Altered Functional Protein Networks in the Prefrontal Cortex and Amygdala of Victims of Suicide

<div><p>Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on <em>post mortem</em> brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of <em>post mortem</em> tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard <em>t</em> test and 1.3 fold change as cut off for significance. Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the <em>q</em>-values for all the <em>t</em> tests performed and by using 0.06 and 0.4 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively. From these we identified 59 proteins in the cortex and 11 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies. The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis.</p> </div

Description of participants in the present study.

<p>NA: not available; S: suicide; C: control.</p

Representative gel image.

<p>The first dimension was carried out in pH 3–10 NL IPG strip and the second dimension was 24×20 cm 10% SDS PAGE. Part A shows the overlaid image, part B shows the standardized log abundance of a representative spot (2406, prefrontal cortex) on the different gels, part C shows 3D views of the individual spots (C1–C6: control brains; S1–S6: suicide brains).</p

Functionally clustered protein changes in the prefrontal cortex.

*<p>: proteins involved in schizophrenia; +: proteins involved in depression; S: proteins involved in suicide.</p>*<p>: proteins involved in schizophrenia; +: proteins involved in depression; S: proteins involved in suicide. Bold-italic gene names highlighting those proteins that were found in those differently expressed protein spots that proved significant with both statistical tests.</p><p>↑ or ↓: the direction of the spot intensity change of a given spot compared to control.</p

Functionally clustered changes in proteins of the amygdala.

*<p>proteins involved in schizophrenia; +: proteins involved in depression; S: proteins involved in suicide.</p>*<p>proteins involved in schizophrenia; +: proteins involved in depression; S: proteins involved in suicide. Bold-italic gene names highlighting those proteins that were found in those differently expressed protein spots that proved significant with both statistical tests. ↑ or ↓: the direction of the spot intensity change of a given spot compared to control.</p

Western blot validation of GFAP, cathepsin and actin expressions in the cortex and amygdala of suicide and control subjects.

<p>The expressions of GFAP and cathepsin were significantly decreased in the suicide prefrontal cortex compared to the control samples while in the amygdala their expressions were significantly increased. In case of the actin similar but non-significant changes were found. The loading control was Ponceau, mean ± SEM.</p

The protein network of altered cytoskeleton proteins in the brains of suicide victims (green) is connected to the receptor-interaction network of glutamate and serotonin (red) via NEFL and GFAP.

<p>Abbreviations: GRIA1– Glutamate receptor, ionotropic, AMPA1, GRIA3 - glutamate receptor, ionotrophic, AMPA 3, GRIK1– Glutamate receptor, ionotropic, kainate 1, GRIN1– Glutamate receptor, ionotropic, N-methyl-D-aspartate, HTR1A –5-Hydroxytryptamin (serotonin) receptor 1A, HTR2A (5-hydroxytryptamine (serotonin) receptor 2A, HTR1B (5-hydroxytryptamine (serotonin) receptor 1B, CKB - Creatine kinase B-type, ACTB - Actin, cytoplasmic 1, TUBA1A – Tubulin alpha-1B chain, NEFL – Neurofilament, light polypeptide 68 kDa, NEFM – Neurofilament, medium polypeptide, INA – Alpha-internexin, GFAP – Glial fibrillary acidic protein, CTSD - Cathepsin D, HSPA8 - Heat shock 70 kDa protein 8.</p

Altered proteins in the prefrontal cortex and amygdala.

<p>Proteins labelled by * were changed in both the cortex and the amygdala, but the directions of the changes were in reverse directions. Bold-italic gene name as in previous tables. ↑ or ↓: the direction of the spot intensity change of a given spot compared to control, for details see the Suppl. Materials <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050532#pone-0050532-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050532#pone-0050532-t004" target="_blank">4</a>, Suppl.</p

Image_7_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p

Image_2_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p