Molecular evidence for BDNF- and GABA-related dysfunctions in the amygdala of female subjects with major depression

Abstract

Women are twice as likely as men to develop major depressive disorder (MDD) and are more prone to recurring episodes. Hence, we tested the hypothesis that the illness may associate with robust molecular changes in female subjects, and investigated large-scale gene expression in the post-mortem brain of MDD subjects paired with matched controls (n=21 pairs). We focused on the lateral/basolateral/basomedian complex of the amygdala as a neural hub of mood regulation affected in MDD. Among the most robust findings were downregulated transcripts for genes coding for γ-aminobutyric acid (GABA) interneuron-related peptides, including somatostatin (SST), tachykinin, neuropeptide Y (NPY) and cortistatin, in a pattern reminiscent to that previously reported in mice with low brain-derived neurotrophic factor (BDNF). Changes were confirmed by quantitative PCR and not explained by demographic, technical or known clinical parameters. BDNF itself was significantly downregulated at the RNA and protein levels in MDD subjects. Investigating putative mechanisms, we show that this core MDD-related gene profile (including SST, NPY, TAC1, RGS4 and CORT) is recapitulated by complementary patterns in mice with constitutive (BDNF-heterozygous) or activity-dependent (exon IV knockout) decreases in BDNF function, with a common effect on SST and NPY. Together, these results provide both direct (low RNA/protein) and indirect (low BDNF-dependent gene pattern) evidence for reduced BDNF function in the amygdala of female subjects with MDD. Supporting studies in mutant mice models suggest a complex mechanism of low constitutive and activity-dependent BDNF function in MDD, particularly affecting SST/NPY-related GABA neurons, thus linking the neurotrophic and GABA hypotheses of depression.

Figure 1. qPCR validation of altered gene expression and reduced BDNF transcript/protein in the amygdala of female MDD subjects

(A) Confirmation of microarray results by qPCR (See also Supplementary Table ST4). (B) BDNF-IXd coding exon transcript expression in relative expression (%) of the control group mean (*, p<0.05). (C) Mature BDNF (m-BDNF) and pro-BDNF protein relative immunoreactivity migrates at the expected ~15 and 25kD size respectively. Examples of 2 sample pairs of control (C) and MDD (D) on the same gel. (D) Relative level of m-BDNF in MDD in function of respective paired CTRL (◆ADD-treated MDD ◇ADD-free MDD Average of population) (*, p<0.05).

Figure 2. Reduced SST mRNA and protein levels in amygdala of female MDD subjects

(A) SST mRNA expression assessed by autoradiographic optical density measures in LBNC sub-regions (*, p<0.05). (B) LBNC details (C) In situ hybridization film autoradiograms of a MDD subject (left) displaying robust SST mRNA downregulation compared to a matched control (right) (D) Immunoblot of prepro-SST in CTRL and MDD subjects. (E) Relative prepro-SST levels in MDD in function of respective paired CTRL (◆Antidepressant-treated MDD ◇Antidepressant-free MDD; Average of population).

Figure 3. A proposed model of a BDNF-SST-GABA molecular intermediate phenotype within the complex multi-module pathophysiology of MDD

Our results suggest that the frequent observation of low SST in MDD is linked to altered BDNF function. The two components of this "molecular intermediate phenotype" (BDNF & SST) are known to be controlled by sets of unique and/or shared upstream etiological factors. Their downstream impact on integrated GABA function will be determined by the extent of SST-expressing cell dysregulation and by the degree of disease effects on other components of the GABA microcircuitry (e.g. PV, not affected here). So, altered GABA circuitry may be considered a one-scale-higher "cellular intermediate phenotype". The outcome of dysregulated BDNF-SST-GABA function in the amygdala and in an extended corticolimbic neural network is hypothesized to alter mood regulation and contribute to the "affect dysregulation" symptom domain of the illness. In this model, sets of additional molecular/cellular intermediate phenotype (or modules) are under the control of their respective etiological factors, display multiple levels of interactions, but potentially impinge on different neural networks, which in turn mediate distinct symptom domains.