Molecular Profiling of Ulcerative Colitis Subjects from the TURANDOT Trial Reveals Novel Pharmacodynamic/Efficacy Biomarkers.

BACKGROUND AND AIMS: To define pharmacodynamic and efficacy biomarkers in ulcerative colitis [UC] patients treated with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody, in the TURANDOT study. METHODS: Transcriptome, proteome and immunohistochemistry data were generated in peripheral blood and intestinal biopsies from 357 subjects in the TURANDOT study. RESULTS: In peripheral blood, C-C motif chemokine receptor 9 [CCR9] gene expression demonstrated a dose-dependent increase relative to placebo, but in inflamed intestinal biopsies CCR9 gene expression decreased with increasing PF-00547659 dose. Statistical models incorporating the full RNA transcriptome in inflamed intestinal biopsies showed significant ability to assess response and remission status. Oncostatin M [OSM] gene expression in inflamed intestinal biopsies demonstrated significant associations with, and good accuracy for, efficacy, and this observation was confirmed in independent published studies in which UC patients were treated with infliximab or vedolizumab. Compared with the placebo group, intestinal T-regulatory cells demonstrated a significant increase in the intermediate 22.5-mg dose cohort, but not in the 225-mg cohort. CONCLUSIONS: CCR9 and OSM are implicated as novel pharmacodynamic and efficacy biomarkers. These findings occur amid coordinated transcriptional changes that enable the definition of surrogate efficacy biomarkers based on inflamed biopsy or blood transcriptomics data.ClinicalTrials.gov identifierNCT01620255.

De Novo Protein Synthesis Mediated by the Eukaryotic Elongation Factor 2 Is Required for the Anxiolytic Effect of Oxytocin.

BACKGROUND: The neuropeptide oxytocin (OXT) mediates its actions, including anxiolysis, via its G protein-coupled OXT receptor. Within the paraventricular nucleus of the hypothalamus (PVN), OXT-induced anxiolysis is mediated, at least in part, via activation of the mitogen-activated protein kinase pathway following calcium influx through transient receptor potential cation channel subfamily V member 2 channels. In the periphery, OXT activates eukaryotic elongation factor 2 (eEF2), an essential mediator of protein synthesis. METHODS: In order to study whether OXT activates eEF2 also in neurons to exert its anxiolytic properties in the PVN, we performed in vivo and cell culture experiments. RESULTS: We demonstrate that OXT, in a protein kinase C-dependent manner, activates eEF2 both in a hypothalamic cell line and in vivo within the PVN. Next, we reveal that OXT stimulates de novo protein synthesis, while inhibition of protein synthesis within the PVN prevents the anxiolytic effect of OXT in male rats. Moreover, activation of eEF2 within the PVN conveyed an anxiolytic effect supporting a role of OXT-induced eEF2 activation and protein synthesis for its anxiolysis. Finally, we show that one of the proteins that is upregulated by OXT is the neuropeptide Y receptor 5. Infusion of a specific neuropeptide Y receptor 5 agonist into the PVN consequently led to decreased anxiety-related behavior, while pretreatment with a neuropeptide Y receptor 5 antagonist prevented the anxiolytic effect of OXT. CONCLUSIONS: Taken together, these results show that OXT recruits several intracellular signaling cascades to induce protein synthesis, which mediates the anxiolytic effects of OXT within the PVN and suggests that eEF2 represents a novel target for anxiety-related disorders.

Discovering in vivo cytokine-eQTL interactions from a lupus clinical trial.

BACKGROUND: Cytokines are critical to human disease and are attractive therapeutic targets given their widespread influence on gene regulation and transcription. Defining the downstream regulatory mechanisms influenced by cytokines is central to defining drug and disease mechanisms. One promising strategy is to use interactions between expression quantitative trait loci (eQTLs) and cytokine levels to define target genes and mechanisms. RESULTS: In a clinical trial for anti-IL-6 in patients with systemic lupus erythematosus, we measure interferon (IFN) status, anti-IL-6 drug exposure, and whole blood genome-wide gene expression at three time points. We show that repeat transcriptomic measurements increases the number of cis eQTLs identified compared to using a single time point. We observe a statistically significant enrichment of in vivo eQTL interactions with IFN status and anti-IL-6 drug exposure and find many novel interactions that have not been previously described. Finally, we find transcription factor binding motifs interrupted by eQTL interaction SNPs, which point to key regulatory mediators of these environmental stimuli and therefore potential therapeutic targets for autoimmune diseases. In particular, genes with IFN interactions are enriched for ISRE binding site motifs, while those with anti-IL-6 interactions are enriched for IRF4 motifs. CONCLUSIONS: This study highlights the potential to exploit clinical trial data to discover in vivo eQTL interactions with therapeutically relevant environmental variables.

Mucosal Gene Expression in Pediatric and Adult Patients With Ulcerative Colitis Permits Modeling of Ideal Biopsy Collection Strategy for Transcriptomic Analysis.

Background: Transcriptional profiling has been performed on biopsies from ulcerative colitis patients. Limitations in prior studies include the variability introduced by inflammation, anatomic site of biopsy, extent of disease, and medications. We sought to more globally understand the variability of gene expression from patients with ulcerative colitis to advance our understanding of its pathogenesis and to guide clinical study design. Methods: We performed transcriptional profiling on 13 subjects, including pediatric and adult patients from 2 hospital sites. For each patient, we collected 6 biopsies from macroscopically inflamed tissue and 4 biopsies from macroscopically healthy-appearing tissue. Isolated RNA was used for microarray gene expression analysis utilizing Affymetrix Human Primeview microarrays. Ingenuity pathway analysis was used to assess over-representation of gene ontology and biological pathways. RNAseq was also performed, and differential analysis was assessed to compare affected vs unaffected samples. Finally, we modeled the minimum number of biopsies required to reliably detect gene expression across different subject numbers. Results: Transcriptional profiles co-clustered independently of the hospital collection site, patient age, sex, and colonic location, which parallels prior gene expression findings. A small set of genes not previously described was identified. Our modeling analysis reveals the number of biopsies and patients per cohort to yield reliable results in clinical studies. Conclusions: Key findings include concordance, including some expansion, of previously published gene expression studies and similarity among different age groups. We also established a reliable statistical model for biopsy collection for future clinical studies.

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

Anti-MAdCAM Antibody Increases ß7+ T Cells and CCR9 Gene Expression in the Peripheral Blood of Patients With Crohn's Disease.

OBJECTIVE: To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn's disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody. METHODS: In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, ß7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing. RESULTS: A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [-87% to -98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for ß7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for ß7+ effector memory T cells [placebo, 8%; PF-00547659, 84-138%] and ß7+ naïve T cells [8%; 13-50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in ß7+ T cells. CONCLUSIONS: Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes.

QuickMIRSeq: a pipeline for quick and accurate quantification of both known miRNAs and isomiRs by jointly processing multiple samples from microRNA sequencing.

BACKGROUND: Genome-wide miRNA expression data can be used to study miRNA dysregulation comprehensively. Although many open-source tools for microRNA (miRNA)-seq data analyses are available, challenges remain in accurate miRNA quantification from large-scale miRNA-seq dataset. We implemented a pipeline called QuickMIRSeq for accurate quantification of known miRNAs and miRNA isoforms (isomiRs) from multiple samples simultaneously. RESULTS: QuickMIRSeq considers the unique nature of miRNAs and combines many important features into its implementation. First, it takes advantage of high redundancy of miRNA reads and introduces joint mapping of multiple samples to reduce computational time. Second, it incorporates the strand information in the alignment step for more accurate quantification. Third, reads potentially arising from background noise are filtered out to improve the reliability of miRNA detection. Fourth, sequences aligned to miRNAs with mismatches are remapped to a reference genome to further reduce false positives. Finally, QuickMIRSeq generates a rich set of QC metrics and publication-ready plots. CONCLUSIONS: The rich visualization features implemented allow end users to interactively explore the results and gain more insights into miRNA-seq data analyses. The high degree of automation and interactivity in QuickMIRSeq leads to a substantial reduction in the time and effort required for miRNA-seq data analysis.

Tofacitinib attenuates pathologic immune pathways in patients with psoriasis: A randomized phase 2 study.

BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. OBJECTIVE: We sought to elucidate the molecular mechanisms underlying the clinical efficacy of tofacitinib in patients with psoriasis. METHODS: Twelve patients with plaque psoriasis were randomized (3:1) to receive 10 mg of tofacitinib or placebo twice daily for 12 weeks. Biopsy specimens were taken from nonlesional (baseline) and lesional (baseline, days 1 and 3, and weeks 1, 2, 4, and 12) skin. Biopsy specimens were examined for psoriatic epidermal features (thickness, Ki67(+) keratinocytes and keratin 16 [KRT16] mRNA expression, and phosphorylated signal transducer and activator of transcription [pSTAT](+) nuclei) and T-cell and dendritic cell (DC) subsets by using immunohistochemistry, and mRNA transcripts were quantified by using a microarray. RESULTS: In lesional skin keratinocyte pSTAT1 and pSTAT3 staining was increased at baseline but reduced after 1 day of tofacitinib (baseline, median of 1290 pSTAT1(+) cells/µm(2); day 1, median of 332 pSTAT1(+) cells/µm(2); and nonlesional, median of 155 pSTAT1(+) cells/µm(2)). Epidermal thickness and KRT16 mRNA expression were significantly and progressively reduced after days 1 and 3 of tofacitinib administration, respectively (eg, KRT16 decreased 2.74-fold, day 3 vs baseline, P = .016). Decreases in DC and T-cell numbers were observed after weeks 1 and 2, respectively. At week 4, significant decreases in IL-23/TH17 pathways were observed that persisted through week 12. Improvements in clinical and histologic features were strongly associated with changes in expression of psoriasis-related genes and reduction in IL-17 gene expression. CONCLUSIONS: Tofacitinib has a multitiered response in patients with psoriasis: (1) rapid attenuation of keratinocyte Janus kinase/STAT signaling; (2) removal of keratinocyte-induced cytokine signaling, leading to reductions in pathologic DC and T-cell numbers to nonlesional levels; and (3) inhibition of the IL-23/TH17 pathway.

QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization.

BACKGROUND: RNA sequencing (RNA-seq), a next-generation sequencing technique for transcriptome profiling, is being increasingly used, in part driven by the decreasing cost of sequencing. Nevertheless, the analysis of the massive amounts of data generated by large-scale RNA-seq remains a challenge. Multiple algorithms pertinent to basic analyses have been developed, and there is an increasing need to automate the use of these tools so as to obtain results in an efficient and user friendly manner. Increased automation and improved visualization of the results will help make the results and findings of the analyses readily available to experimental scientists. RESULTS: By combing the best open source tools developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we have implemented QuickRNASeq, a pipeline for large-scale RNA-seq data analyses and visualization. The QuickRNASeq workflow consists of three main steps. In Step #1, each individual sample is processed, including mapping RNA-seq reads to a reference genome, counting the numbers of mapped reads, quality control of the aligned reads, and SNP (single nucleotide polymorphism) calling. Step #1 is computationally intensive, and can be processed in parallel. In Step #2, the results from individual samples are merged, and an integrated and interactive project report is generated. All analyses results in the report are accessible via a single HTML entry webpage. Step #3 is the data interpretation and presentation step. The rich visualization features implemented here allow end users to interactively explore the results of RNA-seq data analyses, and to gain more insights into RNA-seq datasets. In addition, we used a real world dataset to demonstrate the simplicity and efficiency of QuickRNASeq in RNA-seq data analyses and interactive visualizations. The seamless integration of automated capabilites with interactive visualizations in QuickRNASeq is not available in other published RNA-seq pipelines. CONCLUSION: The high degree of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption.

Comparison of stranded and non-stranded RNA-seq transcriptome profiling and investigation of gene overlap.

BACKGROUND: While RNA-sequencing (RNA-seq) is becoming a powerful technology in transcriptome profiling, one significant shortcoming of the first-generation RNA-seq protocol is that it does not retain the strand specificity of origin for each transcript. Without strand information it is difficult and sometimes impossible to accurately quantify gene expression levels for genes with overlapping genomic loci that are transcribed from opposite strands. It has recently become possible to retain the strand information by modifying the RNA-seq protocol, known as strand-specific or stranded RNA-seq. Here, we evaluated the advantages of stranded RNA-seq in transcriptome profiling of whole blood RNA samples compared with non-stranded RNA-seq, and investigated the influence of gene overlaps on gene expression profiling results based on practical RNA-seq datasets and also from a theoretical perspective. RESULTS: Our results demonstrated a substantial impact of stranded RNA-seq on transcriptome profiling and gene expression measurements. As many as 1751 genes in Gencode Release 19 were identified to be differentially expressed when comparing stranded and non-stranded RNA-seq whole blood samples. Antisense and pseudogenes were significantly enriched in differential expression analyses. Because stranded RNA-seq retains strand information of a read, we can resolve read ambiguity in overlapping genes transcribed from opposite strands, which provides a more accurate quantification of gene expression levels compared with traditional non-stranded RNA-seq. In the human genome, it is not uncommon to find genomic loci where both strands encode distinct genes. Among the over 57,800 annotated genes in Gencode release 19, there are an estimated 19 % (about 11,000) of overlapping genes transcribed from the opposite strands. Based on our whole blood mRNA-seq datasets, the fraction of overlapping nucleotide bases on the same and opposite strands were estimated at 2.94 % and 3.1 %, respectively. The corresponding theoretical estimations are 3 % and 3.6 %, well in agreement with our own findings. CONCLUSIONS: Stranded RNA-seq provides a more accurate estimate of transcript expression compared with non-stranded RNA-seq, and is therefore the recommended RNA-seq approach for future mRNA-seq studies.

Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226.

Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4(+)FOXP3(+)Helios(+) thymic-derived Tregs and CD4(+)FOXP3(+)Helios(-) T cells, followed by comparison with CD4(+)FOXP3(-)Helios(-) T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4(+)CD25(+)CD127(lo/-) T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226(+)TIGIT(-) population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.

Modeling the clinical phenotype of BTK inhibition in the mature murine immune system.

Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.

Brown fat determination and development from muscle precursor cells by novel action of bone morphogenetic protein 6.

Brown adipose tissue (BAT) plays a pivotal role in promoting energy expenditure by the virtue of uncoupling protein-1 (UCP-1) that differentiates BAT from its energy storing white adipose tissue (WAT) counterpart. The clinical implication of "classical" BAT (originates from Myf5 positive myoblastic lineage) or the "beige" fat (originates through trans-differentiation of WAT) activation in improving metabolic parameters is now becoming apparent. However, the inducers and endogenous molecular determinants that govern the lineage commitment and differentiation of classical BAT remain obscure. We report here that in the absence of any forced gene expression, stimulation with bone morphogenetic protein 6 (BMP6) induces brown fat differentiation from skeletal muscle precursor cells of murine and human origins. Through a comprehensive transcriptional profiling approach, we have discovered that two days of BMP6 stimulation in C2C12 myoblast cells is sufficient to induce genes characteristic of brown preadipocytes. This developmental switch is modulated in part by newly identified regulators, Optineurin (Optn) and Cyclooxygenase-2 (Cox2). Furthermore, pathway analyses using the Causal Reasoning Engine (CRE) identified additional potential causal drivers of this BMP6 induced commitment switch. Subsequent analyses to decipher key pathway that facilitates terminal differentiation of these BMP6 primed cells identified a key role for Insulin Like Growth Factor-1 Receptor (IGF-1R). Collectively these data highlight a therapeutically innovative role for BMP6 by providing a means to enhance the amount of myogenic lineage derived brown fat.

The molecular basis of the cooperation between EGF, FGF and eCB receptors in the regulation of neural stem cell function.

Adult neurogenesis relies on EGF and FGF receptor (EGFR/FGFR) function and endocannabinoid (eCB) signalling. Here we have used a neural stem cell (NSC) line to determine how these systems cooperate to regulate neurogenesis. The results show the EGFR to be solely responsible for maintaining PI3K activation explaining its dominant role in promoting NSC survival. The EGFR and FGFR synergistically regulate the ERK/MAPK pathway, and this explains the requirement for both for optimal cell proliferation. The eCB receptors did not contribute to activation of the PI3K or ERK/MAPK pathways, highlighting the importance of another major proliferation pathway. The EGFR plays the dominant role in maintaining the transcriptome, with significant changes in the expression of over 3500 transcripts seen within hours of inhibition or activation of this receptor. The FGFR has a more modest effect on transcription with evidence for nodal integration with EGFR signalling at the level of the ERK/MAPK pathway. A common set of transcripts are regulated by the CB1 and CB2 receptors, with cooperation between these receptors and the EGFR apparent in the regulation of a pool of transcripts, most likely representing signal integration downstream from an as yet to be identified node. Finally, a first level molecular analysis of the transcriptional response shows regulation of a number of key growth factors, growth factor receptors and GPCRs to be under the control of the EGFR.

Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo.

Foxp3(+) CD4(+) T helper cells called regulatory T (T reg) cells play a key role in controlling reactivity to self-antigens and onset of autoimmunity. T reg cells either arise in thymus and are called natural T reg (nT reg) cells or are generated in the periphery through induction of Foxp3 and are called inducible T reg (iT reg) cells. The relative contributions of iT reg cells and nT reg cells in peripheral tolerance remain unclear as a result of an inability to separate these two subsets of T reg cells. Using a combination of novel TCR transgenic mice with a defined self-antigen specificity and conventional mouse models, we demonstrate that a cell surface molecule, neuropilin-1 (Nrp-1), is expressed at high levels on nT reg cells and can be used to separate nT reg versus iT reg cells in certain physiological settings. In addition, iT reg cells generated through antigen delivery or converted under homeostatic conditions lack Nrp-1 expression. Nrp-1(lo) iT reg cells show similar suppressive activity to nT reg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings. Collectively, our data show that Nrp-1 provides an excellent marker to distinguish distinct T reg subsets and will be useful in studying the role of nT reg versus iT reg cells in different disease settings.

Temporal proteomic profile of memory consolidation in the rat hippocampal dentate gyrus.

Information storage in the brain depends on the ability of neurons to alter synaptic connectivity within key circuitries such as the hippocampus. Memory-associated synaptic plasticity is mediated by a temporal cascade of de novo protein synthesis and altered protein processing. Here, we have used two-dimensional difference in gel electrophoresis (2-D DIGE) to investigate memory-specific protein changes in the hippocampal dentate gyrus at increasing times following spatial learning. We identified 42 proteins that were significantly regulated in the first 24 h of spatial memory consolidation. Two distinct waves of protein expression regulation were evident, at 3 and 12 h post-learning and this is in agreement with studies employing inhibitors of global translation. Functional classification of the memory-associated proteins revealed that the majority of regulated proteins contributed either to cellular structure or cellular metabolism. For example, actins, tubulins and intermediate filament proteins, core proteins of the three major cytoskeletal components, were dynamically regulated at times that suggest a role in memory-associated synaptic reorganization. Increased proteasome-mediated protein degradation was evident in the early post-training period including the down-regulation of phosphoprotein enriched in astrocytes 15 kDa, a key inhibitor of extracellular signal-regulated kinase signaling. Some of the most substantial protein expression changes were observed for secreted carrier proteins including transthyretin and serum albumin at 6-12 h post-learning, regulations that could serve an important role in increasing the supply of retinoic acid and thyroid hormone, key synaptic plasticity-promoting signals in the adult brain. Together these observations provide further insight into protein level regulations occurring in the hippocampus during spatial memory consolidation.

Dynamic changes in the microRNA expression profile reveal multiple regulatory mechanisms in the spinal nerve ligation model of neuropathic pain.

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3'-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.

Temporal dysregulation of cortical gene expression in the isolation reared Wistar rat.

The critical sequence of molecular, neurotransmission and synaptic disruptions that underpin the emergence of psychiatric disorders like schizophrenia remain to be established with progress only likely using animal models that capture key features of such disorders. We have related the emergence of behavioural, neurochemical and synapse ultrastructure deficits to transcriptional dysregulation in the medial prefrontal cortex of Wistar rats reared in isolation. Isolation reared animals developed sensorimotor deficits at postnatal day 60 which persisted into adulthood. Analysis of gene expression prior to the emergence of the sensorimotor deficits revealed a significant disruption in transcriptional control, notably of immediate early and interferon-associated genes. At postnatal day 60 many gene transcripts relating particularly to GABA transmission and synapse structure, for example Gabra4, Nsf, Syn2 and Dlgh1, transiently increased expression. A subsequent decrease in genes such as Gria2 and Dlgh2 at postnatal day 80 suggested deficits in glutamatergic transmission and synapse integrity, respectively. Microdialysis studies revealed decreased extracellular glutamate suggesting a state of hypofrontality while ultrastructural analysis showed total and perforated synapse complement in layer III to be significantly reduced in the prefrontal cortex of postnatal day 80 isolated animals. These studies provide a molecular framework to understand the developmental emergence of the structural and behavioural characteristics that may in part define psychiatric illness.

Sonic Hedgehog signaling in astrocytes is dependent on p38 mitogen-activated protein kinase and G-protein receptor kinase 2.

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh-regulated gene targets, Gli-1 and Patched in a cyclopamine- and forskolin-sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular-signal-regulated kinases or c-jun N-terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh-dependent up-regulation of Gli-1, inter-alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin-like growth factor 2 or a novel Shh target, membrane-associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively-active mutant we show that Shh signaling to p38 MAPK and subsequent Gli-1 transcription requires G-protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G-protein receptor kinase 2-dependent p38 MAPK activity in regulating Shh-mediated gene transcription in astrocytes.

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities.

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.