Mark W. Barnett

Somatic retrotransposition alters the genetic landscape of the human brain

Retrotransposons are mobile genetic elements that use a germline ‘copy-and-paste’ mechanism to spread throughout metazoan genomes. At least 50 per cent of the human genome is derived from retrotransposons, with three active families (L1, Alu and SVA) associated with insertional mutagenesis and disease. Epigenetic and post-transcriptional suppression block retrotransposition in somatic cells, excluding early embryo development and some malignancies. Recent reports of L1 expression and copy number variation in the human brain suggest that L1 mobilization may also occur during later development. However, the corresponding integration sites have not been mapped. Here we apply a high-throughput method to identify numerous L1, Alu and SVA germline mutations, as well as 7,743 putative somatic L1 insertions, in the hippocampus and caudate nucleus of three individuals. Surprisingly, we also found 13,692 somatic Alu insertions and 1,350 SVA insertions. Our results demonstrate that retrotransposons mobilize to protein-coding genes differentially expressed and active in the brain. Thus, somatic genome mosaicism driven by retrotransposition may reshape the genetic circuitry that underpins normal and abnormal neurobiological processes.

Signalling by glial cell line-derived neurotrophic factor (GDNF) requires heparan sulphate glycosaminoglycan

Glial cell line-derived neurotrophic factor, GDNF, is vital to the development and maintenance of neural tissues; it promotes survival of sympathetic, parasympathetic and spinal motor neurons during development, protects midbrain dopaminergic neurons from apoptosis well enough to be a promising treatment for Parkinsons disease, and controls renal and testicular development. Understanding how GDNF interacts with its target cells is therefore a priority in several fields. Here we show that GDNF requires glycosaminoglycans as well as the already-known components of its receptor complex, c-Ret and GFRα-1. Without glycosaminoglcyans, specifically heparan sulphate, c-Ret phosphorylation fails and GDNF cannot induce axonogenesis in neurons, in PC-12 cells, or scatter of epithelial cells. Furthermore, exogenous heparan sulphate inhibits rather than assists GDNF signalling. The involvement of heparan sulphates in GDNF signalling raises the possibility that modulation of heparan expression may modulate signalling by GDNF in vivo.

A gene expression atlas of the domestic pig

BackgroundThis work describes the first genome-wide analysis of the transcriptional landscape of the pig. A new porcine Affymetrix expression array was designed in order to provide comprehensive coverage of the known pig transcriptome. The new array was used to generate a genome-wide expression atlas of pig tissues derived from 62 tissue/cell types. These data were subjected to network correlation analysis and clustering.ResultsThe analysis presented here provides a detailed functional clustering of the pig transcriptome where transcripts are grouped according to their expression pattern, so one can infer the function of an uncharacterized gene from the company it keeps and the locations in which it is expressed. We describe the overall transcriptional signatures present in the tissue atlas, where possible assigning those signatures to specific cell populations or pathways. In particular, we discuss the expression signatures associated with the gastrointestinal tract, an organ that was sampled at 15 sites along its length and whose biology in the pig is similar to human. We identify sets of genes that define specialized cellular compartments and region-specific digestive functions. Finally, we performed a network analysis of the transcription factors expressed in the gastrointestinal tract and demonstrate how they sub-divide into functional groups that may control cellular gastrointestinal development.ConclusionsAs an important livestock animal with a physiology that is more similar than mouse to man, we provide a major new resource for understanding gene expression with respect to the known physiology of mammalian tissues and cells. The data and analyses are available on the websites http://biogps.org and http://www.macrophages.com/pig-atlas.

Synaptic Ras GTPase activating protein regulates pattern formation in the trigeminal system of mice

The development of ordered connections or “maps” within the nervous system is a common feature of sensory systems and is crucial for their normal function. NMDA receptors are known to play a key role in the formation of these maps; however, the intracellular signaling pathways that mediate the effects of glutamate are poorly understood. Here, we demonstrate that SynGAP, a synaptic Ras GTPase activating protein, is essential for the anatomical development of whisker-related patterns in the developing somatosensory pathways in rodent forebrain. Mice lacking SynGAP show only partial segregation of barreloids in the thalamus, and thalamocortical axons segregate into rows but do not form whisker-related patches. In cortex, layer 4 cells do not aggregate to form barrels. In Syngap+/− animals, barreloids develop normally, and thalamocortical afferents segregate in layer 4, but cell segregation is retarded. SynGAP is not necessary for the development of whisker-related patterns in the brainstem. Immunoelectron microscopy for SynGAP from layer 4 revealed a postsynaptic localization with labeling in developing postsynaptic densities (PSDs). Biochemically, SynGAP associates with the PSD in a PSD-95-independent manner, and Psd-95−/− animals develop normal barrels. These data demonstrate an essential role for SynGAP signaling in the activity-dependent development of whisker-related maps selectively in forebrain structures indicating that the intracellular pathways by which NMDA receptor activation mediates map formation differ between brain regions and developmental stage.

Involvement of Protein Kinase A in Patterning of the Mouse Somatosensory Cortex

Patterning of the mouse somatosensory cortex is unusually evident because of the presence of a “barrel field.” Presynaptic serotonin and postsynaptic glutamate receptors regulate barrel formation, but little is known of the intracellular signaling pathways through which they act. To determine whether protein kinase A (PKA) plays a role in the development of the barrel field, we examined five viable PKA subunit-specific knock-out (KO) mouse lines for barrel field abnormalities. Barrels are present in these mice, but those lacking the RIIβ subunit display significantly reduced contrast between the cell densities of barrel hollows and sides compared with wild-type animals. Thalamocortical afferent segregation in the posterior medial barrel subfield appeared normal, suggesting a postsynaptic site of gene action for the RIIβ protein. Immunoelectron microscopy confirmed that RIIβ was selectively localized to dendrites and dendritic spines. Mice lacking RIIβ show reduced glutamate receptor A (GluRA) subunit insertion into the postsynaptic density in postnatal day 7 somatosensory cortex; however, GluRA KO mice developed normal barrels. Our results clearly demonstrate a role for postsynaptic PKA signaling pathways in barrel differentiation. They also demonstrate a clear dissociation between the regulation of GluRA trafficking by PKA and its role in barrel formation. Finally, although a role for PKA downstream of cAMP cannot be ruled out, these data suggest that PKA may not be the principle downstream target because none of the mutants showed a barrelless phenotype similar to that observed in adenylate cyclase type 1 KO mice. These results give insight into activity-dependent mechanisms that regulate barrel formation.

SynGAP isoforms exert opposing effects on synaptic strength

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5′ rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing.

Analysis of the transcriptional networks underpinning the activation of murine macrophages by inflammatory mediators

Macrophages respond to the TLR4 agonist LPS with a sequential transcriptional cascade controlled by a complex regulatory network of signaling pathways and transcription factors. At least two distinct pathways are currently known to be engaged by TLR4 and are distinguished by their dependence on the adaptor molecule MyD88. We have used gene expression microarrays to define the effects of each of three variables—LPS dose, LPS versus IFN‐β and ‐γ, and genetic background—on the transcriptional response of mouse BMDMs. Analysis of correlation networks generated from the data has identified subnetworks or modules within the macrophage transcriptional network that are activated selectively by these variables. We have identified mouse strain‐specific signatures, including a module enriched for SLE susceptibility candidates. In the modules of genes unique to different treatments, we found a module of genes induced by type‐I IFN but not by LPS treatment, suggesting another layer of complexity in the LPS‐TLR4 signaling feedback control. We also observe that the activation of the complement system, in common with the known activation of MHC class 2 genes, is reliant on IFN‐γ signaling. Taken together, these data further highlight the exquisite nature of the regulatory systems that control macrophage activation, their likely relevance to disease resistance/susceptibility, and the appropriate response of these cells to proinflammatory stimuli.

USP18 restricts PRRSV growth through alteration of nuclear translocation of NF-κB p65 and p50 in MARC-145 cells

Although the functions of porcine respiratory and reproductive syndrome virus (PRRSV) proteins are increasingly understood, the roles of host factors in modifying infection are less well understood. Growing evidence places deubiquitination at the core of a multitude of regulatory processes, ranging from cell growth to innate immune response and health, such as cancer, degenerative and infectious diseases. This report provides further information on the functional role of the porcine ubiquitin-specific peptidase 18 (USP18) during innate immune responses to PRRSV. We have shown that constitutive overexpression of the porcine USP18 in MARC-145 cells restricts PRRSV growth, at least in part via early activation of NF-κB. Viral growth of PRRSV may be perturbed by increasing and decreasing nuclear translocation of p65 and p50, respectively. Our data highlight USP18 as a host restriction factor during innate immune response to PRRSV.

Modelling the Structure and Dynamics of Biological Pathways

There is a need for formalised diagrams that both summarise current biological pathway knowledge and support modelling approaches that explain and predict their behaviour. Here, we present a new, freely available modelling framework that includes a biologist-friendly pathway modelling language (mEPN), a simple but sophisticated method to support model parameterisation using available biological information; a stochastic flow algorithm that simulates the dynamics of pathway activity; and a 3-D visualisation engine that aids understanding of the complexities of a system’s dynamics. We present example pathway models that illustrate of the power of approach to depict a diverse range of systems.

Pathways to Barrel Development

Understanding the cellular mechanism by which glutamate receptors mediate changes in neuronal phenotype is key to understanding activity-dependent development of the nervous system. The primary somatosensory cortex (S1) of rodents offers a unique opportunity to identify key molecules that regulate glutamate-dependent cortical development because of its unique cytoarchitectonic structures in layer 4 termed “barrels”. Analysis of knockout mice has revealed that both NMDA receptors, and metabotropic glutamate receptor 5 (mGluR5) activation of phospholipase C-β1 (PLC-β1), are necessary for normal barrel development (Erzurumlu and Kind, 2001). Over the last several years, we have been using analysis of barrel cortex development in knockout (KO) mice to identify the signalling pathways downstream of glutamate receptors that regulate cortical development. This approach has been greatly helped by the isolation and characterisation of proteins associated with the postsynaptic density (Walikonis et al., 2000; (Husi et al., 2000). To date we have analysed more than 35 mice with selective deletion of key PSD components. Two of these mutants, those lacking Syngap (Barnett et al., 2006) and those lacking the RIIβ subunit of PKA (PKARIIβ-/-, Watson et al., in press), also showed defects in barrel development. This chapter reviews the principle cellular processes involved in barrel development. It also reviews the current state of knowledge of the intracellular signalling pathways, initiated by glutamate neurotransmission, that regulate barrel development, with specific focus on SynGAP and mGluR5 activation of PLC-β1. Finally, we examine how the analysis of mutant mice has increased our knowledge about the cellular processes that underlie barrel development.