Cotinine administration improves impaired cognition in the mouse model of Fragile X syndrome

Abstract

Cotinine is the major metabolite of nicotine and has displayed some capacity for improving cognition in mouse models following chronic administration. We tested if acute cotinine treatment is capable of improving cognition in the mouse model of Fragile X syndrome, Fmr1^-/- knockout mice, and if this is related to inhibition by cotinine treatment of glycogen synthase kinase-3β (GSK3β), which is abnormally active in Fmr1^-/- mice. Acute cotinine treatment increased the inhibitory serine-phosphorylation of GSK3β and the activating phosphorylation of AKT, which can mediate serine-phosphorylation of GSK3β, in both wild-type and Fmr1^-/- mouse hippocampus. Acute cotinine treatment improved cognitive functions of Fmr1^-/- mice in coordinate and categorical spatial processing, novel object recognition, and temporal ordering. However, cotinine failed to restore impaired cognition in GSK3β knockin mice, in which a serine9-to-alanine9 mutation blocks the inhibitory serine phosphorylation of GSK3β, causing GSK3β to be hyperactive. These results indicate that acute cotinine treatment effectively repairs impairments of these four cognitive tasks in Fmr1^-/- mice, and suggest that this cognition-enhancing effect of cotinine is linked to its induction of inhibitory serine-phosphorylation of GSK3. Taken together, these results show that nicotinic receptor agonists can act as cognitive enhancers in a mouse model of Fragile X syndrome and highlight the potential role of inhibiting GSK3β in mediating the beneficial effects of cotinine on memory.

Keywords: Fragile X syndrome; cotinine; glycogen synthase kinase-3; novel object recognition; spatial memory.

Figure 1

Acute cotinine treatment increases in phosphorylation of GSK3 and AKT in the hippocampus of wild-type mice. Immunoblots from representative mice in each group, at 30 and 90 min after treatment, showing the increase in the hippocampus of phosphorylation of (A) Ser-9-GSK3β, (B) Ser-21-GSK3α, (C) Thr-308-Akt and (D) Ser-473-Akt. Data was normalized relative to control (vehicle (0 mg/kg)). Values are means ±SEM. *p<0.05 **p<0.01 compared to vehicle-treated mice. (n=4–8 per group).

Figure 2

Cotinine treatment increases phosphorylation of GSK3 and AKT in the hippocampus of Fmr1^−/− mice. Immunoblots from representative mice in each group, at 30 and 90 min after treatment, showing the increase in the hippocampus of phosphorylation of (A) Ser-9-GSK3β, (B) Ser-21-GSK3α, (C) Thr-308-Akt and (D) Ser-473-Akt. Data was normalized relative to control (vehicle (0 mg/kg)). Values are means ±SEM. *p<0.05 **p<0.01 compared to vehicle treated mice. (n=5–8 per group).

Figure 3

Comparisons of the phosphorylation of GSK3 and AKT in the hippocampus of wild-type (WT) mice and Fmr1^−/− mice. Immunoblots from representative mice in each group, at 30 min after treatment, showing the basal levels and cotinine-induced increase in the hippocampus of phosphorylation of (A) Ser-9-GSK3β, (B) Ser-21-GSK3α, (C) Thr-308-Akt and (D) Ser-473-Akt. Data was normalized relative to vehicle treated wild-type mice. Values are means ±SEM. *p<0.05 **p<0.01 compared to wild-type vehicle-treated mice; #p<0.05 ##p<0.01 compared to Fmr1^−/− vehicle (0 mg/kg) mice. (n=4–5 per group).

Figure 4

Cotinine effects on performance of Fmr1^−/− mice in four cognitive tasks. (A) Performance of wild-type (WT) mice (n=15) and Fmr1^−/− mice (n=14) in the coordinate spatial processing task (Means±SEM; *p<0.05 compared to vehicle-treated WT mice). (B) Performance of WT mice (n=16) and Fmr1^−/− mice (n=15) in the categorical spatial processing task (Means±SEM; *p<0.05 compared to vehicle-treated WT mice). (C,D) Performance of wild-type (WT) mice (n=16), and Fmr1^−/− mice (n=15) on novel object recognition. (C) Percent time spent exploring the novel (N) and familiar (F) object. (Means±SEM; *p<0.05 compared to time spent with familiar object). (D) Discrimination index (Means±SEM; *p<0.05 compared to vehicle-treated WT mice). (E,F) Performance of WT mice (n=16) and Fmr1^−/− mice (n=15) on the temporal order task. (E) Percent time spent exploring the first (1) and last (3) object presented. (Means±SEM; *p<0.05 compared to time spent with object 1). (F) Discrimination index (Means±SEM; *p<0.05 compared to vehicle-treated WT mice).

Figure 5

Cotinine effects on performance of GSK3β KI mice in two cognitive tasks. A) Immunoblots from 3 mice in each group showing the absence of serine-9 phosphorylation of GSK3β in the hippocampus of male GSK3β KI mice. Total levels of GSK3β are equal in GSK3β KI and wild-type (WT) mice. (B) Performance of wild-type (WT) mice (n=15) and GSK3β KI (GSK3β KI) mice (n=18) in the coordinate spatial processing task (Means±SEM; *p<0.05 compared to vehicle-treated WT mice). (C,D) Performance of WT mice (n=16) and GSK3β KI mice (n=19) on novel object recognition. (C) Percent time spent exploring the novel (N) and familiar (F) object. (Means±SEM; *p<0.05 compared to time spent with familiar object). (D) Discrimination index (one-way ANOVA, F(3,34)=5.38, p<0.01; Means±SEM; *p<0.05 compared to vehicle-treated WT mice).

Figure 6

Signaling pathway stimulated by cotinine. Stimulation of α7 nicotinic receptors activates PI3K leading to the activating phosphorylations on Akt, which phosphorylates the inhibitory Ser-9 of GSK3β, leading to pro-cognitive effects.