Homocysteine Mediates Cognitive Inflexibility Induced by Stress via Targeting PIN1

Abstract

Background: Increasing evidence shows that HCY plays an important role in stress-induced cognitive dysfunction, and HCY significantly promotes the decline of cognitive function. Stress has been reported to cause elevated HCY in the hippocampus of mice. Cognitive flexibility refers to the ability of individuals to quickly adjust their neurobehavioral strategies to different situations or to solve different tasks.

Aims: This study aims to explore the role of HCY in the impairment of cognitive flexibility induced by stress and its possible regulatory mechanism.

Methods and results: First, we examined changes in the protein and mRNA levels of the cognitive flexibility effector molecule, PIN1, during stress in mice. The results show that stress can cause a decline in cognitive flexibility in mice and lead to an increase in PIN1. Moreover, through the use of in vitro experiments, we found that HCY could induce an increase in PIN1 expression in neurons. Further in vivo experiments were used to investigate the effect of VitB on HCY and PIN1 and evaluated the therapeutic effect of VitB on stress-induced impairment of cognitive flexibility. The results show that VitB decreased the levels of HCY in plasma and the hippocampus, alleviated the stress-induced impairment of cognitive flexibility, and reduced the expression of PIN1.

Conclusions: These results suggest that the impairment of cognitive flexibility induced by stress can be inhibited by regulating the content of HCY. Collectively, our findings highlight therapeutic strategies aimed at improving HCY treatment for impairments in cognitive flexibility.

Keywords: HCY; PIN1; cognitive flexibility; stress.

Figure 1

Cognitive flexibility impairment induced by restraint stress. (A) Time course of restraint stress and MWM test. (B) Changes in escape latency in sMWM and rMWM learning tasks in the control, RS, and RS1 groups (n = 7, two-way ANOVA with Tukey’s post hoc test vs. Rs; # p < 0.05 vs. RS1 * p < 0.05). (C) In the rMWM test task, the proportion of time that the mouse spent in the target quadrant compared to the total time, (n = 7, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01). (D) In the rMWM test task, the proportion of distance traveled by the mouse in the target quadrant compared to the total distance traveled (n = 7, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, * p < 0.05). (E) The number of times the platform was crossed in the target quadrant during the rMWM test task (n = 7, one-way ANOVA with Tukey’s post hoc test, * p < 0.05). (F) The average moving speed of the mice during the rMWM test task (n = 7, one-way ANOVA with Tukey’s post hoc test, ns p > 0.05).

Figure 2

The decrease in C-fos activation and high PIN1 expression in the hippocampus of mice exposed to stress. (A) Representative images of C-fos staining in control, RS, and RS1 mice. Scale bar, 50 μm. (* p < 0.05, ns: no significance) (B) The mRNA expression of PIN1 in hippocampal lysates from mice (n = 6, Student’s t-test, ** p < 0.01). (C) The protein expression of PIN1 in hippocampus lysates from mice (n = 4, Student’s t-test, * p < 0.05).

Figure 3

HCY accumulation is associated with stress-induced decline in cognitive flexibility. (A) Concentrations of HCY in the plasma of mice (n = 7, Student’s t-test, ** p < 0.01). (B,C) Correlation analysis of time spent in target quadrant, distance traveled, and plasma HCY in mice during rMWM test task (n = 7, simple linear regression). (D) Concentration of HCY in the hippocampus of mice (n = 7, Student’s t-test, ** p < 0.01). (E,F) Correlation analysis of time spent in target quadrant, distance traveled, and HCY in mouse hippocampus during rMWM test task (n = 7, simple linear regression).

Figure 4

HCY induces an increase in PIN1 expression in HT22 cells. (A) HT22 cells were treated with the indicated concentrations of HCY for 24 h, and cell growth was evaluated using a cell counting kit-8 (CCK-8) assay, in regard to three independent experiments. The values are presented as means ± SD (n = 3). Statistical analysis was performed using a one-way ANOVA; ** p < 0.01. (B) HCY induced lactate dehydrogenase (LDH) release in HT22 cells. The values are presented as means ± SD (n = 3). Statistical analysis was performed using a one-way ANOVA; ** p < 0.01. (C) The mRNA expression of PIN1 in HT22 cells (n = 3, Student’s t-test, * p < 0.05). (D) The protein expression of PIN1 in HT22 cells (n = 3, Student’s t-test, * p < 0.05).

Figure 5

HCY increases PIN1 expression in mice with cognitive impairment. (A) Concentrations of HCY in the plasma of mice (n = 7, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, * p < 0.05). (B) Concentrations of HCY in the hippocampus of mice (n = 7, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, * p < 0.05). (C) The mRNA expression of PIN1 in hippocampus lysates (n = 6, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, * p < 0.05). (D) The protein expression of PIN1 in hippocampus lysates (n = 3, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, * p < 0.05).

Figure 6

VitB ameliorates cognitive flexibility impairment induced by restraint stress by regulating HCY. (A) Representative trajectory plots of mice in regard to the rMWM learning and testing task. (B) Escape latency in regard to rMWM test (n = 7, one-way ANOVA with Tukey’s post hoc test, RS + VitB vs. RS, # p < 0.05, control vs. RS, ** p < 0.01). (C) In the rMWM test task, the proportion of time that the mice spent in the target quadrant compared to the total time (n = 7, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, * p < 0.05). (D) In the rMWM test task, the proportion of the distance traveled by the mouse in the target quadrant compared to the total distance traveled (n = 7, one-way ANOVA with Tukey’s post hoc test, ** p < 0.01, * p < 0.05). (E) The number of times the mice crossed the platform in the target quadrant during the rMWM test task (n = 7, one-way ANOVA with Tukey’s post hoc test, * p < 0.05). (F) The average moving speed of the mice in the rMWM test task (n = 7, one-way ANOVA with Tukey’s post hoc test).