Differential expression of gene co-expression networks related to the mTOR signaling pathway in bipolar disorder

Abstract

Bipolar disorder (BPD) is a severe mental illness characterized by episodes of depression and mania. To investigate the molecular mechanisms underlying the pathophysiology of bipolar disorder, we performed transcriptome studies using RNA-seq data from the prefrontal cortex (PFC) of individuals with BPD and matched controls, as well as data from cell culture and animal model studies. We found 879 differentially expressed genes that were also replicated in an independent cohort of post-mortem samples. Genes involving the mechanistic target of rapamycine (mTOR) pathway were down-regulated, while genes interrelated with the mTOR pathway such as Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway were up-regulated. Gene co-expression network analyses identified a module related to the mTOR pathway that was up-regulated in BPD and also enriched for markers of endothelial cells. We also found a down-regulated co-expression module enriched for genes involved in mTOR signalling and in mTOR related pathways and enriched with neuronal markers. The mTOR related modules were also replicated in the independent cohort of samples. To investigate whether the expression of the modules related to mTOR signalling pathway could be differentially regulated in different cell types we performed comparative network analyses in experimental models. We found both up-regulated modules in the PFC significantly overlapped with an up-regulated module in the brain endothelial cells from mice treated with lipopolysaccharides (LPS) and mTOR related pathways such as JAK-STAT, PI3K-Akt and ribosome were enriched in the common genes. In addition, the down-regulated module in the PFC significantly overlapped with a down-regulated module from neurons treated with the mTOR inhibitor, Torin1 and mTOR signalling, autophagy, and synaptic vesicle cycles were significantly enriched in the common genes. These results suggest that co-expression networks related to mTOR signalling pathways may be up- or down-regulated in different cell types in the PFC of BPD. These results provide novel insights into the molecular mechanisms underlying the pathophysiology of BPD.

Fig. 1. Co-expression modules differentially expressed between BPD and controls in PFC.

The eigengene adjacency heatmap of the modules (A), the eigengene values across samples and major KEGG pathways significantly enriched in the genes in the PFC_M4 module (B), the PFC_M5 module (C) and the PFC_M23 module (D).

Fig. 2. Transcription factors and their target genes in the PFC_M4 and the PFC_M5 module.

Visualization of STAT3-target gene network in the PFC_M4 module (A), Biological processes significantly enriched in the network (B). Visualization of p53 -target gene network in the PFC_M5 module (C) and biological processes significantly enriched in the network (D).

Fig. 3. Effects of Torin1 and LY2584702 in primary neuronal cells.

Neuronal cells were treated with Torin1 (A) and LY2584702 (B) for 24 h at 250 nM concentration. Cell lysates were analysed by SDS-PAGE and Western blot analyses with each of the primary antibodies. Western blot analyses revealed the levels of phospho-Ser473-Akt, phospho-Ser2448-mTORC1, phospho-Thr389-S6K, phospho-Ser240/244-S6, PSD-95, GluA1, LC3B-II, and Beclin 1.Representative images and quantitative analyses normalized to the total levels for each protein or α-tubulin are shown. The original images for crude blot are shown in Supplementary Fig. 4 (Torin1) and in Supplementary Fig. 6 (LY2584702). Values (n = 4–6) are shown as the mean ± SEM expressed as a percentage of the control cells (no drug) values. *p < 0.05, **p < 0.01, unpaired Student t test.

Fig. 4. Comparison of the differentially expressed modules in the PFC of BPD to those from Torin1 treated neurons.

Pairwise comparisons of the modules differentially expressed in BPD and the differentially expressed modules in Torin1-treated neurons (A). The color code of the heatmap encodes −log (P-value). The P-values were calculated by permutation test for the overlap of the two modules. The numbers in the heatmap indicate gene counts in the intersection of two modules. Venn diagram shows the number of common and unique genes between the PFC_M23 and the Torin1_M6 module (B). KEGG pathways significantly enriched in the genes common to both co-expression modules (C) and visualization of the network connections of the common genes between the two modules using Cytoscape [25] (D). The hub genes are larger circles in the network. Genes related to mTOR signaling pathway are in red and genes related to synaptic vesicle cycle are in blue.

Fig. 5. Comparison of differentially expressed modules in the PFC of BPD to that from cerebral endothelial cells of mice treated with LPS.

A The eigengene values of a differentially expressed module, Endo_LPS_M7 in the endothelial cells of mice treated with LPS for 2 h. B, Pairwise comparisons of the modules differentially expressed in BPD and the differentially expressed module in the endothelial cells. The color code of the heatmap encodes −log (P value). The P-values were calculated by Fisher exact test for the overlapping of the two modules. The numbers in the heatmap indicate gene counts in the intersection of two modules. Major KEGG pathways significantly enriched in the genes common between PFC_M4 and Endo_LPS_M7 (C) and between PFC_M5 and Endo_LPS_M7 (D).