Dysregulated mitochondrial genes and networks with drug targets in postmortem brain of patients with posttraumatic stress disorder (PTSD) revealed by human mitochondria-focused cDNA microarrays

Abstract

Posttraumatic stress disorder (PTSD) is associated with decreased activity in the dorsolateral prefrontal cortex (DLPFC), the brain region that regulates working memory and preparation and selection of fear responses. We investigated gene expression profiles in DLPFC Brodmann area (BA) 46 of postmortem patients with (n=6) and without PTSD (n=6) using human mitochondria-focused cDNA microarrays. Our study revealed PTSD-specific expression fingerprints of 800 informative mitochondria-focused genes across all of these 12 BA46 samples, and 119 (+/->1.25, p<0.05) and 42 (+/->1.60, p<0.05) dysregulated genes between the PTSD and control samples. Quantitative RT-PCR validated the microarray results. These fingerprints can essentially distinguish the PTSD DLPFC BA46 brains from controls. Of the 119 dysregulated genes (+/-> or =125%, p<0.05), the highest percentages were associated with mitochondrial dysfunction (4.8%, p=6.61 x 10(-6)), oxidative phosphorylation (3.8%, p=9.04 x 10(-4)), cell survival-apoptosis (25.2%, p<0.05) and neurological diseases (23.5%, p<0.05). Fifty (50) dysregulated genes were present in the molecular networks that are known to be involved in neuronal function-survival and contain 7 targets for neuropsychiatric drugs. Thirty (30) of the dysregulated genes are associated with a number of neuropsychiatric disorders. Our results indicate mitochondrial dysfunction in the PTSD DLPFC BA46 and provide the expression fingerprints that may ultimately serve as biomarkers for PTSD diagnosis and the drugs and molecular targets that may prove useful for development of remedies for prevention and treatment of PTSD.

Keywords: Biomarker; Brain dorsolateral prefrontal cortex BA46; Canonical pathways; Gene expression pattern; Mitochondria; Molecular networks; Neuropsychiatric drug targets; PTSD.

Fig 1

Box plots of expression data before and after normalization. The quantile normalization algorithms were used to adjust the values of the background-subtracted mean pixel intensities of each and every set of 800 genes that were selected from the hMitChip3 triplicate microarray experiments hybridized with PTSD and control prefrontal cortex RNA samples as described previously. In contrast to the pre-normalization boxplots (top panel), the post-normalized boxplots distribute in the same intervals with the same density center, indicating successful adjustment of data. The post-normalized data were used for clustering analysis. S-E: Sample and independent microarray experiment.

Fig 2

Dendrograms and heat maps of gene expression in human brain DLPFC BA46 tissue samples. (A) Dendrogram of unsupervised cluster of the PTSD and control DLPFC BA46 tissue samples based on expression similarities of 800 human mitochondria-focused genes (Supplemental Table 1); (B) Dendrograms and heat maps of 119 (±≥1.25, p<0.05) and (C) 42 (±≥1.60, p<0.05) dysregulated genes across all of these 12 BA46 samples. The results revealed the PTSD (P) and non-PTSD (N) groups. The control case N2 is clustered in the PTSD group, while the PTSD case P4 in the control group. Green: down-regulation; red: up-regulation; black: no change. The case numbers and gene symbols are indicated. The color map indicates degrees of up- and down-regulation of gene expression.

Fig 3

The consistency in RNA levels measured by microarray and qRT-PCR. Relative RNA levels of 5 genes were measured and analyzed by both microarray and qRT-PCR experiments. The arrows indicate obvious inconsistent results between these two experiments. Out of these 5 genes in 12 tissues compared, 8 of 12 (66.7%) are consistent for DNAJC19, SLC1A6 and ESR2, 11 of 12 (92%) for APP, and 9 of 12 (75%) for SLC9A6. PM: mean and SD of PTSD cases; NM: mean and SD of non-PTSD controls.

Fig 4

The bar graphs indicate canonical pathways and neuropsychiatric and systemic disorders associated with dysregulated genes in the PTSD DLPFC BA46. (A) 10 canonical biochemical and molecular biology pathways with significantly (p<0.05, Fisher's Exact Test) dysregulated genes, in the PTSD DLPFC BA46 group (P1, P2, P3, P5, P6 and N2) in comparison to the non-PTSD controls (N1, N3, N4, N5, N6 and P4). The ratio is calculated as the number of genes in a given pathway divided by a total number of genes that make up that pathway. The mitochondrial dysfunction pathway contains 8 (4.8%) dysregulated genes (p=6.61x10^-6) including upregulated APP, CAT, NDUFA10 and UCP2 and downregulated COX8A, NDUFB5, NDUFS2 and PDHA1. (B) Six categories of abnormalities in which the significant (p<0.05, Fisher's Exact Test) number of the dysregulated genes in the PTSD DLPFC BA46 are known to involve, by searching the literature in the NCBI PUBMED, DAVID, OMIM and Ingenuity databases. Red: upregulation; green downregulation.

Fig 5

The molecular networks for neuron function and survival. The networks have a total of 75 elements including 27 (36%) upregulated genes, 27 (36%) downregulated genes and 21 (28%) unchanged ones. Twenty-one elements in white color include 7 genes present and 11 genes absent in the hMitChip3 microarray, and 3 small molecules. The symbols in Legend illustrate the networks. The genes in the blue boxes are the known targets for neuropsychiatric drugs. For example: the ESR2 is a target of 17β-estradiol or related ligands. The APP is a target for Bapineuzumab (AAB-001) that is currently used for the Phase 3 clinical trials for the treatment of patients with Alzheimer's disease. The ESR2 is a target for Tamoxifen that is used for the treatment of mania in patients with bipolar disorder. The PRKCD is a target for both Tamoxifen, and rottlerin that has been used in preclinical treatment of Parkinson's disease. The PP2A is a target for Fostriecin that antagonize dopamine activities. The VAMP1 is a target of the neurotoxin protein Dysport that is used in minute doses to treat painful muscle spasms. The SLC1A6 is a target of Riluzole used to treat amyotrophic sclerosis. The UCP2 is a target for protection of dopaminergic neurons from oxidative stress caused by 1,2,3,6-methyl-phenyl-tetrahydropyridine (MPTP) toxicity.