Neuregulin 1 and susceptibility to schizophrenia

To access full text version of this article. Please click on the hyperlink "View/Open" at the bottom of this pageThe cause of schizophrenia is unknown, but it has a significant genetic component. Pharmacologic studies, studies of gene expression in man, and studies of mouse mutants suggest involvement of glutamate and dopamine neurotransmitter systems. However, so far, strong association has not been found between schizophrenia and variants of the genes encoding components of these systems. Here, we report the results of a genomewide scan of schizophrenia families in Iceland; these results support previous work, done in five populations, showing that schizophrenia maps to chromosome 8p. Extensive fine-mapping of the 8p locus and haplotype-association analysis, supplemented by a transmission/disequilibrium test, identifies neuregulin 1 (NRG1) as a candidate gene for schizophrenia. NRG1 is expressed at central nervous system synapses and has a clear role in the expression and activation of neurotransmitter receptors, including glutamate receptors. Mutant mice heterozygous for either NRG1 or its receptor, ErbB4, show a behavioral phenotype that overlaps with mouse models for schizophrenia. Furthermore, NRG1 hypomorphs have fewer functional NMDA receptors than wild-type mice. We also demonstrate that the behavioral phenotypes of the NRG1 hypomorphs are partially reversible with clozapine, an atypical antipsychotic drug used to treat schizophrenia

Particulate matter exposure during pregnancy is associated with birth weight, but not gestational age, 1962-1992: a cohort study

<p>Abstract</p> <p>Background</p> <p>Exposure to air pollutants is suggested to adversely affect fetal growth, but the evidence remains inconsistent in relation to specific outcomes and exposure windows.</p> <p>Methods</p> <p>Using birth records from the two major maternity hospitals in Newcastle upon Tyne in northern England between 1961 and 1992, we constructed a database of all births to mothers resident within the city. Weekly black smoke exposure levels from routine data recorded at 20 air pollution monitoring stations were obtained and individual exposures were estimated via a two-stage modeling strategy, incorporating temporally and spatially varying covariates. Regression analyses, including 88,679 births, assessed potential associations between exposure to black smoke and birth weight, gestational age and birth weight standardized for gestational age and sex.</p> <p>Results</p> <p>Significant associations were seen between black smoke and both standardized and unstandardized birth weight, but not for gestational age when adjusted for potential confounders. Not all associations were linear. For an increase in whole pregnancy black smoke exposure, from the 1^st(7.4 μg/m³) to the 25^th(17.2 μg/m³), 50^th(33.8 μg/m³), 75^th(108.3 μg/m³), and 90^th(180.8 μg/m³) percentiles, the adjusted estimated decreases in birth weight were 33 g (SE 1.05), 62 g (1.63), 98 g (2.26) and 109 g (2.44) respectively. A significant interaction was observed between socio-economic deprivation and black smoke on both standardized and unstandardized birth weight with increasing effects of black smoke in reducing birth weight seen with increasing socio-economic disadvantage.</p> <p>Conclusions</p> <p>The findings of this study progress the hypothesis that the association between black smoke and birth weight may be mediated through intrauterine growth restriction. The associations between black smoke and birth weight were of the same order of magnitude as those reported for passive smoking. These findings add to the growing evidence of the harmful effects of air pollution on birth outcomes.</p

Comparison of Strong Cation Exchange and SDS-PAGE Fractionation for Analysis of Multiprotein Complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels

Enriched classes of proteins repressed by KSHV miRNAs.

<p>A list of the 5% most repressed proteins (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003584#ppat-1003584-g001" target="_blank">Fig. 1C</a>) was analyzed for the most enriched networks of interacting gene products using Metacore (GeneGo). This is a similar analysis to gene ontology term enrichment analysis.</p

Comparison of Strong Cation Exchange and SDS-PAGE Fractionation for Analysis of Multiprotein Complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. While affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes pre-fractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Pre-fractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data shows that pre-fractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels

Proteomic screening for KSHV miRNA targets.

<p>(A) Experimental design shows HUVECs transfected with control or KSHV miRNAs, then labeled with stable isotope-labeled amino acids (normal/light “L”, medium-heavy “M”, and heavy “H”), cells from both conditions were combined and LC-MS/MS was used to measure relative abundance of peptides corresponding to the labeled amino acids. Green proteins symbolize proteins that were translated before the amino acid labeling and/or do not contain stable isotope-labeled amino acids. (B) Argonaute2 (AGO2) was immunoprecipitated from HUVECs. Western blot shows unbound lysate (flow-through, “FT”) and immunoprecipitated material (IP) probed with AGO2 antibody. Graph shows RT-PCR miRNA data from AGO2-immunoprecipiated material from at least three immunoprecipitations per sample from either KSHV-infected HUVECs (black) or HUVECs transfected with miRNA mimics (gray) as in pSILAC assay. (C). Known miRNA targets (TWEAKR and BCLAF1) are repressed when 16 miRNA mimics are co-transfected. Shown is two-color quantitative Western blot analysis from three biological replicates. (D) Range of relative changes in protein expression of all proteins detected with at least two peptides per protein and found in two biological replicates. (E) Table shows the most repressed proteins in the KSHV miRNA samples. Protein levels were determined by pulsed SILAC and mRNA levels were determined by microarray.</p

Proteomic Screening of Human Targets of Viral microRNAs Reveals Functions Associated with Immune Evasion and Angiogenesis

<div><p>Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.</p></div

KSHV miRNAs increase HIF1α and HMOX1 protein levels.

<p>(A) Western blot analysis of cells transfected with miRNAs and exposed to hypoxia (blots above, quantitation below). (B) Luciferase reporters containing hypoxia responsive elements (HRE-luc) or parental control (ctl-luc) in the promoter were transfected into 293 cells with miRNAs and exposed to hypoxia (1% oxygen for 16 hr.). Luciferase activity was normalized to an internal control reporter as well as the condition without hypoxia and transfected with the negative control miRNA. (C) Cells were treated as in (A) and HIF1α mRNA was measured using qPCR. (D) The same samples used in (A) were analyzed by Western blot analysis for proteins shown. Data were analyzed and presented as in (A). (E) Cells were transfected with KSHV miRNAs or controls and analyzed for BACH1 and HMOX1 protein levels using Western blot analysis. Average relative protein expression changes are shown with error bars representing S.D. from ≥3 biological replicates. Asterisks denote P<0.05 using a T-test.</p

Validation of predicted miRNA targets with Western blotting and <i>de novo</i> infection.

<p>(A) Primary HUVECs were transfected without miRNAs (no), a negative control miRNA (ctl), or KSHV miRNAs. Whole cells lysates were analyzed using Western blotting and normalized to actin (loading control) and the negative control miRNA (ctl). MirVana miRNAs mimics for miR-K8 in STAT3 assays are shown. Other mirVana miRNA mimic results are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003584#ppat.1003584.s002" target="_blank">Figure S2</a>. Average relative protein expression changes are shown with error bars showing S.D. from ≥3 biological replicates. (B) Primary HUVECs were <i>de novo</i> infected with KSHV (3 or 7 days post infection) and whole cell lysates were analyzed using Western blot analysis as in (A). Asterisks denote P<0.05, n≥3 using a T-test. (C) Plot showing average changes in protein expression on the horizontal axis from SILAC data and mRNA changes from microarray data (vertical axis) from the same transfections. Gray open circles are gene products found in both assays and black filled circles represent gene products from six validated targets (HMGCS1, STAT3, GRB2, ROCK2, AKAP9, TSPAN3). Multiple microarray probes are indicated for a subset of genes, yielding multiple vertically-aligned circles from multiple microarray probes, but one protein measurement (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003584#ppat-1003584-g001" target="_blank">Figure 1E</a> for examples).</p

Analysis of miRNA seed-matching sites.

<p>(A) Proteins identified in screen were analyzed for KSHV miRNA seed-matching sites in their corresponding 3′UTRs using TargetScan. Histogram shows the distribution of the number of sites per 3′UTR. (B) Graph displays the fraction of proteins whose transcripts contain no or at least one seed-matching site in the transcripts of proteins with indicated repression levels in the presence of KSHV miRNAs. (C–D) Empirical cumulative distribution graph showing protein expression changes whose transcripts contain at least one miRNA seed-matching site (C) or classes of multiple sites (D).</p