Estrogen receptor β deficiency impairs BDNF-5-HT2A signaling in the hippocampus of female brain: A possible mechanism for menopausal depression

Abstract

Depression currently affects 350 million people worldwide and 19 million Americans each year. Women are 2.5 times more likely to experience major depression than men, with some women appearing to be at a heightened risk during the menopausal transition. Estrogen signaling has been implicated in the pathophysiology of mood disorders including depression; however, the underlying mechanisms are poorly understood. In this study, the role of estrogen receptor (ER) subtypes, ERα and ERβ, in the regulation of brain-derived neurotrophic factor (BDNF) and serotonin (5-HT) signaling was investigated; two pathways that have been hypothesized to be interrelated in the etiology of depression. The analyses in ERα^-/- and ERβ^-/- mouse models demonstrated that BDNF was significantly downregulated in ERβ^-/- but not ERα^-/- mice, and the ERβ^-/--mediated effect was brain-region specific. A 40% reduction in BDNF protein expression was found in the hippocampus of ERβ^-/- mice; in contrast, the changes in BDNF were at a much smaller magnitude and insignificant in the cortex and hypothalamus. Further analyses in primary hippocampal neurons indicated that ERβ agonism significantly enhanced BDNF/TrkB signaling and the downtream cascades involved in synaptic plasticity. Subsequent study in ERβ mutant rat models demonstrated that disruption of ERβ was associated with a significantly elevated level of 5-HT_2A but not 5-HT_1A in rat hippocampus, indicating ERβ negatively regulates 5-HT_2A. Additional analyses in primary neuronal cultures revealed a significant association between BDNF and 5-HT_2A pathways, and the data showed that TrkB activation downregulated 5-HT_2A whereas activation of 5-HT_2A had no effect on BDNF, suggesting that BDNF/TrkB is an upstream regulator of the 5-HT_2A pathway. Collectively, these findings implicate that the disruption in estrogen homeostasis during menopause leads to dysregulation of BDNF-5-HT_2A signaling and weakened synaptic plasticity, which together predispose the brain to a vulnerable state for depression. Timely intervention with an ERβ-targeted modulator could potentially attenuate this susceptibility and reduce the risk or ameliorate the clinical manifestation of this brain disorder.

Keywords: 5-HT(1A); 5-HT(2A); Brain-derived neurotrophic factor (BDNF); Estrogen receptor β (ERβ); Hippocampus; Menopausal depression.

Figure 1. ERβ, not ERα, knockout downregulates BDNF and TrkB in female mouse hippocampus

a) Validation of specificity of BDNF antibody. Western blots show both the monomeric (14 kDa) and trimeric (42 kDa) forms of mature BDNF in mouse hippocampal protein lysates. b) BDNF regulation by ERs was examined in three brain regions of ERα^-/- and ERβ^-/- female mice. Data indicate that: b-c) BDNF is not regulated by either ER in mouse cortex and hypothalamus; d-e) ERβ but not ERα regulates BDNF and TrkB protein expression in mouse hippocampus. Data are shown as mean ± standard deviation, n=5. *p<0.05, **p<0.01.

Figure 2. ERβ disruption downregulates BDNF and TrkB in female rat hippocampus

BDNF and TrkB regulation by ERβ was further examined in the hippocampus of a-b) 6-month-old and c-d) 10-month-old ERβ mutant female rats. ERβ mutant rats with deletions of exon 3 or exon 4 leads to a significant reduction in the expression levels of BDNF and TrkB in rat hippocampus. Data are shown as mean ± standard deviation, n=5. *p<0.05, **p<0.01.

Figure 3. BDNF is more significantly impacted by ERβ status than aging

BDNF regulation by ERβ and aging was comparatively examined in the hippocampus of 6 and 10-month-old ERβ mutant female rats. Data indicate that: a-c) Aging decreases BDNF and TrkB protein expression in rat hippocampus irrespective of ERβ status; d) ERβ disruption exerts a more prominent impact on BDNF in comparison to aging. Data are shown as mean ± standard deviation, n=5. *p<0.05, **p<0.01. ^#p<0.05 comparison between 6 Months and 10 Months group.

Figure 4. ERβ activation upregulates BDNF/TrkB signaling and synaptic markers in primary hippocampal neurons

Primary hippocampal neurons were treated with PPT (100 nM), DPN (100 nM), or DPN (100 nM) in combination with PHTPP (1 μM). Data indicate that: a) Activation of ERβ but not ERα results in a significant increase in BDNF protein expression in primary hippocampal neurons; b-c) Activation of ERβ also leads to an increase in the expression levels of pTrkB and synaptic proteins synaptophysin, VAMP2, and PSD95; ERβ agonism-mediated increase was reverted back to basal levels in neurons co-treated with ERβ antagonist PHTPP. Data are shown as mean ± standard deviation, n=3. *p<0.05, **p <0.01, ***p <0.001.

Figure 5. a-b) ERβ disruption upregulates 5-HT_2A but has no effect on 5-HT_1A in female rat hippocampus

ERβ regulation of serotonergic receptors, 5-HT_1A and 5-HT_2A, was examined in the hippocampus of ERβ mutant female rats. Data indicate that ERβ mutant rats with deletions of exon 3 or exon 4 leads to an increase in 5-HT_2A protein expression but has no effect on 5-HT_1A in the hippocampus of both 6 and 10-month-old rats. c-d) TrkB activation downregulates 5-HT_2A in primary hippocampal neurons. Interaction between BDNF/TrkB and 5-HT_2A signling was examined in primary hippocampal neurons. Data indicate that: c) Activation of BDNF/TrkB negatively regulates 5-HT_2A; d) Activation of 5-HT_2A does not regulate BDNF/TrkB. Data are shown as mean ± standard deviation, n=3. *p<0.05, **p <0.01, ***p <0.001.

Figure 6. ERβ regulation of BDNF–5-HT_2A signaling in the hippocampus of female brain

Based on the above findings, we hypothesize that: (1) the onset of menopause results in (2) an irregular profile of estrogen in the brain. Disruption of estrogen homeostasis (3) decreases the activation of ERβ signaling in the brain. Decresed ERβ signaling results in reduced transcription of the BDNF gene, (4) decreasing BDNF protein levels in the hippocampus. Decreased BDNF protein levels leads to (5) decreased TrkB signaling, which through unknown mechanisms (6) increases 5-HT_2A protein expression and (7) 5-HT_2A-mediated signaling. The decrease in BDNF/TrkB signaling also leads to (8) downregulation of synaptic proteins, thus weakening the synaptic strength. Decreased BDNF/TrkB signaling and increased 5-HT_2A signaling along with decreased neuroplasticity predispose the brain to (9) an increased risk of developing depression, which could be exacerbated by environmental stressors leading to (10) the onset of depression.