Running exercise-induced up-regulation of hippocampal brain-derived neurotrophic factor is CREB-dependent
Michael J Chen, Amelia A Russo-Neustadt, Michael J Chen, Amelia A Russo-Neustadt
Abstract
The past decade has witnessed burgeoning evidence that antidepressant medications and physical exercise increase the expression of hippocampal brain-derived neurotrophic factor (BDNF). This phenomenon has gained widespread appeal, because BDNF is one of the first macromolecules observed to play a central role not only in the treatment of mood disorders, but also in neuronal survival-, growth-, and plasticity-related signaling cascades. Thus, it has become critical to understand how BDNF synthesis is regulated. Much evidence exists that changes in BDNF expression result from the activation/phosphorylation of the transcription factor, cAMP-response-element binding protein (CREB) following the administration of antidepressant medications. Utilizing a mouse model genetically engineered with an inducible CREB repressor, our current study provides evidence that increases in BDNF expression and cellular survival signaling resulting from physical exercise are also dependent upon activation of this central transcription factor. The transcription and expression of hippocampal BDNF, as well as the activation of Akt, a key survival signaling molecule, were measured following acute exercise, and also following short-term treatment with the norepinephrine reuptake inhibitor, reboxetine. We found that both interventions led to a marked increase in hippocampal BDNF mRNA, BDNF protein, and Akt phosphorylation (as well as CREB phosphorylation) in wild-type mice. As expected, activation of the CREB repressor in mutant mice sharply decreased CREB phosphorylation. In addition, all measures noted above remained at baseline levels when mutant mice exercised or received reboxetine. Increases in BDNF and phospho-Akt were also prevented when mutant mice received a combination of exercise and antidepressant treatment. The results are discussed in the context of what is currently known about BDNF signaling.
Copyright 2008 Wiley-Liss, Inc.
Figures
In CREBIR mice, P-CREB expression is repressed. (a) Representative 1.5 % agarose gel of PCR products of 6 mice in duplicate. Lanes 1 and 2, WT; Lanes 3 and 4, CREBIR, Lanes 5 and 6, CREBIR where the tube containing one of the duplicates (lane 5) largely evaporated during the PCR cycling process; Lanes 7 and 8, WT; Lanes 9 and 10, WT; Lanes 11 and 12, CREBIR. Note the presence of the repressor at ∼400 bp in the 3 CREBIR mice (Lanes 3, 4; 5, 6; and 11, 12). (b) WT mice express significantly more P-CREB than CREBIR mice across all treatments. Additionally, the conditionally activated mutation prevents any increase in P-CREB due to our interventions. *Among the WT mice, those who exercised (p < .0001, n = 29), received reboxetine (p = .067, n = 9), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more P-CREB than those that were sedentary (n = 12); and those that received reboxetine (n = 9) expressed significantly less P-CREB than exercising mice (p = .001, n = 29) or the combination-treated mice (p = .007, n = 7). Among the CREBIR mice, there were no significant differences among treatments in P-CREB immunoreactivity. Note the dramatic decrease in P-CREB immunoreactivity of CREBIR mice (black bars), compared to that of WT (white bars). Anti-P-CREB was very insensitive and required 60 μg protein for detection. P-CREB was undetectable in all 4 treatment groups and both genotypes, when less than 60 μg protein was applied to the gels. This large amount of protein, however, resulted in overloaded lanes for the much more sensitive anti-CREB. Because of the very small sample volumes, each mouse hippocampus was randomly assigned to a particular gel/Western film, thereby controlling for film-to-film variability. Therefore, the row of P-CREB bands and their respective CREB and GAPDH bands are a composite from several different Western blotting films. However, each P-CREB band and its respective CREB band and GAPDH band all represent the same mouse hippocampal sample (same gel lane).
In CREBIR mice, P-CREB expression is repressed. (a) Representative 1.5 % agarose gel of PCR products of 6 mice in duplicate. Lanes 1 and 2, WT; Lanes 3 and 4, CREBIR, Lanes 5 and 6, CREBIR where the tube containing one of the duplicates (lane 5) largely evaporated during the PCR cycling process; Lanes 7 and 8, WT; Lanes 9 and 10, WT; Lanes 11 and 12, CREBIR. Note the presence of the repressor at ∼400 bp in the 3 CREBIR mice (Lanes 3, 4; 5, 6; and 11, 12). (b) WT mice express significantly more P-CREB than CREBIR mice across all treatments. Additionally, the conditionally activated mutation prevents any increase in P-CREB due to our interventions. *Among the WT mice, those who exercised (p < .0001, n = 29), received reboxetine (p = .067, n = 9), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more P-CREB than those that were sedentary (n = 12); and those that received reboxetine (n = 9) expressed significantly less P-CREB than exercising mice (p = .001, n = 29) or the combination-treated mice (p = .007, n = 7). Among the CREBIR mice, there were no significant differences among treatments in P-CREB immunoreactivity. Note the dramatic decrease in P-CREB immunoreactivity of CREBIR mice (black bars), compared to that of WT (white bars). Anti-P-CREB was very insensitive and required 60 μg protein for detection. P-CREB was undetectable in all 4 treatment groups and both genotypes, when less than 60 μg protein was applied to the gels. This large amount of protein, however, resulted in overloaded lanes for the much more sensitive anti-CREB. Because of the very small sample volumes, each mouse hippocampus was randomly assigned to a particular gel/Western film, thereby controlling for film-to-film variability. Therefore, the row of P-CREB bands and their respective CREB and GAPDH bands are a composite from several different Western blotting films. However, each P-CREB band and its respective CREB band and GAPDH band all represent the same mouse hippocampal sample (same gel lane).
Representative autoradiograms showing hippocampal BDNF mRNA by in situ hybridization. (a) WT sedentary; (b) WT exercising; (c) CREBIR exercising.
The TAM-activated CREB repressor prevented the BDNF mRNA-increasing effects of exercise, reboxetine or exercise/reboxetine in all hippocampal regions examined. (a) CA1: *Among the WT mice, those who exercised (p = .012, n = 19), received reboxetine (p = .006, n = 8), and received the combination of the two treatments (p = .004, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 4.48, p = .008, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .791, p = .507, n = 40]. (b) CA2: *Among the WT mice, those who exercised (p = .002, n = 19), received reboxetine (p = .039, n = 8), and received the combination of the two treatments (p = .001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 5.26, p = .004, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.67, p = .192, n = 40]. (c) CA3: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 20.81, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.34, p = .276, n = 40]. (d) CA4/hilus: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 18.70, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .346, p = .792, n = 40]. (e) DG: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 32.10, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .516, p = .674, n = 40].
The TAM-activated CREB repressor prevented the BDNF mRNA-increasing effects of exercise, reboxetine or exercise/reboxetine in all hippocampal regions examined. (a) CA1: *Among the WT mice, those who exercised (p = .012, n = 19), received reboxetine (p = .006, n = 8), and received the combination of the two treatments (p = .004, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 4.48, p = .008, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .791, p = .507, n = 40]. (b) CA2: *Among the WT mice, those who exercised (p = .002, n = 19), received reboxetine (p = .039, n = 8), and received the combination of the two treatments (p = .001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 5.26, p = .004, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.67, p = .192, n = 40]. (c) CA3: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 20.81, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.34, p = .276, n = 40]. (d) CA4/hilus: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 18.70, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .346, p = .792, n = 40]. (e) DG: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 32.10, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .516, p = .674, n = 40].
The TAM-activated CREB repressor prevented the BDNF mRNA-increasing effects of exercise, reboxetine or exercise/reboxetine in all hippocampal regions examined. (a) CA1: *Among the WT mice, those who exercised (p = .012, n = 19), received reboxetine (p = .006, n = 8), and received the combination of the two treatments (p = .004, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 4.48, p = .008, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .791, p = .507, n = 40]. (b) CA2: *Among the WT mice, those who exercised (p = .002, n = 19), received reboxetine (p = .039, n = 8), and received the combination of the two treatments (p = .001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 5.26, p = .004, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.67, p = .192, n = 40]. (c) CA3: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 20.81, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.34, p = .276, n = 40]. (d) CA4/hilus: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 18.70, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .346, p = .792, n = 40]. (e) DG: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 32.10, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .516, p = .674, n = 40].
The TAM-activated CREB repressor prevented the BDNF mRNA-increasing effects of exercise, reboxetine or exercise/reboxetine in all hippocampal regions examined. (a) CA1: *Among the WT mice, those who exercised (p = .012, n = 19), received reboxetine (p = .006, n = 8), and received the combination of the two treatments (p = .004, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 4.48, p = .008, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .791, p = .507, n = 40]. (b) CA2: *Among the WT mice, those who exercised (p = .002, n = 19), received reboxetine (p = .039, n = 8), and received the combination of the two treatments (p = .001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 5.26, p = .004, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.67, p = .192, n = 40]. (c) CA3: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 20.81, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.34, p = .276, n = 40]. (d) CA4/hilus: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 18.70, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .346, p = .792, n = 40]. (e) DG: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 32.10, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .516, p = .674, n = 40].
The TAM-activated CREB repressor prevented the BDNF mRNA-increasing effects of exercise, reboxetine or exercise/reboxetine in all hippocampal regions examined. (a) CA1: *Among the WT mice, those who exercised (p = .012, n = 19), received reboxetine (p = .006, n = 8), and received the combination of the two treatments (p = .004, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 4.48, p = .008, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .791, p = .507, n = 40]. (b) CA2: *Among the WT mice, those who exercised (p = .002, n = 19), received reboxetine (p = .039, n = 8), and received the combination of the two treatments (p = .001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 5.26, p = .004, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.67, p = .192, n = 40]. (c) CA3: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 20.81, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = 1.34, p = .276, n = 40]. (d) CA4/hilus: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 18.70, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .346, p = .792, n = 40]. (e) DG: *Among the WT mice, those who exercised (p < .0001, n = 19), received reboxetine (p < .0001, n = 8), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more BDNF mRNA than those that were sedentary (n = 12) [F(3,42) = 32.10, p < .0001, n = 46]. # WT mice also expressed significantly more BDNF mRNA than CREBIR mice for each intervention, except for sedentary mice. Among the CREBIR mice, there were no significant differences among treatments [F(3,36) = .516, p = .674, n = 40].
Exercise and antidepressant interventions increased hippocampal mature BDNF immunoreactivity in WT mice. No BDNF response to interventions was evident CREBIR mice. *Among the WT mice, those who exercised (p = .019, n = 27) and received the combination of the two treatments (p = .004, n = 7), but not those that received reboxetine (p = .078, n = 7), expressed significantly more mature BDNF immunoreactivity (electrophoresing as a ∼17-kDal band) than those that were sedentary (n = 12) [F(3,49) = 3.395, p = .025]. # WT mice also expressed significantly more BDNF immunoreactivity than CREBIR mice for each intervention, except for sedentary mice, who expressed the same amount between genotypes. Among the CREBIR mice, exercising mice (n = 23) expressed significantly more BDNF immunoreactivity than those that received just reboxetine (p = .006, n = 5) and the combination treatment (p = .007, n = 7). Because of the very small sample volumes, each mouse hippocampus was randomly assigned to a particular gel/Western film, thereby controlling for film-to-film variability. Therefore, the row of BDNF bands and their respective GAPDH bands are a composite from several different Western blotting films. However, each BDNF band and its respective GAPDH band all represent the same mouse hippocampal sample (same gel lane).
In WT mice, all interventions led to a significant activation of the signaling molecule, Akt, whereas no change from baseline was evident in CREBIR mice. *Among the WT mice, those who exercised (p < .0001, n = 27), received reboxetine (p = .027, n = 7), and received the combination of the two treatments (p < .0001, n = 7) expressed significantly more P-Akt immunoreactivity than those that were sedentary (p < .0001, n = 12). #WT mice also expressed significantly more P-Akt immunoreactivity than CREBIR mice for each intervention, except for sedentary mice, who expressed the same amount between genotypes. Among the CREBIR mice, there were no significant differences among treatments in P-Akt immunoreactivity. Because of the very small sample volumes, each mouse hippocampus was randomly assigned to a particular gel/Western film, thereby controlling for film-to-film variability. Therefore, the row of P-Akt bands and their respective Akt and GAPDH bands are a composite from several different Western blotting films. However, each P-Akt band and its respective Akt band and GAPDH band all represent the same mouse hippocampal sample (same gel lane).
Source: PubMed