Controlling the Outcome of the Toll-Like Receptor Signaling Pathways

The Toll-Like Receptors (TLRs) are proteins involved in the immune system that increase cytokine levels when triggered. While cytokines coordinate the response to infection, they appear to be detrimental to the host when reaching too high levels. Several studies have shown that the deletion of specific TLRs was beneficial for the host, as cytokine levels were decreased consequently. It is not clear, however, how targeting other components of the TLR pathways can improve the responses to infections. We applied the concept of Minimal Cut Sets (MCS) to the ihsTLR v1.0 model of the TLR pathways to determine sets of reactions whose knockouts disrupt these pathways. We decomposed the TLR network into 34 modules and determined signatures for each MCS, i.e. the list of targeted modules. We uncovered 2,669 MCS organized in 68 signatures. Very few MCS targeted directly the TLRs, indicating that they may not be efficient targets for controlling these pathways. We mapped the species of the TLR network to genes in human and mouse, and determined more than 10,000 Essential Gene Sets (EGS). Each EGS provides genes whose deletion suppresses the network's outputs

Monoallelic Expression of Multiple Genes in the CNS

The inheritance pattern of a number of major genetic disorders suggests the possible involvement of genes that are expressed from one allele and silent on the other, but such genes are difficult to detect. Since DNA methylation in regulatory regions is often a mark of gene silencing, we modified existing microarray-based assays to detect both methylated and unmethylated DNA sequences in the same sample, a variation we term the MAUD assay. We probed a 65 Mb region of mouse Chr 7 for gene-associated sequences that show two distinct DNA methylation patterns in the mouse CNS. Selected genes were then tested for allele-specific expression in clonal neural stem cell lines derived from reciprocal F1 (C57BL/6×JF1) hybrid mice. In addition, using a separate approach, we directly analyzed allele-specific expression of a group of genes interspersed within clusters of OlfR genes, since the latter are subject to allelic exclusion. Altogether, of the 500 known genes in the chromosomal region surveyed, five show monoallelic expression, four identified by the MAUD assay (Agc1, p (pink-eyed dilution), P4ha3 and Thrsp), and one by its proximity to OlfR genes (Trim12). Thrsp (thyroid hormone responsive SPOT14 homolog) is expressed in hippocampus, but the human protein homolog, S14, has also been implicated in aggressive breast cancer. Monoallelic expression of the five genes is not coordinated at a chromosome-wide level, but rather regulated at individual loci. Taken together, our results suggest that at least 1% of previously untested genes are subject to allelic exclusion, and demonstrate a dual approach to expedite their identification

Schizophrenia susceptibility alleles are enriched for alleles that affect gene expression in adult human brain

It is widely thought that alleles that influence susceptibility to common diseases, including schizophrenia, will frequently do so through effects on gene expression. As only a small proportion of the genetic variance for schizophrenia has been attributed to specific loci, this remains an unproven hypothesis. The International Schizophrenia Consortium (ISC) recently reported a substantial polygenic contribution to that disorder, and that schizophrenia risk alleles are enriched among single-nucleotide polymorphisms (SNPs) selected for marginal evidence for association (P<0.5) from genome-wide association studies (GWAS). It follows that if schizophrenia susceptibility alleles are enriched for those that affect gene expression, those marginally associated SNPs, which are also expression quantitative trait loci (eQTLs), should carry more true association signals compared with SNPs that are not marginally associated. To test this, we identified marginally associated (P<0.5) SNPs from two of the largest available schizophrenia GWAS data sets. We assigned eQTL status to those SNPs based upon an eQTL data set derived from adult human brain. Using the polygenic score method of analysis reported by the ISC, we observed and replicated the observation that higher probability cis-eQTLs predicted schizophrenia better than those with a lower probability for being a cis-eQTL. Our data support the hypothesis that alleles conferring risk of schizophrenia are enriched among those that affect gene expression. Moreover, our data show that notwithstanding the likely developmental origin of schizophrenia, studies of adult brain tissue can, in principle, allow relevant susceptibility eQTLs to be identified

Nucleic acid chaperone properties of ORF1p from the non-LTR retrotransposon, LINE-1

Long interspersed element-1 (LINE-1 or L1) is a non-long terminal repeat (LTR) retrotransposon that has amplified to hundreds of thousands of copies in mammalian evolution. A small number of the individual copies of L1 are active retrotransposons which are presently replicating in most species, including humans and mice. L1 retrotransposition begins with transcription of an active element and ends with a newly inserted cDNA copy, a process which requires the two element-encoded proteins to act in cis on the L1 RNA. The ORF1 protein (ORF1p) is a high-affinity, non-sequence-specific RNA binding protein with nucleic acid chaperone activity, whereas the ORF2 protein (ORF2p) supplies the enzymatic activities for cDNA synthesis. This article reviews the nucleic acid chaperone properties of ORF1p in the context of L1 retrotransposition

1.4 The Cerebral Tricarboxylic Acid Cycles

We review the operation of the cerebral tricarboxylic acid (TCA) cycles in the neuronal and glial compartments of the adult rat brain, with an emphasis on the mechanisms underlying intercellular oxidative coupling during glutamatergic neurotransmission. We begin with an update of the enzymatic properties, gene location, regulation, and regional distribution of the enzymes involved. Then, we describe the main methodologies used to investigate TCA cycle activity in vitro and in vivo such as autoradiography, positron emission tomography (PET), nuclear magnetic resonance (NMR) imaging or spectroscopy, and dual photon fluorescence microscopy. Previous interpretations conceived cerebral glucose metabolism during glutamatergic neurotransmission as a coupled process, involving exclusively anaerobic metabolism in the astrocytes and oxidative metabolism in the neurons. The glutamine cycle was proposed to be stoichiometrically coupled to astrocytic glucose uptake, glutamine synthesis being supported by astrocytic glycolysis only and glutamine being the main precursor of cerebral glutamate. Compelling evidences have accumulated since then, showing that astrocytes display significant oxidative capacity in vivo, more than 60% of the glutamine is produced from ATP synthesized by astroglial oxidative phosphorylation, and approximately 40% of cerebral glutamate is not derived from glutamine. Together, these findings suggest that the coupling mechanisms between astrocytic and neuronal oxidative and nonoxidative metabolisms are more complex than initially envisioned. In this review, we propose a novel mechanism based on the operation of intracellular redox switches and the transcellular coupling of the NAD(P)/NAD (P)H redox states between both cell types through lactate transfers. The redox switch/redox coupling hypothesis is compatible with the simultaneous operation of glycolytic and oxidative metabolisms in both neural cell types. Transcellular redox coupling through lactate transfers mimics the intracellular coupling existing between cytosolic NADH production and mitochondrial NADH oxidation, as seen from the redox shuttles exchanging reducing equivalents through the inner mitochondrial membrane of neural cells.This work was supported in part by grants SAF 2001‐224, SAF 2004‐03197, FISss C03/08, G03/155, C03/10 and PI051530 to S.C. JUSTESA IMAGEN S.A. provided the core support of LISMAR during this work. T.B. R was supported by a fellowship from Fundaçâo para a Ciência e Tecnologia, Portugal (SFRH/BD/5407/2001).Peer reviewe