Amygdala-Anterior Cingulate Cortex Altered Gene Expression in Human Major Depression
Large-scale gene expression profiles were generated from subdissected amygdala and anterior cingulate cortex () in postmortem brains of male subjects with familial major depression and matched controls (). Overall correlations of gene transcript levels were significantly higher in matched pairs compared to nonmatched major depression-control pairs (), thus validating the pairing protocol at controlling non-disease-related factors and reducing signal variability. Three hundred ninety-five genes in anterior cingulate cortex and 191 genes in amygdala were identified by paired statistics or ANOVA models as differentially expressed in major depression (Figure S1 and 1D
). A qPCR survey on adjacent tissue sections yielded highly concordant results (array-qPCR Pearson correlation r= 0.88, p<e−5
, N=16 genes; see Figure S1 in the online data supplement
and for selected genes), confirming the sampling and technical reliability of the array data. Nevertheless, as results are expected to contain false positives, we hypothesized that identifying relevant findings within this large pool of genes would benefit from a comparison with equivalent data obtained in an animal model that recapitulates behavioral and pharmacological aspects of depression.
Core Genes Significantly Affected in Human Major Depression and Mouse UCMS
Profiles of Altered Gene Transcripts Are Conserved Between Human Major Depression and the UCMS Mouse Model of Depression and Are Reversed by Antidepressant Treatments in Mice
We previously reported (9
) that UCMS induces a depressive-like syndrome in mice, consisting of progressive deterioration in coat state, reduced weight gain, and increased agonistic and emotion-related behaviors and that both symptom dimensions were reversed by chronic administration of an effective (fluoxetine) or putative (Crf1r antagonist) antidepressant. UCMS also induced region-specific patterns of altered gene expression in cingulate cortex and in the lateral/basolateral nuclei of the amygdala, which were reversed by both drug treatments. These behavioral and molecular results are summarized in , and the array results from that study were used here to support the analysis of the human data. Specifically, we hypothesized that if cellular mechanisms underlying mood regulation were conserved across species, then altered transcriptome in human major depression would predict similar changes in mice after UCMS, and that the effects of antidepressants in mice would help separate the effects of major depression from those of drug exposure in humans.
We investigated the degree of conservation of altered transcript levels for ortholog genes between major depression and UCMS by analysis of directional correlations. Confirming our hypothesis, highly significant, reciprocal, and consistent predictions of molecular changes were identified in amygdala ( and ). Specifically, of 191 genes with altered transcript levels in major depression, expression levels in mice were available for 105 ortholog probesets. Changes for these 105 mouse probesets were significantly correlated with human major depression-related changes (r=0.29, p<0.005). Conversely, of 299 genes with altered transcripts in amygdala of UCMS mice, the 213 identified human orthologs revealed a reciprocal mouse-to-human significant correlation of similar amplitude (r=0.29, p<0.00001). Analysis of 1,000 bootstrap resamplings demonstrated that the probability of obtaining the observed reciprocal concordance levels by chance was very low (p<0.001 for individual directional correlations, p<0.001 for concurrent positive findings in both directions). Markedly, the human-to-mouse correlations disappeared after successful antidepressant treatments in UCMS-exposed mice (). Thus, the pharmacological reversal of the major depression-UCMS correlation by two different antidepressants (i.e., targeting serotonergic or neuroendocrine stress pathways) demonstrated that the molecular changes supporting the major depression-UCMS correlations in amygdala were specific to the altered mood phenotype.
FIGURE 2 Reciprocal Prediction of Altered Amygdala Gene Expression Between Human Major Depression and the Mouse UCMS Model of Depression, and Reversal by Antidepressant Drug Treatmentsa
Toxicological screens identified the presence of antidepressants in five human subjects (four in the amygdala cohort), although these subjects were depressed at time of death, suggesting a lack of efficacy, suboptimal treatment, or treatment resistance. Similar correlations were observed between that patient subgroup and UCMS (r values ~0.35), thus supporting the clinical evidence of a lack of antidepressant efficacy in these subjects, at least for genes underlying the UCMS-to-major depression correlation.
|Enrichment||qPCR (p)||qPCR (alr)||Synapse Function|
Conversely, UCMS-induced changes in mouse cingulate cortex did not predict corresponding changes in human anterior cingulate cortex (r=0.10), while human major-depression-related changes were also unrelated to changes in mouse cingulate cortex (r=0.02) (). These low and nonsignificant anterior cingulate cortex-cingulate cortex correlations could result from differential involvement of that brain area in major depression and UCMS, or reflect a low conservation of cingulate structure and function across species. To partly address this question, we took advantage of the robust differences in transcriptomes between anterior cingulate cortex and amygdala in human subjects (~20% of genes; >2-fold change, p<0.01) and between mouse cingulate cortex and amygdala (~10% of genes; >2-fold change, p<0.01) to estimate the degree of similarity in “molecular structure” between areas across species. We found highly significant and reciprocal correlations between human anterior cingulate cortex/amygdala and mouse cingulate cortex/amygdala differences (mouse-to-human, r=0.63, p<0.0001; human-to-mouse, r=0.55, p<0.0001). These values did not differ between major depression subjects, UCMS-treated mice, or control samples (r values ~0.60, all comparisons, not shown). These results suggested that the lack of conserved depression-related findings in anterior cingulate cortex was not due to overall differences in “molecular structures” of the amygdala/anterior cingulate cortex network across species, thus also highlighting the amygdala specificity of the human-rodent correlation of the molecular impacts of major depression and UCMS.
Amygdala Cross-Species Correlations of Depression-Related Molecular Changes Identified a Subgroup of Human Major Depression Subjects
Absent or weak mouse-human correlations in cingulate cortices could also arise from variable or opposite effects in subgroups of human subjects, resulting in a null group effect. Indeed, despite our efforts to reduce the heterogeneity of the human cohort, major depressive disorder is by its clinical definition a heterogeneous disorder, and one may reasonably expect differences in molecular pathologies across subjects. Moreover, since the current analyses rely on large numbers of genes (178 in anterior cingulate cortex and 213 in amygdala), different gene sets may weigh differently across subjects; thus, correlation analyses in individual subjects may reveal features of cross-species predictions otherwise not available using combined group values. Here, using subjectwise changes in transcript levels for the identified genes (, step 3), we confirmed the lack of conserved major depression-UCMS effect in anterior cingulate cortex, as most individual human subjects displayed no cross-species correlation ( and , left panel). In amygdala, however, directional correlations revealed a large heterogeneity in cross-species predictions, with half of the subjects displaying positive correlations and the rest displaying either absent or negative correlations (, right panel). This difference from the anterior cingulate distribution was not explained by baseline changes, as the variability in gene expression of controls was comparable between anterior cingulate and amygdala (p>0.2). Rather, it was due to a selective increase in gene expression variability in the amygdala in subjects with major depression (amygdala: 50.3% higher gene transcript variance versus controls, p<0.01; anterior cingulate: 1% increase, p=0.97).
Notably, the subgroup of major depression subjects with positive UCMS correlation in amygdala (denoted here MDDUCMS
) did not differ in demographic parameters from controls or other subjects with major depression (all p values >0.05) and was not explained by differences in death by suicide, disease recurrence, antidepressant exposure, or alcohol dependence relative to other subjects with major depression (, bottom grid). The four major depression subjects with evidence of antidepressant exposure at time of death were all included in the MDDUCMS
subgroup, confirming the presence of a depression-related molecular profile in these subjects (i.e., positive correlation with UCMS), consistent with their clinical profile. Overall, MDDUCMS
subjects displayed a trend toward more depressive symptoms (7.4 versus 4.6 in the remaining major depression subjects, p=0.07). Interestingly, the two subjects with large negative correlations were among the only three major depression subjects who met requirements for remission or partial remission due to fewer depressive symptoms at time of death (, circles with red borders). Together, these findings suggest that the degree of correlation between UCMS and major depression molecular changes in amygdala may predict the severity of depression in human subjects. Indeed, a positive and significant correlation was observed between symptom numbers and UCMS-major depression correspondence (r=0.62, p=0.02; N=14 pairs), although this effect was partly driven by two remitted subjects (see Figure S2 in the online data supplement
). Finally, restricting the analysis to MDDUCMS
subjects (, step 4), we identified a larger number of genes with altered transcript levels in amygdala (N≈ 2,100; 1,139 orthologs), suggesting a greater homogeneity in molecular profiles within this subgroup. In the absence of demographic identifiers, we interpreted these findings as evidence for a subgroup of major depression subjects (MDDUCMS
) with a consistent amygdala pathology, potentially reflecting a more severe form of the illness, and for which the UCMS rodent model provided significant predictability at the gene expression level.
Two Distinct Oligodendrocyte and Neuronal Depression-Related Phenotypes in Amygdala
To characterize putative biological events underlying the cross-species correlations of changes and to address the presence of false positives in single data sets, we focused on genes with confirmed changes across species. Selected genes had to be significantly affected by UCMS and major depression and reversed by antidepressant treatments in the mouse model (, step 4), thus tracking the altered mood phenotype and controlling for drug effects. Of 299 gene transcripts affected by UCMS in amygdala, 61 were also significantly affected in major depression, mostly corresponding to changes in MDDUCMS subjects. Several of these transcript changes corresponded to the same genes and were combined, reducing the selection to 44 genes. Thirty-eight of the 44 genes displayed changes in the same direction in both species. Finally, antidepressant treatments reversed changes for 32 of these genes in rodents (), together identifying a core set of genes, characterized by concordant major depression and UCMS effects and effective reversal by antidepressant treatments.
qPCR analysis on RNA extracted from adjacent tissue sections for 17 of these genes revealed a very high correlation with array results in the MDDUCMS group (r=0.95, p<0.00001; ), even if individual statistical significances for some genes were only at the trend level (p=0.1). To determine whether this represented a quantitative limitation of the qPCR assay or a lack of biological effect, we assessed changes in protein levels for CNP, one of the three genes with trend-level significance by qPCR. Quantitative Western blot analysis revealed stable CNP protein levels over the postmortem interval covered in our study (PMI/ protein, r=0.01; not shown), a high concordance with RNA levels (r=0.76 for all 14 pairs, p=0.002), and a significant down-regulation in MDDUCMS subjects (−21.5%, p=0.01; ). In concert with qPCR, these findings provided supporting evidence for the technical reliability and biological validity of the identified molecular profile described in .
FIGURE 3 Concordant CNP RNA and Protein Down-Regulation in MDDUCMS Subjectsa
Within the group of genes with suggested glial enrichment of transcripts, genes were almost exclusively related to oligodendrocyte structure and function and were all down-regulated (, bottom rows). This striking convergence of gene function and direction of biological effects strongly suggests the presence of a conserved phenotype negatively affecting oligodendrocytes in amygdala under major depression and UCMS conditions. Conversely, genes with suggested neuronal enrichment of transcripts were mostly up-regulated and related to cellular maturation and synaptic development, neurotransmission and signaling, and cell-cell and cell-matrix interactions (, upper rows), suggesting a putative increase in neuronal structure and function in amygdala of major depression subjects.
Genes With Conserved Major Depression- and UCMS-Related Changes Participate in a Highly Cohesive and Interactive Gene Coexpression Network
We next investigated whether the identified genes represented various unrelated molecular findings or if they participate in shared cellular and biological functions (known as functional modules). It is possible to test these hypotheses by simultaneously inferring the interactions, or “links,” between our identified genes. These links are based on synchronized fluctuations in gene expression across samples (i.e., “coexpression” link), which have been shown to correspond to shared biological functions (30
). Indeed, gene networks built on coexpression links typically cluster in functional modules that correspond to specific cellular activities (31
), and this organization persists across species (33
). Hence, biological networks built on coexpression links are a useful means of determining whether genes share common functions, and here they represent a bias-free and data-driven way to investigate putative unifying major-depression-related cellular processes shared by our identified genes.
Accordingly, we used Pearson correlations to determine pairwise coexpression links between the 32 identified genes, which were then used to build gene networks (see the online data supplement
). To ensure that the coexpression links represented robust markers of biological gene interactions, we used clustering coefficient analysis and jackknife resampling methods to optimize our criteria for inclusion in the networks. Clustering coefficients estimate the density of local connections within functional modules and represent measures of network structure with wide applicability in brain networks (34
). Here, local modules were more connected than randomized networks (i.e., higher clustering coefficient; ), indicating that the identified genes participate in shared biological functions. Pearson correlation values resulting in networks with the largest differences in clustering coefficients compared to permutated networks provide the most biological information and were retained here as optimized cutoff points to build gene networks (dashed line in ) (35
). Additionally, we used a jackknife resampling approach to remove spurious links and maximize the biological reliability of the network. The obtained bimodal distributions clearly segregated links as robust (i.e., survive jackknife resampling; , right bars) or spurious (i.e., do not survive jackknife; left bars) in both species. Thus, gene networks were built using 100 links from the most robust groups in the jackknife histograms (), corresponding to clustering coefficients within the suggested range of optimized values (; >0.65 in human; >0.75 in mouse).
FIGURE 4 A Conserved and Tightly Clustered Gene Coexpression Network With Distinct Glial and Neuronal Components Underlies the Identified Molecular Signature of Depressiona
The 32 identified genes formed a tightly clustered network () with ~7 times more connections than random networks of similar size (p<0.01). Moreover, the overall clustering coefficients for each network were on average 77% higher than degree-matched randomly selected reference networks (p<0.001). Results were highly similar for all conditions and in both species, thus strongly supporting the biological validity and reliability of the identified network. Although the organic representation of the networks showed some differences (), the internal topology was well conserved, with a ~40% concordance of individual links across species, or ~57% using correlation of “betweenness centrality,” a more general measure of network similarity (36
). Within this network, genes with suggested glial or neuronal enrichment of cellular origin of transcripts naturally segregated (), which was quantitatively reflected by higher intraconnections (glial-glial and neuronal-neuronal) than interconnections (glial-neuronal) ().
In summary, these results demonstrate that genes forming the identified molecular signature of depression belong to an existing and tightly connected gene network that is conserved across species and that reflects the interactive glial/neuronal cellular compartments of gray matter tissue, together suggesting an abnormal recruitment by the illness of existing cellular pathways.