A Gene Co-Expression Network in Whole Blood of Schizophrenia Patients Is Independent of Antipsychotic-Use and Enriched for Brain-Expressed Genes

Despite large-scale genome-wide association studies (GWAS), the underlying genes for schizophrenia are largely unknown. Additional approaches are therefore required to identify the genetic background of this disorder. Here we report findings from a large gene expression study in peripheral blood of schizophrenia patients and controls. We applied a systems biology approach to genome-wide expression data from whole blood of 92 medicated and 29 antipsychotic-free schizophrenia patients and 118 healthy controls. We show that gene expression profiling in whole blood can identify twelve large gene co-expression modules associated with schizophrenia. Several of these disease related modules are likely to reflect expression changes due to antipsychotic medication. However, two of the disease modules could be replicated in an independent second data set involving antipsychotic-free patients and controls. One of these robustly defined disease modules is significantly enriched with brain-expressed genes and with genetic variants that were implicated in a GWAS study, which could imply a causal role in schizophrenia etiology. The most highly connected intramodular hub gene in this module (ABCF1), is located in, and regulated by the major histocompatibility (MHC) complex, which is intriguing in light of the fact that common allelic variants from the MHC region have been implicated in schizophrenia. This suggests that the MHC increases schizophrenia susceptibility via altered gene expression of regulatory genes in this network

WNT signaling enhances breast cancer cell motility and blockade of the WNT pathway by sFRP1 suppresses MDA-MB-231 xenograft growth

ABSTRACT: INTRODUCTION: In breast cancer deregulation of the WNT signaling pathway occurs by autocrine mechanisms. WNT ligands and Frizzled (FZD) receptors are coexpressed in primary breast tumors and cancer cell lines. Moreover, many breast tumors show hypermethylation of secreted Frizzled-related protein 1 (sFRP1)'s promoter region, causing low expression of this WNT antagonist. We have previously shown that the WNT pathway influences proliferation of breast cancer cell lines via activation of canonical signaling and epidermal growth factor receptor (EGFR) transactivation, and that interference with WNT signaling reduces proliferation. Here we examine the role of WNT signaling in breast tumor cell migration and on xenograft outgrowth. METHODS: The breast cancer cell line MDA-MB-231 was used to study WNT signaling. We examined the effects of activating or blocking the WNT pathway on cell motility by treatment with WNT ligands or by ectopic sFPR1 expression, respectively. The ability of sFRP1 expressing MDA-MB-231 cells to grow as xenografts was also tested. Microarray analyses were carried out to identify targets with roles in MDA-MB-231/sFRP1 tumor growth inhibition. RESULTS: We show that WNT stimulates the migratory ability of MDA-MB-231 cells. Furthermore, ectopic expression of sFRP1 in MDA-MB-231 cells blocks canonical WNT signaling and decreases their migratory potential. Moreover, the ability of MDA-MB-231/sFRP1 expressing cells to grow as xenografts in mammary glands and to form lung metastases is dramatically impaired. Microarray analyses led to the identification of two genes, CCND1 and CDKN1A, whose expression level is selectively altered in vivo in sFRP1 expressing tumors. The encoded proteins, Cyclin D1 and p21Cip1 were down- and up-regulated, respectively, in sFRP1 expressing tumors, suggesting that they are downstream mediators of WNT signaling. CONCLUSIONS: Our results show that the WNT pathway influences multiple biological properties of MDA-MB-231 breast cancer cells. WNT stimulates tumor cell motility; conversely sFRP1 mediated WNT pathway blockade reduces motility. Moreover, ectopic sFRP1 expression in MDA-MB-231 cells has a strong negative impact on tumor outgrowth and blocked lung metastases. These results suggest that interference with WNT signaling using sFRP1 to block the ligand-receptor interaction may be a valid therapeutic approach in breast cancer

Runx1 Loss Minimally Impacts Long-Term Hematopoietic Stem Cells

RUNX1 encodes a DNA binding subunit of the core-binding transcription factors and is frequently mutated in acute leukemia, therapy-related leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia. Mutations in RUNX1 are thought to confer upon hematopoietic stem cells (HSCs) a pre-leukemic state, but the fundamental properties of Runx1 deficient pre-leukemic HSCs are not well defined. Here we show that Runx1 deficiency decreases both apoptosis and proliferation, but only minimally impacts the frequency of long term repopulating HSCs (LT-HSCs). It has been variously reported that Runx1 loss increases LT-HSC numbers, decreases LT-HSC numbers, or causes age-related HSC exhaustion. We attempt to resolve these discrepancies by showing that Runx1 deficiency alters the expression of several key HSC markers, and that the number of functional LT-HSCs varies depending on the criteria used to score them. Finally, we identify genes and pathways, including the cell cycle and p53 pathways that are dysregulated in Runx1 deficient HSCs

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Novel Cell and Tissue Acquisition System (CTAS): Microdissection of Live and Frozen Brain Tissues

<div><p>We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS - Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.</p> </div

Sequential microdissection of granular cells from the 9^th lobe of cerebellum using fresh frozen and unstained moue brain sections (thickness = 20 µm). A.

<p>Red dotted line outlines the area of interest. <b>B.</b> Dissection of granular cells from the middle part of the granular layer. <b>C.</b> Additional dissection from the same area. Abbreviations: Sf – the secondary fissure of the cerebellum; 9Cb –9^th area of cerebellum, Ecu – external cuneate nucleus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041564#pone.0041564-Paxinos1" target="_blank">[50]</a>.</p

Representative collection of individual CA1 interneurons in stratum orience (A

<p>–<b>D) and Purkinje cells (E</b>–<b>F) from sucrose treated mouse coronal brain sections.</b> Images before (<b>A</b>, <b>C</b>, <b>E</b>) and after (<b>B</b>, <b>D</b>, <b>F</b>) collection are shown. Red arrows and dashed circles show collected cells. Tissue thickness  = 20 µm. DCU ID = 20 µm. Magnification: 400X.</p

Capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) v.4.1.

<p><b>A.</b> Representative image of CTAS v.4.1. The system is attached to an inverted microscope (TCM400, Labomed) and consists of the following key components: sample collection assembly with collection unit, LED lights, side chassis vacuum module and DCU controls. The latter incorporates electronic controls and a vacuum pump. The two dials at the front of the side chassis control the vacuum strength from 2″Hg (1) to maximum of 22″Hg (10) and the vacuum duration from 100 ms (1) to maximum of 1 second (10). Depending on the tissue type and section thickness, various vacuum strength and duration may be used. Green button turns the power on/off. Three DCU control buttons include two white buttons, which bring the DCU up or down during the calibration procedure and an orange button that sets the Home position of the DCU and brings it to its Standby position. Black “Sample” button initiates sample collection by bringing the DCU down to the Home position and activating the vacuum at the selected strength and duration; <b>B.</b> CTAS sample collection assembly in its lifted position for DCU attachment/removal. DCU attached to a collection unit with connectors for multiple cables and a vacuum tube. Calibration LED source for illuminating the tip of the capillary is shown. In this position, the green horizontal and red vertical calibration LED lights are automatically turned off. The lights are automatically turned on when the CTAS head is in its upright position. The x–y position of the DCU is controlled by the knobs on the linear stages (x–y controls).</p

Dissection of subanatomical regions (marked with red dotted line or arrows) from fresh frozen mouse coronal brain sections (20 µm thickness) using CTAS v.4.1. A.

<p>Collection of the left hilus of the dentate gyrus (hDG). <b>B.</b> Dissection of granular cells (G), molecular layer (M), and white matter (W) from mouse cerebellum. Intact (<b>C</b>) and dissected (<b>D</b>) anterior commissure, anterior (ACA) and right piriform cortex (Rpir). <b>E.</b> Dissected (left) and intact (right) thalamic and hypothalamic areas including posterior thalamic nucleus (1), part of ventral posteromedial thalamic nucleus (2), ventromedial thalamic nucleus (3), dorsomedial hypothalamic nucleus (4) and arcuate hypothalamic nucleus (5). Homotopical intact areas are outlined with dashed red lines. Tissues were stained with Toluidine Blue. Scale bar  = 250 µm. DCU ID = 50 µm; vacuum pulse duration: 100 ms; <b>F.</b> Representative microdissection of middle molecular layer of the dentate gyrus (a) and cellular layer of the subiculum (b) from fresh frozen nonstained mouse brain sections (20 µm thickness). Abbreviations: CA1–CA1 area of hippocampus; Sub – subiculum; DG – dentate gyrus; hf – hippocampal fissure. Scale bar  = 100 µm.</p