Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

A loss-of-function screen for siRNAs that arrest human cells in metaphase reveals genes involved in mitotic spindle integrity

Customization of the lysis and reaction setup steps of FastLane Cell kits for an automated workflow

Automated workflows can be optimized for enhanced flexibility by determining the steps within the protocol that tolerate extended storage of assay components. We determined the tolerated stopping points and storage conditions in a multiplex real-time RT-PCR workflow using the FastLane Cell Multiplex Kit. Breaks were introduced within the procedure and the effect of incubation times and temperatures on the observed CT values were measured. Using these parameters, we provide guidance on adapting the protocol to enable an automated workflow. We also suggest steps for achieving optimal results when using FastLane Cell kits with TaqMan Gene Expression Assays

Establishment of an automated process based on FastLane technology for screening of chemically modified siRNAs

Optimizing the conditions for transfecting siRNA is a laborious and time-consuming process that involves testing many different parameters. The combination of FastLane technology and a Beckman Coulter robotic system enabled automated screening of transfection conditions, significantly shortening the time to establish protocols for automated “high-content” siRNA screening. This novel method is of great benefit to applications such as identification of potent chemically modified siRNAs for therapeutic purposes

The lysosomal transmembrane protein 9B regulates the activity of inflammatory signaling pathways.

The intracellular signaling pathway by which tumor necrosis factor (TNF) induces its pleiotropic actions is well characterized and includes unique components as well as modules shared with other signaling pathways. In addition to the currently known key effectors, further molecules may however modulate the biological response to TNF. In our attempt to characterize novel regulators of the TNF signaling cascade, we have identified transmembrane protein 9B (TMEM9B, c11orf15) as an important component of TNF signaling and a module shared with the interleukin 1beta (IL-1beta) and Toll-like receptor (TLR) pathways. TMEM9B is a glycosylated protein localized in membranes of the lysosome and partially in early endosomes. The expression of TMEM9B is required for the production of proinflammatory cytokines induced by TNF, IL-1beta, and TLR ligands but not for apoptotic cell death triggered by TNF or Fas ligand. TMEM9B is essential in TNF activation of both the NF-kappaB and MAPK pathways. It acts downstream of RIP1 and upstream of the MAPK and IkappaB kinases at the level of the TAK1 complex. These findings indicate that TMEM9B is a key component of inflammatory signaling pathways and suggest that endosomal or lysosomal compartments regulate these pathways

Basal and induced sphingosine kinase 1 activity in A549 carcinoma cells: function in cell survival and IL-1beta and TNF-alpha induced production of inflammatory mediators.

Sphingosine-1-phosphate, a lipid mediator produced by sphingosine kinases, regulates diverse cellular processes, ranging from cell growth and survival to effector functions, such as proinflammatory mediator synthesis. Using human A549 epithelial lung carcinoma cells as a model system, we observed transient upregulation of sphingosine kinase type 1 (SPHK1) enzyme activity upon stimulation with both TNF-alpha or IL-1beta. This transient activation of SPHK1 was found to be required for cytokine-induced COX-2 transcription and PGE2 production, since not only specific siRNA (abolishing both basal and induced SPHK1 enzyme activity), but also a dominant-negative SPHK1 mutant (suppressing induced SPHK1 activity only) both reduced COX-2 and PGE2. Furthermore, TNF-alpha- or IL-1beta-induced transcription of selected cytokines, chemokines, and adhesion molecules (IL-6, RANTES, MCP-1, and VCAM-1) was found to require SPHK1 activation. Suppression of SPHK1 activation led to reduction of cytokine-induced IkappaBalpha phosphorylation and consequently diminished NFkappaB activity due to reduced nuclear translocation of RelA (p65), explaining the dependence of inflammatory mediator production on SPHK1 activation. Inhibition of basal SPHK1 activity by N,N-dimethylsphingosine or by downregulation of its expression using siRNA induced spontaneous apoptosis in A549 cells, an effect that can be explained through interference with constitutive NFkappaB activity in this cell type. In contrast, expression of the dominant-negative mutant did not induce apoptosis. Taken together, these findings demonstrate a role of SPHK1 activation in proinflammatory signalling and of SPHK1 basal activity in survival of A549 lung carcinoma cells

Assessing the function of human UNC-93B in Toll-like receptor signaling and major histocompatibility complex II response.

The high sequence identity observed between UNC-93B of mouse and human imply common evolutionary ancestors and a conserved function. A nonconservative point mutation in the mouse Unc93b1 gene has been associated with defective Toll-like receptor (TLR) signaling and impaired major histocompatibility complex (MHC) I and II restricted antigen responses. Like murine UNC-93B, the human homologue is predicted to form 12 transmembrane domains, and it localizes to the endoplasmic reticulum. In human beings its expression is highest in professional antigen-presenting cells such as dendritic cells and macrophages. Interestingly, UNC-93B itself is specifically induced by TLR3 signaling in monocyte-derived dendritic cells and macrophages. To study the effect of UNC-93B deficiency in TLR signaling and antigen-presentation in human beings, UNC-93B message was knocked down in monocyte-derived dendritic cells and a reduced TNFalpha production in response to TLR3 agonists was observed. In the same experiment, the achieved knockdown had no effect on an MHC II-dependent antigen response, suggesting that the reduced quantity of human UNC-93B was still capable of supporting class II antigen presentation or that UNC-93B is not required for class II antigen presentation in human antigen-presenting cells

siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain.

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressant drugs that increase the extracellular levels of serotonin by blocking the reuptake activity of the serotonin transporter (SERT). Although SSRIs elevate brain serotonergic neurotransmission acutely, their full therapeutic effects involve neurochemical adaptations that emerge following chronic drug administration. The adaptive downregulation of SERT has recently been implicated in the therapeutic response of SSRIs. Interestingly, studies using SERT-knockout mice reveal somewhat paradoxical depression-related effects, probably specific to the downregulation of SERT during early development. However, the behavioral significance of SSRI-mediated downregulation of SERT during adulthood is still unknown. We investigated whether somatic gene manipulation, triggered by infusing short interfering RNA (siRNA) into the ventricular system, would enable the downregulation of SERT in the adult mouse brain. Infusing the SERT-targeting siRNA, for 2 weeks, significantly reduced the mRNA levels of SERT in raphe nuclei. Further, a significant, specific and widespread downregulation of SERT-binding sites was achieved in the brain. In contrast, 2-week infusion of the SSRI, citalopram, produced a widespread downregulation of SERT-binding sites, independent of any alterations at the mRNA level. Irrespective of their mechanisms for downregulating SERT in the brain, infusions of SERT-siRNA or citalopram elicited a similar antidepressant-related behavioral response in the forced swim test. These results signify a role for the downregulation of SERT in mediating the antidepressant action of SSRIs in adults. Further, these data demonstrate that siRNA-induced widespread knockdown of gene expression serves as a powerful tool for assessing the function of endogenous genes in the adult brain

Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference.

Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions

The tyrosine kinase BMX is an essential mediator of inflammatory arthritis in a kinase-independent manner

The inflammatory cytokine TNF plays a central role in autoimmune disorders such as rheumatoid arthritis (RA). We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of the TNF signaling pathway in genome-wide siRNA screens. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β or TLR agonists. BMX was required for full phosphorylation of the p38 MAPK, ERK and JNK. The following epistasis analysis indicated that BMX acts downstream or at the same level as the complex of TGFβ activated kinase 1 (TAK1) and TAK1 binding protein (TAB). At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology (PH) domain of BMX and that could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter in vitro required the catalytic activity of BMX. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component inflammatory cytokine signaling and that catalytic, as well as non-catalytic functions of BMX are involved

Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders.

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions