependent learning and memory deficits is not clear. Male BALB/C mice received 6-formylindolo[3,2-b]carbazole (an AhR agonist), CH223191(an AhR antagonist), DAPT (an inhibitor of Notch signaling), and XAV-939 (a WNT pathway inhibitor) at a single dose of 100 μg/kg, 1, 5 , and 5 mg/kg of body weight, respectively, via intraperitoneal injection alone or in combination. Gene expression analyses and protein assay were performed on the 7th and 29th days. To assess the hippocampal-dependent memory, all six mice also underwent contextual fear conditioning on the 28th day after treatments. Our results showed that endogenous ligand of AhR has a regulatory effect on APP gene. Also, the interaction of AhR/WNT/CTNNB1 has a positive regulatory effect, but Notch has a negative regulatory effect on the mRNA and protein expression of APP, which have a correlation with mice's learning skills and memory.

Keywords: 2-b]carbazole (FICZ); 6-formylindolo[3; Notch; WNT/CTNNB1; amyloid beta precursor protein (APP); aryl hydrocarbon receptor (AhR); learning and memory.

Graphical abstract

Figure 1

FICZ induced APP gene expression measured in the mouse hippocampus; animals were treated with olive oil (vehicle-treated controls), FICZ (100 μg/kg), CH223191(1 mg/kg), and XAV-939 (5 mg/kg), and mRNA expression pattern of APP on the 7th and 29th days after injection were measured; the bar plots show the transcriptional levels of APP mRNA on the 7th and 29th days; values are expressed as means ± SD; asterisks denote significant differences (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001) between control and other treated groups; pluses denote significant differences (⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001) between FICZ and other treated groups; ^Ψ denotes significant differences (^ΨP < .05) between FICZ+ XAV-939- and FICZ+ XAV-939+ CH223191-treated groups.

Figure 2

immunoblots performed using whole hippocampal tissues of mouse exposed to olive oil (vehicle-treated controls), FICZ, CH223191, and XAV-939 for the detection of β-catenin and APP proteins; (A) 9 and 140 kDa bands show positive results for CTNNB1 and APP proteins, respectively, on the 7th day; (B) 95 and 140 kDa bands show negative results for β-catenin and APP proteins, respectively, on the 29th day; values are expressed as means ± SD; asterisks denote significant differences (^***P < 0.001) between FICZ (β-catenin protein level) and other treated groups; pluses denote significant differences (⁺⁺⁺P < 0.001) between FICZ (APP protein level) and other treated groups.

Figure 3

the effect of different FICZ, CH223191, and DAPT treatments on the expression of AhR. (A) Hes1,5; olive oil was used as a vehicle; (B) APP (C) genes by real-time PCR on the mouse hippocampus in the first week; mice were treated with a single dose of FICZ 100 μg/kg, CH223191 1 mg /kg, and DAPT 1 mg/kg; and a week later, the expression levels of the relevant genes were measured according to the Materials and Methods section; data are presented as mean ± SD for the average number of five mice in each group; asterisks denote significant differences (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001) between control and other treated groups; pluses denote significant differences (⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001) between FICZ and other treated groups.

Figure 4

immunoblots performed using whole mouse hippocampal tissues of mice exposed to olive oil (vehicle-treated controls), FICZ, CH223191, and DAPT for the detection of HES1 and APP proteins; (A) 30 and 140 kDa bands show positive results for Hes1 and APP proteins, respectively, on the 7th day; (B) 30 and 140 kDa bands show results for Hes1 and APP proteins, respectively, on the 29th day; values are expressed as means ± SD; asterisks denote significant differences (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001) between FICZ (APP protein level) and other treated groups; pluses denote significant differences (⁺⁺⁺P < 0.001) between FICZ (HES1 protein level) and other treated groups; $ denotes significant differences (^$$$P < 0.05) between FICZ+DAPT- (HES1 protein level) and DAPT- as well as FICZ+ CH223191-treated groups.

Figure 5

hippocampal-dependent learning and memory were affected by FICZ, XAV-939, and CH223191 in two passive avoidance task shuttle box and step-down; (A) passive avoidance memory assessment on the 28th day after incubation with olive oil (vehicle-treated controls), FICZ (100 μg/kg), CH223191(1 mg/kg), and XAV-939 (5 mg/kg); learning latency in the training time and STL after 2 min and 24 h after shock delivery were measured using shuttle box apparatus; (B) learning latency in the training time and STL after 30 min and 24 h after shock delivery were measured using step-down apparatus; the bar plots show the values as means ± SD; asterisks denote significant differences (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001) between control and other treated groups; pluses denote significant differences (⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001) between FICZ and other treated groups.

Figure 6

effect of FICZ, CH223191, and DAPT on the hippocampal-dependent learning and using two passive avoidance task shuttle box and step-down; (A) passive avoidance memory assessment on the 28th day after incubation with olive oil (vehicle-treated controls), FICZ (100 μg/kg), CH223191(1 mg/kg), and DAPT (5 mg/kg); learning latency in the training time and STL after 2 min and 24 h after shock delivery were measured using shuttle box apparatus; (B) learning latency in the training time and STL after 30 min and 24 h after shock delivery were measured using step-down apparatus; the bar plots show the values as means ± SD; asterisks denote significant differences (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001) between control and other treated groups; pluses denote significant differences (⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001) between FICZ and other treated groups.