Imbalance in Sirt1 Alternative Splicing in Response to Chronic Stress during the Adolescence Period in Female Mice

Abstract

Stressful unpredictable life events have been implicated in numerous diseases. It is now becoming clear that some life periods are more vulnerable than others. As adolescence is a sensitive period in brain development, the long-term effects of stress during this period could be significant. We investigated the long-term effects of exposure to unpredictable chronic mild stress in adolescent mice on alternative splicing of Sirtuin 1. One-month-old mice were exposed to 4 weeks of UCMS and examined for anxiety and cognition at the age of 2, 4 and 6 months. We found a rise in anxious behavior immediately after the exposure to stress. Notably, there was a long-term impairment of performance in cognitive tasks and an imbalance in Sirtuin 1 and TrkB receptor alternative splicing in the stress-exposed mice compared with controls. To conclude, our results show that exposure to unpredictable chronic mild stress during adolescence affects cognition in adulthood. Understanding pathways affiliated with stress may help minimize the long-term emotional effects of an unpredictable, stressful event.

Keywords: Sirt1; TrkB; cognition; senescence; unpredictable stress.

Figure 1

Stressed mice exhibit anxiety-like behavior in the EPM. Male and female stressed mice present higher levels of anxiety at the age of two (A) and four (B) months, which is manifested in spending significantly less time in the open arm of the EPM compared to naïve mice (p (Males UCMS vs. naive, 2M) < 0.0001, p (Females UCMS vs. naive, 2M) = 0.0001, p (Males UCMS vs. naive, 4M) = 0.0294, p (Females UCMS vs. naive, 4M) = 0.0003. At the age of six months, no significant differences in anxiety-like behavior between stressed and naïve mice were found (A) n(Male 2M naïve) = 10, n(Male 2M UCMS) = 15; n(Female 2M naïve) = 8, n(Female 2M UCMS) = 15. (B) n(Male 4M naïve) = 8, n(Male 4M UCMS) = 18. n(Female 4M naïve) = 9, n(Female 4M UCMS) = 16. (C) n(Male 6M naïve) = 16, n(Male 6M UCMS) = 14. n(Female 6M naïve) = 13, n(Female 6M UCMS) = 10. Data are presented as mean ± SEM and scatter dot plot. *** indicates p < 0.0001, * indicates p < 0.05.

Figure 2

Stressed mice exhibit increased anxiety-like behavior in the OFT. Male and female stressed mice present higher levels of anxiety at the age of two ((A), F (1, 46) = 56.24, p < 0.0001) and four months ((B), F (1, 43) = 16.55, p = 0.0002), which manifested in spending significantly less time in the center of the OF compared to naïve mice At the age of six months, no significant differences were found between stressed and naïve mice (A) Male 2M n(naïve) = 10, n(UCMS) = 17. Female 2M, n(naïve) = 9, n(UCMS) = 14) (B) Male 4M t(df = 23) = 2.92, n(naïve) = 9, n(UCMS) = 16. Female 4M n(naïve) = 6, n(UCMS) = 16). (C) Male 6M n(naïve) = 9, n(UCMS) = 12. Female 6M n(naïve) = 6, n(UCMS) = 9). Data are presented as mean ± SEM and scatter dot plot. *** indicates p < 0.0002.

Figure 3

Stressed mice exhibit spatial memory impairment in the Y-maze. At the age of 4 months (A), male and female stressed mice show no significant differences in their preference index of the Y-maze compared to naïve mice. At the age of 6 months (B), both male and female stressed mice spent significantly less time in the novel arm of the Y-maze compared to naïve mice, resulting in decreased preference index. (p (Males UCMS vs. naive) < 0.0001, p (Females UCMS vs. naive) < 0.0001)). Data are presented as mean ± SEM and scatter dot plot. **** indicates p < 0.0001.

Figure 4

Stressed female mice show an imbalance in sirt1 50 kD/110 kD ratio in specific brain regions. Representative Western blot images of each region are shown above. (A) Cortical sirt1 50 kD/110 kD protein expression ratio. A significant increase in sirt1 50 kD/110 kD ratio among 6-month-old stressed female mice compared to naïve age-matched mice (* p = (0.0406), n = 3). A significant increase in sirt1 50 kD/110 kD ratio between 4 and 6 months old UCMS female mice (* p = (0.0151), n = 3). (A) Two-way ANOVA analysis of the effect of age and UCMS paradigm on sirt1 50 kD/110 kD ratio showed a statistically significant simple main effect of age (** p = (0.0069)) and of the UCMS paradigm (* p = (0.0322)). (B) Cortical sirt1 short/long mRNA expression ratio increased in the cortex of stressed mice (p = 0.009, t = 1.552, df = 7). (C) Hippocampal sirt1 50 kD/110 kD protein expression ratio. Stressed female mice show no significant differences in sirt1 50 kD/110 kD ratio hippocampal levels compared to naïve age-matched mice (p > 0.05, n = 3). (D) Stressed female mice show a trend toward an increase in the sirt1 short/long mRNA expression ratio in the hippocampus (p = 0.08). (E) Correlation of cortical sirt1 50 kD/110 kD vs. Y-maze preference index. A Pearson correlation coefficient was computed to assess the linear relationship between cortical sirt1 50 kD/110 kD ratio and Y-maze Preference Index scores. There was a negative correlation between the two variables, r(8) = (−0.821), ** p = (0.0018).

Figure 5

Stressed female mice show an imbalance in sirt1 Trkb.T1/TrkB.full ratio in specific brain regions. A significant increase in TrkB.t1 receptor/TrkB.FL receptor among 6-month-old stressed female mice compared to naïve age-matched mice in the Hipp (A) (* p = (0.03), n = 6), indicating a higher tendency to express TrkB.t1 over the Trk.B full receptor. There was no difference in the CTX (B) (* p = (0.07), n = 6). A correlation coefficient was computed to assess the linear relationship between the long sirt1 in the PFC and the TrkB.t receptor in the hippocampus (C). There was a negative correlation between the two variables, r(11) = (−0.64), * p = (0.02).