Etienne Sibille

Using the Gene Ontology for Microarray Data Mining: A Comparison of Methods and Application to Age Effects in Human Prefrontal Cortex

One of the challenges in the analysis of gene expression data is placing the results in the context of other data available about genes and their relationships to each other. Here, we approach this problem in the study of gene expression changes associated with age in two areas of the human prefrontal cortex, comparing two computational methods. The first method, “overrepresentation analysis” (ORA), is based on statistically evaluating the fraction of genes in a particular gene ontology class found among the set of genes showing age-related changes in expression. The second method, “functional class scoring” (FCS), examines the statistical distribution of individual gene scores among all genes in the gene ontology class and does not involve an initial gene selection step. We find that FCS yields more consistent results than ORA, and the results of ORA depended strongly on the gene selection threshold. Our findings highlight the utility of functional class scoring for the analysis of complex expression data sets and emphasize the advantage of considering all available genomic information rather than sets of genes that pass a predetermined “threshold of significance.”

Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide.

Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n=11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission.

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

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.

Corticolimbic transcriptome changes are state-dependent and region-specific in a rodent model of depression and of antidepressant reversal.

Gene microarrays may enable the elucidation of neurobiological changes underlying the pathophysiology and treatment of major depression. However, previous studies of antidepressant treatments were performed in healthy normal rather than ‘depressed’ animals. Since antidepressants are devoid of mood-changing effects in normal individuals, the clinically relevant rodent transcriptional changes could remain undetected. We investigated antidepressant-related transcriptome changes in a corticolimbic network of mood regulation in the context of the unpredictable chronic mild stress (UCMS), a naturalistic model of depression based on socio-environmental stressors. Mice subjected to a 7-week UCMS displayed a progressive coat state deterioration, reduced weight gain, and increased agonistic and emotion-related behaviors. Chronic administration of an effective (fluoxetine) or putative antidepressant (corticotropin-releasing factor-1 (CRF1) antagonist, SSR125543) reversed all physical and behavioral effects. Changes in gene expression differed among cingulate cortex (CC), amygdala (AMY) and dentate gyrus (DG) and were extensively reversed by both drugs in CC and AMY, and to a lesser extent in DG. Fluoxetine and SSR125543 also induced additional and very similar molecular profiles in UCMS-treated mice, but the effects of the same drug differed considerably between control and UCMS states. These studies established on a large-scale that the molecular impacts of antidepressants are region-specific and state-dependent, revealed common transcriptional changes downstream from different antidepressant treatments and supported CRF1 targeting as an effective therapeutic strategy. Correlations between UCMS, drug treatments, and gene expression suggest distinct AMY neuronal and oligodendrocyte molecular phenotypes as candidate systems for mood regulation and therapeutic interventions.

Brain-Derived Neurotrophic Factor Signaling and Subgenual Anterior Cingulate Cortex Dysfunction in Major Depressive Disorder

OBJECTIVE The subgenual anterior cingulate cortex is implicated in the pathology and treatment response of major depressive disorder. Low levels of brain-derived neurotrophic factor (BDNF) and reduced markers for GABA function, including in the amygdala, are reported in major depression, but their contribution to subgenual anterior cingulate cortex dysfunction is not known. METHOD Using polymerase chain reaction, we first assessed the degree to which BDNF controls mRNA expression (defined as BDNF dependency) of 15 genes relating to GABA and neuropeptide functions in the cingulate cortex of mice with reduced BDNF function (BDNF-heterozygous [Bdnf(+/-)] mice and BDNF exon-IV knockout [Bdnf(KIV)] mice). Gene expression was then quantified in the subgenual anterior cingulate cortex of 51 postmortem subjects with major depressive disorder and comparison subjects (total subjects, N=102; 49% were women) and compared with previous amygdala results. RESULTS Based on the results in Bdnf(+/-) and Bdnf(KIV) mice, genes were sorted into high, intermediate, and no BDNF dependency sets. In postmortem human subjects with major depression, BDNF receptor (TRKB) expression, but not BDNF, was reduced. Postmortem depressed subjects exhibited down-regulation in genes with high and intermediate BDNF dependency, including markers of dendritic targeting interneurons (SST, NPY, and CORT) and a GABA synthesizing enzyme (GAD2). Changes extended to BDNF-independent genes (PVALB and GAD1). Changes were greater in men (potentially because of low baseline expression in women), displayed notable differences from prior amygdala results, and were not explained by demographic or clinical factors other than sex. CONCLUSIONS These parallel human/mouse analyses provide direct (low TRKB) and indirect (low expression of BDNF-dependent genes) evidence in support of decreased BDNF signaling in the subgenual anterior cingulate cortex in individuals with major depressive disorder, implicate dendritic targeting GABA neurons and GABA synthesis, and, together, suggest a common BDNF-/GABA-related pathology in major depression with sex- and brain region-specific features.

Gene Expression Profiling of Depression and Suicide in Human Prefrontal Cortex

Mood disorders are a major cause of disability. Etiology includes genetic and environmental factors, but the responsible genes have yet to be identified. Using DNA microarrays, we have conducted a large-scale gene expression analysis, in two regions of the human prefrontal cortex from post-mortem matched groups of subjects with major depression who had died by suicide, and control subjects who died from other causes and were free from psychiatric disorders. Bioinformatic analysis was used to investigate molecular and cellular pathways potentially involved in depression and suicidal behavior. We tested several hypotheses of disease pathology and of their putative molecular impact, including changes in single genes, the existence of subgroups of patients or disease subtypes, or the possibility of common biological pathways being affected in the disease process. Within the analytical limits of this relatively large genomic study, we found no evidence for molecular differences that correlated with depression and suicide, suggesting a pathology that is below the detection level of current genomic approaches, or that is either localized to other brain areas, or more associated with post-transcriptional effects and/or changes in protein levels or functions, rather than altered transcriptome in the prefrontal cortex.

GABA-related transcripts in the dorsolateral prefrontal cortex in mood disorders

Reduced cortical γ-aminobutyric acid (GABA) levels and altered markers for subpopulations of GABA interneurons have been reported in major depressive disorder (MDD) by in-vivo brain imaging and post-mortem histological studies. Subgroups of GABA interneurons exert differential inhibitory control on principal pyramidal neurons and can be identified based on the non-overlapping expression of the calcium-binding proteins parvalbumin (PV) or calretinin (CR) or the neuropeptide somatostatin (SST). As altered markers of GABAergic functions may also be present in bipolar disorder (BPD), the specificity of particular GABA-related molecular deficits in mood disorders is not known. We used real-time quantitative polymerase chain reaction (qPCR) to assess expression levels of two GABA synthesizing enzymes (glutamate decarboxylase; GAD65 and GAD67) and of three markers of GABA neuron subpopulations (PV, CR, SST) in the dorsolateral prefrontal cortex (DLPFC; Brodmann area 9) in triads (n=19) of control subjects and matched subjects with BPD or MDD. BPD subjects demonstrated significantly reduced PV mRNA, trend level reduction in SST mRNA and no alterations in GAD67, GAD65, or CR mRNA levels; MDD subjects demonstrated reduced SST mRNA expression without alterations in the other transcripts. The characteristic age-related decline in SST expression was not observed in MDD, as low expression was detected across age in MDD subjects. After controlling for age, MDD subjects demonstrated significantly reduced SST mRNA expression. Decreased SST levels in MDD were confirmed at the protein precursor level. Results were not explained by other clinical, demographic or technical parameters. In summary, MDD was characterized by low DLPFC SST, whereas decreased PV mRNA appears to distinguish BPD from MDD.

An R package suite for microarray meta-analysis in quality control, differentially expressed gene analysis and pathway enrichment detection

SUMMARY With the rapid advances and prevalence of high-throughput genomic technologies, integrating information of multiple relevant genomic studies has brought new challenges. Microarray meta-analysis has become a frequently used tool in biomedical research. Little effort, however, has been made to develop a systematic pipeline and user-friendly software. In this article, we present MetaOmics, a suite of three R packages MetaQC, MetaDE and MetaPath, for quality control, differentially expressed gene identification and enriched pathway detection for microarray meta-analysis. MetaQC provides a quantitative and objective tool to assist study inclusion/exclusion criteria for meta-analysis. MetaDE and MetaPath were developed for candidate marker and pathway detection, which provide choices of marker detection, meta-analysis and pathway analysis methods. The system allows flexible input of experimental data, clinical outcome (case-control, multi-class, continuous or survival) and pathway databases. It allows missing values in experimental data and utilizes multi-core parallel computing for fast implementation. It generates informative summary output and visualization plots, operates on different operation systems and can be expanded to include new algorithms or combine different types of genomic data. This software suite provides a comprehensive tool to conveniently implement and compare various genomic meta-analysis pipelines. AVAILABILITY http://www.biostat.pitt.edu/bioinfo/software.htm CONTACT ctseng@pitt.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Beyond genotype: serotonin transporter epigenetic modification predicts human brain function

We examined epigenetic regulation in regards to behaviorally and clinically relevant human brain function. Specifically, we found that increased promoter methylation of the serotonin transporter gene predicted increased threat-related amygdala reactivity and decreased mRNA expression in postmortem amygdala tissue. These patterns were independent of functional genetic variation in the same region. Furthermore, the association with amygdala reactivity was replicated in a second cohort and was robust to both sampling methods and age.

Molecular heterogeneity along the dorsal–ventral axis of the murine hippocampal CA1 field: a microarray analysis of gene expression

There has been increasing interest in functional heterogeneity along the septotemporal, dorsal-ventral (D-V) axis of the hippocampus. Although anatomical connectivity and lesion studies point to discrete roles for these sub-regions, the contribution of differential gene expression across this axis has not been systematically studied. Here we present findings from an Affymetrix microarray screen aimed at identifying genes in the CA1 region of the adult murine hippocampus that show significant differential expression along the D-V axis. Our results indicate that the vast majority of monitored genes (>90%) had tissue expression levels that differed by less than 20% between regions, while less than 0.1% of genes had expression levels that varied more than three-fold by sub-region. Only 23 probes showed a CA1 dorsoventral signal intensity ratio greater than three: 18 enriched dorsally and five enriched ventrally. Probes with the greatest difference in expression levels represent a range of genes with known functions in patterning and signaling, as well as genes without known function. Selective screening with digoxigenin-labeled in situ hybridization confirms the existence of CA1 sub-regionalized expression, with some genes exhibiting a graded expression pattern across the D-V axis, and others restricted to a discrete region. Our findings demonstrate that there are gene expression differences across the D-V axis of the adult murine hippocampus within traditionally recognized cytoarchitecturally defined boundaries. Combined with the previously recognized differences in connectivity and results from lesion studies, our data further confirm the existence of functional heterogeneity along the D-V axis.