FoxO1, A2M, and TGF-beta 1 : three novel genes predicting depression in gene X environment interactions are identified using cross-species and cross-tissues transcriptomic and miRNomic analyses

To date, gene-environment (GxE) interaction studies in depression have been limited to hypothesis-based candidate genes, since genome-wide (GWAS)-based GxE interaction studies would require enormous datasets with genetics, environmental, and clinical variables. We used a novel, cross-species and cross-tissues "omics" approach to identify genes predicting depression in response to stress in GxE interactions. We integrated the transcriptome and miRNome profiles from the hippocampus of adult rats exposed to prenatal stress (PNS) with transcriptome data obtained from blood mRNA of adult humans exposed to early life trauma, using a stringent statistical analyses pathway. Network analysis of the integrated gene lists identified the Forkhead box protein O1 (FoxO1), Alpha-2-Macroglobulin (A2M), and Transforming Growth Factor Beta 1 (TGF-beta 1) as candidates to be tested for GxE interactions, in two GWAS samples of adults either with a range of childhood traumatic experiences (Grady Study Project, Atlanta, USA) or with separation from parents in childhood only (Helsinki Birth Cohort Study, Finland). After correction for multiple testing, a meta-analysis across both samples confirmed six FoxO1 SNPs showing significant GxE interactions with early life emotional stress in predicting depressive symptoms. Moreover, in vitro experiments in a human hippocampal progenitor cell line confirmed a functional role of FoxO1 in stress responsivity. In secondary analyses, A2M and TGF-beta 1 showed significant GxE interactions with emotional, physical, and sexual abuse in the Grady Study. We therefore provide a successful 'hypothesis-free' approach for the identification and prioritization of candidate genes for GxE interaction studies that can be investigated in GWAS datasets.Peer reviewe

The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation.

FoxO (forkhead box O; forkhead members of the O class) are transcription factors that function under the control of insulin/insulin-like signalling. FoxO factors have been associated with a multitude of biological processes, including cell-cycle, cell death, DNA repair, metabolism and protection from oxidative stress. Central to the regulation of FoxO factors is a shuttling system, which confines FoxO factors to either the nucleus or the cytosol. Shuttling of FoxO requires protein phosphorylation within several domains, and association with 14-3-3 proteins and the nuclear transport machinery. Description of the FoxO-shuttling mechanism contributes to the understanding of FoxO function in relation to signalling and gene regulation

Pitx3-Cre driven eYfp expression starts between E12.5 and E13.5 and marks the ventral tegmentum, overlapping with Th.

<p>A) Th and eYfp co-localization in sagittal sections of the ventral mesodiencephalic region. Note that at this stage eYfp is not detected. B) Th and eYfp seperate as well as co-localization in sagittal sections of the ventral mesodiencephalic region. At this stage co-localization can be found but also some areas where Th and eYfp are uncoupled. C) Similar analysis as in B, on E14.5 material, confirming the Yfp and Th distribution. The white arrows indicates a medial/rostral region where Yfp is expressed in cells that do not express Th. L: lateral; M: medial.</p

Pitx3-Cre driven expression of eYfp co-localizes with all Th positive neurons.

<p>A) Th and eYfp expression in the control LoxP-stop-LoxP-eYfp animal. Note that only Th is expressed in these animals. B) Co-localization of Th and eYfp in the mdDA neuronal region. Note the complete overlap of Th positive neurons with eYfp. C) Enlarged figures of boxes as marked in B. White arrows: indication of the regions and cells where there is eYfp expression with no Th co-localization.</p

Pitx3-Cre driven eYfp expression outside the brain maps to the muscle.

<p>A) Representation of the eYfp muscle expression in the developing embryo at 2 stages of development. B) Enlarged figures of boxes marked in A. C) Schematic representation of the recombinant knock-in construct indicating the position and the 3′-restricted flank of the alternative exon1 position. Figure details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042641#pone-0042641-g001" target="_blank">figure 1</a>.</p

Pitx3-Cre driven expression of eYfp marks transient expression of Pitx3 in a medial non-dopaminergic cell-group.

<p>A) In depth analysis through Th and eYfp co-localization of non-dopaminergic regions that express eYfp. B) Enlarged figures of boxes marked in A. C) Co-localistion of Yfp with 5-Ht staining at 4 separate A/P positions from left to right (top 4 panels). Higher magnification of the boxed areas are presented in the lower 2 panels, clearly showing that the Yfp positive neurons do not co-localise with 5-HT neurons. IF: interfascicular nucleus; IPF: interpeduncular fossa; RLi: rostral linear nucleus of the raphe; CLi:caudal linear nucleus of the raphe.</p

Functional connectivity and network analysis during hypoactive delirium and recovery from anesthesia

OBJECTIVE: To gain insight in the underlying mechanism of reduced levels of consciousness due to hypoactive delirium versus recovery from anesthesia, we studied functional connectivity and network topology using electroencephalography (EEG). METHODS: EEG recordings were performed in age and sex-matched patients with hypoactive delirium (n=18), patients recovering from anesthesia (n=20), and non-delirious control patients (n=20), all after cardiac surgery. Functional and directed connectivity were studied with phase lag index and directed phase transfer entropy. Network topology was characterized using the minimum spanning tree (MST). A random forest classifier was calculated based on all measures to obtain discriminative ability between the three groups. RESULTS: Non-delirious control subjects showed a back-to-front information flow, which was lost during hypoactive delirium (p=0.01) and recovery from anesthesia (p<0.01). The recovery from anesthesia group had more integrated network in the delta band compared to non-delirious controls. In contrast, hypoactive delirium showed a less integrated network in the alpha band. High accuracy for discrimination between hypoactive delirious patients and controls (86%) and recovery from anesthesia and controls (95%) were found. Accuracy for discrimination between hypoactive delirium and recovery from anesthesia was 73%. CONCLUSION: Loss of functional and directed connectivity were observed in both hypoactive delirium and recovery from anesthesia, which might be related to the reduced level of consciousness in both states. These states could be distinguished in topology, which was a less integrated network during hypoactive delirium. SIGNIFICANCE: Functional and directed connectivity are similarly disturbed during a reduced level of consciousness due to hypoactive delirium and sedatives, however topology was differently affected

FoxO6 affects Plxna4-mediated neuronal migration during mouse cortical development

The forkhead transcription factor FoxO6 is prominently expressed during development of the murine neocortex. However, its function in cortical development is as yet unknown. We now demonstrate that cortical development is altered in FoxO6+/- and FoxO6-/- mice, showing migrating neurons halted in the intermediate zone. Using a FoxO6-directed siRNA approach, we substantiate the requirement of FoxO6 for a correct radial migration in the developing neocortex. Subsequent genome-wide transcriptome analysis reveals altered expression of genes involved in cell adhesion, axon guidance, and gliogenesis upon silencing of FoxO6. We then show that FoxO6 binds to DAF-16-binding elements in the Plexin A4 (Plxna4) promoter region and affects Plxna4 expression. Finally, ectopic Plxna4 expression restores radial migration in FoxO6+/- and siRNA-mediated knockdown models. In conclusion, the presented data provide insights into the molecular mechanisms whereby transcriptional programs drive cortical development

FoxO6 transcriptional activity is regulated by Thr(26) and Ser(184), independent of nucleo-cytoplasmic shuttling

Forkhead members of the ‘O’ class (FoxO) are transcription factors crucial for the regulation of metabolism, cell cycle, cell death and cell survival. FoxO factors are regulated by insulin-mediated activation of PI3K (phosphoinositide 3-kinase)–PKB (protein kinase B) signalling. Activation of PI3K–PKB signalling results in the phosphorylation of FoxO factors on three conserved phosphorylation motifs, which are essential for the translocation of FoxO factors from the nucleus to the cytosol. FoxO6, however, remains mostly nuclear due to the fact that its shuttling ability is dramatically impaired. FoxO1, FoxO3 and FoxO4 all contain an N- and C-terminal PKB motif and a motif located in the forkhead domain. FoxO6 lacks the conserved C-terminal PKB motif, which is the cause of the shuttling impairment. Since FoxO6 can be considered constitutively nuclear, we investigated whether it is also a constitutively active transcription factor. Our results show that FoxO6 transcriptional activity is inhibited by growth factors, independent of shuttling, indicating that it is not constitutively active. The PKB site in the forkhead domain (Ser(184)) regulated the DNA binding characteristics and the N-terminal PKB site acted as a growth factor sensor. In summary, FoxO6 is not a constitutively active transcription factor and can be regulated by growth factors in a Thr(26)- and Ser(184)-dependent manner, independent of shuttling to the cytosol