Branched-chain amino acids mediate resilience to chronic social defeat stress by activating BDNF/TRKB signaling

Abstract

How individuals respond to chronic stress varies. Susceptible individuals ultimately develop depression; whereas resilient individuals live normally. In this study, our objective was to examine the effect of branched-chain amino acids (BCAA), commonly used by athletes, on susceptibility to stress. Male C57BL/6 mice were subjected to daily defeat sessions by a CD1 aggressor, for 10 days. On day11, the behavior of mice was assessed using the social interaction test, elevated plus maze and open field. Mice received the BCAA leucine, isoleucine or valine before each defeat session. Furthermore, we examined whether BCAA regulate brain derived neurotrophic factor (BDNF) signaling by using a brain-permeable tropomyosin receptor kinase B (TRKB) inhibitor, ANA-12. We also tested the effect of voluntary exercise and high protein diets on susceptibility to stress. Mice exposed to chronic stress displayed increased susceptibility and social avoidance. BCAA promoted resilience to chronic stress, rescued social avoidance behaviors and increased hippocampal BDNF levels and TRKB activation. Inhibition of TRKB signaling abolished the ability of BCAA to promote resilience to stress and to rescue social avoidance. Interestingly, we found that BCAA activate the exercise-regulated PGC1a/FNDC5 pathway known to induce hippocampal BDNF signaling. Although both voluntary exercise and BCAA promoted resilience to stress, combining them did not yield synergistic effects confirming that they affect similar pathways. We also discovered that high protein diets mimic the effect of BCAA by rescuing social deficits induced by chronic stress and increase Bdnf expression in the hippocampus. Our data indicate that BCAA, exercise and high protein diets rescue susceptibility to stress by activating the hippocampal BDNF/TRKB signaling.

Keywords: BDNF; Branched chain amino acids; Chronic social defeat stress; Exercise; FNDC5; High protein diet.

Fig. 1

BCAA mediate resilience to chronic social defeat stress and rescue social avoidance behavior. (A) The CSDS paradigm consists of ten days of daily defeat sessions. Each day, the experimental mouse is subjected to direct physical contact with an aggressor mouse for 7 min. On the eleventh day, behavioral tests and brain tissue collection are conducted. Mice receive daily intraperitoneal injections of either saline, Leu, Ile, or Val 15 min before each defeat session. (B) Leu, Ile and Val increase resilience to stress. In the group of mice (n = 24) receiving saline and subjected to CSDS, 33.3% are resilient to stress, whereas 66.7% are susceptible to stress. In the group of mice (n = 17) receiving Leu (32.5 mg/kg, daily for ten days) and subjected to CSDS, 76.5% are resilient to stress, whereas 23.5% are susceptible to stress. In the group of mice (n = 12) receiving Ile (32.5 mg/kg, daily for ten days) and subjected to CSDS, 91.7% are resilient to stress, whereas 8.3% are susceptible to stress. In the group of mice (n = 13) receiving Val (28.5 mg/kg, daily for ten days) and subjected to CSDS, 84.6% are resilient to stress, whereas 15.4% are susceptible to stress. (C) Intraperitoneal injections of Leu, Ile and Val reverse the social avoidance phenotype induced by CSDS as shown by the increase in the time spent in interaction zone of the social interaction test. Statistical significance was measured by 2way Anova followed by Bonferroni posttests. Significance was measured versus the defeat groups. ***p < 0.001. The n numbers for the control, defeat, defeat + Leu, defeat + Ile and defeat + Val are 16, 16, 17,12 and 13 respectively. (D) Intraperitoneal injections of Ile and Val, but not Leu decrease anxiety. defeat + saline and defeat + Leu animals exhibit anxiety-like behavior as measured by the significant increase in the time spent in the closed arm of the elevated plus maze (EPM), whereas defeat + Ile and defeat + Val behave similar to Control. Statistical significance was measured by one-way anova followed by Dunnett's multiple comparison test. Significance was measured versus the control groups *p < 0.05. The n numbers for control + saline, defeat + saline, defeat + Leu, defeat + Ile and defeat + Val are 16, 16, 17, 12 and 13 respectively. (E) There was no significant difference in the distance travelled in the open field between the different mice groups.

Fig. 2

BCAA restore control levels of BDNF protein and BDNF signaling in the hippocampus. (A) CSDS significantly decrease BDNFI expression in the hippocampus as measured by real-time RTPCR. Intraperitoneal injections of Ile and Val reverse this effect and restore control hippocampal BDNFI expression in animals subjected to CSDS. The number of animal used for control + saline, defeat = saline, defeat + Leu, defeat + Ile and defeat + Val are 13, 13 4, 3 and 3 respectively. Statistical significance was measured by one-way anova followed by Dunnett's multiple comparison test. *p < 0.05. Defeat vs control: p = 0.0228, defeat vs defeat + Leu: p = 0.9700, defeat vs defeat + Ile: p = 0.0264 and defeat vs defeat + Val: p = 0.0362. (B) Representative Western blot images depicting BDNF levels in hippocampus of control + saline, defeat + saline, defeat + Leu, defeat + Ile and defeat + Val animals. (C) Quantification of the BDNF western blots. Significance was measured versus defeat * p < 0.05. The n of hippocampi analyzed are 7,5,4,3, and 2 for control + saline, defeat + saline, defeat + Leu, defeat + Ile and defeat + Val groups respectively. Defeat vs control: p = 0.0940 and df = 10, defeat vs defeat + Leu: p = 0.1068 and df = 7, defeat vs defeat + Ile: p = 0.0082 and df = 6 and defeat vs defeat + Val: p < 0.0001 and df = 5. (D) Representative Western blot images depicting phosphorylated TRKB levels in hippocampus of control, defeat, defeat + Leu, defeat + Ile and defeat + Val animals. (E) Quantification of the phosphorylated TRKB western blots. Significance was measured versus defeat * p < 0.05. The n of hippocampi analyzed are 6,4,5,6, and 2 for control + saline, defeat + saline, defeat + Leu, defeat + Ile and defeat + Val groups respectively. Defeat vs control: p = 0.5853 and df = 9, defeat vs defeat + Leu: p = 0.0191 and df = 7, defeat vs defeat + Ile: p = 0.0022 and df = 8 and defeat vs defeat + Val: p = 0.0331 and df = 4.

Fig. 3

BCAA mediate resilience to stress and rescue social avoidance behavior by activating BDNF/TRKB signaling. (A) The combined treatment of Leu, Ile and Val with the TRKB inhibitor ANA-12 failed to increase resilience to stress. In the group of mice receiving saline and subjected to CSDS, 25% are resilient to stress, whereas 75% are susceptible to stress. In the group of mice receiving Leu + ANA-12 and subjected to CSDS, 10% are resilient to stress, whereas 90% are susceptible to stress. In the group of mice receiving Ile + ANA-12 and subjected to CSDS, 18% are resilient to stress, whereas 82% are susceptible to stress. In the group of mice receiving Val + ANA-12 and subjected to CSDS, 0% are resilient to stress, whereas 100% are susceptible to stress. The n numbers for the control + vehicle, defeat + vehicle, defeat + Leu + ANA-12, defeat + Ile + ANA-12 and defeat + Val + ANA- 32, 10, 11, 11. (B) Intraperitoneal injections of Leu, Ile and Val in combination with ANA-12 do not rescue the social avoidance phenotype associated with CSDS as shown by the lack of increase in the time spent in interaction zone of the social interaction test. Statistical significance was measured by 2way Anova followed by Tukey's multiple comparison test. Significance was measured versus both control + vehicle or defeat + vehicle groups. *p < 0.05. The n numbers for the control + vehicle, defeat + vehicle, defeat + Leu + ANA-12, defeat + Ile + ANA-12 and defeat + Val + ANA-12 are 6, 8, 10, 11 and 11. Interaction: (defeat + vehicle vs control + vehicle: p = 0.0268, defeat + vehicle vs defeat + Leu: p = 0.6559, defeat + vehicle vs defeat + Ile: p = 0.9930 and defeat + vehicle vs defeat + Val: p > 0.9999) and No Interaction: (defeat + vehicle vs control + vehicle: p = 0.0227, defeat + vehicle vs defeat + Leu: p = 0.0630 defeat + vehicle vs defeat + Ile: p = 0.2149 and defeat + vehicle vs defeat + Val: p = 0.9659).

Fig. 4

BCAA activate the PGC1A/FNDC5 pathway known to activate BDNF signaling in the hippocampus (A) BCAA restore control hippocampal levels of PGC1a and FNDC5 in animals subjected to CSDS. Representative Western blot image depicting hippocampal PGC1a and FNDC5 in control, defeat, defeat + Leu, defeat + Ile and defeat + Val mice. (B) Quantification of the PGC1A western blots. Statistical significance was measured by the unpaired t-test. Significance was measured versus defeat * p < 0.05. The n of hippocampi analyzed are 3 for control + saline, defeat + saline, defeat + Leu, defeat + Ile and defeat + Val groups respectively. Defeat + saline vs control = saline: p = 0.0102 and df = 4, defeat + saline vs defeat + Leu: p = 0.0063 and df = 4, defeat + saline vs defeat + Ile: p = 0.0175 and df = 4 and defeat + saline vs defeat + Val: p = 0.0213 and df = 4 (C) Quantification of the FNDC5 western blots. Statistical significance was measured by the unpaired t-test. Significance was measured versus defeat * p < 0.05. The n of hippocampi analyzed are 4,4,3,3, and 3 for control + saline, defeat + saline, defeat + Leu, defeat + Ile and defeat + Val groups respectively. Defeat vs control: p = 0.1572 and df = 6, defeat vs defeat + Leu: p = 0.0226 and df = 5, defeat vs defeat + Ile: p = 0.0226 and df = 5 and defeat vs defeat + Val: p = 0.0090 and df = 5. (D) VWR and intraperitoneal injections of Ile alone or combined reverse the chronic social defeat phenotype as shown by the increase in the time spent in interaction zone of the social interaction test. Statistical significance was measured by 1way Anova followed by Dunnett's multiple comparison test. Significance was measured versus the defeat groups. *p < 0.05 and **p < 0.01. The n numbers for the control + saline, exercise + saline, defeat + saline, defeat + Exercise (Ex), defeat + Ile and defeat + Ex + Ile are 7, 6, 7, 8, 3 and 4 respectively. Defeat + saline vs control + saline: p = 0.0013, defeat + saline vs Ex + saline: p = 0.0244, defeat + saline vs defeat + Ex + saline: p = 0.0090, defeat + saline vs defeat + Ile: p = 0.0375 and defeat + saline vs defeat + Ex + Ile: p = 0.0071.

Fig. 5

HPD promotes resilience to CSDS, rescues social avoidance behaviors and induces hippocampal Bdnf expression. (A) Animals were fed either STD or HPD for two weeks before being subjected to CSDS. Animals continued to receive the different diets during the CSDS. On day 24, behavioral tests and brain tissue collection were conducted. (B) HPD increase resilience to stress. In the group of mice (n = 19) receiving a STD and subjected to CSDS, 26% are resilient to stress. In the group of mice (n = 7) receiving a HPD and subjected to CSDS, 85.7% are resilient to stress. (C) HPD reverses the social avoidance phenotype as shown by the increase in the time spent in interaction zone of the social interaction test. Statistical significance was measured by 2way Anova followed by Tukey's multiple comparison test. Interaction: F(1,43) = 4.886, p = 0.0324, Row Factor F(1,43) = 6.864, p = 0.0121 and column factor F(1,43) = 10.84, p = 0.02. STD + defeat vs HPD + Defeat: p = 0.0153, STD + defeat vs STD: p = 0.001 and STD + defeat vs HPD: p = 0.0002. (D) No significant changes in the weight were observed between the mice receiving a STD or HPD at the end of the experiment. (E) There was no significant difference in the distance travelled in the open field between the different mice groups. (F) Mice receiving a HPD for 4 weeks had significantly increased BdnfI expression levels as compared to mice receiving a STD. Statistical significance was measured by unpaired t-test groups. *p < 0.05. The n number for mice receiving STD and HPD is 10 and 12 respectively.

Fig. 6

BCAA mediate resilience to stress by inducing the hippocampal BNDF/TRKB signaling through the PGC1a/FNDC5 pathway.